U.S. patent application number 15/699556 was filed with the patent office on 2018-08-09 for angiogenically effective unit dose of fgf and method of administering.
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 W. Michael Kavanaugh, Martha J. Whitehouse.
Application Number | 20180221446 15/699556 |
Document ID | / |
Family ID | 35733125 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180221446 |
Kind Code |
A1 |
Whitehouse; Martha J. ; et
al. |
August 9, 2018 |
Angiogenically Effective Unit Dose Of FGF And Method Of
Administering
Abstract
The present invention provides a unit dose comprising 0.2
.mu.g/kg to 36 .mu.g/kg of a recombinant FGF or an angiogenically
active fragment or mutein thereof. Also provided is a
pharmaceutical composition comprising an angiogenically effective
dose of an FGF or an angiogenically active fragment or mutein
thereof, and a pharmaceutically acceptable carrier. Also provided
is a method for treating a human patient for coronary artery
disease, comprising administering into at least one coronary vessel
of said patient a safe and angiogenically effective dose of a
recombinant FGF of any of SEQ ID NOS: 1-3, 5, 8-10, or 12-14, or an
angiogenically active fragment or mutein thereof.
Inventors: |
Whitehouse; Martha J.; (San
Francisco, CA) ; Kavanaugh; W. Michael; (Mill Valley,
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: |
35733125 |
Appl. No.: |
15/699556 |
Filed: |
September 8, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14867645 |
Sep 28, 2015 |
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15699556 |
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14099143 |
Dec 6, 2013 |
9149507 |
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14867645 |
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12947939 |
Nov 17, 2010 |
8618052 |
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14099143 |
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12391956 |
Feb 24, 2009 |
7858584 |
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12947939 |
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11238936 |
Sep 29, 2005 |
7511019 |
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12391956 |
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10131965 |
Apr 25, 2002 |
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11238936 |
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09417721 |
Oct 13, 1999 |
6451303 |
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10131965 |
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60104103 |
Oct 13, 1998 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/726 20130101;
C07K 14/50 20130101; A61P 9/10 20180101; A61K 38/1825 20130101;
A61P 9/00 20180101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 31/726 20060101 A61K031/726; C07K 14/50 20060101
C07K014/50 |
Claims
1. A method for treating a human patient for coronary artery
disease, comprising administering into one or more coronary vessels
in a human patient in need of treatment for coronary artery disease
a therapeutically effective amount of an angiogenically active
fragment or angiogenically active mutein of a recombinant
fibroblast growth factor (FGF) having the sequence set forth in SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID
NO:13.
2. The method of claim 1, wherein said therapeutically effective
amount administered to said patient is a unit dose of about 0.008
mg to about 6.1 mg of said angiogenically active fragment or
angiogenically active mutein of said recombinant FGF.
3. The method of claim 2, wherein said therapeutically effective
amount administered to said patient is a unit dose of 0.3 mg to 3.5
mg of said angiogenically active fragment or angiogenically active
mutein of said recombinant FGF.
4. The method of claim 1, comprising administering into one or more
coronary vessels of said patient about 0.2 .mu.g/kg to about 36
.mu.g/kg of said angiogenically active fragment or angiogenically
active mutein of said recombinant FGF.
5. The method of claim 4, comprising administering into one or more
coronary vessels of said patient about 0.2 .mu.g/kg to about 2
.mu.g/kg of said angiogenically active fragment or angiogenically
active mutein of said recombinant FGF.
6. The method of claim 4, comprising administering into one or more
coronary vessels of said patient about 2 .mu.g/kg to about 20
.mu.g/kg of said angiogenically active fragment or angiogenically
active mutein of said recombinant FGF.
7. The method of claim 4, comprising administering into one or more
coronary vessels of said patient about 20 .mu.g/kg to about 36
.mu.g/kg of said angiogenically active fragment or angiogenically
active mutein of said recombinant FGF.
8. A method for treating a human patient for coronary artery
disease, comprising administering into one or more coronary vessels
in a human patient in need of treatment for coronary artery disease
a therapeutically effective amount of a recombinant fibroblast
growth factor (FGF) having the sequence set forth in SEQ ID NO: 10,
12, or 14 or an angiogenically active fragment or an angiogenically
active mutein thereof.
9. The method of claim 8, wherein said therapeutically effective
amount administered to said patient is a unit dose of about 0.008
mg to about 6.1 mg of said recombinant FGF or said angiogenically
active fragment or said angiogenically active mutein thereof.
10. The method of claim 9, wherein said therapeutically effective
amount administered to said patient is a unit dose of 0.3 mg to 3.5
mg of said recombinant FGF or said angiogenically active fragment
or said angiogenically active mutein thereof.
11. The method of claim 8, comprising administering into one or
more coronary vessels of said patient about 0.2 .mu.g/kg to about
36 .mu.g/kg of said recombinant FGF or said angiogenically active
fragment or said angiogenically active mutein thereof.
12. The method of claim 11, comprising administering into one or
more coronary vessels of said patient about 0.2 .mu.g/kg to about 2
.mu.g/kg of said recombinant FGF or said angiogenically active
fragment or said angiogenically active mutein thereof.
13. The method of claim 11, comprising administering into one or
more coronary vessels of said patient about 2 .mu.g/kg to about 20
.mu.g/kg of said recombinant FGF or said angiogenically active
fragment or said angiogenically active mutein thereof.
14. The method of claim 11, comprising administering into one or
more coronary vessels of said patient about 20 .mu.g/kg to about 36
.mu.g/kg of said recombinant FGF or said angiogenically active
fragment or said angiogenically active mutein thereof.
15. A method for inducing angiogenesis in the heart of a patient,
comprising administering into one or more coronary vessels of a
human patient in need of coronary angiogenesis a therapeutically
effective amount of a recombinant fibroblast growth factor (FGF)
having the sequence set forth in SEQ ID NO:3, 5, 10, 12, or 14 or
an angiogenically active fragment or an angiogenically active
mutein thereof, or a therapeutically effective amount of an
angiogenically active fragment or an angiogenically active mutein
of a recombinant fibroblast growth factor (FGF) having the sequence
set forth in SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:13.
16. The method of claim 15, wherein said therapeutically effective
amount administered to said patient is a unit dose of about 0.008
mg to about 6.1 mg of said recombinant FGF or said angiogenically
active fragment or said angiogenically active mutein thereof.
17. The method of claim 15, wherein said unit dose is administered
into two coronary vessels of said patient.
18. The method of claim 15, further comprising the step of
administering to said patient an effective amount of a
glycosaminoglycan within 30 minutes of administering said
recombinant FGF or said angiogenically active fragment or said
angiogenically active mutein thereof.
19-21. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/099,143, filed Dec. 6, 2013, which is a divisional of U.S.
application Ser. No. 12/947,939, filed Nov. 17, 2010, now U.S. Pat.
No. 8,618,052, which is a divisional of U.S. application Ser. No.
12/391,956, filed Feb. 24, 2009, now U.S. Pat. No. 7,858,584, which
is a divisional of U.S. application Ser. No. 11/238,936, filed Sep.
29, 2005, now U.S. Pat. No. 7,511,019, which is a continuation of
U.S. application Ser. No. 10/131,965, filed Apr. 25, 2002, now
abandoned, which is a continuation of U.S. application Ser. No.
09/417,721, filed Oct. 13, 1999, now U.S. Pat. No. 6,451,303, which
claims the benefit of U.S. Application Ser. No. 60/104,103, filed
Oct. 13, 1998, which applications are herein incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to a unit dose comprising
an FGF or an angiogenically active fragment or mutein thereof for
inducing cardiac angiogenesis in a human. The present invention is
also directed to a pharmaceutical composition comprising the unit
dose of the FGF and to a method for administering this
pharmaceutical composition to a human to induce cardiac
angiogenesis while minimizing systemic risk to the patient. The
present invention is useful because the unit dose, pharmaceutical
composition and the method for its administration provide an
alternative to surgical intervention for the treatment of coronary
artery disease (CAD) and may 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 (atherosclerosis) is a progressive
disease in humans wherein one or more coronary arteries gradually
become occluded through the buildup of plaque. 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, 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 preclude
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 a fibroblast growth
factor, such as of SEQ ID NOS:1-3, 5, 8-9, or 12-14 or an
angiogenically active fragment or mutein thereof, when administered
as a unit dose of about 0.2 .mu.g/kg to about 36 .mu.g/kg into one
or more coronary vessels (IC) of a human patient in need of
coronary angiogenesis, unexpectedly provides the human patient with
a rapid and therapeutic cardiac angiogenesis sufficient to obviate
surgical intervention and results in an unexpectedly superior
increase in the treated patient's exercise tolerance time (ETT). 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. By the term "cardiac angiogenesis"
or "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.
[0010] FGFs that are suitable for use in the present invention
include human FGF-1 (SEQ ID NO:1), bovine FGF-1 (SEQ ID NO:2),
human FGF-2 (SEQ ID NO:3), bovine FGF-2 (SEQ ID NO:5), human FGF-4
(SEQ ID NO:8), human FGF-5 (SEQ ID NO:9), human FGF-6 (SEQ ID
NO:10), human FGF-8 (SEQ ID NO:12), human FGF-9 (SEQ ID NO:13), and
human FGF-98 (SEQ ID NO:14). In one embodiment, FGF molecules are
human FGF-1 (SEQ ID NO:1), bovine FGF-1 (SEQ ID NO:2), human FGF-2
(SEQ ID NO:3), bovine FGF-2 (SEQ ID NO:5), human FGF-4 (SEQ ID
NO:8), and human FGF-5 (SEQ ID NO:9). In an alternative embodiment,
the FGF molecules are human FGF-6 (SEQ ID NO:10), murine FGF-8 (SEQ
ID NO:12), human FGF-9 (SEQ ID NO:13) or human FGF-98 (SEQ ID
NO:14).
[0011] Typically, the angiogenically active fragments of the
present invention retain the distal two thirds of the mature FGF
molecule (i.e., the two thirds of the molecule at the carboxy end
that have the cell binding sites). For convenience, the terms
"human FGF," "bovine FGF," and murine FGF are used herein in
abbreviated form as "hFGF," "bFGF," and "mFGF," respectively.
[0012] The Applicants also discovered that a single unit dose of an
FGF or an angiogenically active fragment thereof, when administered
as a unit dose into one or more coronary vessels (IC) of a human
patient in need of coronary angiogenesis (e.g., a human patient
with coronary artery disease despite optional medical management),
unexpectedly provides the human patient with a therapeutic benefit
that is seen as early as two weeks after the single unit dose is
administered (as reflected in symptoms), and that lasts at least 60
days after the single unit dose is administered (as reflected in
ETT and the "Seattle Angina Questionnaire" (SAQ)). For example,
when 28 human patients diagnosed as having CAD were assessed by the
SAQ both before and 57 days after being administered IC a single
unit dose of 0.33 .mu.g/kg to 48 .mu.g/kg of FGF-2 of SEQ ID NO:5,
the mean increase in their scores on the five criteria assessed
ranged from 13 to 36 points, which is about 1.6-4.5 times greater
than the 8 point change which was considered to be "clinically
significant" in alternative modes of treatment. See Table 2. When
the scores of the 15 first patients were broken down between those
receiving a low dose (less than 2 .mu.g/kg) and those receiving a
higher dose (greater than or equal to 2 .mu.g/kg) of FGF-2 of SEQ
ID NO:5, and assessed by the SAQ, both doses were found to provide
scores that had "clinically significant" increases ranging from
12.3 to 58.1 and 10.9 to 32.1, respectively. Thus, whether the
patients were administered the lower doses or the higher doses of
the invention, their increased scores were about 1.4-7.2 times
greater than the 8 point change which was considered to be
"clinically significant" in alternative modes of treatment. See
Table 3.
[0013] As part of this study, MRI was performed on 23 human
patients diagnosed with CAD to assess ejection fraction, regional
myocardial function and perfusion (delayed arrival zone). The
patients were administered IC a single unit dose of 0.33 .mu.g/kg
to 12 .mu.g/kg of FGF-2 of SEQ ID NO:5. Their cardiac and coronary
functions were objectively assessed by magnetic resonance imaging
(MRI) both before and after treatment. The MRI results demonstrated
significant improvement in regional wall motion (%) and wall
thickening (%) during systole. The results also showed a
significant reduction in the delayed arrival zone (% LV). The
results did not demonstrate any significant change in ejection
fraction (EF). Thus, the Applicants have demonstrated the clinical
efficacy in humans of a single unit dose of an FGF when
administered IC in accordance with the present invention.
[0014] Accordingly, in one aspect, the Applicants' invention is
directed to a unit dose of FGF comprising a safe and
therapeutically effective amount of an FGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or
mutein thereof. Typically, the safe and therapeutically effective
amount comprises about 0.2 .mu.g/kg to about 36 l.mu.g/kg of an FGF
of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an
angiogenically active fragment or mutein thereof. 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, 2.0 .mu.g/kg to
20 .mu.g/kg, or 20 .mu.g/kg to 36 .mu.g/kg of an FGF of any one of
SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active
fragment or mutein thereof. Expressed in absolute terms for the
majority of human CAD patients, the unit dose of the present
invention comprises 0.008 mg to 6.1 mg, more typically 0.3 mg to
3.5 mg, of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14
or an angiogenically active fragment or mutein thereof.
[0015] In another aspect, the present invention is directed to a
pharmaceutical composition comprising a safe and therapeutically
effective amount of an FGF or an angiogenically active fragment or
mutein thereof, and a pharmaceutically acceptable carrier.
Typically, the safe and therapeutically effective amount of an FGF
comprises about 0.2 .mu.g/kg to about 36 .mu.g/kg of an FGF of any
one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically
active fragment or mutein thereof, and a pharmaceutically
acceptable carrier. In other embodiments of the pharmaceutical
composition, the safe and therapeutically effective amount of an
FGF comprises 0.2 .mu.g/kg to 2 .mu.g/kg, 2 .mu.g/kg to 20
.mu.g/kg, or 20 .mu.g/kg to 36 .mu.g/kg of an FGF, such as an FGF
of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an
angiogenically active fragment or mutein thereof, and a
pharmaceutically acceptable carrier.
[0016] In yet another aspect, the present invention is directed to
a method of using the above described unit dose or pharmaceutical
composition to treat a human patient for CAD or to induce coronary
angiogenesis therein. The method comprises administering into one
or more coronary vessels 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 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. More typically, the
safe and therapeutically effective amount comprises about 0.2
.mu.g/kg to about 36 .mu.g/kg of an FGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14 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 20 .mu.g/kg, or
20 .mu.g/kg to 36 .mu.g/kg of the FGF of any one of SEQ ID NOS:1-3,
5, 8-10, or 12-14 or an angiogenically active fragment or mutein
thereof in a pharmaceutically acceptable carrier.
[0017] Because FGF is a glycosoaminoglycan (e.g., heparin) binding
protein and the presence of a glycosoaminoglycan (also known as a
"proteoglycan" or a "mucopolysaccharide") optimizes activity and
AUC, the IC dosages of the FGF of the present invention typically
are administered within 20 minutes of the IV administration of a
glycosoaminoglycan, such as a heparin.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is a plot of the mean rFGF-2 plasma concentration
versus time (hours) for six different doses of rFGF-2 (SEQ ID NO:5)
administered by IC infusion in humans over a 20 minute period. The
six doses of rFGF-2 in FIG. 1 are 0.33 .mu.g/kg, 0.65 .mu.g/kg, 2
.mu.g/kg, 6 .mu.g/kg, 12 .mu.g/kg, and 24 .mu.g/kg of lean body
mass (LBM).
[0019] FIG. 2 is a plot of each individual patient's rFGF-2 area
under the curve (AUC) in pghr/ml for FIG. 1 for the six doses of
rFGF-2, and shows the dose linearity of systemic rFGF-2 exposure
following IC infusion.
[0020] FIG. 3 is a plot of individual human patient rFGF-2 dose
normalized 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.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The Applicants have discovered that single dose of an FGF or
an angiogenically active fragment or mutein thereof, when
administered in a safe and therapeutically effective amount into
one or more coronary vessels 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 6 months before a further treatment is needed. In fact, the
Applicants' method for treating CAD, when assessed by the standard
objective criterion employed in the art (i.e., ETT), provided an
unexpectedly superior increase of one and a half to two minutes in
the treated patient's ETT. This compares exceptionally well when
compared to the increase of 30 seconds that is deemed clinically
significant for the current mode of treatment, i.e.,
angioplasty.
[0022] By the phrase "safe and therapeutically effective amount" as
used herein in relation to FGF is meant an amount of an FGF 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, with no other side
effects is considered "safe" for the purpose of this invention.
Typically, the safe and therapeutically effective amount of an FGF
comprises about 0.2 .mu.g/kg to about 36 .mu.g/kg of the FGF of any
one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically
active fragment or mutein thereof.
[0023] Accordingly, the present invention has multiple aspects. In
its first aspect, the present invention is directed to a unit dose
of the FGF medicament that has produced unexpectedly superior
results in treating CAD in humans when compared to angioplasty. In
particular, the unit dose comprises a safe and therapeutically
effective amount of an FGF or an angiogenically active fragment or
mutein thereof. Typically, the unit dose comprises about 0.2
.mu.g/kg to about 36 .mu.g/kg of the FGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or
mutein thereof. In other embodiments of the unit dose, the 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 20 .mu.g/kg, or about
20 .mu.g/kg to about 36 .mu.g/kg of the FGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or
mutein thereof. It is convenient to provide the unit dose of the
present invention in a formulation comprising in absolute terms
from 0.008 mg to 6.1 mg of the FGF of any one of SEQ ID NOS:1-3, 5,
8-10, or 12-14 or an angiogenically active fragment or mutein
thereof. In this embodiment, the unit dose contains a sufficient
amount of FGF to accommodate dosing any one of the majority of 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., 170 kg) at about the highest dosage (about 36 jg/kg). More
typically, the unit dose comprises 0.3 mg to 3.5 mg of the FGF of
any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically
active fragment or mutein thereof. The unit dose is typically
provided in solution or lyophilized form containing the above
referenced amount of FGF and an effective amount of one or more
pharmaceutically acceptable buffers, stabilizers and/or other
excipients as later described herein.
[0024] The active agent in the above described unit dose is a
recombinant FGF or an angiogenically active fragment or mutein
thereof. Typically, the active agent of the unit dose is the FGF of
any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14. More typically, the
active agent in the unit dose is hFGF-1 (SEQ ID NO:1), bFGF-1 (SEQ
ID NO:2), hFGF-2 (SEQ ID NO:3), bFGF-2 (SEQ ID NO:5), hFGF-4 (SEQ
ID NO:8) or hFGF-5 (SEQ ID NO:9). In an alternative embodiment, the
active agent in the unit dose is hFGF-6 (SEQ ID NO:10), mFGF-8 (SEQ
ID NO:12), hFGF-9 (SEQ ID NO:13) or hFGF-98 (SEQ ID NO:14).
[0025] The amino acid sequences and methods for making many of the
mammalian FGFs that are employed in the unit dose, pharmaceutical
composition and method of the present invention are well known in
the art. In particular, references disclosing the amino acid
sequence and recombinant expression of FGF 1-9 and FGF-98 are
discussed sequentially below.
[0026] FGF-1: The amino acid sequence of hFGF-1 (SEQ ID NO:1) and
its recombinant expression are disclosed in U.S. Pat. No. 5,604,293
(Fiddes), entitled "Recombinant Human Basic Fibroblast Growth
Factor," which issued on Feb. 18, 1997. See FIG. 2d of the '293
patent. This reference and all other references herein, whether
cited before or after this sentence, are expressly incorporated
herein by reference in their entirety. The amino acid sequence and
recombinant expression of bFGF-1 (SEQ ID NO:2) are also disclosed
in U.S. Pat. No. 5,604,293 (Fiddes) which has been incorporated
herein by reference. See, FIG. 1b of the '293 patent. Both hFGF-1
(SEQ ID NO:1) and bFGF-1 (SEQ ID NO:2) have 140 amino acid
residues. bFGF-1 differs from hFGF-1 at 19 residue positions:
5(Pro.fwdarw.Leu), 21 (His.fwdarw.Tyr), 31
(Tyr.fwdarw..fwdarw.Val), 35(Arg.fwdarw.Lys), 40(Gln.fwdarw.Gly),
45(Gln.fwdarw.Phe), 47(Ser.fwdarw.*Cys), 51(Tyr.fwdarw.Ile),
54(Tyr.fwdarw.Val), 64(Tyr.fwdarw.Phe), 80(Asn.fwdarw.Asp),
106(Asn.fwdarw.His), 109(Tyr.fwdarw.Val), 116(Ser.fwdarw.*Arg),
117(Cys.fwdarw.Ser), 119(Arg.fwdarw.Leu), 120(Gly.fwdarw.Glu),
125(Tyr.fwdarw.Phe), and 137(Tyr.fwdarw.Val). In most instances,
the differences are conserved. Further, the differences at residue
positions 116 and 119 merely interchange the position of the
Arg.
[0027] FGF-2: The amino acid sequence of hFGF-2 (SEQ ID NO:3) and
methods for its recombinant expression are disclosed in U.S. Pat.
No. 5,439,818 (Fiddes) entitled "DNA Encoding Human Recombinant
Basic Fibroblast Growth Factor," which issued on Aug. 8, 1995 (see
FIG. 4 therein), and in U.S. Pat. No. 5,514,566 (Fiddes), entitled
"Methods of Producing Recombinant Fibroblast Growth Factors," which
issued on May 7, 1996 (see FIG. 4 therein). The amino acid sequence
of bFGF-2 (SEQ ID NO:5) and various methods for its recombinant
expression are disclosed in U.S. Pat. No. 5,155,214, entitled
"Basic Fibroblast Growth Factor," which issued on Oct. 13, 1992.
When the 146 residue forms of hFGF-2 and bFGF-2 are compared, their
amino acid sequences are nearly identical with only two residues
that differ. In particular, in going from hFGF-2 to bFGF-2, the
sole differences occur at residue positions 112(Thr.fwdarw.Ser) and
128(Ser.fwdarw.Pro).
[0028] FGF-3: FGF-3 (SEQ ID NO:7) was first identified as an
expression product of a mouse int-2 mammary tumor and its amino
acid sequence is disclosed in Dickson et al., "Potential Oncogene
Product Related to Growth Factors," Nature 326:833 (Apr. 30, 1987).
FGF-3 (SEQ ID NO:7), which has 243 residues when the N-terminal Met
is excluded, is substantially longer than both FGF-1 (human and
bovine) and FGF-2 (human and bovine). A comparison of amino acid
residues for mFGF-3 (SEQ ID NO:7) relative to bFGF-1 (SEQ ID NO:2)
and bFGF-2 (SEQ ID NO:5) is presented in overlap fashion in Dickson
et al. (1987). When the amino acid sequence of mFGF-3 (SEQ ID NO:7)
is compared to bFGF-1 (SEQ ID NO 2): and bFGF-2 (SEQ ID NO:5),
FGF-3 has 5 locations containing residue inserts relative to both
FGF-1 and FGF-2. The most significant of these inserts is a 12 and
14 residue insert relative to FGF-2 and FGF-1, respectively,
beginning at residue position 135 of FGF-3. Allowing for the
inserts, Dickson discloses that mFGF-3 has 53 residue identities
relative to FGF-1 and 69 residue identities relative to FGF-2. In
addition, FGF-3 contains a hydrophobic N-terminal extension of 10
residues relative to the N-terminus of the signal sequence in both
FGF-1 and FGF-2. Relative to the C-terminus of bFGF-1 and bFGF-2,
mFGF-3 contains an approximately 60 residue extension. It is
unlikely that the C-terminal extension of mFGF-3 is necessary for
activity. More likely, it is a moderator of activity by conferring
receptor specificity on the FGF.
[0029] FGF-4: The amino acid sequence for the hst protein, now
known as hFGF-4 (SEQ ID NO:8), was first disclosed by 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).
Including its leader sequence, hFGF-4 (SEQ ID NO:8) has 206 amino
acid residues. When the amino acid sequences of hFGF-4 (SEQ ID
NO:7), hFGF-1 (SEQ ID NO:1), hFGF-2 (SEQ ID NO:3), and mFGF-3 (SEQ
ID NO:7) are compared, residues 72-204 of hFGF-4 have 43% homology
to hFGF-2 (SEQ ID NO:5); residues 79-204 have 38% homology to
hFGF-1 (SEQ ID NO:1); and residues 72-174 have 40% homology to
mFGF-3 (SEQ ID NO:7). A comparison of these four sequences in
overlap form is shown in Yoshida (1987) at FIG. 3. Further, the Cys
at residue positions 88 and 155 of hFGF-4 are highly conserved
among hFGF-1, hFGF-2, mFGF-3, and hFGF-4 and are found in a
homologous region.
[0030] The two putative cell binding sites of hFGF-2 (SEQ ID NO:3)
occur at residue positions 36-39 and 77-81 thereof. See Yoshida
(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 substantial similarity between the
amino acid sequences for human and bovine FGF-2, we would expect
the cell binding sites for bFGF-2 (SEQ ID NO:5) to also be at
residue positions 36-39 and 77-81 thereof, and the heparin binding
sites to be at residue positions 18-22 and 107-111 thereof. In
relation to hFGF-1 (SEQ ID NO:1), the putative cell binding sites
occur at residues 27-30 and 69-72, and the putative heparin binding
sites occur at residues 9-13 and 98-102. Insofar as bFGF-1 (SEQ ID
NO:2) has the identical amino acids at residue positions 9-13,
27-30, 69-72, and 98-102 as does hFGF-1 (SEQ ID NO:1), bFGF-1 would
be expected to have the same cell and heparin binding sites as does
hFGF-1.
[0031] FGF-5: The amino acid sequence and method for cloning hFGF-5
(SEQ ID NO: 15) are disclosed in Zhan, et al., "The Human FGF-5
Oncogene Encodes a Novel Protein Related to Fibroblast Growth
Factors," Molec. and Cell. Biol., 8(8):3487-3495 (August 1988). The
Applicants also sequenced the FGF-5 and obtained the amino acid
sequence of SEQ ID NO:9, which differed from Zhan's sequence at
residue position 236 (having a Lys instead of the Zhan's Asn) and
at residue position 243 (having a Pro instead of Zhan's Ser). Both
hFGF-5 (SEQ ID NO:9) and hFGF-5 (SEQ ID NO:15) have 266 amino acid
residues that include a leader sequence of 67 residues upstream of
the first residue of the FGF-2 of SEQ ID NO:5 and a tail sequence
that extends about 47 residues beyond the C-terminus of hFGF-2. A
comparison between the amino acid sequences of hFGF-1 (SEQ ID
NO:1), hFGF-2 (SEQ ID NO:3), mFGF-3 (SEQ ID NO:7), hFGF-4 (SEQ ID
NO:8), and FGF-5 (SEQ ID NO:9) is presented in FIG. 2 of Zhan
(1988). In FIG. 2 of Zhan, hFGF-1, hFGF-2, mFGF-3, and hFGF-4 are
identified as aFGF (i.e., acidic FGF), bFGF (i.e., basic FGF),
int-2, and hstKS3, respectively, i.e., by their original names. In
the above referenced comparison, two blocks of FGF-5 amino acid
residues (90 to 180 and 187-207) showed substantial homology to FGF
1-4, i.e., 50.4% with FGF-4, 47.5% with FGF-3, 43.4% with FGF-2 and
40.2% with hFGF-1. See Zhan (1988) at FIG. 2. U.S. Pat. No.
5,155,217 (Goldfarb) and U.S. Pat. No. 5,238,916 (Goldfarb), which
correspond to the Zhan publication, refer to the FGF-5 of Zhan as
FGF-3. However, the art (as evidenced by Coulier below) has come to
recognize that the hFGF of Zhan (and Goldfarb) as FGF-5 and not as
FGF-3. The two Goldfarb patents contain the same amino acid
sequence for hFGF-5 (SEQ ID NO: 15) as reported above by Zhan.
[0032] FGF-6: The amino acid sequence and method for cloning hFGF-6
(SEQ ID NO: 10) are disclosed in Coulier et al., "Putative
Structure of the FGF-6 Gene Product and Role of the Signal
Peptide," Oncogene 6:1437-1444 (1991). hFGF-6 is one of the largest
of the FGFs, having 208 amino acid residues. When the amino acid
sequences of human FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, and
FGF-7 are compared, there are strong similarities in the C-terminal
two-thirds of the molecules (corresponding e.g., to residues 78-208
of hFGF-6 (SEQ ID NO:10)). In particular, 23 residues, including
two cysteines (at positions 90-157 of hFGF-6 of SEQ ID NO:10) were
identical between the seven members of the family. This number
increases to 33 residues when conserved amino acid residues are
considered. The overall similarities between these seven human FGFs
range from 32 to 70% identical residues and 48 to 79% conserved
residues for the C-terminal two-thirds of the molecules. The
sequence comparisons, relative to FGF-6, are shown in Table 1
herein.
TABLE-US-00001 TABLE 1 Amino Acid Sequence Comparison of hFGF-6
With Other hFGFs Identical Conserved SEQ ID Identical Conserved
Residues* Residues** NO: Residues* Residues** (%) (%) hFGF-4 8 91
103 70 79 hFGF-5 9 64 82 49 63 hFGF-3 7 55 78 42 60 hFGF-2 3 54 69
42 53 hFGF-7 11 47 68 36 52 hFGF-1 1 42 62 32 48 *Number and
percentages of identical or conserved residues were calculated for
the C-terminal two-thirds of the hFGF6 molecule (residues 78-208).
**Conserved residues are defined according to the structure-genetic
matrix of Feng et al., J. Mol. Evol., 21: 112-125 (1985).
[0033] Referring to Table 1, FGF-6 has the highest correspondence
(91 identical residues/103 conserved residues) with FGF-4. This
amounts to 70% identical and 79% conserved residues. hFGF-6 (SEQ ID
NO: 10) differed most from hFGF-3 (SEQ ID NO:2); hFGF-2 (SEQ ID
NO:3), hFGF-7 (SEQ ID NO: 11), and FGF-1 (SEQ ID NO:1), with 42,
42, 36, and 32 identical residues, respectively.
[0034] An overlayed comparison of the amino acid sequences of FGFs
1-7 is shown in FIG. 3 of incorporated Coulier (1991). FIG. 3 of
Coulier shows that when the C-terminal two thirds of the FGF
molecules are aligned, there are 23 residue positions wherein the
residues from all seven FGF members are identical. There are also
ten residue positions wherein residues from all seven FGF members
are conserved. Coulier (1991) at FIG. 3. In combination, these
identical and conserved residues form about 6 locations of three to
five residues on the terminal two thirds of each of the FGFs 1-7,
wherein three to five residues are grouped together in all seven
species of human FGF (i.e., hFGF 1-7).
[0035] FGF-7: The amino acid sequence of hFGF-7 (SEQ ID NO: 11) is
disclosed in Miyamoto, et al., "Molecular Cloning of a Novel
Cytokine cDNA Encoding the Ninth Member of the Fibroblast Growth
Factor Family, Which Has a Unique Secretion Property," Mol. and
Cell. Biol. 13(7):4251-4259 (1993). In Miyamoto, the hFGF-7 was
referred to by its older name KGF. As reflected in SEQ ID NO:11,
FGF-7 has 191 amino acid residues. Miyamoto compared hFGF-7 (SEQ ID
NO: 11) to hFGF 1-6 and hFGF-9 shows that the carboxy terminal two
thirds of the FGF-7 has comparable homology with the distal two
thirds of the other members of the group. See Miyamoto (1993) at
page 4254 (FIG. 2).
[0036] FGF-8: The amino acid sequence of mFGF-8 (SEQ ID NO:12) and
a method for its recombinant expression are disclosed in Tanaka et
al., "Cloning and Characterization of an Androgen-induced Growth
Factor Essential for the Growth of Mouse Mammary Carcinoma Cells,"
PNAS USA, 89:8928-8932 (1992). The mFGF-8 of Tanaka has 215 amino
acid residues. MacArthur, et al., "FGF-8 Isoforms Activate Receptor
Splice Forms that Are Expressed in Mesenchymal Regions of Mouse
Development," Development, 121:3603-3613 (1995) discloses that
FGF-8 has 8 different isoforms that differ at the mature N-terminus
but that are identical over the C-terminal region. The 8 isoforms
arise because FGF-8 has 6 exons of which the first four (which
correspond to the first exon of most other FGF genes) result in
alternative splicing.
[0037] FGF-9: The amino acid sequence of hFGF-9 and a method for
its recombinant expression are disclosed in Santos-Ocampo, et al.,
"Expression and Biological Activity of Mouse Fibroblast Growth
Factor," J. Biol. Chem., 271(3):1726-1731 (1996). Notwithstanding
its title, Ocampo discloses the amino acid sequence of both hFGF-9
(SEQ ID NO:13) and mFGF-9. Both the human and murine FGF-9
molecules have 208 amino acid residues and sequences that differ by
only two residues. In particular, the hFGF-9 has Asn and Ser at
residues 9 and 34, respectively, whereas the mFGF-9 has Ser and
Asn, respectively. FGF-9 has complete preservation of the conserved
amino acids that define the FGF family. Santos-Ocampo (1996) at
page 1726. Half-maximal activation of FGF-9 is seen at 185 ng/ml
heparin, whereas half-maximal activation of FGF-1 is seen at 670
ng/ml heparin. Santos-Ocampo (1996) at page 1730. When compared to
FGF-1, both FGF-2 and FGF-9 require lower heparin concentrations
for optimal activity.
[0038] FGF-98: The amino acid sequence of hFGF-98 (SEQ ID NO:14)
and a method for its recombinant expression are disclosed in
provisional patent application Ser. No. 60/083,553 which is hereby
incorporated herein by reference in its entirety. hFGF-98, which is
also known as hFGF-18, has 207 amino acid residues. Thus, hFGF-6
(207 residues), hFGF-9 (208 residues), and hFGF-98 (207 residues)
are similar in size.
[0039] FGFs differentially bind to and activate one or more of four
related transmembrane receptors which in turn mediate a biological
response. The FGF receptors ("FGFR") are members of the tyrosine
kinase receptor superfamily. The extracellular domain of the FGFR
comprises 2-3 immunoglobulin-like ("IG-like") domains that are
differentially expressed as a result of alternative splicing.
Another alternative splicing event can also alter the sequence of
the carboxy-terminal half of the Ig-like domain III without
altering the reading frame. Santos-Ocampo (1996). The two splice
forms, which are referred to as "b" and "c", occur for FGFRs 1, 2,
3 but not 4. A more detailed description of the FGFR is found in
Mathieu, et al, "Receptor Binding and Mitogenic Properties of Mouse
Fibroblast Growth Factor 3," J. Biol. Chem., 270(41):24197-24203
(1995). The ability of FGF 1-9 to differentially stimulate FGFRs
was receptor dependent as reported by Ornitz et al., J. Biol.
Chem., 271(25):15292-15297 (1996). In Ornitz, the cell line BaF3
was divided into fractions and each fraction was transfected to
express one of the following FGF receptors: FGFR1b, FGFR1c, FGFR2b,
FGFR2c, FGFR3b, FGFR3c, and FGF4 (minus one Ig-like domain).
Thereafter, the transformed cell lines were exposed to one of FGF
1-9 (5 nM) and heparin (2 .mu.g/ml) as a cofactor. The mitogenic
response was then measured by incorporation of [.sup.3H] thymidine.
The results in cpm are as follows:
[0040] 1. FGFR1b: similar mitogenic responses were produced by
hFGF-1 (32,000 cpm) and hFGF-2 (28,000 cpm) with the next highest
responses by mFGF-3 (about 16,000 cpm) and hFGF-4 (15,000 cpm);
[0041] 2. FGFR1c: similar mitogenic responses were produced by
hFGF-1, hFGF-2, hFGF-4, hFGF-5, and hFGF-6 (about 36,000 cpm), with
mFGF-9 producing the only other significant response (about 19,000
cpm); 3. FGFR2b: best mitogenic responses were by hFGF-7 (14,000
cpm), hFGF-1 (12,500 cpm), and mFGF-3 (9,500 cpm);
[0042] 4. FGFR2c: best mitogenic responses were by hFGF-4 (21,000
cpm), mFGF-9 (20,000 cpm), hFGF-6 (16,500 cpm), hFGF-1 (16,000
cpm), hFGF-2 (14,500 cpm), hFGF-5 (9,500 cpm), and mFGF-8 (9,000
cpm);
[0043] 5. FGFR3b: mitogenic responses only by hFGF-1 (37,000 cpm)
and mFGF-9 (26,000 cpm);
[0044] 6. FGFR3c: best mitogenic responses by hFGF-1 (39,000 cpm),
hFGF-2 (34,000 cpm), hFGF-4 (33,000 cpm), mFGF-8 (32,500 cpm),
mFGF-9 (31,000 cpm), hFGF-5 (16,000 cpm), and hFGF-6 (13,000
cpm);
[0045] 7. FGFR4A: best mitogenic responses by hFGF-2 (29,000 cpm),
hFGF-4 and hFGF-6 (27,000 cpm), mFGF-8 (25,000 cpm), mFGF-1 (24,000
cpm), and hFGF-9 (20,000 cpm) with all others being 6,000 cpm or
less.
[0046] As reflected above, only FGF-1 induces a significant
mitogenic response in all of the receptors tested. Thus, FGF-1 can
be thought of as a universal ligand with N- and C-terminal
additions to the molecule giving rise to receptor specificity
associated with the other FGF. Given the potential for diverse
responses in vivo by systemically administered FGF, the present
invention minimizes the potential for systemic responses by
localized administration, and by discovering the appropriate dosage
for the localized administration, i.e., by administering a
therapeutically effective amount of an FGF into at least one
coronary artery of a patient in need of treatment for CAD.
[0047] In the Examples that follow, bFGF-2 (SEQ ID NO:5) was
administered in vivo to rats, pigs, and humans, and tested for
angiogenic activity. The bFGF-2 of the Examples was made as
described in U.S. Pat. No. 5,155,214. In the '214 patent, a DNA of
SEQ ID NO:4, which encodes a bFGF (hereinafter "FGF-2") of SEQ ID
NO:5, is inserted into a cloning vector, such as pBR322, pMB9, Col
E1, pCRI, RP4 or .lamda.-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:5. Although the FGF-2 of SEQ ID NO:5
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.
[0048] The FGF-2 of SEQ ID NO:5, which is of bovine origin, 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 compared to
the full-length 155-residue bovine FGF-2 of Abraham, Applicants'
FGF-2 of SEQ ID NO:5 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. As discussed above, the
FGF-2 of SEQ ID NO:5 differs from human FGF-2 in two residue
positions. In particular, the amino acids at residue positions 112
and 128 of the bFGF-2 of SEQ ID NO:5 are Ser and Pro, respectively,
whereas in hFGF-2, they are Thr and Ser, respectively. Given this
substantial structural identity, the in vivo clinical results
provided in the Examples and discussed elsewhere herein on bFGF-2
(SEQ ID NO:5) should be directly applicable to hFGF-2 (SEQ ID
NO:3).
[0049] The recombinant bFGF-2 (SEQ ID NO:5) of the Examples 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 was incorporated herein by reference in its
entirety. In particular, the first two steps employed in the
purification of the recombinant bFGF-2 of Applicants' unit dose 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 1.4 M and 1.95 M 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.
[0050] In addition to the FGF of any one of SEQ ID NOS:1-3, 5,
8-10, or 12-14, the active agent in the unit dose of the present
invention also comprises an "angiogenically active fragment
thereof." By the term "angiogenically active fragment thereof" is
meant a fragment of any one of the FGF of SEQ ID NOS:1-3, 5, 8-10,
or 12-14 that has about 80% of the residues sequence of SEQ ID NO:5
and that retains the angiogenic effect of the corresponding mature
FGF. A common truncation is the removal of the N-terminal
methionine, using well known techniques such as treatment with a
methionine aminopeptidase. A second desirable truncation comprises
the FGF without its leader sequence. Those skilled in the art
recognize the leader sequence as the series of hydrophobic residues
at the N-terminus of a protein that facilitate its passage through
a cell membrane but that are not necessary for activity and that
are not found on the mature protein.
[0051] Preferred truncations are determined relative to hFGF-2 (or
the analogous bFGF-2). As a general rule, the amino acid sequence
of an FGF is aligned with FGF-2 to obtain maximum homology.
Portions of the FGF that extend beyond the corresponding N-terminus
of the aligned FGF-2 (SEQ ID NO:3) are suitable for deletion
without adverse effect. Likewise, portions of the FGF that extend
beyond the C-terminus of the aligned FGF-2 (SEQ ID NO:3) are also
capable of being deleted without adverse effect.
[0052] Fragments of FGF that are smaller than those described above
are also within the scope of the present invention so long as they
retain the cell binding portions of FGF and at least one heparin
binding segment. As already discussed above, the heparin binding
segments of FGF-2 (human or bovine) occur at residues 18-22 and
107-111, whereas the cell binding portions occur at residues 36-39
and 77-81. For example, it is well known in the art that N-terminal
truncations of bFGF-2 do not eliminate its angiogenic activity in
cows. In particular, the art discloses several naturally occurring
and biologically active fragments of bFGF-2 of SEQ ID NO:5 that
have N-terminal truncations relative to the bFGF-2 of SEQ ID NO:5.
An active and truncated bFGF-2 having residues 12-146 of SEQ ID
NO:5 was found in bovine liver and another active and truncated
bFGF-2, having residues 16-146 of SEQ ID NO:5 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:5 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 bFGF-2 (SEQ ID NO:5)
and one of the heparin binding segments (residues 107-111).
Accordingly, the angiogenically active fragments of an FGF
typically encompass those terminally truncated fragments of an FGF
that when aligned to an FGF-2 to maximize homology, have at least
residues that correspond to residues 30-110 of bFGF-2 of SEQ ID
NO:5 (or the hFGF-2 of SEQ ID NO:3); more typically, at least
residues that correspond to residues 18-146 of bFGF-2 of SEQ ID
NO:5.
[0053] The unit dose of the present invention also comprises an
"angiogenically active . . . mutein" of the FGF of any one of SEQ
ID NOS:1-3, 5, 8-10, or 12-14. By the term "angiogenically active .
. . mutein" is meant a mutated form of the FGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14 that structurally retains at least 80%,
preferably 90%, of the residues of any one of SEQ ID NOS:1-3, 5,
8-10, or 12-14 in their respective positions, and that functionally
retains the angiogenic activity of the FGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14. Preferably, the mutations are
"conservative substitutions" using L-amino acids, wherein one amino
acid is replaced by another biologically similar amino acid.
Examples of conservative substitutions include the substitution of
one hydrophobic residue such as Ile, Val, Leu, Pro, or Gly for
another, or PheTyr, SerThr, or the substitution of one polar
residue for another, such as between Arg and Lys, between Glu and
Asp, or between Gln and Asn, and the like. Generally, the charged
amino acids are considered interchangeable with one another.
However, to make the substitution more conservative, one takes into
account both the size and the likeness of the charge, if any, on
the side chain. Other suitable substitutions include the
substitution of serine for one or both of the cysteines at residue
positions which are not involved in disulfide formation, such as
residues 87 and 92 in hFGF-2 (SEQ ID NO:3) or bFGF-2 (SEQ ID NO:5).
Preferably, substitutions are introduced at the N-terminus, which
is not associated with angiogenic activity. However, as discussed
above, conservative substitutions are suitable for introduction
throughout the molecule.
[0054] One skilled in the art, using art known techniques, is able
to make one or more point mutations in the DNA encoding an FGF of
any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 to obtain expression
of an FGF polypeptide mutein (or fragment mutein) having angiogenic
activity for use within the unit dose, compositions and method of
the present invention. To prepare an angiogenically active mutein
of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14, 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), to introduce one or more
point mutations into the cDNA that encodes the FGF of any one of
SEQ ID NOS:1-3, 5, 8-10, or 12-14.
[0055] In a second aspect, the present invention is directed to a
pharmaceutical composition comprising a safe and an angiogenically
effective dose of an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or
12-14 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
36 .mu.g/kg of an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or
12-14 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 20 .mu.g/kg, or 20 .mu.g/kg to 36
.mu.g/kg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14
or an angiogenically active fragment or mutein thereof, and a
pharmaceutically acceptable carrier.
[0056] By the term "pharmaceutically acceptable carrier" as used
herein is meant any of the carriers or diluents known in the art
for the stabilization and/or administration of a proteinaceous
medicament, such as the FGF of any one of SEQ ID NOS:1-3, 5, 8-10,
or 12-14 disclosed herein, that does not itself induce the
production of antibodies harmful to the individual receiving the
composition, and which may be administered without undue toxicity.
Within another aspect of the invention, pharmaceutical compositions
are provided, comprising a recombinant FGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or
mutein thereof in combination with a pharmaceutically acceptable
carrier or diluent. Such pharmaceutical compositions may be
prepared either as a liquid solution, or as a solid form (e.g.,
lyophilized) which is dissolved in a solution prior to
administration. In addition, the composition may be prepared with
suitable carriers or diluents for IC injection or administration.
Pharmaceutically acceptable carriers or diluents are nontoxic to a
human recipient at the dosages and concentrations employed.
Representative examples of suitable carriers or diluents for
injectable or infusible solutions include sterile water or isotonic
saline solutions, which are preferably buffered at a suitable pH
(such as phosphate-buffered saline or Tris-buffered saline), and
optionally contain mannitol, dextrose, glycerol, ethanol, and/or
one or more polypeptides or proteins such as human serum albumin
(HSA). Stabilizers, such as trehalose, thioglycerol, and
dithiothreitol (DTT), may also be added.
[0057] A typical pharmaceutical composition comprises 0.001 to 10
mg/ml, more typically 0.03 to 0.5 mg/ml, of a rFGF of any one of
SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active
fragment or mutein thereof, 10 mM thioglycerol, 135 mM NaCl, 10 mM
Na citrate, and 1 mM EDTA, pH 5. A suitable diluent or flushing
agent for the above described composition is any of the above
described carriers. Typically, the diluent is the carrier solution
itself comprising 10 mM thioglycerol, 135 mM NaCl, 10 mM Na
citrate, and 1 mM EDTA, pH 5. The rFGF of any one of SEQ ID
NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or
mutein thereof is unstable for long periods of time in liquid form.
To maximize stability and shelf life, the pharmaceutical
composition of the present invention comprising an effective amount
of rFGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an
angiogenically fragment or mutein thereof, in a pharmaceutically
acceptable aqueous carrier 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 5-10 ml of the
above described composition having 1.5-8 mg of the FGF of any one
of SEQ ID NOS:1-3, 5, 8-10, or 12-14.
[0058] In another embodiment, the pharmaceutical composition
comprises a unit dose of FGF of any one of SEQ ID NOS:1-3, 5, 8-10,
or 12-14 or an angiogenically active fragment or mutein thereof in
lyophilized (freeze-dried) form. In this form, the unit dose of FGF
would be 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 under reduced pressure of a plurality of vials, each
containing a unit dose of the FGF of the present invention therein.
Lyophilizers, which perform the above described lyophilization, are
commercially available and readily operable by those skilled in the
art. Prior to administration to a patient, the lyophilized product
is reconstituted to a known concentration, preferably in its own
vial, with an appropriate sterile aqueous diluent, typically 0.9%
(or less) sterile saline solution, or a compatible sterile buffer,
or even sterile deionized water. Depending upon the weight of the
patient in kg, a single dose comprising from 0.2 .mu.g/kg to 36
.mu.g/kg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14
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.24 .mu.g/kg) 1680 .mu.g (i.e., 1.680 mg).
[0059] In its third aspect, the present invention is directed to a
method for treating a patient in need of treatment for CAD or MI,
using the above described unit dose or pharmaceutical composition
to treat a human patient for coronary artery disease (CAD). In
particular, 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 of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or
an angiogenically active fragment or mutein thereof to one or more,
typically two, coronary vessels of a human patient in need of
treatment for coronary artery disease. A preferred coronary vessel
is the coronary artery, although grafted saphenous veins and
grafted internal mammary arteries, as provided by coronary
angioplasty, are also suitable.
[0060] The method of the present invention provides clinical
treatment of the underlying condition (i.e., CAD or MI) and not
merely a treatment of 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 36
.mu.g/kg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14
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 20 .mu.g/kg, or .mu.g/kg to 36 .mu.g/kg of
the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an
angiogenically 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 6.1 mg of the FGF of
any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically
fragment or mutein thereof; more typically, 0.3 mg to 3.5 mg of the
FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an
angiogenically fragment or mutein thereof.
[0061] The therapeutically effective amount of the rFGF-2 of the
present invention is administered to at least one coronary vessel
of a human patient diagnosed with CAD, symptomatic despite optimal
medical management, 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. Signs of coronary angiogenesis are apparent in
a matter of days following IC administration of the unit dose.
Therapeutic benefit is seen as early as two weeks following the IC
FGF administration. Clinically significant improvement is readily
demonstrable by objective criterion (ETT and/or SAQ) 30 days
following IC administration of the unit dose. In certain patients
with progressive CAD disease, it may be necessary or appropriate to
administer a unit dose of the FGF, for example, every six months or
annually, to overcome the progression of the CAD during that
interim period.
[0062] 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 as a unit
dose into one or more coronary vessels of a human patient in need
of coronary angiogenesis about 0.2 .mu.g/kg to about 36 .mu.g/kg
(or in absolute terms about 0.008 mg to about 6.1 mg) of a
recombinant FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or
an angiogenically active fragment or mutein thereof.
[0063] Fifty-two (52) human patients diagnosed with CAD, who
satisfied the criteria of Example 2 herein, were administered a
unit dose of 0.33 .mu.g/kg to 48 fig/kg of the FGF-2 of SEQ ID NO:5
by IC infusion over about a 20 minute period. The 52 treated
patients were then assessed by the Seattle Angina Questionnaire,
which provides an assessment based upon a mixed combination of
objective and subjective criteria. See Table 2. The Seattle Angina
Questionnaire is a validated, disease-specific instrument with the
following five subscales that are assessed both before and after
treatment: 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
for each of the five subscales is 0 to 100 with the higher scores
indicating a better quality of life. Moreover, a mean change of 8
points or more between the mean baseline scores (before treatment)
and the post-treatment scores is recognized as being "clinically
significant." Table 2 reports that the 28 patients, who were
pretested and then administered a single unit dose of 0.33 .mu.g/kg
to 24 .mu.g/kg of the FGF-2 of SEQ ID NO:5 by IC infusion,
exhibited a mean score increase of 13 to 36 points for the five
"quality of life" criteria assessed by the "Seattle Angina
Questionnaire." See Table 2 herein. These 13 to 36 point increases
were about 1.6 to 4.5 times greater than the 8 point change which
is recognized in the art as being "clinically significant" in
alternative modes of treatment. See Table 2 herein. Moreover, when
the combined results for the first 15 patients of Table 2 were
broken down between low dose (less than or equal to 2 .mu.g/kg) and
high (more than 2 .mu.g/kg) doses of FGF-2 of SEQ ID NO:5, and
assessed by the "Seattle Angina Questionnaire," both doses were
found to provide increased scores that ranged from about 12.3 to
58.1 and about 10.9 to 32.1, respectively. See Table 3 herein. The
increased scores were about 1.4 to 7.2 times greater than the 8
point change which is considered to be "clinically significant" in
alternative modes of treatment.
[0064] In the same Phase I trial, fifty two human patients who were
diagnosed with CAD and who satisfied the criteria of Example 2
herein, were administered IC a single unit dose of 0.33 .mu.g/kg to
48 .mu.g/kg of FGF-2 of SEQ ID NO:5. The maximum tolerated dose
(MTD) in humans was defined as 36 .mu.g/kg based upon the
occurrence of severe but transient hypotension in 2/10 patients at
48 .mu.g/kg. (In contrast, the MTD in pigs was defined as 6.5
.mu.g/ml.) At one of the sites, the hearts of 23 human patients
were assessed both before ("baseline") and 30 and 60 days after
treatment by magnetic resonance imaging (MRI) for objective signs
of improved coronary sufficiency. Among the objective criteria
assessed by MRI are the following: 1) left ventricular (LV)
ejection fraction (EF); 2) normal wall thickness (NWT); 3) normal
wall motion (NWM); 4) collateral extent; 5) ischemic area zone; 6)
targeted wall thickness (TWT); 7) targeted
TABLE-US-00002 TABLE 2 COMPARISON OF QUALITY OF LIFE BEFORE AND 57
DAYS AFTER IC FGF-2 Seattle Angina Questionnaire (SAQ) Baseline
(Pre FGF-2) 57 Days Post FGF-2 Subscales Mean Score .+-. SD Mean
Score .+-. SD Mean Change.sup.1 p Value n Exertional Capacity 55
.+-. 23 68 .+-. 25 13* 0.02 28 Angina Frequency 42 .+-. 32 66 .+-.
28 24* <0.001 28 Angina Stability 46 .+-. 26 82 .+-. 20 36*
<0.001 27 Disease Perception 40 .+-. 21 61 .+-. 26 19* <0.001
28 Treatment Satisfaction 74 .+-. 24 88 .+-. 16 14* 0.002 28
*Significantly different from baseline to fifty-seven days. .sup.1A
mean change of 8 points or more is considered clinically
significant.
TABLE-US-00003 TABLE 3 IMPROVEMENTS IN THE QUALITY OF LIFE AT DAY
57 (POST IC rFGF-2) AT LOWER AND HIGHER DOSES Seattle Angina
Questionnaire (SAQ) Subscales Dose <2 .mu.g/kg IC Dose >2
.mu.g/kg IC rFGF-2 (n = 7) rFGF-2 (n = 8) Subscales Mean Change in
Score Mean Change in Score (Day 57 score-screen (Day 57
score-screen Independent score) score) Samples t-test Exertional
Capacity 12.30 (23.3) 15.98 (28.7) t = -.27 p = .79 Disease
Perception 26.19 (26.9) 24.47 (21.2) t = .14 p = .89 Treatment
22.32 (27.7) 10.93 (17.3) t = .97 p = .35 Satisfaction Angina
Frequency 28.57 (27.3) 13.75 (22.6) t = 1.15 p = .27 Angina
Stability 58.13 (12.9) 32.14 (34.5) t = 1.75 p = .108 1. Possible
range for each subscale is 0 to 100 with higher scores indicating
better quality of life. 2. Standard deviation noted in
parentheses.
wall motion (TWM); and 8) perfusion or delayed arrival zone (% LV).
The patients were also assessed for angina, treadmill exercise
duration, rest/exercise nuclear perfusion. The results are
summarized in Table 4. Table 4 reflects that the baseline angina
class decreased from 2.6 to 1.4 and 1.2 at 30 and 60 days,
respectively post IC FGF-2. The mean treadmill exercise time
increased from a baseline of 8.5 minutes to 9.4 and 10.0 minutes at
30 and 60 days, respectively, post treatment. No significant
difference was observed in the left ventricular ejection fraction
(LV EF). However, the target wall motion increased significantly,
moving from a baseline of 15.4% to 23.5% (day 30) and 24.1% (day
60) post FGF-2 treatment. Likewise the target wall thickening
increased significantly from a baseline of 28.7% to 34.7% (day 30)
and 45.9% (day 60) post FGF-2 treatment. There was also a
significant increase in perfusion, as measured by a decrease in the
delayed arrival zone (% LV), with the delayed arrival zone
decreasing from a baseline of 18.9% to 7.1% (day 30) and 1.82% (day
60) post FGF-2 treatment. Thus, providing CAD patients with a
single IC infusion of FGF-2 in accordance with the present
invention provided the patients with a significant physical
improvement as objectively measured by MRI and other conventional
criteria.
Pharmacokinetics and Metabolism
[0065] 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).
Because the endothelium is responsible for binding FGF-2 and acts
as a sink after injection, we believed that rFGF-2 will undergo a
fast biodistribution phase right after administration. Accordingly,
we targeted the intracoronary as opposed to the intravenous route
of administration.
[0066] 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, the FGF-2 of SEQ ID NO:5 has a molecular weight of
TABLE-US-00004 TABLE 4 MEAN DATA AND DATA RESULTS AS A FUNCTION OF
TIME AND DOSE Baseline 30 Day 60 Day Angina Class 2.6 .+-. 0.7 1.4
.+-. 0.9*** 1.2 .+-. 0.8*** Exercise Time (min.) 8.5 .+-. 2.6 9.4
.+-. 1.9*** 10.0 .+-. 2.5** LV EF (%) 47.4 .+-. 12.3 47.4 .+-. 10.6
48.6 .+-. 11.0 Target Wall Motion (%) 15.4 .+-. 10.1 23.5 .+-.
12.0* 24.1 .+-. 10.1** Target Wall Thickening 28.7 .+-. 14.0 34.7
.+-. 14.1 45.9 .+-. 11.7** (%) Delayed Arrival Zone 18.9 .+-. 8.3
7.1 .+-. 3.6*** 1.82 .+-. 2.4*** (% LV) *= p < 0.05 **= p <
0.01 ***= p < 0.001 (2-tailed, paired)
about 16 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 the same study, FGF-2 appeared to bind to red
blood cells, however these results were not confirmed by in vitro
analysis of the whole blood. 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). More particularly, it
is 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. When FGF-2 was administered in
combination with heparin, the renal excretion of FGF-2 was
increased. Whalen et al. (1989). The FGF-2 molecule, which is
cationic when not complexed with heparin, is likely repelled by the
cationic heparin sulfate of the glomerular basement membrane. The
FGF-2/heparin complex is more neutrally charged, and therefore is
more easily filtered and excreted by the kidney.
[0067] We determined the pharmacokinetics of rFGF-2 (SEQ ID NO:5)
after intravenous (IV) and intracoronary (IC) administration in
domestic Yorkshire pigs, after IV dosing in Sprague Dawley ("SD")
rats, and after IC administration in CAD human patients. In all
species, the rFGF-2 plasma concentrations after IV and/or IC
injection 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. 1 provides a plasma
concentration versus time curve showing these phases in humans
after IC administration of rFGF-2 of SEQ ID NO:5 as a function of
the following doses: 0.33 .mu.g/kg, 0.65 .mu.g/kg, 2 .mu.g/kg, 6
.mu.g/kg, 12 .mu.g/kg, and 24 .mu.g/kg of lean body mass (LBM). The
plasma concentrations of rFGF-2 of SEQ ID NO:5 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:5. 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.
[0068] The design of these pharmokinetic studies, pharmacokinetic
parameters, and conclusions are listed in Tables 5 and 6 for
studies in pigs and rats, respectively. The reader is referred to
these tables for the specific details. However, among the points to
be noted are that the half-life (T.sub.1/2) was 2.8.+-.0.8 to 3.5
hours following a single IC infusion for the single component model
for animals having a clearance (CL) of 702.+-.311 to 609.+-.350
ml/hr/kg. The results of this study show that the pharmacokinetics
of the recombinant bFGF-2 of SEQ ID NO:5 were substantially
identical regardless of whether the animals were dosed via the IC
or IV routes. See Table 5. In pigs, the maximum tolerated dose of
recombinant bFGF-2 was 6.5 .mu.g/kg. Among the other
pharmacokinetic results to be taken from Tables 5 and 6 of these
studies is that there is a fast distribution phase followed by a
more moderate elimination phase, and dose linearity as reported in
FIG. 1 for humans. Also, there were no gender differences. Further,
the three compartment model was analyzed for pigs receiving 70 U/kg
of heparin approximately (".about.") 15 minutes before receiving
0.65-6.5 .mu.g/kg by 5-10 minute IC infusion. The half lives
(T.sub.1/2.alpha., T.sub.1/2.beta. and T.sub.1/2.gamma.) for the
three compartments were 1.5 minutes, 17 minutes, and 6.6 hours,
respectively. In these animals, the initial volume ("V.sub.I") was
approximately the plasma volume, and the steady state volume
("V.sub.ss") was approximately 10-fold the plasma volume. See Table
5. In pigs, the binding of recombinant bFGF-2 of SEQ ID NO:5 to
circulating heparin appears to decrease biodistribution and
elimination. Likewise, in rats, both the volume of distribution and
the clearance of rFGF-2 were smaller when heparin was administered.
See Table 6. Further, the greatest and most favorable changes on
clearance of FGF-2 were found when heparin was administered within
+15 minutes, preferably immediately prior to rFGF-2 IC infusion.
See Table 6.
TABLE-US-00005 TABLE 5 Pharmacokinetics (PK) and Pharmacodynamics
of rFGF-2 in Pigs Animals Dosing Regimen PK Parameters Results
Domestic Yorkshire pigs 2-20 .mu.g/kg IV bolus CL = 702 .+-. 311
mL/hr/kg Systemic PK identical between IV and IC route under
general anesthesia 2-20 .mu.g/kg IC bolus T1/2 = 2.8 .+-. 0.8 hr.
Fast distribution phase (n = 13; 30 .+-. 5 kg) 20 .mu.g/kg by
10-min IC Dose-linearity infusion Transient decreases of MAP 70
U/kg heparin ~15 min before rFGF-2 Domestic Yorkshire pigs 0.65-6.5
.mu.g/kg by 5-min CL = 609 .+-. 350 ml/hr/kg No gender difference
in PK under general anesthesia IC infusion T1/2 = ~3.5 hr Biphasic
decline of plasma rFGF-2 (n = 17; 26 .+-. 4 kg) 70 U/kg heparin ~15
min 3-Comp. Model: Dose-linearity before rFGF-2 T1/2.alpha. = 1.5
min V.sub.1 equal to ~plasma volume T1/2.beta. = 17 min V.sub.55
equal to ~10-fold plasma volume T1/2.gamma. = 6.6 hr Magnitude and
duration of MAP decrease correlated CL = 580 ml/hr/kg with rFGF-2
dose and peak plasma level V.sub.1 = 55 ml/kg V.sub.55 = 523 ml/kg
Domestic Yorkshire pigs 6.5 .+-. .mu.g/kg weekly by 5 min Without
Heparin The rFGF-2 distribution phase was less steep, the volume
under general anesthesia IV infusion for 6 (Doses 1-6): of
distribution smaller, and clearance was slower with (n = 6; 2.5
.+-. 5 kg) weeks T1/2 = 2-6 hr heparin-pretreatment 70 U/kg heparin
10 min CL = 777-2749 ml/hr/kg Binding of rFGF-2 to circulating
heparin appears to before rFGF-2 (n = 3), or V.sub.55 = 871-12,500
ml/kg decrease biodistribution and elimination rFGF-2 alone (n = 3)
With Heparin Both volume and clearance of rFGF-2 increased at
(Doses 1-6): later doses (potential receptor upregulation), but
more T1/2 = 2-3 hr so in the absence of heparin CL = 235-347
ml/hr/kg Magnitude and duration of MAP decreases were similar
V.sub.55 = 71-153 ml/kg with or without heparin
TABLE-US-00006 TABLE 6 Pharmacokinetics (PK) of rFGF-2 in Rats
Animals Dosing Regimen PK Parameters Results Conscious SD rats
3-100 .mu.g/kg bolus IV T1/2 = 1.1 .+-. 0.51 hr Fast distribution
phase (n = 18; 322 .+-. 93 g) injection CL = 4480 .+-. 2700
ml/hr/kg Apparent dose-linearity V.sub.55 = 1924 .+-. 1254 ml/kg
conscious SD rats 30-300 .mu.g/kg weekly by T1/2 = 1.4 .+-. 0.13 hr
Time-invariant PK; plasma profiles, PK parameters and (n = 54; 149
.+-. 12 g) bolus IV injection for 6 CL = 1691 .+-. 169 ml/hr/kg
AUCs were similar over time weeks V.sub.55 = 1942 .+-. 358 ml/kg
Dose-linearity No heparin pretreatment Conscious SD rats 30
.mu.g/kg bolus IV Time-Averaged PK In all cases, heparin increased
the rFGF-2 plasma levels (27 males; 381 .+-. 48 g; injection
Parameters: Both volume of distribution and clearance of 20
females; No heparin T1/2 CL V.sub.55 rFGF-2 were smaller with
heparin 268 .+-. 22 g) 40 U/kg IV Heparin: hr ml/hr/kg ml/kg
Greatest changes on CL and V.sub.55 were observed when at ~min 0.75
4332 2389 heparin was administered immediately prior to rFGF-2 just
prior to rFGF-2 0.91 1728 844 at +15 min 1.3 516 147 at +3 hr 1.2
1158 626 0.93 1338 1351
[0069] The pharmacokinetics of the rFGF-2 of SEQ ID NO:5 was
studied in humans, diagnosed with CAD despite optimal medical
management, in a Phase 1 clinical study supporting this filing. The
doses of rFGF-2 employed in that Phase 1 study were 0.33 .mu.g/kg,
0.65 .mu.g/kg, 2 .mu.g/kg, 6 .mu.g/kg, 12 .mu.g/kg, and 24 .mu.g/kg
of lean body mass (LBM), and all doses were administered by a 20
minute IC infusion (10 minutes into each of two patent coronary
vessels) after pretreating the patient with 40 U/kg heparin which
was administered IV or IC 1-95 minutes before rFGF-2 infusion.
FIGS. 1-3 herein summarize the data underlying those results. In
particular, FIG. 1 is a plot of the mean rFGF-2 plasma
concentration versus time (hours) for the six different doses of
rFGF-2 (SEQ ID NO:5) administered by IC infusion as described above
over a 20 minute period. FIG. 1 shows dose linearity and a biphasic
plasma level decline, i.e., a fast distribution phase during the
first hour, followed by an elimination phase with T.sub.1/2 of
1.9.+-.2.2 hours. The dose linearity is more readily seen in FIG.
2, which is a plot of the individual patient rFGF-2 area under the
curve (AUC) in pghr/ml for FIG. 1 for each of the six doses of
rFGF-2 administered. FIG. 3 is a plot individual human patient
rFGF-2 dose normalized AUCs versus time of heparin dose in "minutes
prior to rFGF-2 infusion" and shows the influence of timing of
heparin administration on rFGF-2 AUC. FIG. 3 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 rFGF-2 infusion. Typically, an effective
amount of a glycosoaminoglycan is 40-70 U/kg heparin. These
pharmacokinetic results are summarized in Table 7 herein.
[0070] The rFGF-2 distribution phase was less steep with heparin,
the volume of distribution smaller, and the clearance slower, as
compared to rFGF-2 without heparin. It appears that the complex of
rFGF-2 with circulating heparin decreases the biodistribution and
elimination of rFGF-2. 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 the FGF-2
receptor is approximately two orders of magnitude higher
(dissociation constant .about.2.times.10.sup.-11 M). Moscatelli et
al., (1991).
TABLE-US-00007 TABLE 7 Pharmacokinetics of rFGF-2 in Human Subjects
Dosing Regimen PK Parameters Results Patients with CAD 0.33-24
.mu.g/kg LBM* Preliminary PK data Biphasic plasma level decline:
fast distribution phase by 20-min IC injection from 0.33-24
.mu.g/kg during 1 hr; followed by elimination phase with (10 min in
left main doses (n = 32) T1/2 approximately (~) 2 hr coronary
artery + 10 min T1/2 = 1.9 .+-. 2.2 hr Dose-linearity in right main
CL = 264 .+-. 150 ml/hr/kg Greater rFGF-2 exposure (as measured by
the coronary artery) V.sub.55 = 184 .+-. 74 ml/kg AUC) was found
when heparin pretreatment was 0.33 .mu.g/kg, n = 4 given closer to
the start of the rFGF-2 infusion, 0.65 .mu.g/kg, n = 4 preferably
within 20 minutes 2 .mu.g/kg, n = 8 6 .mu.g/kg, n = 4 12 .mu.g/kg,
n = 4 24 .mu.g/kg, n = 8 36 .mu.g/kg, n = 10 48 .mu.g/kg, n = 10 40
U/kg heparin pretreatment, 1-95 min before rFGF-2 infusion *LBM =
lean body mass
[0071] In addition, the complexation of the rFGF-2 of SEQ ID NO:5
with a glycosoaminoglycan, such as a heparin, might increase signal
transduction and mitogenesis, and/or protect the rFGF-2 from
enzymatic degradation.
[0072] 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
[0073] The rFGF-2 of SEQ ID NO:5 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.
[0074] 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:5 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.
[0075] 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 20
to 8.degree. C.
[0076] 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 ml. In order to keep the EDTA
concentration below the limit of 100 .mu.g/ml, the total infusion
volume was increased to 20 ml when proportionately higher absolute
amounts of FGF-2 were administered to patients with high body
weights.
Example 2
Selection Criteria for Patients with Coronary Artery Disease for
Treatment with rFGF-2
[0077] 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:
[0078] Inclusion Criteria:
[0079] Subject is eligible if: [0080] Male or female, greater than
or equal to 18 years of age [0081] Diagnosis of coronary artery
disease (CAD) [0082] Suboptimal candidates for approved
revascularization procedures, e.g., angioplasty, stents, coronary
artery bypass graft (CABG) (or refuses those interventions) [0083]
Able to exercise at least three minutes using a modified Bruce
protocol and limited by coronary ischemia [0084] Inducible and
reversible defect of at least 20% myocardium on pharmacologically
stressed thallium sestamibi scan [0085] CBC, platelets, serum
chemistry within clinically acceptable range for required cardiac
catheterization [0086] Normal INR, or if anticoagulated with
Coumadin, INR <2.0 [0087] Willing and able to give written
informed consent to participate in this study, including all
required study procedures and follow-up visits
[0088] Exclusion Criteria:
[0089] Subject is not eligible if: [0090] Malignancy: any history
of malignancy within past ten years, with the exception of
curatively treated basal cell carcinoma [0091] 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 [0092] Renal function: creatinine clearance below
normal range adjusted for age; protein >250 mg or microalbumin
>30 mg/24 h urine [0093] Class IV heart failure (New York Heart
Association) [0094] Ejection fraction <20% by echocardiogram,
thallium scan, MRI or gated pooled blood scan (MUGA) [0095]
Hemodynamically relevant arrhythmias (e.g., ventricular
fibrillation, sustained ventricular tachycardia) [0096] Severe
valvular stenosis (aortic area <1.0 cm.sup.2, mitral area
<1.2 cm.sup.2), or severe valvular insufficiency [0097] Marked
increase in angina or unstable angina within three weeks [0098]
History of myocardial infarction (MI) within three months [0099]
History of transient ischemic attack (TIA) or stroke within six
months [0100] History of CABG, angioplasty or stent within six
months [0101] History of treatment with transmyocardial laser
revascularization, rFGF-2, or vascular enodothelial growth factor
(VEGF) within six months [0102] Females of child-bearing potential
or nursing mothers [0103] Any pathological fibrosis, e.g.,
pulmonary fibrosis, scleroderma [0104] Known vascular malformation,
e.g., AV malformation, hemangiomas [0105] Coexistence of any
disease which might interfere with assessment of symptoms of CAD,
e.g., pericarditis, costochondritis, esophagitis, systemic
vasculitis, sickle cell disease [0106] 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) [0107] Participation in clinical trials of
investigational agents, devices or procedures within thirty days
(or scheduled within sixty days of study drug) [0108] Known
hypersensitivity to rFGF-2 or related compounds [0109] 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:1)
Administered to Humans
[0110] Recombinant bFGF-2 of SEQ ID NO:5 was administered to 52
human patients with severe CAD, who remained symptomatic despite
optimal medical management and who refused or were suboptimal
candidates for surgical or percutaneous revascularization, in a
Phase I open label, single administration, dose escalation,
two-site trial. 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). The doses (.mu.g/kg) of rFGF-2 administered 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:5. Angina frequency
and quality of life was assessed by the Seattle Angina
Questionnaire (SAQ) at a baseline (before rFGF-2 administration)
and at about 60 days after rbFGF-2 administration. Exercise
tolerance time (ETT) was assessed by the threadmill test.
Rest/exercise nuclear perfusion and gated sestamibi-determined rest
ejection fraction (EF), and magnetic resonance imaging (MRI) were
assessed at baseline, and at 30 days and 60 days post FGF-2
administration. Other end points that were evaluated included MRI
(to objectively measure ejection fraction (EF), normal wall motion
(NWM), targeted wall motion (TWM), normal wall thickness (NWT),
targeted wall thickness (TWT), ischemic area zone and collateral
extent). See Tables 2-4, respectively.
[0111] The preliminary safety results indicate that serious events
were not dose related. Thus far, of the eight dosage groups, there
were three deaths in the lowest 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). There were six hospitalizations for acute myocardial
infarction (MI) in three patients, i.e., one patient from each of
groups 1 (0.33 .mu.g/kg), 3 (2.0 .mu.g/kg), and 4 (6.0 .mu.g/kg).
One of the three patients accounted for four of the six
hospitalizations for acute MI. There was also one large B cell
lymphoma that was diagnosed three weeks after dosing in a patient
in group 4. The patient died at two months post dosing. Acute
hypotension, seen at higher doses during or just subsequent to
infusion, was managed by administration of fluids without need for
a vasopressor. The maximum tolerated dose of rFGF-2 (SEQ ID NO:2)
was defined as 36 .mu.g/kg. Doses of rFGF-2 up to 48 .mu.g/kg IC
were managed in patients with aggressive fluid management. However,
they were not tolerated due to acute and/or orthostatic hypotension
in two out of ten patients. The half-life in humans of the IC
infused rFGF-2 was about one hour.
[0112] The human patients in this study that were treated with a
single IC infusion of rFGF-2 of SEQ ID NO:5 exhibited a mean
increase in ETT of 1.5 to 2 minutes. This is especially significant
because an increase in ETT of >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 57 days in all
five subscales for the 28 patients (n=28) tested. See Tables 2 and
3. In particular, the mean changes in scores for the five criteria
evaluated by the SAQ ranged from 13 to 36 with a mean change of 8
or more considered "clinically significant." See Table 2.
[0113] Magnetic resonance imaging (MRI) showed objective
improvements following administration of a single unit dose of the
bFGF-2 of SEQ ID NO:5, including increased targeted wall motion at
30 and 60 days (p<0.05), and increased targeted wall thickening
at 60 days (p<0.01). MRI further showed improved regional wall
motion, and increased myocardial perfusion and collateral
development in the targeted area for both the lower dose (0.33
.mu.g/kg and 0.65 .mu.g/kg) and higher dose (2.0 .mu.g/kg and 12.0
.mu.g/kg) groups in an 11 patient test group (n=11).
[0114] Abnormal perfusion zone, which was assessed at one of the
sites on 28 patients, decreased significantly at 30 and 60 days
(p<0.001).
[0115] 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 for
57-60 days in all dosage groups. (See Tables 2-4.) Based upon the
results already obtained, it is expected that the angiogenic
effects would last twelve months or more but at least six months,
at which time the procedure could be repeated, if necessary.
Example 4
Proposed Phase II Clinical Study on Recombinant FGF-2 (SEQ ID NO:1)
Administered to Humans to Treat Coronary Artery Disease
[0116] 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 .mu.g/kg, and 30 .mu.g/kg administered IC.
Example 5
Unit Dose and Pharmaceutical Composition of rFGF-2 for the Phase II
Human Clinical Trial
[0117] The rFGF-2 of SEQ ID NO:5 was formulated as a unit dose and
pharmaceutical composition for administration to humans in the
Phase II clinical trial referenced herein. The various formulations
are described below.
[0118] 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:5 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 confirmed to produce a unit dose of 36 mg/kg for the Phase
II study.
[0119] The rFGF 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. Like the unit dose, the rFGF-2 placebo is stored at
2.degree. to 8.degree. C.
[0120] 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.
Example 6
Selection Criteria for CAD Patients for the Phase II Human Clinical
Trial of IC rFGF-2
[0121] Accordingly, the above described evidence of an unexpectedly
superior improvement in quality of life and of increased angiogenic
efficacy in humans who were administered a single unit dosage of
rFGF-2 in accordance with this invention, supports the
patentability of the Applicants' unit dose, pharmaceutical
composition and method of using the same.
[0122] 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.
[0123] 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
151140PRTHuman FGF-1 1Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro
Lys Leu Leu Tyr Cys 1 5 10 15Ser Asn Gly Gly His Phe Leu Arg Ile
Leu Pro Asp Gly Thr Tyr Asp 20 25 30Gly Thr Arg Asp Arg Ser Asp Gln
His Ile Gln Leu Gln Leu Ser Ala 35 40 45Glu Ser Tyr Gly Glu Tyr Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60Leu Ala Met Asp Thr Asp
Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80Glu Glu Cys Leu
Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95Tyr Ile Ser
Lys Lys His Ala Glu Lys Asn Trp Phe Tyr Gly Leu Lys 100 105 110Lys
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 115 120
125Ala Ile Leu Phe Leu Pro Leu Pro Tyr Ser Ser Asp 130 135
1402140PRTbovine FGF-1 2Phe Asn Leu Pro Leu Gly Asn Tyr Lys Lys Pro
Lys Leu Leu Tyr Cys 1 5 10 15Ser Asn Gly Gly Tyr Phe Leu Arg Ile
Leu Pro Asp Gly Thr Val Asp 20 25 30Gly Thr Lys Asp Arg Ser Asp Gly
His Ile Gln Leu Phe Leu Cys Ala 35 40 45Glu Ser Ile Gly Glu Val Tyr
Ile Lys Ser Thr Glu Thr Gly Gln Phe 50 55 60Leu Ala Met Asp Thr Asp
Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asp 65 70 75 80Glu Glu Cys Leu
Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95Tyr Ile Ser
Lys Lys His Ala Glu Lys His Trp Phe Val Gly Leu Lys 100 105 110Lys
Asn Gly Arg Ser Lys Leu Glu Pro Arg Thr His Phe Gly Gln Lys 115 120
125Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135
1403146PRTHuman FGF-2 3Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala
Phe Pro Pro Gly His 1 5 10 15Phe Lys Asp Pro Lys Arg Leu Tyr Cys
Lys Asn Gly 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 Thr 100 105 110Ser
Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser 115 120
125Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala
130 135 140Lys Ser1454442DNAbovine FGF-2CDS(1)..(438) 4cca gcc cta
cca gaa gat ggg ggg tcc ggg gcc ttc cca cca ggg cac 48Pro Ala Leu
Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His 1 5 10 15ttc
aaa gat cca aaa cga cta tat tgt aaa aac ggg ggg ttc ttc cta 96Phe
Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25
30cga atc cac cca gat ggg cga gta gat ggg gta cga gaa aaa tcc gat
144Arg Ile His Pro Asp 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 Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val
Ser 50 55 60atc aaa ggg gta tgt gcc aac cga tat cta gcc atg aaa gaa
gat ggg 240Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu
Asp Gly 65 70 75 80cga cta cta gcc tcc aaa tgt gta acc gat gaa tgt
ttc ttc ttc gaa 288Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys
Phe Phe Phe Glu 85 90 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 ggg cca 384Ser Trp Tyr Val Ala Leu Lys Arg Thr
Gly Gln Tyr Lys Leu Gly Pro 115 120 125aaa acc ggg cca ggg caa aaa
gcc atc cta ttc cta 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
Ser1455146PRTbovine FGF-2 5Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly
Ala Phe Pro Pro Gly His 1 5 10 15Phe Lys Asp Pro Lys Arg Leu Tyr
Cys Lys Asn Gly 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 Ser14569PRTBovis bovinus 6Met Ala Ala Gly
Ser Ile Thr Thr Leu 1 57240PRTMurine FGF-3 7Met Gly Leu Ile Trp Leu
Leu Leu Leu Ser Leu Leu Glu Pro Ser Trp 1 5 10 15Pro Thr Thr Gly
Pro Gly Thr Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30Gly Gly Val
Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45Tyr Cys
Ala Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60Asn
Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 65 70
75 80Val Glu Val Gly Val Val Ala Ile Lys Gly Leu Phe Ser Gly Arg
Tyr 85 90 95Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Asp His
Tyr Asn 100 105 110Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu
Gly Tyr Asn Thr 115 120 125Tyr Ala Ser Arg Leu Tyr Arg Thr Gly Ser
Ser Gly Pro Gly Ala Gln 130 135 140Arg Gln Pro Gly Ala Gln Arg Pro
Trp Tyr Val Ser Val Asn Gly Lys145 150 155 160Gly Arg Pro Arg Arg
Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser 165 170 175Leu Phe Leu
Pro Arg Val Leu Gly His Lys Asp His Glu Met Val Arg 180 185 190Leu
Leu Gln Ser Ser Gln Pro Arg Ala Pro Gly Glu Gly Ser Gln Pro 195 200
205Arg Gln Arg Arg Gln Lys Lys Gln Ser Pro Gly Asp His Gly Lys Met
210 215 220Glu Thr Leu Ser Thr Arg Ala Thr Pro Ser Thr Gln Leu His
Thr Gly225 230 235 2408205PRTHuman FGF-4 8Ser Gly Pro Gly Thr Ala
Ala Val Ala Leu Leu Pro Ala Val Leu Leu 1 5 10 15Ala Leu Leu Ala
Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro Thr 20 25 30Ala Pro Asn
Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu Ser 35 40 45Leu Val
Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro Lys 50 55 60Glu
Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile Lys 65 70
75 80Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu
Gln 85 90 95Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His Ala Asp Thr
Arg Asp 100 105 110Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val
Val Ser Ile Phe 115 120 125Gly Val Ala Ser Arg Phe Phe Val Ala Met
Ser Ser Lys Gly Lys Leu 130 135 140Tyr Gly Ser Pro Phe Phe Thr Asp
Glu Cys Thr Phe Lys Glu Ile Leu145 150 155 160Leu Pro Asn Asn Tyr
Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met 165 170 175Phe Ile Ala
Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg Val 180 185 190Ser
Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200
2059266PRTHuman FGF-5 9Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser
His Leu Ile Leu Ser 1 5 10 15Ala Trp Ala His Gly Glu Lys Arg Leu
Ala Pro Lys Gly Gln Pro Gly 20 25 30Pro Ala Ala Thr Asp Arg Asn Pro
Arg Gly Ser Ser Ser Arg Gln Ser 35 40 45Ser Ser Ser Ala Met Ser Ser
Ser Ser Ala Ser Ser Ser Pro Ala Ala 50 55 60Ser Leu Gly Ser Gln Gly
Ser Gly Leu Glu Gln Ser Ser Phe Gln Trp 65 70 75 80Ser Leu Gly Ala
Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly Ile Gly 85 90 95Phe His Leu
Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser His Glu 100 105 110Ala
Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln Gly Ile 115 120
125Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met Ser Lys
130 135 140Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys
Lys Phe145 150 155 160Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr
Tyr Ala Ser Ala Ile 165 170 175His Arg Thr Glu Lys Thr Gly Arg Glu
Trp Tyr Val Ala Leu Asn Lys 180 185 190Arg Gly Lys Ala Lys Arg Gly
Cys Ser Pro Arg Val Lys Pro Gln His 195 200 205Ile Ser Thr His Phe
Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro Glu 210 215 220Leu Ser Phe
Thr Val Thr Val Pro Glu Lys Lys Lys Pro Pro Ser Pro225 230 235
240Ile Lys Pro Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr Asn Ser
245 250 255Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260
26510207PRTHuman FGF-6 10Ala Leu Gly Gln Lys Leu Phe Ile Thr Met
Ser Arg Gly Ala Gly Arg 1 5 10 15Leu Gln Gly Thr Leu Trp Ala Leu
Val Phe Leu Gly Ile Leu Val Gly 20 25 30Met Val Val Pro Ser Pro Ala
Gly Thr Arg Ala Asn Asn Thr Leu Leu 35 40 45Asp Ser Arg Gly Trp Gly
Thr Leu Leu Ser Arg Ser Arg Ala Gly Leu 50 55 60Ala Gly Glu Ile Ala
Gly Val Asn Trp Glu Ser Gly Tyr Leu Val Gly 65 70 75 80Ile Lys Arg
Gln Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His 85 90 95Leu Gln
Val Leu Pro Asp Gly Arg Ile Ser Gly Thr His Glu Glu Asn 100 105
110Pro Tyr Ser Leu Leu Glu Ile Ser Thr Val Glu Arg Gly Val Val Ser
115 120 125Leu Phe Gly Val Arg Ser Ala Leu Phe Val Ala Met Asn Ser
Lys Gly 130 135 140Arg Leu Tyr Ala Thr Pro Ser Phe Gln Glu Glu Cys
Lys Phe Arg Glu145 150 155 160Thr Leu Leu Pro Asn Asn Tyr Asn Ala
Tyr Glu Ser Asp Leu Tyr Gln 165 170 175Gly Thr Tyr Ile Ala Leu Ser
Lys Tyr Gly Arg Val Lys Arg Gly Ser 180 185 190Lys Val Ser Pro Ile
Met Thr Val Thr His Phe Leu Pro Arg Ile 195 200 20511193PRTHuman
FGF-7 11Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr
Arg 1 5 10 15Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser
Leu Ala Cys 20 25 30Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val
Asn Cys Ser Ser 35 40 45Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met
Glu Gly Gly Asp Ile 50 55 60Arg Val Arg Arg Leu Phe Cys Arg Thr Gln
Trp Tyr Leu Arg Ile Asp 65 70 75 80Lys Arg Gly Lys Val Lys Gly Thr
Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95Ile Met Glu Ile Arg Thr Val
Ala Val Gly Ile Val Ala Ile Lys Gly 100 105 110Val Glu Ser Glu Phe
Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125Ala Lys Lys
Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140Glu
Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly145 150
155 160Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg
Gly 165 170 175Lys Lys Thr Lys Lys Gln Lys Thr Ala His Phe Leu Pro
Met Ala Ile 180 185 190Thr12215PRTMurine FGF-8 12Met Gln Ser Pro
Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15Val Leu
Cys Leu Gln Ala Gln Val Thr Val Gln Ser Ser Pro Asn Phe 20 25 30Thr
Gln His Val Arg Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg 35 40
45Arg Leu Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His
50 55 60Val Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Phe Asp
Gln 65 70 75 80Asp Pro Phe Ala Lys Leu Ile Val Glu Tyr Asp Thr Phe
Gly Ser Arg 85 90 95Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile
Cys Met Asn Lys 100 105 110Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly
Lys Gly Lys Asp Cys Val 115 120 125Phe Thr Phe Ile Val Ile Glu Asn
Asn Tyr Thr Ala Leu Gln Asn Ala 130 135 140Lys Tyr Glu Gly Trp Tyr
Met Ala Phe Thr Ala Lys Gly Arg Pro Arg145 150 155 160Lys Gly Ser
Lys Thr Arg Gln His Gln Arg Glu Val His Phe Met Lys 165 170 175Arg
Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu 180 185
190Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg
195 200 205Thr Trp Ala Pro Glu Pro Arg 210 21513208PRTHuman FGF-9
13Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala 1
5 10 15Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val
Leu 20 25 30Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro
Arg Gly 35 40 45Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu
Arg Arg Arg 50 55 60Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile
Phe Pro Asn Gly 65 70 75 80Thr Ile Gln Gly Thr Arg Lys Asp His Ser
Arg Phe Gly Ile Leu Glu 85 90 95Phe Ile Ser Ile Ala Val Gly Leu Val
Ser Ile Arg Gly Val Asp Ser 100 105 110Gly Leu Tyr Leu Gly Met Asn
Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125Lys Leu Thr Gln Glu
Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140Tyr Asn Thr
Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg145 150 155
160Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr
165 170 175Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg
Pro Val 180 185 190Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile
Leu Ser Gln Ser 195 200 20514207PRTHuman FGF-98 14Met Tyr Ser Ala
Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15Leu Leu
Cys Phe Gln Val Gln Val Leu Val Ala Glu Glu Asn Val Asp 20 25 30Phe
Arg Ile His Val Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40
45Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys
50 55 60His Ile Gln Val Leu Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp
Gly 65 70 75 80Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe
Gly Ser
Gln 85 90 95Val Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met
Asn Arg 100 105 110Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser
Lys Glu Cys Val 115 120 125Phe Ile Glu Lys Val Leu Glu Asn Asn Tyr
Thr Ala Leu Met Ser Ala 130 135 140Lys Tyr Ser Gly Trp Tyr Val Gly
Phe Thr Lys Lys Gly Arg Pro Arg145 150 155 160Lys Gly Pro Lys Thr
Arg Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175Arg Tyr Pro
Lys Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190Thr
Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Ala 195 200
20515266PRTHuman FGF-5 15Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe
Ser His Leu Ile Leu Ser 1 5 10 15Ala Trp Ala His Gly Glu Lys Arg
Leu Ala Pro Lys Gly Gln Pro Gly 20 25 30Pro Ala Ala Thr Asp Arg Asn
Pro Arg Gly Ser Ser Ser Arg Gln Ser 35 40 45Ser Ser Ser Ala Met Ser
Ser Ser Ser Ala Ser Ser Ser Pro Ala Ala 50 55 60Ser Leu Gly Ser Gln
Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln Trp 65 70 75 80Ser Leu Gly
Ala Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly Ile Gly 85 90 95Phe His
Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser His Glu 100 105
110Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln Gly Ile
115 120 125Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met
Ser Lys 130 135 140Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp
Asp Cys Lys Phe145 150 155 160Arg Glu Arg Phe Gln Glu Asn Ser Tyr
Asn Thr Tyr Ala Ser Ala Ile 165 170 175His Arg Thr Glu Lys Thr Gly
Arg Glu Trp Tyr Val Ala Leu Asn Lys 180 185 190Arg Gly Lys Ala Lys
Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His 195 200 205Ile Ser Thr
His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro Glu 210 215 220Leu
Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro Ser Pro225 230
235 240Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr Asn
Ser 245 250 255Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265
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