U.S. patent application number 10/131965 was filed with the patent office on 2002-11-07 for angiogenically effective unit dose of fgf and method of administering.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Whitehouse, Martha J..
Application Number | 20020165160 10/131965 |
Document ID | / |
Family ID | 22298692 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020165160 |
Kind Code |
A1 |
Whitehouse, Martha J. |
November 7, 2002 |
Angiogenically effective unit dose of FGF and method of
administering
Abstract
The present invention has multiple aspects. In particular, in
one aspect, the present invention is directed to 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. In another
aspect, the present invention is directed to a pharmaceutical
composition comprising an angiogenically effective dose of an FGF
or an angiogenically active fragment or mutein thereof, and a
pharmaceutically acceptable carrier. Typically, the angiogenically
effective dose comprises 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. In yet another aspect, the
present invention is directed to a method for treating a human
patient for coronary artery disease, comprising administering into
at least one coronary vessel of a human patient in need of
treatment for coronary artery disease a safe and angiogenically
effective dose 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.
Inventors: |
Whitehouse, Martha J.; (San
Francisco, CA) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
Emeryville
CA
|
Family ID: |
22298692 |
Appl. No.: |
10/131965 |
Filed: |
April 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10131965 |
Apr 25, 2002 |
|
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09417721 |
Oct 13, 1999 |
|
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60104103 |
Oct 13, 1998 |
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Current U.S.
Class: |
514/9.1 ;
514/13.3; 514/16.4 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 9/00 20180101; A61P 9/10 20180101; A61K 38/1825 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/18 |
Claims
That which is claimed is:
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 of a recombinant fibroblast growth factor (FGF) having the
sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.
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 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 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 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 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 82
g/kg of said angiogenically active fragment 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 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 an angiogenically active
mutein of a recombinant fibroblast growth factor (FGF) having the
sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.
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 angiogenically active mutein of said
recombinant FGF.
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 angiogenically active mutein of said recombinant
FGF.
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 angiogenically active mutein of said
recombinant FGF.
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 angiogenically active mutein of said recombinant
FGF.
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 angiogenically active mutein of said recombinant
FGF.
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 angiogenically active mutein of said recombinant
FGF.
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 or SEQ ID NO: 5 or an
angiogenically active fragment or an angiogenically active mutein
thereof.
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. A unit dose of a fibroblast growth factor (FGF), comprising
about 0.008 mg to about 6.1 mg of an FGF having the amino acid
sequence of SEQ ID NO: 3 or SEQ ID NO: 5 or an angiogenically
active fragment or an angiogenically active mutein thereof.
20. The unit dose of claim 19, comprising 0.3 mg to 3.5 mg of said
FGF or said angiogenically active fragment or said angiogenically
active mutein thereof.
21. The unit dose of claim 19, wherein said FGF has the sequence of
SEQ ID NO: 3 or SEQ ID NO: 5.
22. A pharmaceutical composition comprising (i) a therapeutically
effective amount of a fibroblast growth factor (FGF) having the
sequence of SEQ ID NO: 3 or SEQ ID NO: 5 or an angiogenically
active fragment or an angiogenically active mutein thereof, and
(ii) a pharmaceutically acceptable carrier, said composition being
in a form and a size suitable for administration to a human
patient.
23. The pharmaceutical composition of claim 22, wherein said
therapeutically effective amount comprises about 0.2 .mu.g/kg to
about 36 .mu.g/kg of said FGF or said angiogenically active
fragment or said angiogenically active mutein thereof.
24. The pharmaceutical composition of claim 23, wherein said
therapeutically effective amount comprises about 0.2 .mu.g/kg to
about 2 .mu.g/kg of said FGF or said angiogenically active fragment
or said angiogenically active mutein thereof.
25. The pharmaceutical composition of claim 23, wherein said
therapeutically effective amount comprises about 2 .mu.g/kg to
about 20 .mu.g/kg of said FGF or said angiogenically active
fragment or said angiogenically active mutein thereof.
26. The pharmaceutical composition of claim 23, wherein said
therapeutically effective amount comprises about 20 .mu.g/kg to
about 36 .mu.g/kg.
27. The pharmaceutical composition of claim 22, wherein said
therapeutically effective amount comprises about 008 mg to about
6.1 mg of said FGF or said angiogenically active fragment or said
angiogenically active mutein thereof.
28. The pharmaceutical composition of claim 27, wherein said
therapeutically effective amount comprises 0.3 mg to 3.5 mg of said
FGF or said angiogenically active fragment or said angiogenically
active mutein thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/417,721, filed Oct. 13, 1999, which claims the benefit of
U.S. Application Serial No. 60/104,103, filed Oct. 13, 1998, which
applications are herein incorporated by reference in their
entirety.
[0002] 1. Field of the Invention
[0003] 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 fuirther provide an adjunct for
reducing post myocardial infarct (MI) injury in humans.
[0004] 2. Background of the Invention
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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 ofBasic Fibroblast Growth Factor Enhances
Angiogenesis in Infarcted Swine Myocardium, " JACC,
22(7):2001-6(Dec. 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
[0011] 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.
[0012] 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).
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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 .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 .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.
[0017] 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.
[0018] 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.
[0019] 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
[0020] 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).
[0021] FIG. 2 is a plot of each individual patient's rFGF-2 area
under the curve (AUC) in pg.multidot.hr/ml for FIG. 1 for the six
doses of rFGF-2, and shows the dose linearity of systemic rFGF-2
exposure following IC infusion.
[0022] FIG. 3 is a plot 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
[0023] 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.
[0024] 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.
[0025] 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 .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./kg) through the larger patients
(e.g., 170 kg) at about the highest dosage (about 36 .mu.g/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.
[0026] 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).
[0027] 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.
[0028] 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-1differs from hFGF-1at 19 residue positions:
5(Pro.fwdarw.Leu), 21(His.fwdarw.Tyr), 31(Tyr.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.
[0029] 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. 08, 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 07, 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).
[0030] 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-3has 5 locations containing residue inserts relative to both
FGF-1and FGF-2. The most significant of these inserts is a 12 and
14 residue insert relative to FGF-2and 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.
[0031] 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-I (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.
[0032] 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- I (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-l (SEQ ID NO: 1), bFGF-1
would be expected to have the same cell and heparin binding sites
as does hFGF- 1.
[0033] 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 hstKS 3, 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. Nos. 5,155,217 (Goldfarb) and 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.
[0034] 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.
1TABLE 1 Amino Acid Sequence Comparison of hFGF-6 With Other hFGFs
Identical Conserved Identical Conserved SEQ ID NO: Residues*
Residues** 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).
[0035] 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.
[0036] 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).
[0037] 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).
[0038] 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 Essentialfor 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.
[0039] 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.
[0040] 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 Serial 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. 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 Imf 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 FGF1-9 (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:
[0041] 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);
[0042] 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);
[0043] 3. FGFR2b: best mitogenic responses were by hFGF-7 (14,000
cpm), hFGF-1(12,500 cpm), and mFGF-3 (9,500 cpm);
[0044] 4. FGFR 2c: 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);
[0045] 5. FGFR 3b: mitogenic responses only by hFGF-1 (37,000 cpm)
and mFGF-9 (26,000 cpm);
[0046] 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);
[0047] 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.
[0048] 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.
[0049] 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 X-phage, and the cloning vector is used to
transform either a eukaryotic or prokaryotic cell, wherein the
transformed cell expresses the FGF-2. In one embodiment, the host
cell is a yeast cell, such as Saccharomyces cerevisiae. The
resulting full-length FGF-2 that is expressed has 146 amino acids
in accordance with SEQ ID NO: 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.
[0050] 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).
[0051] 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-SEPHAROSER.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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] A typical pharmaceutical composition comprises 0.001 to 10
.mu.mg/ml, more typically 0.03 to 0.5 .mu.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 NaCI, 10 mM
Na citrate, and 1 mM EDTA, pH 15. 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.
[0060] 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).
[0061] 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.
[0062] 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 20 .mu.g/kg to 36 .mu./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.
[0063] 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.
[0064] 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.
[0065] 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 .mu.g/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.
[0066] 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
2TABLE 2 COMPARISON OF QUALITY OF LIFE BEFORE AND 57 DAYS AFTER IC
FGF-2 Baseline Seattle Angina (Pre FGF-2) 57 Days Post
Questionnaire Mean FGF-2 Mean Mean (SAQ) Subscales Score .+-. SD
Score .+-. SD Change.sup.1 p Value n Exertional 55 .+-. 23 68 .+-.
25 13* 0.02 28 Capacity Angina 42 .+-. 32 66 .+-. 28 24* <0.001
28 Frequency Angina Stability 46 .+-. 26 82 .+-. 20 36* <0.001
27 Disease 40 .+-. 21 61 .+-. 26 19* <0.001 28 Perception
Treatment 74 .+-. 24 88 .+-. 16 14* 0.002 28 Satisfaction
*Significantly different from baseline to fifty-seven days. .sup.1A
mean change of 8 points or more is considered clinically
significant.
[0067]
3TABLE 3 IMPROVEMENTS IN THE QUALITY OF LIFE AT DAY 57 (POST IC
rFGF-2) AT LOWER AND HIGHER DOSES Dose <2 .mu.g/kg Dose <2
.mu.g/kg IC rFGF-2 IC rFGF-2 (n = 7) (n = 8) Mean Change Mean
Change Seattle Angina in Score in Score Questionnaire (Day 57
score- (Day 57 score- Independent (SAQ) Subscales screen score)
screen score) Samples t-test Exertional 12.30 (23.3) 15.98 (28.7) t
= -.27 p = .79 Capacity Disease 26.19 (26.9) 24.47 (21.2) t = .14 p
= .89 Perception Treatment 22.32 (27.7) 10.93 (17.3) t = .97 p =
.35 Satisfaction Angina 28.57 (27.3) 13.75 (22.6) t = 1.15 p = .27
Frequency 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.
[0068] 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 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
[0069] 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
4TABLE 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)
[0070] 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.
[0071] 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 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.
[0072] 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.
[0073] 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 (Ty,) 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
pharinacokinetic 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.1") 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
5TABLE 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
.about. 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 = .about.3.5 hr
Biphasic decline of plasma rFGF-2 (n = 17; 26 .+-. 4 kg) 70 U/kg
heparin .about. 15 3-Comp. Model; Dose-linearity min before rFGF-2
T1/2.alpha. = 1.5 min V.sub.1 equal to .about. plasma volume
T1/2.beta. = 17 min V.sub.55 equal to .about. 10-fold 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 Without Heparin The rFGF-2 distribution phase
was less steep, the under general anesthesia min IV infusion for 6
(Doses 1-6): volume of distribution smaller, and clearance was (n =
6; 25 .+-. 5 kg) weeks T1/2 = 2-6 hr slower with
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 V.sub.55 = 71-153 ml/kg similar with or without
heparin
[0074] 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.
[0075] 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 pg.multidot.hr/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.
[0076] 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
6TABLE 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
Time-Averaged PK In all cases, heparin increased the rFGF-2 plasma
levels (27 males; 381 .+-. 48 g; Parameters: Both volume of
distribution and clearance of 20 females; 268 .+-. 22 g) T1/2 CL
V.sub.55 rFGF-2 were smaller with heparin hr. ml/hr/kg ml/kg
Greatest changes on CL and V.sub.55 were observed when 30 .mu.g/kg
bolus IV 0.75 4332 2389 heparin was administered immediately prior
to rFGF-2 injection No heparin 40 U/kg IV Heparin: at .about. 15
min 0.91 1728 844 just prior to rFGF-2 1.3 516 147 at +15 min 1.2
1158 626 at +3 hr 0.93 1338 1351
[0077]
7TABLE 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 by 20-min IC injection from 0.33-24 .mu.g/kg phase
during 1 hr; followed by elimination phase with (10 min in left
main doses (n = 32) T1/2 approximately (.about.) 2 hr coronary
artery + 10 T1/2 = 1.9 .+-. 2.2 hr Dose-linearity min 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
[0078] FGF-2 receptor is approximately two orders of magnitude
higher (dissociation constant .about.2.times.10.sup.-11 M).
Moscatelli et al., (1991).
[0079] 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.
[0080] 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"
[0081] 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.
[0082] 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.
[0083] The rFGF Diluent was supplied in 5 cc type I glass vials
with a laminated gray butyl rubber stopper and red flip-off
overseal. The rFGF-2 diluent contains 10 mM sodium citrate, 10 mM
monothioglycerol, 135 mM sodium chloride, pH 5.0. Each vial
contained 5.2 ml of rFGF-2 diluent solution that was stored at
2.degree. to 8.degree. C.
[0084] 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 infuision
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"
[0085] 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:
Inclusion criteria: Subject is eligible if:
[0086] Male or female, greater than or equal to 18 years of age
[0087] Diagnosis of coronary artery disease (CAD)
[0088] Suboptimal candidates for approved revascularization
procedures, e.g., angioplasty, stents, coronary artery bypass graft
(CABG) (or refuses those interventions)
[0089] Able to exercise at least three minutes using a modified
Bruce protocol and limited by coronary ischemia
[0090] Inducible and reversible defect of at least 20% myocardium
on pharmacologically stressed thallium sestamibi scan
[0091] CBC, platelets, serum chemistry within clinically acceptable
range for required cardiac catheterization
[0092] Normal INR, or if anticoagulated with Coumadin, INR
<2.0
[0093] Willing and able to give written informed consent to
participate in this study, including all required study procedures
and follow-up visits
Exclusion criteria: Subject is not eligible if:
[0094] Malignancy: any history of malignancy within past ten years,
with the exception of curatively treated basal cell carcinoma
[0095] 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
[0096] Renal function: creatinine clearance below normal range
adjusted for age; protein >250 mg or microalbumin >30 mg/24 h
urine
[0097] Class IV heart failure (New York Heart Association)
[0098] Ejection fraction <20% by echocardiogram, thallium scan,
MRI or gated pooled blood scan (MUGA)
[0099] Hemodynamically relevant arrhythmias (e.g., ventricular
fibrillation, sustained ventricular tachycardia)
[0100] Severe valvular stenosis (aortic area <1.0 cm.sup.2,
mitral area <1.2 cm.sup.2), or severe valvular insufficiency
[0101] Marked increase in angina or unstable angina within three
weeks
[0102] History of myocardial infarction (MI) within three
months
[0103] History of transient ischemic attack (TIA) or stroke within
six months
[0104] History of CABG, angioplasty or stent within six months
[0105] History of treatment with transmyocardial laser
revascularization, rFGF-2, or vascular enodothelial growth factor
(VEGF) within six months
[0106] Females of child-bearing potential or nursing mothers
[0107] Any pathological fibrosis, e.g., pulmonary fibrosis,
scleroderma
[0108] Known vascular malformation, e.g., AV malformation,
hemangiomas
[0109] Coexistence of any disease which might interfere with
assessment of symptoms of CAD, e.g. pericarditis, costochondritis,
esophagitis, systemic vasculitis, sickle cell disease
[0110] 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)
[0111] Participation in clinical trials of investigational agents,
devices or procedures within thirty days (or scheduled within sixty
days of study drug)
[0112] Known hypersensitivity to rFGF-2 or related compounds
[0113] 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"
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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).
[0118] Abnormal perfusion zone, which was assessed at one of the
sites on 28 patients, decreased significantly at 30 and 60 days
(p<0.001).
[0119] In addition to the above criterion (ie., 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"
[0120] 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"
[0121] 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. 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.
[0122] 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.
[0123] 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"
[0124] 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.
[0125] 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.
[0126] 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
15 1 140 PRT Human FGF-1 1 Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys
Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg
Ile Leu Pro Asp Gly Thr Tyr Asp 20 25 30 Gly Thr Arg Asp Arg Ser
Asp Gln His Ile Gln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Tyr Gly
Glu Tyr Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala
Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn 65 70 75 80
Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85
90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Tyr Gly Leu
Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr
Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu Pro Tyr Ser Ser
Asp 130 135 140 2 140 PRT bovine FGF-1 2 Phe Asn Leu Pro Leu Gly
Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly
Tyr Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr
Lys Asp Arg Ser Asp Gly His Ile Gln Leu Phe Leu Cys Ala 35 40 45
Glu Ser Ile Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Phe 50
55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro
Asp 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His
Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu Lys His Trp
Phe Val Gly Leu Lys 100 105 110 Lys Asn Gly Arg Ser Lys Leu Glu Pro
Arg Thr His Phe Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro Leu
Pro Val Ser Ser Asp 130 135 140 3 146 PRT Human FGF-2 3 Pro Ala Leu
Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His 1 5 10 15 Phe
Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25
30 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp
35 40 45 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val
Val Ser 50 55 60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met
Lys Glu Asp Gly 65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp
Glu Cys Phe Phe Phe Glu 85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn
Thr Tyr Arg Ser Arg Lys Tyr Thr 100 105 110 Ser Trp Tyr Val Ala Leu
Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser 115 120 125 Lys Thr Gly Pro
Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala 130 135 140 Lys Ser
145 4 442 DNA bovine FGF-2 CDS (1)..(438) 4 cca gcc cta cca gaa gat
ggg ggg tcc ggg gcc ttc cca cca ggg cac 48 Pro Ala Leu Pro Glu Asp
Gly Gly Ser Gly Ala Phe Pro Pro Gly His 1 5 10 15 ttc aaa gat cca
aaa cga cta tat tgt aaa aac ggg ggg ttc ttc cta 96 Phe Lys Asp Pro
Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25 30 cga atc
cac cca gat ggg cga gta gat ggg gta cga gaa aaa tcc gat 144 Arg Ile
His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp 35 40 45
cca cac atc aaa cta caa cta caa gcc gaa gaa cga ggg gta gta tcc 192
Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 50
55 60 atc aaa ggg gta tgt gcc aac cga tat cta gcc atg aaa gaa gat
ggg 240 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp
Gly 65 70 75 80 cga cta cta gcc tcc aaa tgt gta acc gat gaa tgt ttc
ttc ttc gaa 288 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe
Phe Phe Glu 85 90 95 cga cta gaa tcc aac aac tat aac acc tat cga
tcc cga aaa tat tcc 336 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg
Ser Arg Lys Tyr Ser 100 105 110 tcc tgg tat gta gcc cta aaa cga acc
ggg caa tat aaa cta ggg cca 384 Ser Trp Tyr Val Ala Leu Lys Arg Thr
Gly Gln Tyr Lys Leu Gly Pro 115 120 125 aaa acc ggg cca ggg caa aaa
gcc atc cta ttc cta cca atg tcc gcc 432 Lys Thr Gly Pro Gly Gln Lys
Ala Ile Leu Phe Leu Pro Met Ser Ala 130 135 140 aaa tcc taag 442
Lys Ser 145 5 146 PRT bovine FGF-2 5 Pro Ala Leu Pro Glu Asp Gly
Gly Ser Gly Ala Phe Pro Pro Gly His 1 5 10 15 Phe Lys Asp Pro Lys
Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25 30 Arg Ile His
Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp 35 40 45 Pro
His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 50 55
60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly
65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe
Phe Glu 85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser
Arg Lys Tyr Ser 100 105 110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly
Gln Tyr Lys Leu Gly Pro 115 120 125 Lys Thr Gly Pro Gly Gln Lys Ala
Ile Leu Phe Leu Pro Met Ser Ala 130 135 140 Lys Ser 145 6 9 PRT
Bovis bovinus 6 Met Ala Ala Gly Ser Ile Thr Thr Leu 1 5 7 240 PRT
Murine FGF-3 7 Met Gly Leu Ile Trp Leu Leu Leu Leu Ser Leu Leu Glu
Pro Ser Trp 1 5 10 15 Pro Thr Thr Gly Pro Gly Thr Arg Leu Arg Arg
Asp Ala Gly Gly Arg 20 25 30 Gly Gly Val Tyr Glu His Leu Gly Gly
Ala Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Ala Thr Lys Tyr His
Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60 Asn Gly Ser Leu Glu
Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 65 70 75 80 Val Glu Val
Gly Val Val Ala Ile Lys Gly Leu Phe Ser Gly Arg Tyr 85 90 95 Leu
Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Asp His Tyr Asn 100 105
110 Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn Thr
115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Gly Ser Ser Gly Pro Gly
Ala Gln 130 135 140 Arg Gln Pro Gly Ala Gln Arg Pro Trp Tyr Val Ser
Val Asn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr
Arg Arg Thr Gln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val Leu
Gly His Lys Asp His Glu Met Val Arg 180 185 190 Leu Leu Gln Ser Ser
Gln Pro Arg Ala Pro Gly Glu Gly Ser Gln Pro 195 200 205 Arg Gln Arg
Arg Gln Lys Lys Gln Ser Pro Gly Asp His Gly Lys Met 210 215 220 Glu
Thr Leu Ser Thr Arg Ala Thr Pro Ser Thr Gln Leu His Thr Gly 225 230
235 240 8 205 PRT Human FGF-4 8 Ser Gly Pro Gly Thr Ala Ala Val Ala
Leu Leu Pro Ala Val Leu Leu 1 5 10 15 Ala Leu Leu Ala Pro Trp Ala
Gly Arg Gly Gly Ala Ala Ala Pro Thr 20 25 30 Ala Pro Asn Gly Thr
Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu Ser 35 40 45 Leu Val Ala
Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro Lys 50 55 60 Glu
Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile Lys 65 70
75 80 Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu
Gln 85 90 95 Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His Ala Asp
Thr Arg Asp 100 105 110 Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly
Val Val Ser Ile Phe 115 120 125 Gly Val Ala Ser Arg Phe Phe Val Ala
Met Ser Ser Lys Gly Lys Leu 130 135 140 Tyr Gly Ser Pro Phe Phe Thr
Asp Glu Cys Thr Phe Lys Glu Ile Leu 145 150 155 160 Leu Pro Asn Asn
Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met 165 170 175 Phe Ile
Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg Val 180 185 190
Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205 9
266 PRT Human FGF-5 9 Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser
His Leu Ile Leu Ser 1 5 10 15 Ala Trp Ala His Gly Glu Lys Arg Leu
Ala Pro Lys Gly Gln Pro Gly 20 25 30 Pro Ala Ala Thr Asp Arg Asn
Pro Arg Gly Ser Ser Ser Arg Gln Ser 35 40 45 Ser Ser Ser Ala Met
Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala Ala 50 55 60 Ser Leu Gly
Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln Trp 65 70 75 80 Ser
Leu Gly Ala Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly Ile Gly 85 90
95 Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser His Glu
100 105 110 Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln
Gly Ile 115 120 125 Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu
Ala Met Ser Lys 130 135 140 Lys Gly Lys Leu His Ala Ser Ala Lys Phe
Thr Asp Asp Cys Lys Phe 145 150 155 160 Arg Glu Arg Phe Gln Glu Asn
Ser Tyr Asn Thr Tyr Ala Ser Ala Ile 165 170 175 His Arg Thr Glu Lys
Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys 180 185 190 Arg Gly Lys
Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His 195 200 205 Ile
Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro Glu 210 215
220 Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Lys Pro Pro Ser Pro
225 230 235 240 Ile Lys Pro Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn
Thr Asn Ser 245 250 255 Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260
265 10 207 PRT Human FGF-6 10 Ala Leu Gly Gln Lys Leu Phe Ile Thr
Met Ser Arg Gly Ala Gly Arg 1 5 10 15 Leu Gln Gly Thr Leu Trp Ala
Leu Val Phe Leu Gly Ile Leu Val Gly 20 25 30 Met Val Val Pro Ser
Pro Ala Gly Thr Arg Ala Asn Asn Thr Leu Leu 35 40 45 Asp Ser Arg
Gly Trp Gly Thr Leu Leu Ser Arg Ser Arg Ala Gly Leu 50 55 60 Ala
Gly Glu Ile Ala Gly Val Asn Trp Glu Ser Gly Tyr Leu Val Gly 65 70
75 80 Ile Lys Arg Gln Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe
His 85 90 95 Leu Gln Val Leu Pro Asp Gly Arg Ile Ser Gly Thr His
Glu Glu Asn 100 105 110 Pro Tyr Ser Leu Leu Glu Ile Ser Thr Val Glu
Arg Gly Val Val Ser 115 120 125 Leu Phe Gly Val Arg Ser Ala Leu Phe
Val Ala Met Asn Ser Lys Gly 130 135 140 Arg Leu Tyr Ala Thr Pro Ser
Phe Gln Glu Glu Cys Lys Phe Arg Glu 145 150 155 160 Thr Leu Leu Pro
Asn Asn Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr Gln 165 170 175 Gly Thr
Tyr Ile Ala Leu Ser Lys Tyr Gly Arg Val Lys Arg Gly Ser 180 185 190
Lys Val Ser Pro Ile Met Thr Val Thr His Phe Leu Pro Arg Ile 195 200
205 11 193 PRT Human FGF-7 11 Met His Lys Trp Ile Leu Thr Trp Ile
Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Ile Ile Cys
Leu Val Gly Thr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro
Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg
His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60 Arg
Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 70
75 80 Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr
Asn 85 90 95 Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala
Ile Lys Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys
Glu Gly Lys Leu Tyr 115 120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys
Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr
Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met Phe
Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys
Thr Lys Lys Gln Lys Thr Ala His Phe Leu Pro Met Ala Ile 180 185 190
Thr 12 215 PRT Murine FGF-8 12 Met Gln Ser Pro Arg Ser Ala Leu Ser
Cys Leu Leu Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln Ala Gln
Val Thr Val Gln Ser Ser Pro Asn Phe 20 25 30 Thr Gln His Val Arg
Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg 35 40 45 Arg Leu Ile
Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55 60 Val
Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Phe Asp Gln 65 70
75 80 Asp Pro Phe Ala Lys Leu Ile Val Glu Tyr Asp Thr Phe Gly Ser
Arg 85 90 95 Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys
Met Asn Lys 100 105 110 Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys
Gly Lys Asp Cys Val 115 120 125 Phe Thr Phe Ile Val Ile Glu Asn Asn
Tyr Thr Ala Leu Gln Asn Ala 130 135 140 Lys Tyr Glu Gly Trp Tyr Met
Ala Phe Thr Ala Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Ser Lys
Thr Arg Gln His Gln Arg Glu Val His Phe Met Lys 165 170 175 Arg Leu
Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu 180 185 190
Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg 195
200 205 Thr Trp Ala Pro Glu Pro Arg 210 215 13 208 PRT Human FGF-9
13 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala
1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro
Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly
Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp His Leu Lys
Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe
His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile Gln Gly Thr Arg
Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95 Phe Ile Ser Ile
Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser 100 105 110 Gly Leu
Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125
Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130
135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly
Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro
Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys Phe Thr His
Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val Pro Glu Leu
Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205 14 207 PRT Human FGF-98
14 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu
1 5 10 15 Leu Leu Cys Phe Gln Val Gln Val Leu Val Ala Glu Glu Asn
Val Asp 20 25 30 Phe Arg Ile His Val Glu Asn Gln Thr Arg Ala Arg
Asp Asp Val Ser
35 40 45 Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser
Gly Lys 50 55 60 His Ile Gln Val Leu Gly Arg Arg Ile Ser Ala Arg
Gly Glu Asp Gly 65 70 75 80 Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr
Asp Thr Phe Gly Ser Gln 85 90 95 Val Arg Ile Lys Gly Lys Glu Thr
Glu Phe Tyr Leu Cys Met Asn Arg 100 105 110 Lys Gly Lys Leu Val Gly
Lys Pro Asp Gly Thr Ser Lys Glu Cys Val 115 120 125 Phe Ile Glu Lys
Val Leu Glu Asn Asn Tyr Thr Ala Leu Met Ser Ala 130 135 140 Lys Tyr
Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155
160 Lys Gly Pro Lys Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys
165 170 175 Arg Tyr Pro Lys Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys
Tyr Thr 180 185 190 Thr Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr
His Pro Ala 195 200 205 15 266 PRT Human FGF-5 15 Ser Leu Ser Phe
Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu Ser 1 5 10 15 Ala Trp
Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gln Pro Gly 20 25 30
Pro Ala Ala Thr Asp Arg Asn Pro Arg Gly Ser Ser Ser Arg Gln Ser 35
40 45 Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala
Ala 50 55 60 Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser
Phe Gln Trp 65 70 75 80 Ser Leu Gly Ala Arg Thr Gly Ser Leu Tyr Cys
Arg Val Gly Ile Gly 85 90 95 Phe His Leu Gln Ile Tyr Pro Asp Gly
Lys Val Asn Gly Ser His Glu 100 105 110 Ala Asn Met Leu Ser Val Leu
Glu Ile Phe Ala Val Ser Gln Gly Ile 115 120 125 Val Gly Ile Arg Gly
Val Phe Ser Asn Lys Phe Leu Ala Met Ser Lys 130 135 140 Lys Gly Lys
Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys Lys Phe 145 150 155 160
Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile 165
170 175 His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn
Lys 180 185 190 Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys
Pro Gln His 195 200 205 Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln
Ser Glu Gln Pro Glu 210 215 220 Leu Ser Phe Thr Val Thr Val Pro Glu
Lys Lys Asn Pro Pro Ser Pro 225 230 235 240 Ile Lys Ser Lys Ile Pro
Leu Ser Ala Pro Arg Lys Asn Thr Asn Ser 245 250 255 Val Lys Tyr Arg
Leu Lys Phe Arg Phe Gly 260 265
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