U.S. patent application number 09/886856 was filed with the patent office on 2002-08-22 for methods and compositions for the treatment of peripheral artery disease.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Whitehouse, Martha Jo.
Application Number | 20020115603 09/886856 |
Document ID | / |
Family ID | 27498936 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115603 |
Kind Code |
A1 |
Whitehouse, Martha Jo |
August 22, 2002 |
Methods and compositions for the treatment of peripheral artery
disease
Abstract
Compositions and methods for treating peripheral artery disease
in a patient are provided. Compositions comprise recombinant
fibroblast growth factor-2. Fibroblast growth factor, such as
FGF-2, is administered in therapeutically effective amounts to
treat or prevent peripheral artery disease including claudication
and critical limb ischemia. Pharmaceutical compositions comprising
a therapeutically effective amount of FGF-2 and a pharmaceutically
acceptable carrier are also provided. The methods of the invention
to treat peripheral artery disease and claudication comprise
administering at least a single dose of a pharmaceutical
composition comprising the FGF, such as FGF-2, via intra-arterial,
intravenous, or intramuscular infusion to the patient. It is
recognized that increased benefits may result from multiple dosing,
including intermittent dosing.
Inventors: |
Whitehouse, Martha Jo; (San
Francisco, CA) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property Department
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
|
Family ID: |
27498936 |
Appl. No.: |
09/886856 |
Filed: |
June 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60213504 |
Jun 22, 2000 |
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60264572 |
Jan 26, 2001 |
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60276549 |
Mar 16, 2001 |
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Current U.S.
Class: |
514/9.1 ;
514/13.3; 514/16.4 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 2300/00 20130101; C07K 14/50 20130101; A61P 9/00 20180101;
A61P 25/04 20180101; A61K 38/1825 20130101; A61K 38/1825 20130101;
A61P 5/50 20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/18 |
Claims
That which is claimed:
1. A method for treating peripheral artery disease in a patient,
said method comprising administering to said patient a
therapeutically effective amount of fibroblast growth factor (FGF),
wherein said therapeutically effective amount of FGF is divided
into two doses and a single dose is administered into each leg of
said patient within a one hour period.
2. The method of claim 1, wherein said FGF is administered by
intra-arterial infusion (IA) into at least one artery of each leg
of said patient.
3. The method of claim 2, wherein said FGF is administered into the
common femoral artery of each leg of said patient.
4. The method of claim 3, wherein said FGF is administered via
bilateral delivery using a catheter.
5. The method of claim 3, wherein said FGF is administered via
direct IA infusion into the common femoral artery of each leg of
said patient.
6. The method of claim 1, wherein said FGF is administered by one
or more intramuscular (IM) injections.
7. The method according to claim 1, wherein said peripheral artery
disease is evidenced by claudication.
8. The method according to claim 7, wherein said patient has
critical limb ischemia.
9. The method of claim 1, wherein said FGF is FGF-2.
10. The method of claim 9, wherein said FGF-2 is a recombinant
molecule.
11. The method of claim 10, wherein said FGF-2 comprises the
sequence set forth in FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4),
FIG. 4 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:8) or an angiogenically
active fragment or mutein thereof.
12. The method of claim 11, wherein said mutein comprises an FGF-2
molecule wherein at least one constituent cysteine residue is
replaced by a neutral amino acid.
13. The method of claim 12, wherein the neutral amino acid is
serine or threonine.
14. The method of claim 11, wherein said FGF-2 is administered
simultaneously with another molecule selected from the group
consisting of heparin and other proteoglycan.
15. The method of claim 14, wherein said heparin is a low molecular
weight molecule.
16. The method of claim 14, wherein said heparin is unfractionated
heparin.
17. The method of claim 11, wherein said FGF-2 is administered
within about 5 minutes to about 60 minutes of heparin or
proteoglycan administration to said patient.
18. The method of claim 17, wherein said FGF-2 is administered
within about 20 minutes to about 30 minutes of heparin or other
proteoglycan administration to said patient.
19. The method of claim 11, wherein said FGF-2 is administered in
the absence of administering a molecule selected from the group
consisting of heparin and other proteoglycan.
20. The method of claim 11, wherein said therapeutically effective
amount of FGF-2 is administered to said patient once in a 24 hour
period.
21. The method of claim 11, wherein said therapeutically effective
amount of FGF-2 is administered to said patient once a week.
22. The method of claim 11, wherein said therapeutically effective
amount of FGF-2 is administered to said patient once a month, once
every 2 months, once every 3 months, once every four months, once
every five months, or once every six months.
23. The method of claim 11, wherein said therapeutically effective
amount of FGF-2 is administered as an adjunct to vascular surgery,
mechanical bypass surgery, angioplasty, or angiogram.
24. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 0. 1 .mu.g/kg to about 1 .mu.g/kg.
25. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 1 .mu.g/kg to about 3 .mu.g/kg.
26. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 3 .mu.g/kg to about 5 .mu.g/kg.
27. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 5 .mu.g/kg to about 7 .mu.g/kg.
28. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 7 .mu.g/kg to about 9 .mu.g/kg.
29. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 9 .mu.g/kg to about 10 .mu.g/kg.
30. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 10 .mu.g/kg to about 15 .mu.g/kg.
31. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 15 .mu.g/kg to about 20 .mu.g/kg.
32. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 20 .mu.g/kg to about 25 .mu.g/kg.
33. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 25 .mu.g/kg to about 30 .mu.g/kg.
34. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 30 .mu.g/kg to about 40 .mu.g/kg.
35. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 40 .mu.g/kg to about 50 .mu.g/kg.
36. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 4 .mu.g to about 0.3 mg.
37. The method of claim 11, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 0.3 mg to about 3.5 mg.
38. The method of claim 37, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 1.0 to about 2.0mg.
39. The method of claim 37, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 2.0 to about 3.5mg.
40. The method of claim 9, wherein said FGF-2 is administered to
said patient by intra-arterial (IA) or intravenous (IV)
infusion.
41. The method of claim 9, wherein said FGF-2 is administered to
said patient by one or more intramuscular (IM) injections.
42. The method of claim 9, wherein said FGF-2 is administered to
said patient by subcutaneous (SC) injection.
43. The method of claim 9, wherein said administering of FGF-2
provides an improvement in peak walking time (PWT) in said patient
relative to PWT in the absence of said administering of FGF-2.
44. The method of claim 9, wherein said administering of FGF-2
provides an improvement in anklebrachial index (ABI) in said
patient relative to ABI in the absence of said administering of
FGF-2.
45. The method of claim 9, wherein said administering of FGF-2
results in a reduction in body pain.
46. The method of claim 9, wherein said administering of FGF-2
improves stair climbing ability.
47. The method of claim 9, wherein said administering of FGF-2
reduces the severity of claudication.
48. A method for treating peripheral artery disease in a patient,
said method comprising administering to said patient a
therapeutically effective amount of fibroblast growth factor-2
(FGF-2), wherein said therapeutically effective amount is about 0.1
.mu.g/kg to about 9.9 .mu..mu.g/kg.
49. The method of claim 48, wherein said therapeutically effective
amount of FGF-2 is administered as part of a pharmaceutical
composition.
50. The method of claim 49, wherein said pharmaceutical composition
is a stabilized FGF-2-DTT formulation.
51. The method of claim 48, wherein said FGF-2 is administered
simultaneously with another molecule selected from the group
consisting of heparin and other proteoglycan.
52. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 0.1 .mu.g/kg to about 1 .mu.g/kg.
53. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 1 .mu.g/kg to about 3 .mu.g/kg.
54. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 3 .mu.g/kg to about 5 .mu.g/kg.
55. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 5 .mu.g/kg to about 7 .mu.g/kg.
56. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 7 .mu.g/kg to about 8 .mu.g/kg.
57. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 8 .mu.g/kg to about 9 .mu.g/kg.
58. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 9 .mu.g/kg to about 9.9 .mu.g/kg.
59. The method of claim 48, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 7.0 .mu.g to about 0.7 mg.
60. The method of claim 59, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 9.0 .mu.g to about 0.5 mg.
61. The method of claim 60, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 0.1 mg to about 0.4 mg.
62. The method of claim 61, wherein said therapeutically effective
amount of said FGF-2 or said angiogenically active fragment or
mutein thereof is about 0.1 mg to about 0.2 mg.
63. The method of claim 48, wherein said FGF-2 is administered to
said patient by intra-arterial (IA) or intravenous (IV)
infusion.
64. The method of claim 48, wherein said FGF-2 is administered to
said patient by one or more intramuscular (IM) injections.
65. A method for improving peak walking time in a patient with
intermittent claudication, said method comprising administering to
said patient a therapeutically effective amount of fibroblast
growth factor (FGF), wherein said therapeutically effective amount
of FGF is divided into two doses and a single dose is administered
into each leg of said patient within a one hour period.
66. The method of claim 65, wherein said FGF is FGF-2.
67. The method of claim 66, wherein said therapeutically effective
amount of said FGF-2 is about 0.1 .mu.g/kg to about 1 .mu.g/kg.
68. The method of claim 66, wherein said therapeutically effective
amount of said FGF-2 is about 1 .mu.g/kg to about 3 .mu.g/kg.
69. The method of claim 66, wherein said therapeutically effective
amount of said FGF-2 is about 3 .mu.g/kg to about 5 .mu.g/kg.
70. The method of claim 66, wherein said therapeutically effective
amount of said FGF-2 is about 5 .mu.g/kg to about 9 .mu.g/kg.
71. The method of claim 66, wherein said therapeutically effective
amount of said FGF-2 is about 9 .mu.g/kg to about 10 .mu.g/kg.
72. The method of claim 66, wherein said therapeutically effective
amount of said FGF-2 is about 10 .mu.g/kg to about 20 .mu.g/kg.
73. The method of claim 66, wherein said therapeutically effective
amount of said FGF-2 is about 20 .mu.g/kg to about 30 .mu.g/kg.
74. A method for improving ankle-brachial index in a patient with
intermittent claudication, said method comprising administering to
said patient a therapeutically effective amount of fibroblast
growth factor (FGF), wherein said therapeutically effective amount
of FGF is divided into two doses and a single dose is administered
into each leg of said patient within a one hour period.
75. The method of claim 74, wherein said FGF is FGF-2.
76. The method of claim 75, wherein said therapeutically effective
amount of said FGF-2 is about 0.1 .mu.g/kg to about 1 .mu.g/kg.
77. The method of claim 75, wherein said therapeutically effective
amount of said FGF-2 is about 1 .mu.g/kg to about 3 .mu.g/kg.
78. The method of claim 75, wherein said therapeutically effective
amount of said FGF-2 is about 3 .mu.g/kg to about 5 .mu.g/kg.
79. The method of claim 75, wherein said therapeutically effective
amount of said FGF-2 is about 5 .mu.g/kg to about 9 .mu.g/kg.
80. The method of claim 75, wherein said therapeutically effective
amount of said FGF-2 is about 9 .mu.g/kg to about 10 .mu.g/kg.
81. The method of claim 75, wherein said therapeutically effective
amount of said FGF-2 is about 10 .mu.g/kg to about 20 .mu.g/kg.
82. The method of claim 75, wherein said therapeutically effective
amount of said FGF-2 is about 20 .mu.g/kg to about 30 .mu.g/kg.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Serial Nos. 60/213,504, filed Jun. 22, 2000,
60/264,572, filed Jan. 26, 2001, and 60/276,549, filed Mar. 16,
2001, each of which is entitled "Methods and Compositions for the
Treatment of Peripheral Artery Disease," the contents of which are
herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to methods and pharmaceutical
compositions for treating peripheral artery disease, particularly
the administration of compositions that contain recombinant
fibroblast growth factor-2 (rFGF-2).
BACKGROUND OF THE INVENTION
[0003] Coronary artery disease (CAD) and peripheral artery disease
(PAD) are conditions characterized by insufficient blood flow,
usually secondary to atherosclerosis. Symptoms of ischemia (angina
pectoris for CAD or intermittent claudication for PAD) are brought
on by stress and relieved by rest. In CAD, symptoms may become life
threatening due to myocardial infarction, arrhythmia, and
progressive heart failure. In PAD, symptoms are less likely to be
life threatening except when critical limb ischemia develops, but
the risk of adverse cardiovascular events and death is
increased.
[0004] Identification and management of risk factors are important
in the medical management of both CAD and PAD. Pharmacologic
management of risk factors may include anti-hypertensives,
lipid-lowering agents, and hypoglycemic agents; smoking cessation,
diet, and exercise are often prescribed with variable compliance.
Pharmacologic management aimed at reduction of symptoms of ischemia
often includes vasodilators, anti-anginal, and anti-platelet
therapy. Mechanical revascularization by percutaneous angioplasty
(with or without a stent) and direct surgical reconstruction
improve blood flow and reduce symptoms. However, restenosis after
angioplasty and progression of disease may limit the duration of
the benefit.
[0005] PAD afflicts approximately 11 million patients in the United
States. Approximately one third of these patients experience
intermittent claudication (discomfort, pain, fatigue, or heaviness
in the leg muscles that consistently is brought on by the same
amount of muscular activity and relieved by rest). Claudication is
similar to angina and represents ischemic muscle pain that may be
localized to the hip, buttock, thigh, or calf. It occurs
predictably with the same amount of physical stress.
Atherosclerosis is systemic, but often one lower limb is more
affected than the other. Patients may develop critical limb
ischemia, with rest pain, non-healing ulcers, and/or gangrene. Rest
pain occurs when blood supply is inadequate to meet the basic
nutritional requirements at rest and typically localizes in the
toes or foot of the affected limb.
[0006] The prevalence of CAD and PAD is expected to increase in
countries with aging populations, as aging is a primary risk factor
for atherosclerosis. Less invasive catheter-based treatment methods
and more cost-effective programs and treatment methodologies are
needed to manage these conditions.
SUMMARY OF THE INVENTION
[0007] Compositions and methods for treating peripheral artery
disease (PAD) in a patient are provided. Pharmaceutical
compositions comprising a therapeutically effective amount of
fibroblast growth factor, such as FGF-2, and a pharmaceutically
acceptable carrier are provided. Such compositions when
administered in accordance with the methods of the invention
provide effective treatment for PAD patients including those
suffering intermittent claudication associated with this disease.
Such compositions may also be administered to PAD patients to
prevent progression of critical limb ischemia to amputation.
[0008] The methods of the invention comprise administering
pharmaceutical compositions comprising a therapeutically effective
amount of a growth factor, such as FGF-2, as an intra-arterial
infusion (IA), intravenous infusion (IV), intramuscular injection
(IM), or subcutaneous injection (SC). A single-dose administration
of FGF-2 is efficacious for the treatment of PAD. Therapeutic
benefits may be obtained with multiple doses without compromising
safety. Administration of FGF-2 improves peak walking time in
patients with PAD for at least 90 days after FGF-2 administration.
FGF-2 can be used to treat patients suffering from critical limb
ischemia including those with resting pain with and without
non-healing ulcers. Additionally, FGF-2 can be used to treat PAD
patients suffering from critical limb ischemia. The FGF-containing
composition of the invention can be administered as adjuncts to
vascular surgery involving mechanical bypass and percutaneous
transluminal interventions with balloon catheters, with or without
stents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 sets forth the DNA sequence (SEQ ID NO:1) encoding
fibroblast growth factor-2 (FGF-2) having the amino acid sequence
set forth in FIG. 2; this FGF-2 is of bovine origin. The translated
amino acid sequence (SEQ ID NO:2) is also shown.
[0010] FIG. 2 sets forth the amino acid sequence (SEQ ID NO: 2) for
the 146 amino acid residue bovine FGF-2 encoded by the DNA sequence
set forth in FIG. 1.
[0011] FIG. 3 sets forth the DNA sequence (SEQ ID NO:3) encoding
the translated amino acid sequence (SEQ ID NO:4) for the 146 amino
acid residue FGF-2 of human origin.
[0012] FIG. 4 sets forth the DNA sequence (SEQ ID NO:5) encoding
the translated amino acid sequence (SEQ ID NO:6) for the 155 amino
acid residue FGF-2 of bovine origin.
[0013] FIG. 5 sets forth the DNA sequence (SEQ ID NO:7) encoding
the translated amino acid sequence (SEQ ID NO:8) for the 155 amino
acid residue FGF-2 of human origin.
[0014] FIG. 6 shows the relative change in peak walking time (PWT)
at day 90 with administration of rFGF-2 in patients in a phase II
clinical study. In this study, three patient groups were assessed:
a group administered a placebo on both days 1 and 30; a group
administered a single dose of rFGF-2 (30 .mu.g/kg) on day 1 and a
placebo on day 30; and a group administered a dose of rFGF-2 (30
.mu.g/kg) on both days 1 and 30. The mean and standard error are
indicated for the measured PWT in each of these groups. The ANOVA
analysis excluded patients with missing data and revascularized
patients. The ANOVA of Ranks test included patients with missing
data and revascularized patients by assigning the lowest rank.
Pairwise comparison indicated a p value of 0.026 between the single
dose and placebo groups and a p value of 0.45 between the double
dose and placebo group. The figure provides the primary efficacy
analysis of the clinical trial, which specified the use of
log-transformed data. This is considered appropriate statistical
management of data when the results have skewness or kertosis such
as is often seen in treadmill tests.
[0015] FIG. 7 shows absolute change in PWT at days 90 and 180 for
the patient groups receiving placebo, single-dose rFGF-2, or
double-dose rFGF-2. For each patient the PWT at baseline is
subtracted from the PWT at day 90 and the differences are summed
for each group and a mean determined; the data are analyzed by an
analysis of variance (ANOVA).
[0016] FIG. 8 shows the percent absolute change in PWT in the three
patient groups shown at day 90 and day 180. The percent change in
PWT averaged across the two rFGF-2 groups is also shown (designated
Any FGF).
[0017] FIG. 9 shows the measured ABI (ankle brachial index) for the
three patient groups of the phase II clinical study. A baseline
measurement, a day-90 measurement, and the corresponding change
between the baseline and day-90 measurement are indicated. The mean
change in ABI is also shown for the three patient groups. The ABI
is described in An Office Based Approach to the Diagnosis and
Treatment of Peripheral Arterial Disease (2000) Society of Vascular
Medicine and Biology (Medical Communications Media, Inc.,
Wrightstown, Pa.) herein incorporated by reference. Subjects having
an ABI>1.2 at baseline are excluded from the analysis.
[0018] FIG. 10 shows the results of the WIQ severity of
claudication for the three patient groups in the phase II clinical
study at day 90 and day 180. Values represent the percentage of
patients in each group indicating an improvement, no change, or
worsening of this condition.
[0019] FIG. 11 shows the severity scores at baseline, day 90, and
day 180 for distance, speed, and stair climbing for each group. The
figure demonstrates that the results for the single-dose group were
better than the results for the placebo group for WIQ distance,
speed, and stair climbing. The figure is shown with a scale where
higher scores are better.
[0020] FIG. 12 depicts the physical summary scores from the short
form 36 (SF-36). A change of 1 point is associated with an
increased lifespan of 2 years. The change scores in the figure
indicate an improvement in the single-dose group versus the placebo
group by greater than 2 points at day 90.
[0021] FIG. 13 summarizes the results of the study.
[0022] FIG. 14 shows the measured ABI (ankle-brachial index) for
the three patient groups of the phase II clinical study, when
subjects having an ABI>1.2 at anytime (i.e., baseline, day 90,
and/or day 180) are excluded from the analysis. A baseline
measurement, a day-90 measurement, and the corresponding change
between the baseline and day-90 measurement are indicated. The mean
change in ABI is also shown for the three patient groups.
[0023] FIG. 15 shows a hypothetical plot of peak walking time at
day 90 (PWT90) versus peak walking time at baseline (PWTB) when
absolute change score is assumed to be the correct analysis
variable. Assumptions: (PWT90-PWTB)=1d, then PWT90=1.0*PWTB+d
PWT90, scatter plot is linear, slope=1.0, and intercept is d
(unrestricted).
[0024] FIG. 16 shows a hypothetical plot of PWT90 versus PWTB when
relative change score is assumed to be the correct analysis
variable. Assumptions: (PWT90/PWTB)=1d, then PWT90=1d*PWTB+0.0
(across the full range of PWTB), scatter plot is linear, slope is
Id (unrestricted), and intercept is 0.0.
[0025] FIG. 17 shows a scatter plot of PWT90 versus PWTB plus an
unrestricted spline regression curve for the placebo (.DELTA.),
single-dose (.quadrature.), and double-dose (.smallcircle.)
groups.
[0026] FIG. 18 shows the same scatter plot from FIG. 16 plus curves
representing regression model 2 described in Table 15 as applied to
the placebo (.DELTA.), single-dose (.quadrature.), and double-dose
(.smallcircle.) groups. P=placebo; S=single-dose;
D=double-dose.
[0027] FIG. 19 shows the scatter plot of PWT180 versus PWTB plus an
unrestricted spline regression curve for the placebo (.DELTA.),
single-dose (.quadrature.), and double-dose (.smallcircle.)
groups.
[0028] FIG. 20 shows the effect of single administration by
intra-arterial infusion (IA) or intramuscular injection (IM) and
14-day continuous intra-arterial infusion on total hindlimb blood
flow in a rat bilateral PAD model. Phosphate-buffered solution
(PBS) served as the vehicle control.
DETAILED DESCRIPTION OF THE INVENTION
[0029] One potential new alternative for the treatment of
intermittent claudication due to peripheral artery disease (PAD) is
the use of angiogenic growth factors that promote the formation of
new blood vessels from preexisting ones (angiogenesis) and also
restore endothelial cell function. In angiogenesis, endothelial
cells leave their resting state and start to digest the underlying
basement membrane followed by proliferation, migration, and finally
formation of a hollow tube (Gerwins et al. (2000) Crit. Rev. Oncol.
Hematol. 34(3):185-194). Fibroblast growth factors bind to cell
surface receptors that are ligand-stimulatable tyrosine kinases.
Binding of these growth factors to their receptors leads to
activation of the intrinsic tyrosine kinase and signal transduction
to downstream signaling cascades (Gerwins et al. (2000) Crit. Rev.
Oncol. Hematol. 34(3):185-194). Angiogenesis in ischemic tissues
can be promoted by the transmural delivery of angiogenic growth
factors such as VEGF, FGF, and PDGF using an intravascular infusion
catheter. See, for example, U.S. Pat. No. 5,941,868.
[0030] Compositions and methods for treating PAD in a patient are
provided. The compositions and methods are useful in the treatment
and prevention of claudication and critical limb ischemia due to
PAD. The term "critical limb isehemia" is used for all patients
with chronic ischemic rest pain, ulcers, or gangrene attributable
to objectively proven arterial occlusive disease. The term
"critical limb ischemia" implies chronicity and is to be
distinguished from acute limb ischemia. By "acute limb ischemia" is
intended any sudden decrease or worsening in limb perfusion causing
a threat to extremity viability. See, J. Vasc. Surg. 31:S135, S168,
herein incorporated by reference. The methods of the invention
utilize angiogenic agents, such as angiogenic members, of the
fibroblast growth factor (FGF) family, including preferably FGF-1,
FGF-2, FGF-4, FGF-5, FGF-18, and most preferably FGF-2. It is
recognized that all angiogenic growth factors herein described may
be recombinant molecules. Also, it is recognized that compositions
of the invention may comprise one or more fibroblast growth factors
as angiogenic agents as well as biologically active variants
thereof. Variants of an FGF sequence include, but are not limited
to, angiogenically active fragments, analogues, and derivatives. By
"fragment" is intended a polypeptide consisting of only a part of
the intact FGF sequence and structure, and can be a C-terminal
deletion, N-terminal deletion, or both. By "analogues" is intended
analogues of either the angiogenic agent FGF or fragment thereof
that comprise a native FGF sequence and structure having one or
more amino acid substitutions, insertions, or deletions. Peptides
having one or more peptoids (peptide mimics) and muteins, or
mutated forms of the angiogenic agent, are also encompassed by the
term analogue. By "derivatives" is intended any suitable
modification of the angiogenic agent, fragments of the angiogenic
agent, or their respective analogues, such as glycosylation,
phosphorylation, or other addition of foreign moieties, so long as
the angiogenic activity is retained. Methods for making fragments,
analogues, and derivatives are available in the art. See generally
U.S. Pat. Nos. 4,738,921, 5,158,875, and 5,077,276; International
Publication Nos. WO 85/0083 1, WO 92/04363, WO 87/01038, and WO
89/05822; and European Patent Nos. EP 135094, EP 123228, and EP
128733; herein incorporated by reference.
[0031] Such variants should retain angiogenic activities and thus
be "angiogenically active." The variants may be measured for
angiogenic activity using standard bioassays. Representative assays
include known radioreceptor assays using placental membranes (see,
e.g., U.S. Pat. No. 5,324,639; Hall et al. (1974) J. Clin.
Endocrinol. and Metab. 39:973-976; and Marshall et al. (1974) J.
Clin. Endocrinol. and Metab. 39:283-292). Additional assays include
mitogenic activity as determined in an in vitro assay of
endothelial cell proliferation. This activity is preferably
determined in a human umbilical vein endothelial (HUVE) cell-based
assay, as described, for example, in any of the following
publications: Gospodarowicz et al. (1989) Proc. Natl. Acad. Sci.
USA 87:7311-7315; Ferrara and Henzel (1989) Biochem. Biophys. Res.
Commun. 161:851-858; Conn et al. (1990) Proc. Natl. Acad. Sci. USA
87:1323-1327; Soker et al. (1998) Cell 92:735-745; Waltenberger et
al. (1994) J. Biol. Chem. 269:26988-26995; Siemmeister et al.
(1996) Biochem. Biophys. Res. Commun. 222:249-255; Fiebich et al.
(1993) Eur. J. Biochem. 211:19-26; Cohen et al. (1993) Growth
Factors 7:131-138. A further biological activity is involvement in
angiogenesis and/or vascular remodeling, which can be tested, for
example, in the corneal pocket angiogenesis assay as described in
Connolly et al. (1989) J. Clin. Invest. 84:1470-1478 and Lobb et
al. (1985) Biochemistry 24:4969-4973; the endothelial cell tube
formation assay, as described for example in Pepper et al. (1992)
Biochem. Biophys. Res. Commun. 189:824-831; Goto et al. (1993) Lab.
Invest. 69:508-517; or Koolwijk et al. (1996) Cell Biol.
132:1177-1188; the chick chorioallantoic membrane (CAM)
angiogenesis assay as described for example in Pluet et al. (1989)
EMBO. J. 8:3801-3806; the endothelial cell mitogenesis assay as
described in Bohlen et al. (1984) Proc. Natl. Acad. Sci. USA
81:5364-5368; Presta et al. (1986) Mol. Gen. Biol. 6:4060-4066;
Klagsbrun and Shing (1985) Proc. Natl. Acad. Sci. USA 82:805-809;
Gosodarowicz et al. (1985) J. Cell. Physiol. 122:323-332; or the
endothelial cell migration assay as described in Moscatelli et al.
(1986) Proc. Natl. Acad. Sci. USA 83:2091-2095; and Presta et al.
(1986) Mol. and Cell. Biol. 6:4060-4066; all of which are herein
incorporated by reference. It is recognized that one or more of the
assays may be used. Preferably, the variant has at least the same
activity as the native molecule.
[0032] Fibroblast growth factor-2 (FGF-2), including recombinantly
produced forms (rFGF-2), is a potent mitogen and angiogenic agent
that has utility for treatment of coronary artery disease (angina)
and peripheral artery disease (claudication). Although FGF-2 is
normally made in many body tissues and is involved in the body's
response to certain ischemic conditions, the body's own supply of
FGF-2 may not be sufficient to circumvent the complications of
atherosclerosis and arterial insufficiency/ischemia.
[0033] Compositions and methods of the invention can be used to
treat PAD patients, even those suffering a wide spectrum of related
clinical ailments, including but not limited to coronary artery
disease (CAD), myocardial infarctions, stroke, diabetes,
dyslipidemias, hypertension, and patients who have had surgical or
catheter-based revascularizations. Fibroblast growth factors,
particularly FGF-2, can be used to treat PAD patients suffering
from claudication, including those having critical limb ischemia.
Critical limb ischemia, when left untreated, can progress to acute
limb ischemia and ultimately necessitate amputation of the limb. As
such, the methods of the invention can be used to prevent acute
limb ischemia.
[0034] The FGF-containing compositions of the invention are
administered intra-arterially (IA), intravenously (IV),
intramuscularly (IM), subcutaneously (SC), transmurally, and the
like to a patient in need thereof. By "transmural" administration
is intended localized delivery of the composition into the blood
vessel or body lumen wall including neointimal, intimal, medial,
advential, and periviascular spaces, particularly adjacent to the
target site. By "target site" is intended the area surrounding or
immediately surrounding the blood supply into the extremities, e.g,
legs.
[0035] Intra-arterial administration (IA) involves delivery of the
FGF-containing composition into at least one artery. In an IA
infusion, the infusion is typically divided into several arteries
in the legs, e.g., the left and right common femoral arteries, but
is sometimes administered into a single artery. The infusion can be
administered for about 1 minute, 1 to 5 minutes, 10 to 20 minutes,
or 20 to 30 minutes into each artery in both legs. The infusion can
be repeated from time to time to achieve or sustain the predicted
benefit. The timing for repeat administration is based on the
patient's response as measured by symptoms and hemodynamic
measures. A therapeutically effective dose or amount of FGF, such
as FGF-2, that is to be given as an infusion can be divided into
two doses, and a single dose administered into each leg of a
patient undergoing treatment. In this manner, the total dose is
delivered such that the angiogenic agent is presented to both legs
of the patient.
[0036] Thus in one embodiment, a therapeutically effective dose or
amount of FGF as defined elsewhere herein is administered via IA
infusion using a bilateral delivery method such that the procedure
can be completed with a single puncture. In this manner, one-half
of the therapeutically effective amount or total dose of FGF, such
as rFGF-2, is infused into the common femoral artery of the first
leg, followed by guiding the catheter over the bifurcation of the
aorta to the contralateral iliac artery and common femoral artery
and then infusing the remainder of the total dose into the femoral
artery of the second leg. The rate of each infusion, one into each
leg, is about 1 mL/per minute over about a 10-minute period, with a
short interruption between the first and second infusion. Thus, the
second infusion generally begins within about 1 hour of the first
infusion, but can begin up to 2, 3, or 4 hours after the first
infusion. Preferably the second infusion begins within about 30
minutes, more preferably within about 20 minutes, even more
preferably within about 10 minutes, still more preferably within
about 5 minutes of the completion of the first infusion. Each
infusion can take less than about 10 minutes, such as 3, 4, or 5
minutes, so long as the FGF is not administered as a bolus. It is
recognized by one of skill in the art that the therapeutically
effective dose or amount of FGF, such as rFGF-2, can be divided
between the two legs of the patient such that unequal portions of
the total dose are delivered to each leg, for example, one-third to
one leg, and two-thirds to the other leg. The advantage of the
bilateral delivery method is that the two infusions, one into each
leg, can be accomplished with a single puncture to the subject. In
this embodiment, the sight of the puncture is preferably at groin
level. A brachial approach may be used if deemed preferable by the
treating physician. With this procedure, the catheter can be guided
more distally, such as in the area just above the knee, as long as
the obstruction to blood flow remains distal to the point of
infusion.
[0037] Alternatively, the therapeutically effective amount of FGF,
such as rFGF-2, can be delivered by direct IA puncture into each
common femoral artery. In this manner, one-half of the dose of FGF
is administered into one common femoral artery, while the other
half of the dose of FGF is administered into the other common
femoral artery. Direct IA puncture can be advantageous in that it
avoids the catheterization procedure required with bilateral
delivery, but it necessitates two punctures when the
therapeutically effective dose is to be divided and infused into
both legs. As with bilateral delivery, each infusion is delivered
at a rate of about 1 mL per minute over about a 10-minute period,
with a short interruption between the first and second infusion.
Thus, the second infusion generally begins within about 1 hour of
the first infusion, but can begin up to 2, 3, or 4 hours after the
first infusion. Preferably the second infusion begins within about
30 minutes, more preferably within about 20 minutes, even more
preferably within about 10 minutes, still more preferably within
about 5 minutes of the completion of the first infusion. Each
infusion can take less than about 10 minutes, such as 3, 4, or 5
minutes, so long as the FGF is not administered as a bolus. Again,
it is recognized that the therapeutically effective dose or amount
of FGF, such as rFGF-2, can be divided between the two legs of the
patient such that unequal portions of the total dose are delivered
to each leg.
[0038] Delivery of the FGF-containing compositions in accordance
with the methods of the invention may be accomplished through a
variety of known intravascular drug delivery systems. Such delivery
systems include intravascular catheter delivery systems. A variety
of catheter systems useful for the direct transmural infusion of
angiogenic growth factors into the blood vessel are well known in
the art. For purposes of practicing the invention, any of a variety
of diagnostic or therapeutic type catheters could be used. Where
the FGF is administered in conjunction with an angioplasty, balloon
catheters can be used. Balloon catheters having expandable distal
ends capable of engaging the inner wall of a blood vessel and
infusing an angiogenic growth factor directly therein are well
described in the patent literature. See, for example, U.S. Pat.
Nos. 5,318,531; 5,304,121; 5,295,962; 5,286,254; 5,254,089;
5,213,576; 5,197,946; 5,087,244; 5,049,132; 5,021,044; 4,994,033;
and 4,824,436. Catheters having spaced-apart or helical balloons
for expansion within the lumen of a blood vessel and delivery of a
therapeutic agent to the resulting isolated treatment site are
described in U.S. Pat. Nos. 5,279,546; 5,226,888; 5,181,911;
4,824,436; and 4,636,195. Non-balloon drug delivery catheters are
described in U.S. Pat. Nos. 5,180,366; 5,112,305; and 5,021,044;
and PCT Publication WO 92/11890. Catheters that provide for distal
vessel access, as well as stents also can be used. Ultrasonically
assisted drug delivery catheters (phonophoresis devices) are
described in U.S. Pat. Nos. 5,362,309; 5,318,014; and 5,315,998.
Other iontophoresis and phonophoresis drug delivery catheters are
described in U.S. Pat. Nos. 5,304,120; 5,282,785; and 5,267,985.
Sleeve catheters having drug delivery lumens intended for use in
combination with conventional angioplasty balloon catheters are
described in U.S. Pat. Nos. 5,364,356 and 5,336,178. All of these
references are herein incorporated by reference.
[0039] Direct intramuscular (IM) injections can be used to
administer the angiogenic agents of the invention. The agents for
injection can include the FGF protein or angiogenically active
fragments of the protein as well as the gene or plasmid encoding
the angiogenically active FGF protein or fragment. Injections are
administered to the affected limb(s), in the thigh or calf, in the
vicinity of existing vessels, near collateral flow vessels or
conduit vessels such as arteries and arterials. The therapeutically
effective dose of angiogenic agent is administered as a single
injection, or can be divided and administered as multiple
injections. Preferably the therapeutically effective amount or dose
is delivered as 1 to about 20 injections, 1 to about 15 injections,
more preferably 1 to about 10 injections. A single dose of
angiogenic agent can be administered intramuscularly, and repeated
as needed based on symptoms and/or hemodynamic measures. Local
delivery such as with IM injection can provide the added benefit of
administering lower doses of the angiogenic agent. See Example 4
herein, and the copending application entitled "Dose of an
Angiogenic Factor and Method of Administering to Improve Myocardial
Blood Flow," filed Aug. 11, 2000 and assigned U.S. patent
application Ser. No. 09/637,471, based on U.S. provisional
application no. 60/148,746, filed Aug. 13, 1999, both of which
herein incorporated by reference. The advantage to IM injection(s)
is that it is less likely to result in hypotension, is more likely
to have a longer half-life in the ischemic area, is less invasive,
and therefore, can be repeated more frequently than the IA
infusion. An IA infusion or an IM injection(s) could be "boosted"
by an IM injection(s) every 1-2 months as warranted by clinical
symptoms.
[0040] Recombinant FGF-2 releases nitric oxide, a potent
vasodilator, aggressive fluid management prior to (proactively) and
during the infusion is critical to patient's safety. Administration
of IV fluids (e.g., 500-1000 mL of normal saline) to establish an
estimated wedge pressure of 12 mm Hg prior to infusion and
administration of boluses of IV fluids (e.g., 200 mL normal saline)
for decreases of systolic blood pressure (e.g., <90 mm Hg)
associated with infusion optimized the safety of administration of
rFGF-2 by IC or IV infusion to human patients.
[0041] Because a sudden bolus of rFGF-2 is associated with profound
hypotension in animals, the rate of infusion is critical to
patient's safety. Administration at 0.5 to 2 mL per minute,
typically 1 mL per minute, optimized the safety of administration
of rFGF-2 by IC or IV infusion to human patients.
[0042] In another embodiment of the invention, compositions
comprising fibroblast growth factor (FGF), including but not
limited to FGF-2, can be administered to a patient with peripheral
artery disease, including those with claudication, in conjunction
with vascular or mechanical bypass surgery or angioplasty. The FGF,
including but not limited to FGF-2, can be administered with and
without a stent during surgery. The FGF may thus be administered as
an adjunct to vascular surgery involving mechanical bypass and
angioplasty.
[0043] The compositions of the invention provide a safe and
therapeutically effective amount of fibroblast growth factor to
improve blood flow. By "safe and therapeutically effective amount"
is intended an amount of a fibroblast growth factor such as FGF-2,
or angiogenically active variant or fragment thereof, that when
administered in accordance with the invention is free from major
complications that cannot be medically managed, and that provides
for objective improvement in patients having symptoms of PAD. It is
recognized that the therapeutically effective amount may vary from
patient to patient depending upon age, weight, severity of
symptoms, general health, physical condition, and the like. Other
factors include the mode of administration and the respective
amount of FGF included in the pharmaceutical composition.
Typically, a therapeutically effective amount of an angiogenic
agent of the invention, such as FGF-2, is about 0.1 .mu.g/kg to
about 100 .mu.g/kg, preferably about 0.20 .mu.g/kg to about 75
.mu.g/kg, more preferably about 0.4 .mu.g/kg to about 50 .mu.g/kg,
even more preferably about 0.50 .mu.g/kg to about 35 .mu.g/kg, more
preferably still about 1.0 .mu.g/kg to about 30 .mu.g/kg based on
actual body weight. Thus, when the angiogenic agent is FGF-2, a
therapeutically effective amount of FGF-2 is about 0. 1 .mu.g/kg to
about 1 .mu.g/kg, 0.1 .mu.g/kg to about 1 .mu.g/kg, about 1
.mu.g/kg to 3 .mu.g/kg, about 3 .mu.g/kg to about 5 .mu.g/kg, about
5 .mu.g/kg to about 7 .mu.g/kg, about 7 .mu.g/kg to about 8
.mu.g/kg, about 8 .mu.g/kg to about 9 .mu.g/kg, about 9 .mu.g/kg to
about 9.9 .mu.g/kg, such as about 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,
9.6, 9.7, 9.8, or 9.9 .mu.g/kg, up to about 10 .mu.g/kg, about 10
.mu.g/kg to about 15 .mu.g/kg, about 15 .mu.g/kg to about 20
.mu.g/kg, about 20 .mu.g/kg to about 30 .mu.g/kg, about 30 .mu.g/kg
to about 40 .mu.g/kg, about 40 .mu.g/kg to about 60 .mu.g/kg, about
60 .mu.g/kg to about 80 .mu.g/kg of the rFGF-2, depending upon the
route and the mode of administration.
[0044] As indicated, the compositions and methods of the invention
are useful for treating or preventing PAD and symptoms associated
with PAD, including claudication and critical limb ischemia. In
this manner, the desired therapeutic responses include increased
exercise capacity, improvement in ankle-brachial index, reduction
in body pain and claudication. In cases of PAD patients with
critical limb ischemia, desired therapeutic responses include
resolution of unremitting rest pain that is not controllable by
analgesic, healing of ulcers, and prevention of gangrene and
amputation.
[0045] Methods for monitoring efficacy of administration of FGF,
particularly FGF-2, for treatment of PAD are well known in the art.
See, for example, methods for monitoring increased blood flow into
affected limbs, including, but not limited to, Doppler ultrasound,
plethysmography (Macdonald (1994) J. Vas. Tech. 18:241-248), and
magnetic resonance spectroscopy, ankle-brachial or toe systolic
pressure index at rest and after a period of exercise, and
increased collateral vessel density using angiography. Clinical
indicators of efficacy include total treadmill walk time (i.e.,
peak walking time, PWT) and time to onset of claudication; and
patient quality of life questionnaires.
[0046] The FGF-containing pharmaceutical compositions of the
invention will be delivered for a time sufficient to achieve the
desired physiological effect, i.e., angiogenesis, and/or
restoration of endothelial cell function and the promotion of
collateral blood vessels. The compositions may be administered as a
single bolus, or multiple injections. Typically, the angiogenic
factor will be delivered as an infusion over a period of time. It
is recognized that any means for administration are encompassed
including sustained-release formulations, plasmids, or genes, as
well as other routes of administration. The total amount of time
may vary depending on the delivery rate and drug concentration in
the composition being delivered. For example, for intra-arterial
administration, the time of administration may vary from 1 second
to about 24 hours, more usually from about 1 minute to about 6
hours, specifically from about 5 minutes to about 30 minutes. A
single intra-arterial dose administration is efficacious in the
treatment of PAD.
[0047] When administered in accordance with the methods of the
invention, FGF-containing compositions provide the patient with a
safe and therapeutically efficacious treatment for PAD that lasts
at least 1 month, 2 months, generally 3 months, 4 months, 6 months,
and, in some cases, more than 6 months before a further treatment
is needed. The angiogenic agent, such as FGF-2, can be administered
once or twice per day about every week, preferably every month or
more preferably every other month, even more preferably every 3
months, even more preferably every 4 months, and even more
preferably still about every 6 months.
[0048] As indicated, fibroblast growth factors and related
molecules are able to restore endothelial cell function and to
promote endothelial and/or smooth muscle cell proliferation. The
fibroblast growth factors (FGF) are a family of at least
twenty-three structurally related polypeptides (named FGF-1 to
FGF-23) that are characterized by a high degree of affinity for
proteoglycans, such as heparin. The various FGF molecules range in
size from 15 to at least 32.5 kDa, and exhibit a broad range of
biological activities in normal and malignant conditions including
nerve cell adhesion and differentiation (Schubert et al. (1987) J.
Cell. Biol. 104:635-643); 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 (Elements et al. (1993) Oncogene 8:1311-1316);
and as an angiogenic factor (Harada (1994) J. Clin. Invest.
94:623-630). Thus, the FGF family is a family of pluripotent growth
factors that stimulate to varying extents fibroblasts, smooth
muscle cells, epithelial cells, endothelial cells, myocytes, and
neuronal cells. FGF-like polypeptides are also contemplated for use
in the compositions and methods of the present invention. By
"FGF-like" is intended polypeptides that bind FGF receptor 1,
particularly receptor 1-C, bind to heparin-like molecules, and have
angiogenic activity. By heparin-like molecule is intended heparin,
proteoglycans, and other polyanionic compounds that bind FGF, that
dimerize FGF, and that facilitate receptor activation. Of
particular interest in the practice of the invention is the FGF
designated FGF-2 as well as variants and fragments thereof, which
are known in the art. For example, see U.S. Pat. Nos. 5,989,866;
5,925,528; 5,874,254; 5,852,177; 5,817,485; 5,714,458; 5,656,458;
5,604,293; 5,576,288; 5,514,566; 5,482,929; 5,464,943; and
5,439,818.
[0049] The FGF, more particularly FGF-2, to be administered can be
from any animal species including, but not limited to, avian,
canine, bovine, porcine, equine, and human. Generally, the FGF is
from a mammalian species, preferably bovine or human in the case of
FGF-2. The FGF may be in the native, recombinantly produced, or
chemically synthesized forms as outlined below. Where the FGF is
FGF-2, it may be the 146 amino acid form, the 153-155 amino acid
form, or a mixture thereof depending upon the method of recombinant
production. See U.S. Pat. No. 5,143,829, herein incorporated by
reference. Further, angiogenically active muteins of the FGF-2
molecule can be used. See, for example, U.S. Pat. Nos. 5,859,208
and 5,852,177, herein incorporated by reference.
[0050] Biologically active variants of the FGF polypeptide of
interest, more particularly FGF-2, are also encompassed by the
methods of the present invention. As noted previously, such
variants include fragments, analogues, and derivatives. Such
variants should retain angiogenic activities and thus be
"angiogenically active" as measured using standard bioassays noted
above.
[0051] Variants of the native FGF used in the compositions and
methods of the invention will generally have at least 70%,
preferably at least 80%, more preferably about 90% to 95% or more,
and most preferably about 98% or more amino acid sequence identity
to the amino acid sequence of the reference FGF molecule. By
"sequence identity" is intended the same amino acid residues are
found within the variant and the reference FGF molecule when a
specified, contiguous segment of the amino acid sequence of the
variant is aligned and compared to the amino acid sequence of the
reference FGF molecule, which serves as the basis for comparison.
Thus, for example, where the reference FGF-2 molecule is human
FGF-2, an angiogenically active variant thereof will generally have
at least 70%, preferably at least 80%, more preferably about 90% to
95% or more, most preferably about 98% or more, sequence identify
to the full-length amino acid sequence set forth in FIG. 3 (SEQ ID
NO:3). In addition, other FGF receptor-binding peptides can be used
as described in, for example, WO98/21237 or U.S. application Ser.
No. 09/407,687, filed Sep. 28, 1999, herein incorporated by
reference.
[0052] A polypeptide that is a biologically active variant of a
reference polypeptide molecule of interest may differ from the
reference molecule by as few as 1-15 amino acids, as few as 1-10,
such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid
residue. The percentage sequence identity between two amino acid
sequences is calculated by determining the number of positions at
which the identical amino acid residue occurs in both sequences to
yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the segment
undergoing comparison to the reference molecule, and multiplying
the result by 100 to yield the percentage of sequence identity.
[0053] For purposes of optimal alignment of the two sequences, the
contiguous segment of the amino acid sequence of the variant
polypeptide may have additional amino acid residues or deleted
amino acid residues with respect to the amino acid sequence of the
reference polypeptide molecule. The contiguous segment used for
comparison to the reference amino acid sequence will comprise at
least twenty (20) contiguous amino acid residues, and may be 30,
40, 50, 100, or more residues. Corrections for increased sequence
identity associated with inclusion of gaps in the variant's amino
acid sequence can be made by assigning gap penalties. Methods of
sequence alignment are well known in the art for both amino acid
sequences and for the nucleotide sequences encoding amino acid
sequences.
[0054] Thus, the determination of percent identity between any two
sequences can be accomplished using a mathematical algorithm. One
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller (1988) CABIOS 4:11-17. Such an algorithm is utilized in
the ALIGN program (version 2.0), which is part of the GCG sequence
alignment software package. A PAM120 weight residue table, a gap
length penalty of 12, and a gap penalty of 4 can be used with the
ALIGN program when comparing amino acid sequences. Another
preferred, nonlimiting example of a mathematical algorithm for use
in comparing two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin
and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such
an algorithm is incorporated into the NBLAST and XBLAST programs of
Altschul et al. (1990)J. Mol. Biol. 215:403. BLAST nucleotide
searches can be performed with the NBLAST program, score=100,
wordlength=12, to obtain nucleotide sequences homologous to a
nucleotide sequence encoding the polypeptide of interest. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3, to obtain amino acid sequences homologous
to the polypeptide of interest. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively,
PSI-Blast can be used to perform an iterated search that detects
distant relationships between molecules. See Altschul et al. (1997)
supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See www.ncbi.nlm.nih.gov. Also see the ALIGN
program (Dayhoff (1978) in Atlas of Protein Sequence and Structure
5:Suppl. 3 (National Biomedical Research Foundation, Washington,
D.C.) and programs in the Wisconsin Sequence Analysis Package,
Version 8 (available from Genetics Computer Group, Madison,
Wisconsin), for example, the GAP program, where default parameters
of the programs are utilized.
[0055] When considering percentage of amino acid sequence identity,
some amino acid residue positions may differ as a result of
conservative amino acid substitutions, which do not affect
properties of protein function. In these instances, percent
sequence identity may be adjusted upwards to account for the
similarity in conservatively substituted amino acids. Such
adjustments are well known in the art. See, for example, Myers and
Miller (1988) Computer Applic. Biol. Sci. 4:11-17.
[0056] The art provides substantial guidance regarding the
preparation and use of FGF polypeptide variants. In preparing the
polypeptide variants, one of skill in the art can readily determine
which modifications to the native nucleotide or amino acid sequence
will result in a variant that is suitable for use as a
therapeutically active component of a pharmaceutical composition of
the present invention for use in the methods of the invention
directed to treatment of patients having peripheral artery
disease.
[0057] Fibroblast growth factors, such as FGF-2, are formulated
into pharmaceutical compositions for use in the methods of the
invention. In this manner, a pharmaceutically acceptable carrier
may be used in combination with the angiogenic agent such as FGF-2
and other components in the pharmaceutical composition. By
"pharmaceutically acceptable carrier" is intended a carrier or
diluent that is conventionally used in the art to facilitate the
storage, administration, and/or the desired effect of the
therapeutic ingredients. A carrier may also reduce any undesirable
side effects of the angiogenic agent, i.e., FGF or variant thereof.
A suitable carrier should be stable, i.e., incapable of reacting
with other ingredients in the formulation. It should not produce
significant local or systemic adverse effect in recipients at the
dosages and concentrations employed for therapy. Such carriers are
generally known in the art. Suitable carriers for this invention
are those conventionally used large stable macromolecules such as
albumin, gelatin, collagen, polysaccharide, monosaccarides,
polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polymeric
amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose,
lactose, mannose, dextrose, dextran, cellulose, mannitol, sorbitol,
polyethylene glycol (PEG), heparin alginate, and the like.
Slow-release carriers, such as hyaluronic acid, may also be
suitable. Stabilizers, such as trehalose, thioglycerol, and
dithiothreitol (DTT), may also be added. See, for example,
copending U.S. Application Serial No. 60/229,238, entitled
"Stabilized FGF Formulations Containing Reducing Agents," herein
incorporated by reference. FGF formulations comprising DTT as
described in this application are defined herein as "stabilized
FGF-DTT formulations and include stabilized FGF-2-DTT formatting."
Other acceptable components in the composition include, but are not
limited to, buffers that enhance isotonicity such as water, saline,
phosphate, citrate, succinate, acetic acid, and other organic acids
or their salts. Further, the angiogenic agents of the invention may
be administered using a patch for slow release. Such formulation
may include DMSO.
[0058] Preferred pharmaceutical compositions may incorporate
buffers having reduced local pain and irritation resulting from
injection. Such buffers include, but are not limited to, low
phosphate buffers and succinate buffers. The pharmaceutical
composition may additionally comprise a solubilizing compound that
is capable of enhancing the solubility of an angiogenic agent or
variant.
[0059] For the purposes of this invention, the pharmaceutical
composition comprising the angiogenic agent FGF or angiogenically
active variant thereof should be formulated in a unit dosage and in
an injectable or infusible form such as solution, suspension, or
emulsion. It can also be in the form of lyophilized powder, which
can be converted into solution, suspension, or emulsion before
administration. The pharmaceutical composition may be sterilized by
membrane filtration, which also removes aggregates, and stored in
unit-dose or multi-dose containers such as sealed vials or
ampules.
[0060] The method for formulating a pharmaceutical composition is
generally known in the art. A thorough discussion of formulation
and selection of pharmaceutically acceptable carriers, stabilizers,
and isomolytes can be found in Remington 's Pharmaceutical Sciences
(.sub.18th ed.; Mack Pub. Co.: Eaton, Pa. 1990), herein
incorporated by reference.
[0061] The pharmaceutical compositions of the present invention can
also be formulated in a sustained-release form to prolong the
presence of the pharmaceutically active agent in the treated
patient, generally for longer than one day. Many methods of
preparation of a sustained-release formulation are known in the art
and are disclosed in Remington 's Pharmaceutical Sciences
(18.sup.th ed.; Mack Pub. Co.: Eaton, Pa., 1990), herein
incorporated by reference. Generally, the agent can be entrapped in
semipermeable matrices of solid hydrophobic polymers. The matrices
can be shaped into films or microcapsules. Examples of such
matrices include, but are not limited to, polyesters, copolymers of
L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1983)
Biopolymers 22:547-556), poly-actides (U.S. Pat. No. 3,773,919 and
EP 58,481), polyactate polyglycolate (PLGA), hydrogels (see, for
example, Langer et al. (1981)J. Biomed. Mater. Res. 15:167-277;
Langer (1982) Chem. Tech. 12:98-105), non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the Lupron DepotTm, and poly-D-(-)-3-hydroxybutyr- ic acid (EP
133,988). Suitable microcapsules can also include hydroxymethyl
cellulose or gelatin-microcapsules and poly-methylmethacylate
microcapsules prepared by coacervation techniques or by interfacial
polymerization. Microparticles such as heparin alginate beads may
also be used. In addition, microemulsions or colloidal drug
delivery systems such as liposomes and albumin microspheres, may
also be used See a Remington 's Pharmaceutical Sciences (.sub.18th
ed.; Mack Pub. Co.: Eaton, Pa., 1990).
[0062] In particular, a mammalian fibroblast growth factor of
bovine origin, FGF-2 of FIG. 2 (SEQ ID NO:2) also known as basic
FGF (bFGF), and human FGF-2 of FIG. 3 (SEQ ID NO:4), or an
angiogenically active fragment or mutein thereof, can be utilized
in the practice of the invention. The nucleotide sequence encoding
bovine FGF-2 is set forth in FIG. 1 (SEQ ID NO: 1). The nucleotide
sequence encoding human FGF-2 is set forth in FIG. 3 (SEQ ID NO:3).
See also, U.S. Pat. No. 5,604,293, herein incorporated by
reference. The dose of FGF-2 that is predicted to result in
clinical benefit to a patient whose exercise capacity is limited by
claudication associated with PAD ranges from about 0.1 .mu.g/kg to
about 100 .mu.g/kg of the FGF-2, preferably about 0.20 .mu.g/kg to
about 75 .mu.g/kg, more preferably about 0.4 .mu.g,/kg to about 50
.mu.g/kg, even more preferably about 0.50 .mu.g/kg to about 35
.mu.g/kg, more preferably still about 1.0 .mu.g/kg to about 30
.mu.g/kg, and most likely from 0.3 to 3.5 mg as a standard dose.
Thus, in one embodiment, the therapeutically effective dose of
FGF-2, such as recombinant FGF-2 (rFGF-2), is about 0.1 .mu.g/kg to
about 1 .mu.g/kg, about 1 .mu.g/kg to 3 .mu.g/kg, about 3 .mu.g/kg
to about 5 .mu.g/kg, about 5 .mu.g/kg to about 7 .mu.g/kg, about 7
.mu.g/kg to about 8 .mu.g/kg, about 8 Ag/kg to about 9 .mu.g/kg,
about 9 .mu.g/kg to about 9.9 .mu.g/kg, such as about 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 .mu.g/kg, up to about
.mu.g/kg, about 10 .mu.g/kg to about 15 .mu.g/kg, about 15 .mu.g/kg
to about 20 .mu.g/kg, about 20 .mu.g/kg to about 30 .mu.g/kg, about
30 .mu.g/kg to about 40 .mu.g/kg, about 40 .mu.g/kg to about 60
.mu.g/kg, about 60 .mu.g/kg to about 80 .mu.g/kg of the FGF-2,
depending on the route and mode of administration.
[0063] It is convenient to define the dose of angiogenic agent in
more absolute terms that are not dependent upon the weight of the
patient to be treated. In this embodiment, the dose is referred to
as a "standard" dose. When so defined, the standard dose to be
administered in accordance with the methods of the present
invention ranges from about 4.0 .mu.g to about 7.2 mg, such as
about 4.0 .mu.g to about 0.3 mg, preferably from about 0.3 mg to
about 1.0 mg, even more preferably from about 1.0 mg to about 2.0
mg, more preferably still from about 2.0 mg to about 2.5 mg, from
about 2.5 mg to 3.5 mg, from about 3.5 mg to about 4.5 mg, from
about 4.5 mg to about 5.5 mg, from about 5.5 mg to about 6.5 mg, up
to about 7.2 mg. In this embodiment, the standard dose is a
sufficient amount of FGF-2 to accommodate dosing any one of the
majority of human PAD patients, ranging from the smallest patient
(e.g., 40 kg) at the lowest dosage (about 0.1 .mu.g/kg) through the
larger patients (e.g., 150 kg) at higher dosages (about 48 .mu.g/kg
for this embodiment). For example, when a patient weighs 70 kg the
standard dose ranges from about 0.2 mg to about 3.0 mg, from about
0.5 mg to about 2.5 mg, preferably about 2.1 mg, depending upon the
route and mode of administration.
[0064] Where lower doses of FGF-2 are contemplated, such as between
0.1 .mu.g/kg up to about 10 .mu.g/kg, the standard dose to be
administered in accordance with the methods of the present
invention ranges from about 7.0 jig to about 0.7 mg, about 8 .mu.g
to about 0.6 mg, about 9 .mu.g to about 0.5 mg, about 0.1 mg to
about 0.4 mg, preferably about 0.21 mg for a 70 kg patient. Thus,
in some embodiments, the standard dose for a 70 kg patient ranges
from about 7.0 .mu.g to about 0.7 mg, including 8 .mu.g, 9 .mu.g,
0.1 mg, 0.2 mg. 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.65 mg, up to
about 0.7 mg.
[0065] Because FGF-2 is a glycosaminoglycan- (e.g., heparin)
binding protein and the presence of a glycosaminoglycan (also known
as a "proteoglycan" or a "mucopolysaccharide") optimizes activity
and area under the curve (AUC), the dosages of FGF-2 of the present
invention may be administered within 20 to 30 minutes of an
intravenous (IV) administration of a glycosaminoglycan, such as a
heparin. Various fractionated and unfractionated heparins,
proteoglycans, and sulfated mucopolysaccharides such as chondroitin
sulfate can be used in the practice of the invention. Low molecular
weight heparins (<10,000 d) and unfractionated (i.e., high
molecular weight) heparins (>10,000 d) can be used in the
practice of the invention. These molecules can be administered
together with the rFGF-2 or within 20 to 30 minutes of
administration of the rFGF-2. Heparin is suitably dosed at 20-80
units/kg, and preferably at 40 units/kg.
[0066] In one embodiment, the unit dose contains a sufficient
amount of FGF-2 ranging from about 0.1 .mu.g/kg to about 80
.mu.g/kg. More typically, the systemic unit dose comprises 0.3 mg
to 3.5 mg of the FGF-2 of FIG. 2 (SEQ ID NO:2) or the FGF-2 of FIG.
3 (SEQ ID NO:4), or an angiogenically active fragment or mutein
thereof. Dosages for local delivery comprising about 0.01 .mu.g to
about 500 .mu.g up to about 3 mg may be used. When administered
locally as with IM injections, the dose may be the same as,
one-tenth of, or one-hundredth of the dose administered
intra-arterially. The unit dose is typically provided in solution
or reconstituted lyophilized form containing the above-referenced
amount of FGF-2 and an effective amount of one or more
pharmaceutically acceptable buffers, stabilizers, and/or other
excipients as described elsewhere herein.
[0067] The recombinant FGF-2 having the amino acid sequence of FIG.
2 (SEQ ID NO:2) is made as described in U.S. Pat. No. 5,155,214,
entitled "Basic Fibroblast Growth Factor," which issued on Oct. 13,
1992, and which is incorporated herein by reference in its
entirety. As disclosed in the '214 patent, a DNA of FIG. 1 (SEQ ID
NO: 1), which encodes a bFGF (hereinafter "FGF-2") of FIG. 2 (SEQ
ID NO:2), is inserted into a cloning vector, such as pBR322, pMB9,
Col E 1, pCRI, RP4 or .lambda.-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 FIG. 2 (SEQ ID NO:2). Although the FGF-2 of FIG.
2 (SEQ ID NO:2) has four cysteines, i.e., at residue positions 25,
69, 87 and 92, there are no internal disulfide linkages. ['214 at
col. 6, lines 59-61.] However, in the event that cross-linking
occurred under oxidative conditions, it would likely occur between
the residues at positions 25 and 69.
[0068] The 146-residue mammalian FGF-2 of FIG. 2 (SEQ ID NO:2),
which is of bovine origin, like the corresponding 146-residue human
FGF-2 of FIG. 3 (SEQ ID NO:4) is initially synthesized in vivo as a
polypeptide having 155 amino acids (Abraham et al. (1986) EMBO J.
5(10):2523-2528; FIG. 4 (SEQ ID NO:6) of bovine origin; FIG. 5 (SEQ
ID NO:8) of human origin). When compared to the full-length
155-residue FGF-2 molecules, the 146-residue FGF-2 molecules lack
the first nine amino acid residues,
Met-Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu (SEQ ID NO:9), at the
N-terminus of the corresponding full-length bovine and human
155-residue FGF-2 molecules (FIG. 4 (SEQ ID NO:6) and FIG. 5 (SEQ
ID NO:8), respectively). The 155-residueFGF-2 of human or bovine
origin, and biologically active variants thereof, can also be used
in the compositions and methods of the present invention in the
manner described for the bovine and human 146-residue FGF-2
molecules. Again it is recognized that the 155-residue form may
exist as 153-155 residues, or mixtures thereof, depending upon the
method of recombinant protein production. The mammalian FGF-2 of
FIG. 2 (SEQ ID NO:2) differs from human FGF-2 of FIG. 3 (SEQ ID
NO:4) in two residue positions. In particular, the amino acids at
residue positions 112 and 128 of the mammalian FGF-2 of FIG. 2 (SEQ
ID NO:2) are Ser and Pro, respectively, whereas in human FGF-2
(FIG. 3; SEQ ID NO:4), they are Thr and Ser, respectively. For the
155-residue forms, these differences appear at residue positions
121 and 137 of FIG. 4 (SEQ ID NO:6; FGF-2 of bovine origin) and
FIG. 5 (SEQ ID NO:8; FGF-2 of human origin).
[0069] The recombinant FGF-2 employed in the present compositions
and methods was purified to pharmaceutical quality (90% or greater
purity by weight of total proteins, preferably 92% or greater
purity, more preferably 95% or greater purity, preferably
substantially pure, that is about 98% purity by weight of total
proteins) using the techniques described in detail in U.S. Pat. No.
4,956,455, entitled "Bovine Fibroblast Growth Factor," which issued
on Sep. 11, 1990 and which is incorporated herein by reference in
its entirety. In particular, the first two steps employed in the
purification of the recombinant FGF-2 used in a unit dose of a
pharmaceutical composition of the invention are "conventional
ion-exchange and HPLC purification steps as described previously.
"[U.S. Pat. No. 4,956,455, citing to Bolen et al. (1984) Proc.
Natl. Acad. Sci. USA 81:5364-5368.I'm not sure about these
references.] The third step, which the '455 patent refers to as the
"key purification step" ['455 at col. 7, lines 5-6], is
heparin-SEPHAROSE(.RTM. affinity chromatography, wherein the strong
heparin binding affinity of the FGF-2 is utilized to achieve
several thousand-fold purification when eluting at approximately
1.4 M and 1.95 M NaCl ['455 at col. 9, lines 20-25]. Polypeptide
homogeneity may be confirmed by reverse-phase high pressure liquid
chromatography (RP-HPLC). Buffer exchange was achieved by
SEPHADEX.RTM. G-25(M) gel filtration chromatography.
[0070] In addition to the 146-residue FGF-2 of FIG. 2 (SEQ ID
NO:2), the therapeutically active agent in the unit dose of the
present invention also comprises an angiogenically active fragment"
of the FGF-2 of FIG. 2 (SEQ ID NO:2). By the term "angiogenically
active fragment of the FGF-2 of FIG. 2 (SEQ ID NO:2)" is meant a
fragment of FGF-2 that has about 80% of the 146 residues of FIG. 2
(SEQ ID NO:2) and that retains the angiogenic effect of the FGF-2
of FIG. 2 (SEQ ID NO:2). This definition of "angiogenically active
fragment" also applies to human FGF-2 of FIG. 3 (SEQ ID NO:4). An
"angiogenically active fragment" of the FGF-2 of FIG. 4 (SEQ ID
NO:6) or FIG. 5 (SEQ ID NO:8) is a fragment of FGF-2 that has about
80% of the 155 residues of FIG. 4 (SEQ ID NO:6) or FIG. 5 (SEQ ID
NO:8), respectively.
[0071] To be angiogenically active, the FGF-2 fragment should have
two cell binding sites and at least one of the two heparin binding
sites. The two putative cell binding sites of the analogous
146-residue human FGF-2 (hFGF-2; SEQ ID NO:4) occur at about
residue positions 36-39 and about 77-81 thereof. See Yoshida et al.
(1987) Proc. Natl. Aca. Sci. USA 84:7305-7309, at FIG. 3. The two
putative heparin binding sites of hFGF-2 occur at about 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 FGF-2 (hFGF-2) and bovine FGF-2 (bFGF-2), it is expected
that the cell binding sites for bFGF-2 (FIG. 2 (SEQ ID NO:2)) are
also at about residue positions 36-39 and about 77-81 thereof, and
that the heparin binding sites are at about residue positions 18-22
and about 107-111 thereof. The additional 9 residues of the
155-residue form do not affect the relative positions of these
binding sites with respect to residues 1-146 shown in FIG. 2 (SEQ
ID NO:2; FGF-2 of bovine origin) or FIG. 3 (SEQ ID NO:4; FGF-2 of
human origin). Thus, for the 155-residue form of human FGF-2 (FIG.
5; SEQ ID NO: 8), the two putative cell binding sites occur at
about residue positions 45-48 and about 86-90 thereof, and the two
putative heparin binding sites occur at about residue positions
27-31 and about 116-120 thereof. Again, given the substantial
similarity between the 155-residue bovine and human proteins, it is
expected that the two putative cell binding sites are at about
residue positions 45-48 and about 86-90, and the two putative
heparin binding sites are at about residue positions 27-31 and
about 116-120 of the 155-residue bovine FGF-2 (FIG. 4; SEQ ID
NO:6). Consistent with the above, it is well known in the art that
N-terminal truncations of the FGF-2 of FIG. 2 (SEQ ID NO:2) do not
eliminate its angiogenic activity in cows. In particular, the art
discloses several naturally occurring and biologically active
fragments of the FGF-2 that have N-terminal truncations relative to
the FGF-2 of FIG. 2 (SEQ ID NO:2). An active and truncated bFGF-2
having residues 12-146 of FIG. 2 (SEQ ID NO:2) was found in bovine
liver and another active and truncated bFGF-2, having residues
16-146 of FIG. 2 (SEQ ID NO:2) was found in the bovine kidney,
adrenal glands, and testes. (See U.S. Pat. No. 5,155,214 at col. 6,
lines 41-46, citing to Ueno et al. (1986) Biochem. Biophys. Res.
Comm. 138:580-588.) Likewise, other fragments of the bFGF-2 of FIG.
2 (SEQ ID NO:2) that are known to have FGF activity are FGF-2
(24-120)-OH and FGF-2 (30-110)-N1712-[U.S. Pat. No. 5,155,214 at
col. 6, lines 48-52.] These latter fragments retain both of the
cell binding portions of FGF-2 (FIG. 2 (SEQ ID NO:2)) and one of
the heparin binding segments (residues 107-111). Accordingly, the
angiogenically active fragments of a mammalian FGF typically
encompass those terminally truncated fragments of an FGF-2 that
have at least residues that correspond to residues 30-110 of the
FGF-2 of FIG. 2 (SEQ ID NO:2); more typically, at least residues
that correspond to residues 18-146 of the FGF-2 of FIG. 2 (SEQ ID
NO:2).
[0072] It is recognized that other synthetic peptides based on
native FGF sequences may be used as long as these peptides bind FGF
receptors. Additionally hybrid FGF molecules may be constructed
comprising peptides from different native sequences as well as
combinations of native and synthetic sequences. Again, the hybrid
molecules will retain the ability to bind with FGF receptors.
[0073] The unit dose of the present invention also comprises an
"angiogenically active mutein" of the FGF-2 of FIG. 2 (SEQ ID
NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ
ID NO:8). By the term "angiogenically active mutein" is intended a
mutated form of the FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID
NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ ID NO:8) that
structurally retains at least 80%, preferably 90%, of the 146
residues of the FGF-2 sequence shown in FIG. 2 (SEQ ID NO: 2), the
146 residues of the human FGF-2 sequence shown in FIG. 3 (SEQ ID
NO:4), the 155 residues of the FGF-2 sequence shown in FIG. 4 (SEQ
ID NO:6), or the 155 residues of the FGF-2 sequences shown in FIG.
5 (SEQ ID NO:8), respectively, in their respective positions, and
that functionally retains the angiogenic activity of the respective
unmutated form of FGF-2. 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 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.
Suitable substitutions include the substitution of serine for one
or both of the cysteines at residue positions 87 and 92, which are
not involved in disulfide formation. Other suitable substitutions
include any substitution wherein at least one constituent cysteine
is replaced by another amino acid so that the mutein has greater
stability under acidic conditions, see for example U.S. Pat. No.
5,852,177 which is herein incorporated by reference. One such
substitution is the replacement of cysteine residues with neutral
amino acids such as for example: glycine, valine, alanine, leucine,
isoleucine, tyrosine, phenylalanine, histidine, tryptophan, serine,
threonine, and methionine (U.S. Pat. No. 5,852,177). Preferably,
substitutions are introduced at the FGF-2 N-terminus, which is not
associated with angiogenic activity. However, as discussed above,
conservative substitutions are suitable for introduction throughout
the molecule.
[0074] One skilled in the art, using well-known techniques, is able
to make one or more point mutations in the DNA of FIG. 1 (SEQ ID
NO:1), FIG. 3 (SEQ ID NO:3), FIG. 4 (SEQ ID NO:5), or FIG. 5 (SEQ
ID NO:7) to obtain expression of an FGF-2 polypeptide mutein (or
fragment of a mutein) having angiogenic activity for use within the
unit dose, compositions, and methods of the present invention. To
prepare an angiogenically active mutein of the FGF-2 of FIG. 2 (SEQ
ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5
(SEQ ID NO:8), one uses standard techniques for site-directed
mutagenesis, as known in the art and/or as taught in Gilman et al.
(1979) Gene 8:81 or Roberts et al. (1987) Nature 328:73 1, to
introduce one or more point mutations into the cDNA of FIG. 1 (SEQ
ID NO: 1), FIG. 3 (SEQ ID NO:3) FIG. 4 (SEQ ID NO:5), or FIG. 5
(SEQ ID NO:7) that encodes the FGF-2 of FIG. 2 (SEQ ID NO:2), FIG.
3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ ID NO:8),
respectively.
[0075] Pharmaceutical compositions of the invention comprise an
angiogenically effective dose of a mammalian FGF-2 of FIG. 2 (SEQ
ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), FIG. 5 (SEQ
ID NO:8) 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) 1.0 .mu.g/kg to
30.0 .mu.g/kg of an FGF-2 of FIG. 2 (SEQ ID NO:2) or an
angiogenically active fragment or mutein thereof, (ii) and a
pharmaceutically acceptable carrier. In other embodiments, the safe
and angiogenically effective dose comprises about 0.1 .mu.g/kg to
about 1 .mu.g/kg, about 1 .mu.g/kg to 3 .mu.g/kg, about 3 .mu.g/kg
to about 5 .mu.g/kg, about 5 .mu.g/kg to about 7 .mu.g/kg, about 7
.mu.g/kg to about 8 .mu.g/kg, about 8 .mu.g/kg to about 9 .mu.g/kg,
about 9 .mu.g/kg to about 9.9 .mu.g/kg, such as about 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 .mu.g/kg, up to about 10
.mu.g/kg, about 10 .mu.g/kg to about 15 .mu.g/kg, about 15 .mu.g/kg
to about 20 .mu.g/kg, about 20 .mu.g/kg to about 30 .mu.g/kg, about
30 .mu.g/kg to about 40 .mu.g/kg, about 40 .mu.g/kg to about 60
.mu.g/kg, about 60 .mu.g/kg to about 80 .mu.g/kg of the FGF-2 of
FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6),
FIG. 5 (SEQ ID NO:8) or an angiogenically active fragment or mutein
thereof, and a pharmaceutically acceptable carrier.
[0076] A typical pharmaceutical composition comprises 0.1 mg/ml to
10 mg/ml, more typically 0.3 mg/ml to 0.5 mg/ml, of FGF-2, more
particularly recombinant FGF-2 (rFGF-2), having the sequence set
forth in FIG. 2 (SEQ ID NO:2), or in FIG. 3 (SEQ ID NO:4), or an
angiogenically active fragment or mutein thereof, 10 mM
thioglycerol, 135 mM NaCl, 10 mM Na citrate, and 1 mM EDTA, pH 5.0.
A suitable diluent or flushing agent for the above-described
composition is any of the above-described carriers. Typically, the
diluent is the carrier solution itself comprising 10 mM
thioglycerol, 135 mM NaCl, 10 mM Na citrate, and 1 mM EDTA, pH 5.0.
The rFGF-2 of FIG. 2 (SEQ ID NO:2) or an angiogenically active
fragment or mutein thereof is unstable for long periods of time in
liquid form. To maximize stability and shelf life, the
pharmaceutical composition of the present invention comprising an
effective amount of rFGF-2 or an angiogenically fragment or mutein
thereof, in a pharmaceutically acceptable aqueous carrier should be
stored frozen at -60.degree. C. Thawed, the solution is stable for
1 month at refrigerated conditions. A typical unit dose would
comprise about 5-10 ml of the above described composition having
1.5-8 mg of FGF-2 of FIG. 2 (SEQ ID NO:2), or FIG. 3 (SEQ ID
NO:4).
[0077] In another embodiment, the pharmaceutical composition
comprises a unit dose of FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ
ID NO:4), or an angiogenically active fragment or mutein thereof in
lyophilized (freeze-dried) form. In this form, the unit dose of
FGF-2 would be capable of being stored at room temperature 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-2 of the present invention
therein. Lyophilizers, which perform the above described
lyophilization, are commercially available and readily operable by
those skilled in the art. 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. See,
for example, copending U.S. Application Serial No. 60/229,238,
entitled "Stabilized FGF Formulations containing Reducing Agents,"
herein incorporated by reference. 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-2 of FIG. 2 (SEQ ID NO:2), the FGF-2 of FIG. 3
(SEQ ID NO:4), or an angiogenically active fragment or mutein
thereof is withdrawn from the vial as reconstituted product for
administration to the patient. For example, 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
infusion of (70 kg x 24 .mu.g/kg) 1680 .mu.g (i.e., 1.680 mg).
[0078] The pharmaceutical composition in solution form is generally
administered by infusing the unit dose substantially continuously
over a period of about 10 to about 30 minutes, although it is
recognized that the composition may be administered over a longer
period of time. When the composition is administered into more than
one blood vessel, typically, a portion (e.g., one half) of the unit
dose is administered in a first vessel followed by administration
into a second secondary vessel. Using the above-described
repositioning procedure, portions of the unit dose may be
administered to a plurality of vessels until the entire unit dose
has been administered. After administration, the catheter is
withdrawn using conventional protocols known in the art. Signs of
angiogenesis and a therapeutic benefit, such as reduced
claudication, improvement in ankle-brachial index, improvement in
peak walking time, increase in ability to climb stairs, reduced
body pain, improvement in or prevention of critical limb ischemia,
and improved patient quality of life are seen as early as two weeks
to one month following the FGF-2 administration.
[0079] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Unit Dose of rFGF-2 Employed in a Phase I Clinical Trial
[0080] The recombinantly produced FGF-2 (rFGF-2) having the
sequence shown in FIG. 2 (SEQ ID NO:2) was formulated as a unit
dose and pharmaceutical composition. The various formulations are
described below.
[0081] 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 FIG. 2 (SEQ ID NO:2) in 10 mM sodium citrate, 10 mM
monothioglycerol, 1 mM disodium dihydrate EDTA (molecular weight
372.2), 135 mM sodium chloride, pH 5.0. Thus, in absolute terms,
each vial (and unit dose) contained 0.36 mg rFGF-2. The vials
containing the unit dose in liquid form were stored at 2.degree. to
8.degree. C.
[0082] The 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 to
8 C. Such agents may also be administered to prevent progression of
critical limb ischemia to amputation.
[0083] The rFGF-2 pharmaceutical composition that was infused was
prepared by diluting the rFGF-2 unit dose with the rFGF diluent. In
order to keep the EDTA concentration below the limit of 100
.mu.g/ml, the total infusion volume was increased up to 40 ml when
proportionately higher absolute amounts of FGF-2 were administered
to patients.
Example 2
Phase II PAD Clinical Trial
[0084] Peripheral artery disease (PAD), as defined by resting
anklebrachial index (ABI) less than 0.9, is a common condition
afflicting about 15% of adults greater than 55 years of age. About
33% of these individuals are symptomatic with claudication; about
25% will progress. With worsening blood flow limitation, the
spectrum of PAD runs from mild to moderate to severe claudication,
followed by limb-threatening ischemia, initially characterized by
rest pain, then poor wound healing, and impending or overt
gangrene.
[0085] A phase II trial was undertaken to assess the efficacy of
intra-arterial administration of rFGF-2 on exercise capacity in
patients with intermittent claudication due to infra-inguinal PAD.
The phase II PAD trial was a multicenter, randomized, double-blind,
placebo-controlled, regimen finding study of rFGF-2 to evaluate the
safety, pharmocokinetics, and efficacy by intra-arterial (IA)
infusion over 20 minutes in PAD subjects with moderate to severe
intermittent claudication. Major selection criteria for inclusion
of a patient in the trial were age greater than 40 years, exercise
limited by claudication, index ankle brachial index (ABI) of less
than 0.8 at rest, patent femoral inflow, medically stable for
greater than 4 months, and informed consent. Major selection
criteria for exclusion of a patient from the trial were evidence of
malignancy (according to ACS guidelines), creatinine greater than
2.0 mg/dL, urine protein greater than or equal to 2+or greater than
300 mg/day, proliferative retinopathy, and/or other conditions
impacting safety or compliance. 190 patients participated in the
phase II PAD trial. Baseline characteristics of the patient
population are shown in Tables 1-3.
1TABLE 1 Baseline characteristics of the phase II PAD clinical
trial patient population. Placebo SINGLE DOUBLE Any FGF Number of
Subjects 63 66 61 127 Median Age (yrs) 67 65 68 67 Male 73% 71% 82%
76% Female 27% 29% 18% 24% ABI at Rest (index) 0.55 0.57 0.55 0.56
PWT at Baseline 5.32 5.15 5.81 5.48 COT at Baseline 1.97 2.03 2.20
2.13 Current Smoker 38% 24% 21% 23% Past Smoker 43% 59% 61% 60%
Never Smoker 19% 17% 18% 17% Structured Exercise 56% 50% 49% 50%
ABI (ankle-brachial pressure index); PWT (peak walking time as
measured on a treadmill at 2 mph with the incline increasing every
2 minutes; COT (claudication onset time).
[0086]
2TABLE 2 Concurrent diagnoses of the target patient population in
the phase II PAD clinical trial. Placebo SINGLE DOUBLE Any FGF
CARDIAC History of CAD 62% 58% 62% 60% History of CHF 8% 14% 11%
13% Previous MI 29% 30% 31% 30% CAD Angioplasty (PTCA) 21% 27% 21%
24% S/P CABG 41% 33% 31% 32% Prior PAD Surgery 48% 44% 56% 50%
Prior PTI 21% 27% 21% 24% CEREBROVASCULAR Previous Stroke 8% 6% 7%
6% RISK FACTORS Diabetes Mellitus 36% 27% 38% 33% Hyperlipidemia
75% 77% 75% 76% Hypertension 79% 67% 75% 71% CAD = coronary artery
disease; CHF = congestive heart failure; MI = myochardial
infarction; PTCA = percutaneous transluminal coronary angioplasty;
S/P CABG = coronary artery bypass graft; PTI = percutaneous
transluminal intervention-angioplasty.
[0087]
3TABLE 3 Incidence of peripheral angioplasty and limb
revascularization in the target population of the phase II PAD
clinical trial. Indicators of quality of life as measured by WIQ
and SF-36 are also shown. Placebo SINGLE DOUBLE Any FGF Peripheral
Angioplasty None 71% 71% 71% 71% One 7% 14% 17% 16% >one 22% 14%
12% 13% Limb Revascularization None 72% 68% 66% 67% One 16% 16% 17%
17% >one 12% 16% 17% 17% WIQ - distance score 14% 18% 23% 21%
WIQ - speed score 19% 21% 26% 23% WIQ - stair climbing score 32%
23% 37% 30% SF-36 - PCSS 30.3 29.9 32.9 31.6
[0088] Approximately two-thirds of the patients had a history of
coronary artery disease (CAD), slightly less than one-third had
experienced myocardial infarction, one-third were diabetic,
approximately three-fourths had hypertension, and/or dyslipidemia
(Table 2). Approximately 20-30% of this target population had
undergone greater than one vascularization procedure (Table 3). The
low baseline quality of life scores (WIQ and SF-36) are indicative
of a target PAD patient population with moderate to severe disease.
The scores are based on a scale where 1 or 100% is normal. Thus an
increase in the score represents an improvement. Scores are
tabulated based on a questionnaire where patients perform a
self-evaluation.
[0089] The rFGF-2 was administered by intra-arterial (IA) infusion
over 20 minutes divided between two legs on days 1 and 30. The dose
administered was 30 .mu.g/kg of rFGF-2. The trial patients were
divided into three groups: placebo; single dose (rFGF-2 on day 1);
and double dose (rFGF-2 on days 1 and 30). The primary endpoint
used in the study was a change in peak walking time (PWT) at day 90
on a Gardner graded exercise protocol. Secondary endpoints measured
included: change in PWT at day 180, claudication onset time (COT;
noted as the time at which the patient indicates claudication
and/or pain begins), ankle-brachial pressure index (ABI; as
determined using standard ultrasound device), and health-related
quality of life (QOL) by Walking Impairment Questionnaire (WIQ) and
Short-Form-36 (SF-36) at day 90 and day 180.
[0090] Recombinant FGF-2 (rFGF-2) was formulated in a solution
containing 0.3 mg/ml rFGF-2, 10 mM sodium citrate, 0.3 mM EDTA, 10
mM thioglycerol, 135 mM sodium chloride, pH 5.0. Each 5 ml vial
contained 3.7 ml of clear colorless solution (1. 1 mg rFGF-2 per
vial). Vials containing rFGF-2 were labeled "rFGF-2" and supplied
frozen. Drug product was thawed at room temperature prior to
preparation of dose; detailed instruction for pharmacists were
provided in study manuals. Thawed, undiluted active drug product
could be stored refrigerated at 2-8.degree. C. for 30 days.
[0091] Drug product was diluted with placebo (diluent) and filtered
before administration. The filter was sterile, non-pyrogenic, and
low protein binding. Filtration of the drug product through a 0.22
micron syringe filter (e.g., Millipore, Millex-GV, #SLGVR25LS or
equivalent) would remove particle with no resultant loss in
strength or potency. Thawed, undiluted drug product was used within
8 hours.
[0092] Placebo (diulent) was supplied as a clear, colorless
solution indistinguishable from the drug product. It contained 10
mM sodium citrate, 0.3 mM EDTA, 10 mM thioglycerol, 135 mM sodium
chloride, pH 5.0. Vials containing diluent were labeled "placebo,"
supplied in a liquid state, and stored refrigerated at 2-8.degree.
C.
[0093] The results of the trial indicated that rFGF-2 had an
acceptable safety profile at 90 days for both the single- and
double-dose treatment groups. Dosing at day 1 and day 30 yielded
similar safety data as single dosing at day 1 (data not shown).
[0094] Patient disposition and adverse events for patients at day
180 of the study are shown in Tables 4 and 5, respectively.
4TABLE 4 Patient follow-up. Placebo SINGLE DOUBLE Randomized: 63 66
61 Safety: 180 day FU 57 63 56 PWT: 90/180 days 58/54 62/61 54/53
Premature Termination 6 3 5 Death 1 0 1 Adverse Event 1 0 0
Withdrew Consent 2 1 2 Lost to FU 2 2 2 Revascularized/Amputation 3
2 3
[0095]
5TABLE 5 Safety: Adverse Events Placebo SINGLE DOUBLE Any FGF
Number of Subjects 63 66 61 127 Any AE 41 (65%) 43 (65%) 46 (75%)
Any Cardiac AE 8 6 7 13 Hypotension 2 4 5 9 Proteinuria 2 6 7 13
Serious AEs 13 (21%) 9 (14%) 14 (23%) Deaths 1 0 1 1 Serious
Cardiac AEs 3 4 2 6 Revascularizations/ 3 2 3 5 Amputations
Gangrene (2) 0 0 0 Malignancy 1 0 0 0 Retinal Disorders 1 1 0 1
Pleural/Pericar- 1 0 0 0 dial Effusion
[0096] Data Analysis
[0097] Primary analysis of the data was performed by ANOVA. Ten
subjects with missing PWT and 6 subjects who were revascularized
were excluded from the analysis. Secondary analysis was performed
by ANOVA of Ranks. The 16 subjects excluded from the primary
analysis were assigned lowest rank. This represents a more
conservative approach. See, for example, Table 6.
6TABLE 6 Evaluable vs intent to treat analysis. Hypothetical
subjects Baseline PWT Day 90 PWT ANOVA Ranks n = 6 n = 4 n = 6 a
8:40 10:20 1:40 4 b 5:30 6:55 1:25 3 c 4:30 5:00 .fwdarw. 0:30 2 d
6:20 6:00 -0:20 1 e 4:00 Angioplasty day 60 1 f 2:30 Surgery day 89
1 1.degree. Analysis by ANOVA: "Evaluable" = 174/190 --Excludes 16
subjects who were revascularized (2 placebo, 3 single, 4 double) or
missing data (3 placebo, 3 single, 4 double) 2.degree. Analysis by
ANOVA of Ranks: "Intent to Treat" --Assigns lowest rank to 16
subjects excluded above
[0098] Primary Endpoint: Peak Walking Time at Day 90
[0099] Recombinant FGF-2 was efficacious at treating PAD as
measured by a statistically significant improvement in the PWT
(p=0.026) at day 90 in the patients in the trial receiving a single
dose of rFGF-2 compared to the control placebo group (FIG. 6).
Also, results indicated that a double dose of rFGF-2 (days 1 and
30) was not better than a single dose (day 1).
[0100] Secondary Efficacy Variables
[0101] Secondary efficacy variables included PWT at day 180,
claudication onset time (COT) and ankle brachial index (ABI) at
days 90 and 180, and WIQ and SF-36 quality of life questionnaires
at days 90 and 180. Results at day 180 reflect a large increase in
placebo response.
[0102] FIG. 7 shows absolute change in PWT at days 90 and 180 for
the patient groups receiving placebo, single-dose rFGF-2, or
double-dose rFGF-2. For each patient the PWT at baseline is
subtracted from the PWT at day 90 and the differences are summed
for each group and a mean determined. The data are analyzed by an
analysis of variance (ANOVA).
[0103] FIG. 8 shows the percent absolute change in PWT in the three
patient groups shown at day 90 and day 180. The percent change in
PWT averaged across the two rFGF-2 groups is also shown (designated
Any FGF).
[0104] FIG. 9 shows the measured ABI (ankle brachial index) for the
three patient groups of the phase II clinical study. A baseline
measurement, a day-90 measurement, and the corresponding change
between the baseline and day-90 measurement are indicated. The mean
change in ABI is also shown for the three patient groups. The ABI
is described in An Office Based Approach to the Diagnosis and
Treatment of Peripheral Arterial Disease (2000) Society of Vascular
Medicine and Biology (Medical Communications Media, Inc.,
Wrightstown, Pa.).
[0105] The anklebrachial index is the ratio of the systolic
pressure in the foot to the systolic pressure in the arm as
measured by a Doppler ultrasound device. The normal ABI is 1. An
ABI less than 0.9 is considered diagnostic of PAD. The mean ABI of
the target population enrolled in the trial was .56 in the index
leg at rest. The index leg is the leg with the lower ABI. FIG. 9
shows the mean ABI (top panel) at baseline, day 90, and day 180 for
each group. The bottom panel shows the mean change in ABI at day 90
and at day 180 for each group. There is a positive directional
change in the treatment groups compared to placebo. The difference
achieved statistical significance in the double-dose group at day
180 (mean .DELTA.ABI=0.11; p=0.031 versus placebo). As the ABI
represents an objective measure of blood flow, this change is
consistent with a proposed mechanism of FGF, the formation of new
collateral blood vessels.
[0106] FIG. 10 represents the WIQ severity of claudication at days
90 and 180 for single- and double-dose groups relative to the
placebo group. The bar values represent the percentage of patients
in each group who improved, stayed the same, or became worse in
each group. At day 90, greater than 50% of the patients in the
treatment groups were improved whereas less than 40% of the placebo
patients were improved. At day 180 this apparent treatment benefit
is lost. For further information on the WIQ, see Regensteiner et
al. (1990) J. Vasc. Med. Biol. 2:142-153 herein incorporated by
reference.
[0107] FIG. 11 shows the severity scores at baseline, day 90, and
day 180 for distance, speed, and stair climbing for each group.
While changes are directionally positive for the FGF treatment
groups for distance and speed, the results did not achieve
statistical significance. For stair climbing, there was a trend of
improvement for the single-dose group versus the placebo group
(p=.11). The figure shows that results for the single-dose group
were better than results for placebo group for WIQ distance, speed,
and stair climbing. The figure is shown with a scale where higher
scores are better.
[0108] FIG. 12 depicts the physical summary scores from the short
form 36 (SF-36). The SF-36 is a general validated quality of life
instrument consisting of 36 questions. The SF-36 has 12 domains,
which can be collapsed into two summary scores, physical and
mental. A change of 1 point is associated with an increased
lifespan of 2 years. The change scores in the figure indicate an
improvement in the single-dose group versus the placebo group by
greater than 2 points at day 90.
[0109] Results of the study are summarized in FIG. 13.
[0110] Examination of Subgroups
[0111] The analysis plan provided for the tracking of treatment
response in three pre-specified subgroups of the phase II clinical
trial patient population: diabetes (type I or II, yes vs no),
smoking (current vs non-current, which included those individuals
who had smoked in the past or had never smoked), and median age
(<68 years vs >68 years). The results of the primary efficacy
measure, change in PWT, are presented below for each subgroup. Data
in Tables 7-12 reflect the log-transformed results (to be
consistent with the primary efficacy analysis).
[0112] Diabetes
[0113] The results of PWT at days 90 and 180 are presented in
Tables 7 and 8. There was no statistically significant difference
between the placebo and FGF-treated groups. Changes were greater in
both diabetics and non-diabetics in the FGF-treated groups at day
90. At day 180, changes were smaller in diabetics and similar in
non-diabetics in the FGF-treated groups. The percentage of
diabetics in the single-dose group was slightly lower (27% versus
37% in the placebo group, and 36% in the double-dose group).
7TABLE 7 Change in PWT at day 90 and day 180 in diabetics. Placebo
Single Double Any FGF Day 90 N = 21 N = 17 N = 22 N = 39 Relative
Change in PWT 12.3% 26.8% 18.8% 21.9% Pair-wise P-value .44 .70 .53
Mean Change in PWT 1.08 1.42 1.26 1.33 (min) (+/- SD) (1.84) (2.74)
(3.08) (2.90) Overall P value = .74 Day 180 N = 20 N = 17 N = 21 N
= 38 Relative Change in PWT 18.2% 7.6% 4.6% 5.8% Pair-wise P-value
.64 .52 .52 Mean Change in PWT 1.85 1.32 1.07 1.18 (min) (+/31 SD)
(2.47) (3.27) (2.94) (3.05) Overall P value = .81
[0114]
8TABLE 8 Change in PWT at day 90 and day 180 in non-diabetics.
Placebo Single Double Any FGF Day 90 N = 37 N = 45 N = 32 N = 77
Relative Change in PWT 13.0% 34.9% 18.7% 28.0 Pair-wise P-value
.061 .65 .14 Mean Change in PWT .75 2.12 1.90 2.03 (min) (+/- SD)
(2.60) (2.88) (3.61) (3.18) Overall P value = .14 Day 180 N = 34 N
= 44 N = 32 N = 76 Relative Change in PWT 19.1% 22.7% 20.0% 21.6%
Pair-wise P-value .80 .96 .84 Mean Change in PWT 1.57 2.15 1.89
2.04 (min) (+/- SD) (2.91) (3.97) (2.89) (3.54) Overall P value =
.96
[0115] Smoking
[0116] The results of the change in PWT at days 90 and 180 for
current and non-current smokers are presented in Tables 9 and 10.
For current smokers, no statistically significant difference was
seen at day 90 or day 180. For non-current smokers at day 90,there
was a statistically significant difference overall (p=0.007) and
between the placebo and single-dose groups (P-value=.002); at day
180, no statistically significant difference was seen. The results
of regression analysis suggested smoking was a confounder of
outcome.
9TABLE 9 Change in PWT at day 90 and day 180 in current smokers.
Placebo Single Double Any FGF Day 90 N = 23 N = 14 N = 13 N = 25
Relative Change in PWT 29.3% 22.1% 31.3% 26.2% Pair-wise P-value
.76 .93 .87 Mean Change in PWT 1.55 2.27 2.36 2.31 (min) (+/- SD)
(2.67) (4.22) (3.91) (4.00) Overall P value = .93 Day 180 N= 20 N =
14 N = 11 N = 25 Relative Change in PWT 27.2% 1.0% 43.3% 18.1%
Pair-wise P-value .25 .53 .66 Mean Change in PWT 2.03 1.89 2.19
2.02 (min) (+/- SD) (2.45) (4.55) (3.24) (3.95) Overall P value =
.26
[0117]
10TABLE 10 Change in PWT at day 90 and day 180 in non-current
smokers. Placebo Single Double Any FGF Day 90 N = 35 N = 48 N = 43
N = 91 Relative Change in PWT 5.5% 35.9% 15.8% 26.1% Pair-wise
P-value .002 .27 .019 Mean Change in PWT 0.42 1.83 1.45 1.65 (min)
(+/- SD) (2.02) (2.34) (3.27) (2.81) Overall P value = .007 Day 180
N = 34 N = 47 N = 42 N = 89 Relative Change in PWT 19.8% 29.9 10.0%
20.3% Pair-wise P-value .46 .46 .97 Mean Change in PWT 1.46 1.93
1.40 1.68 (min) (+/- SD) (2.90) (3.57) (2.84) (3.24) Overall P
value = .28
[0118] Age
[0119] The results of the change in PWT at days 90 and 180 for
subjects>68 years of age and .ltoreq.68 years of age are
presented in Tables 11 and 12. There was a greater frequency of
subjects with high PWT (.gtoreq.8 minutes) in the double-dose
group. For subjects >68 years, an unfavorable statistically
significant difference was seen in the double-dose group at day 180
(P-value=0.031). For subjects <68 years, the change in PWT was
higher in the FGF-treated groups, but no statistically significant
difference was seen at day 90 or day 180. The results suggest a
greater improvement in subjects .ltoreq.68 years of age.
11TABLE 11 Change in PWT at day 90 and day 180 in subjects >68
years of age. Placebo Single Double Any FGF Day 90 N = 27 N = 24 N
= 30 N = 54 Relative Change in PWT 16.0% 29.1% 8.0% 17.6% Pair-wise
P-value .27 .53 .87 Mean Change in PWT 1.00 1.58 0.81 1.15 (min)
(+/-SD) (2.18) (2.66) (2.25) (2.45) Overall P value = .22 Day 180 N
= 25 N = 25 N = 29 N = 54 Relative Change in PWT 28.8% 7.1% -4.0%
1.4% Pair-wise P-value .19 .031 .044 Mean Change in PWT 1.74 0.86
1.05 0.96 (min) (+/-SD) (2.73) (2.89) (2.75) (2.79) Overall P value
= .095
[0120]
12TABLE 12 Change in PWT at day 90 and day 180 in subjects
.ltoreq.68 years of age. Placebo Single Double Any FGF Day 90 N =
31 N = 38 N = 24 N = 62 Relative Change in PWT 15.0% 33.1% 40.0%
35.7% Pair-wise P-value .18 .11 .099 Mean Change in PWT 0.76 2.16
2.67 2.36 (min) (+/-SD) (2.51) (2.96) (4.25) (3.49) Overall P value
= .23 Day 180 N = 29 N = 36 N = 24 N = 60 Relative Change in PWT
25.8% 31.5% 48.2% 37.9% Pair-wise P-value .72 .25 .44 Mean Change
in PWT 1.61 2.65 2.19 2.47 (min) (+/-SD) (2.78) (4.17) (3.03)
(3.73) Overall P value = .50
[0121] Post-Hoc Responder Analysis
[0122] Of those subjects in the study whose PWT increased by
.gtoreq.2 minutes at day 90,there was a higher frequency of
subjects with low PWT (.ltoreq.4 minutes) at baseline in the
single-dose group. The strongest predictor of response at day 180
was the response at day 90. The change in PWT for subjects whose
PWT increased by .gtoreq.2 minutes and for subjects whose PWT
increased by <2 minutes are presented in Tables 13 and 14,
respectively. There was a higher percentage of responders at days
90 and 180 and the magnitude of the change was greater in the
single-dose group. About 40% of the patients receiving the
single-dose treatment had an increase in PWT of greater than 2
minutes at both day 90 and day 180, while only about 22% and about
26% of the patients receiving placebo or a double dose of FGF-2
experienced this magnitude of response. In addition, the treatment
effect appears to persist at day 180 by this analysis.
13TABLE 13 Change in PWT at day 90 and day 180 in subjects whose
PWT increased by .gtoreq.2 minutes at day 90. Placebo Single Double
Any FGF Day 90 N = 14 N = 27 N = 16 N = 43 Relative Change in PWT
62.1% 85.8% 68.4% 79.0% Pair-wise P-value .17 .76 .27 Mean Change
in PWT 3.67 4.28 5.37 4.68 (min) (+/-SD) (1.57) (2.54) (4.10)
(3.20) Overall P value = .35 Day 180 N = 14 N = 26 N = 16 N = 42
Relative Change in PWT 55.3% 86.0% 53.2% 72.7% Pair-wise P-value
.17 .90 .37 Mean Change in PWT 3.02 4.28 4.16 4.24 (min) (+/-SD)
(2.39) (3.85) (3.31) (3.61) Overall P value = .24
[0123]
14TABLE 14 Change in PWT at day 90 and day 180 in subjects whose
PWT increased by <2 minutes at day 90. Placebo Single Double Any
FGF Day 90 N = 44 N = 35 N = 38 N = 73 Relative Change in PWT 3.8%
4.4% 3.9% 4.1% Pair-wise P-value .93 .99 .95 Mean Change in PWT
-.02 .12 .07 .09 (min) (+/-SD) (1.78) (1.33) (1.04) (1.18) Overall
P value = 1.00 Day 180 N = 40 N = 35 N = 37 N = 72 Relative Change
in PWT 14.5% -5.8% 2.5% -1.4% Pair-wise P-value .079 .31 .11 Mean
Change in PWT 1.20 0.16 0.44 0.31 (min) (+/-SD) (2.71) (2.62)
(1.84) (2.24) Overall P value = .21
[0124] Post-Hoc Analysis of Anklebrachial Index
[0125] The initial analysis plan pre-specified that subjects having
a baseline ankle brachial index (ABI)>1.2 (consistent with
non-compressible artery) be excluded from the analysis (see FIG.
9). In a post-hoc analysis of the data, subjects having an
ABI>1.2 at anytime (i.e., baseline, day 90, and/or day 180) were
excluded from the analysis. As seen in FIG. 14, this post-hoc
analysis indicates that both the single-dose and double-dose groups
had a statistically significant improvement in ABI compared to the
placebo group at day 90. This significance was not apparent at day
180, though the trend persisted for both the single-dose and
double-dose groups.
[0126] Efficacy Summary
[0127] Primary Efficacy Analysis
[0128] The overall P-value for the primary efficacy analysis
(change in PWT at 90 days) was 0.075 (ANOVA). The overall P-value
for the secondary efficacy analysis by ANOVA of Ranks was
statistically significant (P-value=0.034). The single-dose group
demonstrated a 33.5% increase in PWT at day 90 versus a 20.3%
increase in the double-dose group and a 13.8% increase in the
placebo group. Pair-wise comparison of single-dose versus placebo
was statistically significant (P-value=0.026). This treatment
effect was not maintained at day 180 using the log-transformed
data.
[0129] Secondary Efficacy Variables
[0130] Secondary efficacy variables showed no difference in PWT at
day 180, no difference in COT at days 90 or 180, a favorable trend
in ABI in FGF-treated groups with a statistically significant
change in the double-dose group at day 180, and no difference in
calf plethysmography. Interpretation of changes in the WIQ was
confounded by imbalances at baseline. There was a trend towards
improvement in the PCSS of the SF-36 in the single-dose group
compared to placebo at day 90; this trend was driven by a
statistically significant difference in body-pain score.
[0131] In the pre-specified subgroups, the effect of smoking was
the most interesting. The overall P-value for change in PWT at day
90 was 0.007 for non-current smokers whereas it was 0.93 for
current smokers. The change in PWT in the placebo group was 5.5%
for non-current smokers and 29.3% for current smokers. Regression
modeling suggests that smoking is a confounding variable (see
post-hoc regression analysis below). The over-representation of
current smokers in the placebo group (38%, versus 24% in the
single-dose group and 21% in the double-dose group) made it more
difficult to detect the difference in all evaluable subjects.
[0132] Changes in PWT in non-diabetics paralleled changes seen in
all evaluable subjects at days 90 and 180. Change in PWT in
diabetics paralleled changes seen in all evaluable subjects at day
90 but not at day 180. Regression modeling did not suggest that
diabetes was a covariate. Changes in PWT in subjects over the
median age (>68 years) were less than those in subjects under
the median age at days 90 and 180. Regression modeling did not
suggest that age was a covariate.
[0133] The post-hoc responder analysis shows a higher percentage of
responders in the single-dose group and a greater magnitude of
effect in the single-dose group. In addition, it suggests that the
treatment effect persists at 180 days. The strongest predictor of
response at 180 days is the response at 90 days.
[0134] Post-Hoc Regression Analysis of Peak Walking Time
[0135] Regression models were used to evaluate the criteria
underlying the use of absolute change score, relative change score,
and PWT at day 90 (PWT90) as analysis variables. The models are
discussed below. This post-hoc analysis reveals that both absolute
and relative change scores have shortcomings and do not provide the
most value for assessing FGF treatment effect in the phase II
clinical trial. Using PWT90 as the analysis variable and adjusting
for baseline PWT appears to provide a better basis, and value, for
assessing the treatment effect of FGF.
[0136] Background--Assumptions in the Analysis:
[0137] I. Assume absolute change score is the correct variable to
be used in the analysis. For each subject, the absolute day 90
change score in PWT
[0138] =(PWT Day 90-PWT Baseline) or
[0139] =(PWT90-PWTB).
[0140] Assuming that (PWT90-PWTB)=d, then PWT90=1.0*PWTB+d, (across
the full range of PWTB), and the scatter plot of PWT90 versus PWTB
would be:
[0141] 1. Linear,
[0142] Slope=1.0, and
[0143] 3. Intercept would be d (unrestricted).
[0144] FIG. 15 shows a hypothetical plot of PWT90 versus PWTB when
absolute change score is assumed to be the correct variable.
[0145] II. Assume relative change score is the correct variable to
be used in the analysis. For each subject, the relative day 90
change score in PWT
[0146] =(PWT90/PWTB).
[0147] Assuming that (PWT90/PWTB)=1d, then PWT90=1d*PWTB+0.0
(across the full range of PWTB), and the scatter plot of PWT90
versus PWTB would be:
[0148] 1. Linear,
[0149] 2. Slope would be Id (unrestricted), and
[0150] 3. Intercept would be 0.0.
[0151] FIG. 16 shows a hypothetical plot of PWT90 versus PWTB when
relative change score is assumed correct.
[0152] The original analysis plan used absolute change score as the
analysis variable, because there was not strong initial guidance
from PIs/consultants to use relative score, and the absolute change
score was used in the analysis of a related study directed to
treatment of coronary artery disease (CAD). There was interest in a
potential for a combined indication of FGF for CAD and PAD, which
would be facilitated by consistent use of the same analysis
variable. However, the analysis plan was amended to use the Log10
relative change score (Log10(PWT90/PWTB)=(Log10 PWT90-Log10 PWTB)),
as a blinded preliminary evaluation of the skewness and kurtosis of
the change score indicated the day 90 data did not appear to be
symmetric. The day 180 PWT absolute change score appeared (in a
post-hoc analysis) to be more nearly symmetric, i.e., the absolute
change score appeared to have better distributional properties.
[0153] Results
[0154] The results of the post-hoc regression analysis using the
models described in Tables 15 and 16 are shown in FIGS. 17-19. FIG.
17 shows a scatter plot of PWT90 versus PWTB plus an unrestricted
spline regression curve for each treatment group. FIG. 17 suggests
that the first two criteria for use of absolute change score
(linearity and slope=1) are not fully satisfied, and the criteria
for use of relative change score (linearity and intercept=0.0) are
also not satisfied. Thus, use of absolute change score or relative
change score as the analysis variable does not appear to be fully
consistent with the observed data.
[0155] FIG. 18 shows the same scatter plot plus curves representing
regression model 2 described below. Regression models provide a
more flexible method for assessing the change in PWT at day 90 and
adjusting for baseline PWT. The curved shape of the scatter plot
suggests that a regression model that produces curves will better
represent or fit the study data. To achieve a curved shape,
regression models would have PWT90 as the analysis variable, and
predictor variables that include PWTB and (PWTB).sup.2. Other
baseline variables such as smoking status can also be included in
the regression model, if they are important confounders.
[0156] Table 15 shows three regression models with PWT90 as the
analysis variable and adjusted for PWTB. All models also are
adjusted for center (site). Model 1 for PWT90 includes only the
predictor variable PWTB but is more flexible than using the
absolute change score because model 1 can have any slope (i.e.,
model 1 does not require the slope to be 1.0). Model 1 better fits
the data than either the absolute or relative change scores.
[0157] Model 2 for PWT90 includes both PWTB and (PWTB).sup.2 as
predictor variables, and accommodates the curve shape of the
scatter plot (see FIG. 18). Model 2 appears to provide a better fit
to the study data than model 1, as seen by:
[0158] 1) a p-value=0.027 for the inclusion of PWTB.sup.2 in the
model, and
[0159] 2) an increased R.sup.2=0.52 compared to model 1, which
represents a statistically significant improvement in
R.sup.2=0.025).
[0160] Model 3 for PWT90 includes smoking status (at baseline),
PWTB, and (PWTB).sup.2, and adjusts for current smoking status.
15TABLE 15 Regression models for PWT90. All regression models are
adjusted for Center (Site). 1 2 3 Variables Trt. Single Trt. Single
Trt. Single in Model Trt. Double Trt. Double Trt. Double PWTB PWTB
PWTB (straight lines) PWTB.sup.2 PWTB.sup.2 (curves) Smoker
(curves) Variable Single: 69.8 Single: 71.5 Single: 79.3
Coefficients Double: 56.7 Double: 61.8 Double: 71.5 PWTB: 0.986
PWTB: 1.917 PWTB: 1.93 PWTB.sup.2: -0.0012 PWTB.sup.2: -0.0012
Smoker: -23.8 p-value for Single: 0.032 Single: 0.027 Single: 0.015
each variable Double: 0.096 Double: 0.067 Double: 0.037 PWTB:
<0.0001 PWTB: <0.0001 PWTB: <0.0001 PWTB.sup.2: 0.026
PWTB.sup.2: 0.022 Smoker: 0.14 Trt. Effect (seconds) S-Placebo 69.8
71.5 79.3 D-Placebo 56.7 61.8 71.5 Model R.sup.2 0.50 0.52 0.53
Estimated Placebo value for PWT90 when Non- Smk PWTB equals Smk Smk
100 sec. 139 sec. 75 33 57 300 sec. 336 sec. 363 323 347 600 sec.
632 sec. 615 578 602
[0161] The three regression models with PWT90 suggest:
[0162] The single-dose group had a statistically significant
improvement in PWT at day 90, with pairwise p-values of 0.032,
0.027, and 0.015 (for models 1, 2, and 3, respectively).
[0163] The single-dose group had an average increase in PWT over
the placebo group of 69.8, 71.7, and 79.3 seconds.
[0164] The double-dose group had a trend or statistically
significant improvement in PWT at day 90, with pairwise p-values of
0.096, 0.0678, and 0.037.
[0165] The double-dose group had an average increase in PWT over
the placebo group of 56.7, 61.8, and 71.5 seconds; and thus the
double-dose group is more similar to the single-dose group in
increased PWT than to the placebo group.
[0166] The regression models explain 50% or more of the variation
in PWT at day 90.
[0167] FIG. 19 shows the scatter plot of PWT180 versus PWTB plus an
unrestricted spline regression curve for each treatment group. The
shape of the data also do not support a slope of 1 or an intercept
of 0.0. The shape of the day 180 PWT data are only slightly
curved.
[0168] Table 16 shows the three regression models for PWT180 as the
analysis variable and adjusted for PWTB, and other baseline subject
characteristics. All three models indicate similar results, with
the single-dose group having a 22- to 26-second benefit over the
placebo group, and the double-dose group apparently not different
than the placebo group.
16TABLE 16 Regression models for PWT180. All regression models are
adjusted for Center (Site). 1 2 3 Variables Trt. Single Trt. Single
Trt. Single in Model Trt. Double Trt. Double Trt. Double PWTB PWTB
PWTB (straight lines) PWTB.sup.2 PWTB.sup.2 (curves) Smoker
(curves) Variable Single: 22.3 Single: 22.7 Single: 25.9
Coefficients Double: -7.4 Double: -5.8 Double: -1.7 PWTB: 1.2 PWTB:
1.4 PWTB: 1.4 PWTB.sup.2: -0.003 PWTB.sup.2: -0.0003 Smoker: -11.4
p-value for Single: 0.514 Single: 0.506 Single: 0.454 each variable
Double: 0.836 Double: 0.872 Double: 0.964 PWTB: <0.0001 PWTB:
<0.0031 PWTB: 0.0029 PWTB.sup.2: 0.630 PWTB.sup.2: 0.608 Smoker:
0.508 Trt. Effect (seconds) S-Placebo 22.3 22.7 25.9 D-Placebo -7.4
-5.8 -1.7 Model R.sup.2 0.57 0.57 0.57 Estimated Placebo value for
PWT90 when Non- PWTB equals Smk Smk 100 sec. 157 141 123 134 300
sec. 393 395 381 392 600 sec. 745 731 723 734
[0169] In summary, the post-hoc regression analysis using the PWT
at day 90 as the outcome variable and adjusting for PWT at baseline
showed an increase of about 70 seconds (1.16 minutes) over placebo
in the single-dose group, and about 57 seconds (0.95 minutes) in
the double-dose group (p=.032, .096, respectively). Allowing the
relationship to be non-linear (i.e., curved) increased the
treatment effect to about 72 seconds (1.19 minutes) in the
single-dose group, and about 62 seconds (1.03 minutes) in the
double-dose group (p=.027, .067, respectively). Adjusting for
smoking status further increased the treatment effect to about 79
seconds (1.32 minutes) in the single-dose group, and about 72
seconds (1.19 minutes) in the double-dose group (p=0.015, 0.035,
respectively).
[0170] Conclusions from Phase II Clinical Trial
[0171] This study defined an effective dose, route, and regimen for
treatment of PAD with rFGF-2. A single-dose of 30 .mu.g/kg rFGF-2
given intra-arterially improved PWT of PAD patients. Administering
a double-dose of rFGF-2 was not better than administering a
single-dose of FGF-2. The magnitude of benefit in PWT was greater
than 1 minute, with the duration of benefit observed at both 3 and
6 months. In fact, about 40% of the patients receiving the
single-dose treatment experienced an increase in PWT of greater
than 2 minutes at both day 90 and day 180, compared with only about
22% of patients receiving placebo and about 26% of patients
receiving a double dose of rFGF-2. The data indicated that those
patients who responded at Day 90 were more likely to respond at Day
180.
[0172] Thus, repeated dosing with rFGF-2 is feasible where
necessary without compromising patient safety. The beneficial
effect on PWT seen at both day 90 and day 180 with a single dose of
rFGF-2 coupled with the safety of multiple dosing offers a method
for providing prolonged therapeutic benefit to PAD patients. This
can be achieved, for example, by administering to a patient a
therapeutically effective dose at day 1, and subsequent
therapeutically effective doses as clinically needed, i.e., as
symptoms recur.
Example 3
Phase III PAD Clinical Trial
[0173] A phase III, multicenter (up to 50 sites), double-blind,
placebo-controlled, dose-optimization study is conducted. The
primary objective of this trial is to evaluate safety and efficacy
of an intra-arterial (IA) infusion of 3.0 .mu.g/kg or 30.0 .mu.g/kg
rFGF-2 versus placebo in peripheral artery disease (PAD) subjects
with moderate to severe claudication. The trial enrolls 450
subjects (150 per arm) with moderate to severe claudication
limiting exercise. Inclusion criteria and exclusion criteria are
shown in Table 17. Sample size may be adjusted based on DSMB
evaluation of variability of peak walking time at 90 days after 225
subjects are enrolled.
17TABLE 17 Synopsis of Phase III Clinical Trial. Inclu- Male or
female .gtoreq.40 years of age sion History of moderate to severe
claudication limiting exercise for Cri- >6 months teria ABI
>0.3 and <0.8 in the index limb at rest or >20% decrease
after exercise Peripheral angiogram (contrast or MRA) within 4
months confirming >70% obstruction of one or more infra-
inguinal vessels, patent femoral inflow bilaterally, and absence of
hemodynamically significant supra-inguinal obstruction Able to
exercise >1 minute but <12 minutes on two Gardner treadmill
exercise tests. Exercise time must be limited by claudication at
baseline. Duplicate tests will be performed >24 hours but not
>2 weeks apart. The difference between baseline exercise times
must be .ltoreq.20% of their mean. Medically stable for 3 months
with laboratory parameters within clinically acceptable range for
required procedures. Serum creatinine .ltoreq.2.2 mg/dL and urine
protein/creatinine ratio <.3 Willing and able to give written
informed consent Exclu- Peripheral Artery Disease sion History of
rest pain, non-healing ulcer, or gangrene Cri- within 3 months
teria Evidence of hemodynamically significant aorto-iliac
obstructive disease Peripheral revascularization (PTI or surgery)
within 3 months Malignancy History of malignancy within past 5
years (exceptions: curatively treated basal cell carcinoma,
squamous cell carcinoma of the skin in sun-exposed areas, or
carcinoma of the cervix) Evidence or suspicion of malignancy after
screening according to ACS guidelines Ocular conditions
Proliferative retinopathy or moderate or severe non-proliferative
retinopathy Maculopathy with choroidal neovascularization or
macular edema Intra-ocular surgery within 3 months Cardiovascular
conditions Myocardial infarction, CABG, PTCA within 3 months
Transient ischemic attack or stroke within 3 months General medical
conditions: Pregnancy, nursing mothers Participation in clinical
trials of other investigational agents, intra-arterial devices, or
procedures, for which follow-up visits have not been completed
History of organ transplantation Any combined condition which makes
the subject unsuitable for participation in the opinion of the
Investigator, e.g., concurrent medical illness which limits life
expectancy to <12 months, psychosis, severe mental retardation,
inability to communicate with study personnel, drug or alcohol
abuse Diagnosis of primary pulmonary hypertension, restrictive or
obstructive cardiomyopathy, active vasculitis Previous
participation in any therapeutic angiogenesis trial with any
investigational agent unless subject received placebo
[0174] The study drug, rFGF-2 having the sequence shown in FIG. 2
(SEQ ID NO:2), is contained at 0.35 or 3.5 mg/mL in a lyophilized
powder, to be reconstituted with normal saline; it is formulated in
10 mM sodium citrate, 1 mM EDTA, 10 mM dithiothreitol (DTT), 4%
glycine, 1% glucose at pH 6.0. Treatment consists of infusion of 20
mL at 1 mL/min of placebo, 3.0.mu.g/kg or 30.0 .mu.g/kg rFGF-2,
divided equally between two legs, via the common femoral artery.
Assignment to treatments is randomized 1:1:1 placebo: 3.0 .mu.g/kg
rFGF-2: 30.0 .mu.g/kg rFGF-2. Blood will be drawn at baseline and
at the end of the infusion for analysis of plasma concentration of
FGF-2. Each subject is observed in the hospital for 6 hours
following study drug administration and followed as an outpatient
at specified intervals for 180 days.
[0175] Patients are monitored for acute safety variables including
systolic hypotension associated with IA infusion and any evidence
of allergic reactions, as well as frequency and severity of adverse
events, changes in laboratory parameters (especially urine
protein), evidence of retinal toxicity, and evidence of
seroconversion (antibody formation). DSMB will review SAEs and
abnormal laboratory tests during enrollment.
[0176] Primary efficacy variable is change from baseline in peak
walking time (PWT) at 90 days as measured by Gardner graded
exercise test time, adjusted for baseline PWT, smoking status, and
center. Secondary efficacy is established based on the following
parameters:
[0177] Change from baseline in PWT at 45, 135, and 180 days
adjusted for baseline PWT, smoking status, and center;
[0178] Change from baseline in claudication onset time (COT) at 45,
90, 135, and 180 days;
[0179] Change from baseline in anklebrachial index pressure (ABI)
at 45, 90, 135, and 180 days;
[0180] Change from baseline in severity of claudication, distance,
speed, and stair climbing scores of the WIQ at 45, 90, 135, and 180
days;
[0181] Change from baseline in physical component summary score
(PCSS) of the SF-36 at 45, 90, 135, and 180 days; and
[0182] Percentage of responders at 90 and 180 days.
[0183] Potential substudies include plethysmography, muscle biopsy,
and MR spectroscopy. General protocol and information tests at
follow-up visits are shown in Table 18.
18TABLE 18 General Schedule of Events. Primary Screen Dosing
Endpoint Termination DAY DAY DAY DAY DAY DAY DAY Tests -45 to -1 1
15 45 90 135 180 Complete History and X Physical Exam consistent
with ACS guidelines Limited History and X X X X X Physical Exam
Telephone FU X 12-lead ECG X X X X Chest x-ray X PSA (males only);
X serum pregnancy (females only); screen for HIV, hepatitis and
drugs of abuse if appropriate Laboratory Tests (CBC, X X X X X X X
platelets, chemistry.sup.1, urine.sup.2) FGF-2 Antibodies X X X X X
X Gardner graded exercise X, X X X X X test for PWT and COT; ABIs
Ophthalmologic exam; X X fundus photography only if evidence of
retinopathy Quality of Life: WIQ, X X X X X SF-36 Limited Angiogram
X Study Drug X Administration Blood Sample for FGF-2 X and Infusion
Solution Sample Concomitant X X X X X X X Medications Adverse
events (AEs) X X X X X X .sup.1CBC (no differential), platelets,
electrolytes, BUN, creatinine, cholesterol, liver enzymes, glucose,
cotinine .sup.2Urine for specific gravity, qualitative protein,
protein/creatinine ratio
[0184] Statistical Analysis
[0185] Data are analyzed with an intent to treat analysis using
ANOVA of Ranks with last value after baseline carried forward for
missing data, or lowest rank for subjects without a post-baseline
PWT assessment, adjusting for baseline PWT, smoking status, and
center.
Example 4
Influence of FGF-2 Dosing Regimens on Collateral Blood Flow in Rats
With Peripheral Arterial Insufficiency
[0186] A study was undertaken to compare the efficacy of three
routes of FGF-2 administration (intra-arterial [positive control],
intramuscular, and a route used in humans) to increase collateral
blood in rats with experimental peripheral arterial
insufficiency.
[0187] Previous animal model studies have demonstrated that bFGF is
effective at improving collateral blood flow to the distal calf
muscles following bilateral femoral artery occlusion (Yang et al.
(1996) Circ. Res. 79:62-69; Yang and Feng (2000) Am. J. Physiol.
278:H85-H93). The improved blood flow from .about.50 ml/min/100 g
to 70-80 ml/min/100 g was possible due to a significant decrease in
vascular resistance of the collateral vessels of the upper thigh.
The increase in collateral blood flow to the calf muscles
correlates well with an angiographic score obtained from x-ray
images of the thigh arterial tree. Upper thigh collateral vessels
are the major site of resistance in the circuit following occlusion
of the femoral arteries (Yang et al. (1996) Circ. Res. 79:62-69).
It is not likely that de novo synthesis of new capillaries
(angiogenesis) could develop into large conduit vessels and account
for this vascular response. Rather, the extent of the resistance
change and the short time for vascular development to occur (16
days) makes it probable that enlargement of existing vessels was
the primary change contributing to the greater blood flow. This
increase in collateral blood flow with bFGF is also found in aged
rats (Yang and Feng (2000) Am. J. Physiol. 278:H85-H93) and
enhanced with physical activity (Yang et al. (1998) Am. J. Physiol.
274:H2053-H2061).
[0188] A variety of routes and regimens of bFGF administration have
been shown effective at increasing collateral blood flow in animal
models. These include close-arterial systemic, and subcutaneous
routes with bolus, short-term and relatively long-term delivery
regimens achieved by injections or timed infusions with osmotic
pumps (Yang and Feng (2000) Am. J. Physiol. 278:H85-H93). While
these protocols have been useful to demonstrate the efficacy of
bFGF, some of these regimens are not appropriate for patient
management in therapeutic angiogenesis. Further, the value of
repeated administration of bFGF at timed intervals in patients is
expected, but not feasible with many of the procedures used in
previous experimental studies. For example, it would be extremely
valuable to establish the efficacy of intramuscular injections of
bFGF. However, it is presently unclear whether direct intramuscular
injections of bFGF impart targeted or generalized improvement in
collateral blood flow. Thus, the purpose of the present pilot study
was to evaluate the efficacy of FGF-2 administration via
intramuscular injections and a clinically relevant protocol used in
therapeutic angiogenesis.
[0189] Experimental Design:
19 Animals with peripheral arterial insufficiency were divided into
four groups: Group 1 Intra-arterial, 14 day continuous infusion,
FGF-2 N = 6 (5 .mu.g/kg/day) Group 2 Vehicle group comprised of:
group 2a 14 day intra-arterial continuous N = 2 infusion of vehicle
group 2b Single intra-arterial injection of vehicle N = 2 group 2c
Intramuscular, single bolus of vehicle N = 4 Group 3 Single
Intra-arterial Injection group 3a Dose 1 (1.5 .mu.g/kg total) N = 6
group 3b Dose 2 (15 .mu.g/kg total) N = 6 group 3c Dose 3 (30
.mu.g/kg total) N = 6 Group 4 Intramuscular Injection group 4a Dose
1 (0.15 .mu.g/kg total) N = 6 group 4b Dose 2 (1.5 .mu.g/kg total)
N = 6 group 4c Dose 3 (15 .mu.g/kg total) N = 6
[0190] Because of distinct delivery routes, the study was not
performed in a completely blinded manner. However, animals within a
delivery route received the treatments of dose (i.e., vehicle,
infusion, dose 1, dose 2, or dose 3) in a randomly-assigned blinded
manner.
[0191] General Protocol:
[0192] Adult Sprague-Dawley rats (approximately 325 g) were
conditioned to the treadmill by walking for 5-10 min twice daily
for 5 days. To initiate the experiment, the animals were subjected
to bilateral femoral artery occlusion (cf. Methods). On the same
day, animals began a two-week treatment according to the treatment
groups described above. On day 16 of the experiment,
collateral-dependent blood was determined while the rats were
running on a motor-driven treadmill. Following completion of the
data set, the results were pooled according to treatment group, and
the results were analyzed statistically by ANOVA. It was expected
that vehicle-treated animals from each of the delivery routes could
be grouped into one reference control group. However, data were
assessed to determine whether the intramuscular injection treatment
had introduced a systematic response, for example, caused by an
inflammatory response in the muscle.
[0193] Peripheral Arterial Insufficiency Model:
[0194] Bilateral ligation of the femoral artery is designed to
establish peripheral arterial insufficiency without impairing
resting muscle blood flow. The high blood flow reserve of muscle is
markedly reduced while residual muscle blood flow is sufficient to
support resting blood flow needs; e.g., compare (Yang et al. (1990)
J. Appl. Physiol 69:1353-1359; Yang and Terjung(1993) J. Appl,
Physiol. 75(1):452-457; Mackie and Terjung (1983) Am. J. Physiol.
245:H265-H275). Thus, there is no `rest pain` nor complications
leading to pathological changes, tissue necrosis, or gangrene
observed with more proximal vascular obstructions (Chleboun and
Martin (1994) Aust. N. Z. J. Surg. 64:202-207). It is recognized
that these surgically treated animals do not represent the broad
spectrum of peripheral arterial insufficiency found clinically.
Rather, this model is characteristic of large vessel occlusive
disease that often presents itself with symptoms of intermittent
claudication.
[0195] Methods:
[0196] Animal Care.
[0197] Adult Sprague Dawley rats (approximately 325 g), obtained
from Taconic Farms, Germantown, N.Y., were housed in a temperature
controlled room (20+1.degree. C.), with a 12hr/12hr light/dark
cycle. Animals were given Purina Rat Chow and tap water ad libitum.
Previous studies have established that a complete data set of
approximately 12 animals per group is necessary to definitively
evaluate treatment effects (Yang et al. (1990) J. Appl. Physiol
69:1353-1359). Thus, the present work was a pilot study to assess
the general response for future consideration of the half of the
study. Since .multidot.90% of rats received from the supplier are
willing runners (to be randomly assigned to treatment groups) and
some attrition occurs in the conduct of the experiment, it was
expected that n=5-6 per experimental group would be obtained.
[0198] FGF-2 Delivery.
[0199] FGF-2 delivery was initiated/achieved at the time of femoral
artery ligation by: a) a 14-day continuous intra-arterial infusion;
b) a single intramuscular injection; or c) a single intra-arterial
injection as follows.
[0200] For the positive control, a group of 8 rats received a
14-day infusion from an in-dwelling pump/catheter (rate=0.5
.mu.L/hr). The catheter was placed such that the infusion was
delivered upstream of the ligation point in the femoral artery of
one hindlimb. Six of the rats received FGF-2 at a dose of 5
.mu.g/kg/day for 14 days for a total of 70 .mu.g/kg; the other two
received vehicle alone (PBS). To allow for dead spaces, filling the
pump, filling the tubing, etc., a final volume of 0.435 mL of pump
solution for each rat was calculated based on the initial rat
weight of approximately 325 g. A separate pump solution aliquot was
prepared for each rat. Just prior to use, 43.5 .mu.L of sodium
citrate and 7 .mu.L of glycerol was added to each aliquot, so that
the volume of FGF-2 solution or PBS in each of the prepared tubes
was 0.435-0.0435-0.007=0.385 mL. For the FGF aliquots, the
concentration of FGF-2 was thus 152.5 .mu.g/mL.
[0201] A second group of rats received vehicle or FGF-2 with a
single intra-arterial injection. This injection was given over the
span of 10 minutes, into the femoral artery of one hindlimb,
upstream of the point of ligation. The volume to be injected was
0.35 mL. Six rats received 1.5 .mu.g/kg FGF-2 total dose; six
received 15 .mu.g/kg FGF-2; six received 30 .mu.g/kg FGF-2; and two
received vehicle alone.
[0202] A third group of rats received a single intramuscular
injection. This injection was split between two sites in the medial
hamstring on one hindlimb, in the region of collateral formation.
The volume to be injected was 100 .mu.L per site, for a total
volume of 0.2 mL. Six rats received 0. 15 .mu.g/kg FGF-2 total
dose; six received 1.5 .mu.g/kg FGF-2; six received 15 .mu.g/kg
FGF-2; and four received vehicle alone.
[0203] Ligation Surgery.
[0204] Under ether anesthesia each femoral artery was isolated just
distal to the inguinal ligament. A ligature was placed tightly
around the femoral vessel to assure total obstruction to blood
flow. Topical antibiotic powder (Neo-Predef, Upjohn) was placed on
the wound prior to closure with skin clips. The surgical procedure
was brief, was achieved with a 100% success rate, and the animals
recovered rapidly. As done routinely (Yang and Terjung(1993) J.
Appl, Physiol. 75(1):452-457; Yang et al. (1995a) Circ. Res.
76.448-456; Yang et al. (1995b) Am J. Physiol. 268:H1 174-HI 180),
visual inspection at the time of autopsy verified the success of
surgery.
[0205] Blood Flow Determination during Treadmill Running In
Vivo.
[0206] Muscle blood flow was determined in a blinded manner,
utilizing radiolabeled microspheres during treadmill running, as
used extensively (Yang and Terjung(1993) J. Appl, Physiol.
75(1):452-457; Mackie and Terjung (1983) Am. J. Physiol.
245:H265-H275; Mathien and Terjung (1986) Am. J. Physiol.
245:H1050-H1059; Mathien and Terjung (1990) Am J. Physiol.
258:H759-H765; Yang et al. (1990) J. Appl. Physiol 69:1353-1359;
Yang et al. (1995) Circ. Res. 76.448-456; Yang et al. (1995) Am J.
Physiol. 268:H1174-H1180; Yang et al. (1996) Circ. Res. 79:62-69).
Microspheres (15.mu.m diameter), labeled with .sup.85Sr or
.sup.141Ce (-10 mCi/g), were obtained commercially (NEN, Boston) in
a suspension of 10% dextran containing 0.05% Tween 80. A well-mixed
suspension of microspheres was carefully infused into the arch of
the aorta, followed by a saline flush, over a 15-20 second period.
Direct comparisons of injection sites (left ventricle versus aortic
arch) gave the same blood flows to the kidneys and hindlimb
muscles. Approximately 360,000 spheres were infused to establish an
adequate microsphere distribution and to permit statistical
assurance in the data (Mackie and Terjung (1983) Am. J. Physiol.
245:H265-H275; Mathien and Terjung (1986) Am. J. Physiol.
245:H1050-H1059; Mathien and Terjung (1990) Am J. Physiol.
258:H759-H765). Typically, there were well in excess of 400
microspheres per individual muscle sample during exercise.
Withdrawal of the reference blood sample at 500 .mu.l/min (Sage
Instruments Model 355 pump) from the caudal artery was initiated 10
sec prior to infusion of microspheres and continued for
approximately 100 sec. It has been found that reference blood
samples, withdrawn from the right carotid artery, femoral artery,
and caudal artery, match within 5-10% of each other (Mackie and
Terjung (1983) Am. J. Physiol. 245:H265-H275; Unpub. Obs).
[0207] Two blood flow determinations were performed in each animal,
at a moderate and higher treadmill speed, to establish peak
vascular conductance of the muscle so that the upstream collateral
resistance in the upper thigh becomes rate-determining for
downstream blood flow to the calf muscles. This is achieved, since
muscle contraction (exercise) is the most powerful stimulus for
vasodilation. Following exercise, the rats were sacrificed by an
overdose of pentobarbital and the tissue samples obtained as
described below, and counted (LKB Universal Gamma Counter), with
the reference blood sample, to a 1% counting error. Bilateral
sections of kidney (middle 3rd) were taken to verify the adequacy
of microsphere mixing. Appropriate corrections were made for
background and isotopic spillover. Blood flows (ml/min/100 g) were
calculated as follows:
FLOW=(CPM.sub.T.times.FLOW.sub.RBS.times.100)/(CPM.sub.RBS.times.Wt.sub.T)
[0208] where T is tissue and RBS is the reference blood sample.
[0209] Heart rate and arterial pressure were continuously monitored
during exercise.
[0210] Surgical Procedures for Blood Flow Determination. Surgical
preparation for in vivo blood flow determination was a modification
of that described by Laughlin et al. (1982) J. Appl. Physiol
52:1629-1635. Animals were anesthetized with ketamine/ACE-promazine
(100 mg/0.5 mg per kg) and a catheter was inserted into the right
carotid artery to the arch of the aorta for later infusion of the
microspheres. A catheter (PE 50 tapered) was also placed into the
caudal artery for the withdrawal of blood sample. Both catheters
were filled with saline containing heparin (100 IU/ml), led under
the skin, and exteriorized at the back of the neck. Each incision
site was sutured and covered with 1% xylocaine ointment (Astra
Pharm.). Consistent with the experience of others (Gleeson and
Baldwin (1981)J. Appl. Physiol. 50:1205-1211), after 3-4 hr
following placement of the catheters the rats were alert, willing
to run, and exhibited normal exercise tolerance.
[0211] Muscle Sections.
[0212] All tissues of the hindlimb from the hip socket distal, are
dissected, weighed, and counted for radioactivity. Muscles (Greene
(1963) Anatomy of the Rat, New York Hafner Pub. Co.) include:
biceps femoris, semitendinosus, semimembranosus, caudofemoralis,
adductor group, gluteus group, tensor fascias latae, quadriceps
group, soleus, plantaris, gastrocnemius, tibialis anterior,
extensor digitorurm longus, deep lateral and posterior crural
muscles. The tibia, fibula, femur and foot are also weighed and
counted. In addition, sections composed primarily of fast-twitch
red fibers (deep lateral quadriceps and deep lateral
gastrocnemius), fast-twitch white fibers (superficial quadriceps
and superficial medial gastrocnemius), and slow-twitch red fibers
(soleus) are obtained. A thorough biochemical, physiological and
morphological characterization of these muscle fiber sections is
known (cf. Saltin and Gollaick (1983) In: Handbook of
Physiology--Skeletal Muscle, Am. Physiol. Soc., pp. 555-631). Blood
flow to the diaphragm will also be determined to follow the
response of an active muscle that is not subject to ligation.
[0213] Statistical Procedures.
[0214] Statistical evaluation employing repeated measures analysis
of variance, Tukey's comparison of means, and a t-test were
performed as appropriate (Steel and Torrie (1960) Principles &
Procedures of Statistics, McGraw-Hill, New York).
[0215] Results:
[0216] Intramuscular administration of rFGF-2 increased blood flow
in a dose-dependent manner (FIG. 20). When administered
intra-arterially, the 15.mu.g/kg rFGF-2 dose was just as
efficacious as the 30 .mu.g/kg dose. Continuous infusion over a
14-day period did not provide a significantly different efficacy
relative to the single IA infusion or single IM injection mode of
administration.
[0217] These data demonstrate efficacy of single IM injection and
single IA infusion of rFGF-2 in enhancing blood flow to the
hindlimb quarters in a PAD animal model.
[0218] 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. Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be obvious that certain changes
and modifications may be practiced within the scope of the appended
embodiment.
Sequence CWU 1
1
9 1 441 DNA Bos taurus CDS (1)...(441) 1 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 taa 441 Lys
Ser * 145 2 146 PRT Bos taurus 2 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 3 441 DNA
Homo sapiens CDS (1)...(441) 3 ccc gcc ttg ccc gag gat ggc ggc agc
ggc gcc ttc ccg ccc ggc cac 48 Pro Ala Leu Pro Glu Asp Gly Gly Ser
Gly Ala Phe Pro Pro Gly His 1 5 10 15 ttc aag gac ccc aag cgg ctg
tac tgc aaa aac ggg ggc ttc ttc ctg 96 Phe Lys Asp Pro Lys Arg Leu
Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25 30 cgc atc cac ccc gac
ggc cga gtt gac ggg gtc cgg gag aag agc gac 144 Arg Ile His Pro Asp
Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp 35 40 45 cct cac atc
aag cta caa ctt caa gca gaa gag aga gga gtt gtg tct 192 Pro His Ile
Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 50 55 60 atc
aaa gga gtg tgt gct aac cgt tac ctg gct atg aag gaa gat gga 240 Ile
Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly 65 70
75 80 aga tta ctg gct tct aaa tgt gtt acg gat gag tgt ttc ttt ttt
gaa 288 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe
Glu 85 90 95 cga ttg gaa tct aat aac tac aat act tac cgg tca agg
aaa tac acc 336 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg
Lys Tyr Thr 100 105 110 agt tgg tat gtg gca ctg aaa cga act ggg cag
tat aaa ctt gga tcc 384 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln
Tyr Lys Leu Gly Ser 115 120 125 aaa aca gga cct ggg cag aaa gct ata
ctt ttt ctt cca atg tct gct 432 Lys Thr Gly Pro Gly Gln Lys Ala Ile
Leu Phe Leu Pro Met Ser Ala 130 135 140 aag agc tga 441 Lys Ser *
145 4 146 PRT Homo sapiens 4 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 5 468 DNA Bos
taurus CDS (1)...(468) 5 atg gca gcc ggg agc atc acc acg ctg cca
gcc cta cca gaa gat ggg 48 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro
Ala Leu Pro Glu Asp Gly 1 5 10 15 ggg tcc ggg gcc ttc cca cca ggg
cac ttc aaa gat cca aaa cga cta 96 Gly Ser Gly Ala Phe Pro Pro Gly
His Phe Lys Asp Pro Lys Arg Leu 20 25 30 tat tgt aaa aac ggg ggg
ttc ttc cta cga atc cac cca gat ggg cga 144 Tyr Cys Lys Asn Gly Gly
Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 gta gat ggg gta
cga gaa aaa tcc gat cca cac atc aaa cta caa cta 192 Val Asp Gly Val
Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 caa gcc
gaa gaa cga ggg gta gta tcc atc aaa ggg gta tgt gcc aac 240 Gln Ala
Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80
cga tat cta gcc atg aaa gaa gat ggg cga cta cta gcc tcc aaa tgt 288
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85
90 95 gta acc gat gaa tgt ttc ttc ttc gaa cga cta gaa tcc aac aac
tat 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn
Tyr 100 105 110 aac acc tat cga tcc cga aaa tat tcc tcc tgg tat gta
gcc cta aaa 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Ser Ser Trp Tyr Val
Ala Leu Lys 115 120 125 cga acc ggg caa tat aaa cta ggg cca aaa acc
ggg cca ggg caa aaa 432 Arg Thr Gly Gln Tyr Lys Leu Gly Pro Lys Thr
Gly Pro Gly Gln Lys 130 135 140 gcc atc cta ttc cta cca atg tcc gcc
aaa tcc taa 468 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser * 145
150 155 6 155 PRT Bos taurus 6 Met Ala Ala Gly Ser Ile Thr Thr Leu
Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro
Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly
Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly
Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln
Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70
75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys
Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser
Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Ser Ser Trp
Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Pro
Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met
Ser Ala Lys Ser 145 150 155 7 474 DNA Homo sapiens CDS (1)...(468)
7 atg gca gcc ggg agc atc acc acg ctg ccc gcc ttg ccc gag gat ggc
48 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
1 5 10 15 ggc agc ggc gcc ttc ccg ccc ggc cac ttc aag gac ccc aag
cgg ctg 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys
Arg Leu 20 25 30 tac tgc aaa aac ggg ggc ttc ttc ctg cgc atc cac
ccc gac ggc cga 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His
Pro Asp Gly Arg 35 40 45 gtt gac ggg gtc cgg gag aag agc gac cct
cac atc aag cta caa ctt 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro
His Ile Lys Leu Gln Leu 50 55 60 caa gca gaa gag aga gga gtt gtg
tct atc aaa gga gtg tgt gct aac 240 Gln Ala Glu Glu Arg Gly Val Val
Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 cgt tac ctg gct atg aag
gaa gat gga aga tta ctg gct tct aaa tgt 288 Arg Tyr Leu Ala Met Lys
Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 gtt acg gat gag
tgt ttc ttt ttt gaa cga ttg gaa tct aat aac tac 336 Val Thr Asp Glu
Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 aat act
tac cgg tca agg aaa tac acc agt tgg tat gtg gca ctg aaa 384 Asn Thr
Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
cga act ggg cag tat aaa ctt gga tcc aaa aca gga cct ggg cag aaa 432
Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130
135 140 gct ata ctt ttt ctt cca atg tct gct aag agc tga ttttaa 474
Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser * 145 150 155 8 155 PRT
Homo sapiens 8 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro
Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys
Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu
Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys
Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu Glu Arg
Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg Tyr Leu
Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 Val
Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105
110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys
115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly
Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145
150 155 9 9 PRT Bos taurus 9 Met Ala Ala Gly Ser Ile Thr Thr Leu 1
5
* * * * *
References