U.S. patent application number 12/387507 was filed with the patent office on 2009-11-19 for compositions and methods for treatment of neovascular diseases.
This patent application is currently assigned to The Scripps Research Institute. Invention is credited to Hilda Edith Aguilar, Michael I. Dorrell, Martin Friedlander.
Application Number | 20090285792 12/387507 |
Document ID | / |
Family ID | 35463352 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285792 |
Kind Code |
A1 |
Friedlander; Martin ; et
al. |
November 19, 2009 |
Compositions and methods for treatment of neovascular diseases
Abstract
The present invention provides compositions and methods of
treating neovascular diseases, such as a retinal neovascular
diseases and tumors, by administering to a patient suffering from a
neovascular disease or tumor a vascular development inhibiting
amount of a combination of the angiogenesis suppressing drugs
comprising an angiostatic fragment of tryptophanyl-tRNA synthetase
(TrpRS) and at least one compound selected from the group
consisting of a vascular endothelial growth factor (VEGF) signaling
inhibitor and an integrin signaling inhibitor. Compositions for use
in the methods include an admixture of an angiostatic fragment of
tryptophanyl-tRNA synthetase (TrpRS) and at least one of a vascular
endothelial growth factor (VEGF) signaling inhibitor and an
integrin signaling inhibitor, together with a pharmaceutically
acceptable excipient.
Inventors: |
Friedlander; Martin; (Del
Mar, CA) ; Aguilar; Hilda Edith; (San Diego, CA)
; Dorrell; Michael I.; (San Diego, CA) |
Correspondence
Address: |
Olson & Cepuritis, LTD.
20 NORTH WACKER DRIVE, 36TH FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
The Scripps Research
Institute
|
Family ID: |
35463352 |
Appl. No.: |
12/387507 |
Filed: |
May 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11145587 |
Jun 6, 2005 |
7528106 |
|
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12387507 |
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60577156 |
Jun 4, 2004 |
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60585273 |
Jul 1, 2004 |
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60655801 |
Feb 24, 2005 |
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Current U.S.
Class: |
424/94.5 |
Current CPC
Class: |
C12N 9/93 20130101; A61P
43/00 20180101; A61K 31/538 20130101; A61P 9/00 20180101; A61P 9/10
20180101; C12Y 601/01002 20130101; A61K 9/0048 20130101; A61K 38/00
20130101; A61K 45/06 20130101; A61P 27/02 20180101; A61K 31/7072
20130101; A61P 35/00 20180101; A61K 31/538 20130101; A61K 2300/00
20130101; A61K 31/7072 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/94.5 |
International
Class: |
A61K 38/53 20060101
A61K038/53; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0001] A portion of the work described herein was supported by
grant number EY 11254 from the National Institutes of Health. The
United States Government has certain rights in this invention.
SP
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application division of U.S. application Ser. No.
11/145,587, filed on Jun. 6, 2005, now U.S. Pat. No. 7,528,106,
which claims the benefit of U.S. Provisional Application for Patent
Serial No. 60/577,156 filed on Jun. 4, 2004, and claims the benefit
of U.S. Provisional Application for Patent Serial No. 60/585,273
filed on Jul. 1, 2004, and claims the benefit of U.S. Provisional
Application for Patent Serial No. 60/655,801 filed on Feb. 24,
2005, each of which is incorporated herein by reference.
Claims
1. A composition comprising an angiostatic fragment of
tryptophanyl-tRNA synthetase (TrpRS) and at least one
anti-angiogenic agent selected from the group consisting of a VEGF
aptamer and an angiostatic integrin antagonist.
2. The composition of claim 1 wherein the angiostatic fragment of
TrpRS has the amino acid residue sequence of SEQ ID NO: 1 or SEQ ID
NO: 2.
3. The composition of claim 2 wherein the at least one
anti-angiogenic agent comprises a VEGF- 165 aptamer.
4. The composition of claim 3 wherein the VEGF-165 aptamer is a
2'-fluoropyrimidine RNA-based VEGF-165 aptamer.
5. The composition of claim 4 wherein the 2'-fluoropyrimidine
RNA-based VEGF-165 aptamer is pegaptanib sodium.
6. The composition of claim 1 wherein the at least one
anti-angiogenic agent comprises an angiostatic antagonist of
.alpha..sub.v.beta..sub.3 integrin or .alpha..sub.v.beta..sub.5
integrin.
7. The composition of claim 6 wherein the angiostatic antagonist of
.alpha..sub.v.beta..sub.3 integrin or .alpha..sub.v.beta..sub.5
integrin has the formula of Compound (1): ##STR00002##
8. The composition of claim 1 wherein the at least one
anti-angiogenic agent comprises pegaptanib sodium and a compound
having the formula of Compound (1): ##STR00003##
9. The composition of claim 1 further comprising at least one
therapeutic agent selected from the group consisting of an
angiostatic steroid, an anti-neoplastic agent, an anti-bacterial
agent, an anti-viral agent, and an anti-inflammatory agent.
10. The composition of claim 13 wherein the angiostatic fragment of
TrpRS is a dimer.
11. A pharmaceutical composition comprising a composition of claim
1 and a pharmaceutically acceptable carrier therefor.
Description
FIELD OF THE INVENTION
[0003] This invention relates to treatment of neovascular diseases,
such as retinal neovascular diseases. More particularly this
invention relates to methods of treating neovascular disease by
administering a combination of angiostatic and antiangiogenic drugs
to a patient, and to compositions for use in said methods.
BACKGROUND OF THE INVENTION
[0004] The vast majority of diseases that cause catastrophic loss
of vision do so as a result of ocular neovascularization. For
example, age related macular degeneration (ARMD) affects 12-15
million American over the age of 65 and causes visual loss in
10-15% of them as a direct effect of choroidal (sub-retinal)
neovascularization. The leading cause of visual loss for Americans
under the age of 65 is diabetes; 16 million individuals in the
United States are diabetic and 40,000 per year suffer from ocular
complications of the disease, often a result of retinal
neovascularization. While laser photocoagulation has been effective
in preventing severe visual loss in subgroups of high risk diabetic
patients, the overall 10-year incidence of retinopathy remains
substantially unchanged. For patients with choroidal
neovascularization due to ARMD or inflammatory eye disease such as
ocular histoplasmosis, photocoagulation, with few exceptions, is
ineffective in preventing visual loss. While recently developed,
non-destructive photodynamic therapies hold promise for temporarily
reducing individual loss in patients with previously untreatable
choroidal neovascularization, only 61.4% of patients treated every
34 months had improved or stabilized vision compared to 45.9% of
the placebo-treated group.
[0005] ARMD and diabetic retinopathy are the leading causes of
visual loss in industrialized nations and do so as a result of
abnormal retinal neovascularization. Since the retina consists of
well-defined layers of neuronal, glial, and vascular elements,
relatively small disturbances such as those seen in vascular
proliferation or edema can lead to significant loss of visual
function. Inherited retinal degenerations, such as retinitis
pigmentosa (RP), are also associated with vascular abnormalities,
such as arteriolar narrowing and vascular atrophy. While
significant progress has been made in identifying factors that
promote and inhibit angiogenesis, no treatment is currently
available to specifically treat ocular vascular disease.
[0006] Inherited degenerations of the retina affect as many as 1 in
3500 individuals and are characterized by progressive night
blindness, visual field loss, optic nerve atrophy, arteriolar
attenuation, altered vascular permeability and central loss of
vision often progressing to complete blindness (Heckenlively, J.
R., editor, 1988; Retinitis Pigmentosa, Philadelphia: JB Lippincott
Co.). Molecular genetic analysis of these diseases has identified
mutations in over 110 different genes accounting for only a
relatively small percentage of the known affected individuals
(Humphries et al., 1992, Science 256:804-808; Farrar et al. 2002,
EMBO J. 21:857-864.). Many of these mutations are associated with
enzymatic and structural components of the phototransduction
machinery including rhodopsin, cGMP phosphodiesterase, rds
peripherin, and RPE65. Despite these observations, there are still
no effective treatments to slow or reverse the progression of these
retinal degenerative diseases. Recent advances in gene therapy have
led to successful reversal of the rds (Ali et al. 2000, Nat. Genet.
25:306-310) and rd (Takahashi et al. 1999, J. Virol. 73:7812-7816)
phenotypes in mice and the RPE65 phenotype in dogs (Acland et al.
2001, Nat. Genet. 28:92-95) when the wild type transgene is
delivered to photoreceptors or the retinal pigmented epithelium
(RPE) in animals with a specific mutation.
[0007] Angiogenesis is the process by which new blood vessels form.
In response to specific chemical signals, capillaries sprout from
existing vessels, eventually growing in size as needed by the
organism. Initially, endothelial cells, which line the blood
vessels, divide in a direction orthogonal to the existing vessel,
forming a solid sprout. Adjacent endothelial cells then form large
vacuoles and the cells rearrange so that the vacuoles orient
themselves end to end and eventually merge to form the lumen of a
new capillary (tube formation).
[0008] Angiogenesis is stimulated by a number of conditions, such
as in response to a wound, and accompanies virtually all tissue
growth in vertebrate organisms such as mammals. Angiogenesis also
plays a role in certain disease states such as certain cancers. The
growth of tumors, for example, requires blood vessel growth to
provide oxygen and nutrients to the growing tumor tissue. In
addition, ocular neovascularization is associated with the vast
majority of eye diseases that lead to catastrophic loss of
vision.
[0009] Angiogenesis may be arrested or inhibited by interfering
with the chemical signals that stimulate the angiogenic process.
For example, angiogenic endothelial cells produce proteases to
digest the basal lamina that surround the blood vessels, thus
clearing a path for the new capillary. Inhibition of these
proteases, or their formation, can prevent new vessels from
forming. Likewise, the endothelial cells proliferate in response to
chemical signals. Particularly important proliferation signals
include the vascular endothelial growth factor (VEGF), and the
fibroblast growth factor (FGF) families of proteins. VEGF has been
shown to be involved in vascularization of certain tumors.
Interference with these proliferation signaling processes can also
inhibit angiogenesis.
[0010] Several factors are involved in angiogenesis. Both acidic
and basic fibroblast growth factor molecules are mitogens for
endothelial cells and other cell types. A highly selective mitogen
for vascular endothelial cells is VEGF.
[0011] In the normal adult, angiogenesis is tightly regulated, and
is limited to wound healing, pregnancy and uterine cycling.
Angiogenesis is turned on by specific angiogenic molecules such as
basic and acidic fibroblast growth factor (FGF), VEGF, angiogenin,
transforming growth factor (TGF), tumor necrosis factor-.alpha.
(TNF-.alpha.) and platelet derived growth factor (PDGF).
Angiogenesis can be suppressed by inhibitory molecules such as
interferon-.alpha., thrombospondin-1, angiostatin and endostatin.
It is the balance of these naturally occurring stimulators and
inhibitors that controls the normally quiescent capillary
vasculature. When this balance is upset, as in certain disease
states, capillary endothelial cells are induced to proliferate,
migrate and ultimately differentiate.
[0012] Angiogenesis plays a central role in a variety of disease
including cancer and ocular neovascularization. Sustained growth
and metastasis of a variety of tumors has also been shown to be
dependent on the growth of new host blood vessels into the tumor in
response to tumor derived angiogenic factors. Proliferation of new
blood vessels in response to a variety of stimuli occurs as the
dominant finding in the majority of eye disease and that blind
including proliferative diabetic retinopathy, ARMD, rubeotic
glaucoma, interstitial keratitis and retinopathy of prematurity. In
these diseases, tissue damage can stimulate release of angiogenic
factors resulting in capillary proliferation. VEGF plays a dominant
role in iris neovascularization and neovascular retinopathies.
While reports clearly show a correlation between intraocular VEGF
levels and ischemic retinopathic ocular neovascularization, FGF
likely plays a role as well. Basic and acidic FGF are known to be
present in the normal adult retina, even though detectable levels
are not consistently correlated with neovascularization. This may
be largely due to the fact that FGF binds very tightly to charged
components of the extracellular matrix and may not be readily
available in a freely diffusible form that would be detected by
standard assays of intraocular fluids.
[0013] A final common pathway in the angiogenic response involves
integrin-mediated information exchange between a proliferating
vascular endothelial cell and the extracellular matrix. This class
of adhesion receptors, called integrins, are expressed as
heterodimers having an .alpha. and .beta. subunit on all cells. One
such integrin, .alpha..sub.v.beta..sub.3 is the most promiscuous
member of this family and allows endothelial cells to interact with
a wide variety of extracellular matrix components. Peptide and
antibody antagonists of this integrin inhibit angiogenesis by
selectively inducing apoptosis of the proliferating vascular
endothelial cells. Two cytokine-dependent pathways of angiogenesis
exist and may be defined by their dependency on distinct vascular
cell integrins, .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5. Specifically, basic FGF- and
VEGF-induced angiogenesis depend on integrin
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5,
respectively, since antibody antagonists of each integrin
selectively block one of these angiogenic pathways in the rabbit
corneal and chick chorioallantoic membrane (CAM) models. Peptide
antagonists that block all .alpha..sub.v integrins inhibit FGF- and
VEGF-stimulated angiogenesis. While normal human ocular blood
vessels do not display either integrin, .alpha..sub.v.beta..sub.3
and .alpha..sub.v.beta..sub.5 integrins are selectively displayed
on blood vessels in tissues from patients with active neovascular
eye disease. While only .alpha..sub.v.beta..sub.3 was consistently
observed in tissue from patients with ARMD,
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 both were
present in tissues from patients with proliferative diabetic
retinopathy. Systemically administered peptide antagonists of
integrins blocked new blood vessel formation in a mouse model of
retinal vasculogenesis.
[0014] Hence, anti-angiogenic agents have a role in treating
retinal degeneration to prevent the damaging effects of these
trophic and growth factors. Angiogenic agents, also have role in
promoting desirable vascularization to retard retinal degeneration
by enhancing blood flow to cells.
[0015] Immense research efforts have contributed to our
understanding of the mechanisms of angiogenesis during disease
progression, and as a result of these studies, a large number of
angiostatic molecules have been, or are currently being, tested in
clinical trials. However, to date, the results from these clinical
trials have been disappointing, and the benefits from these
antiangiogenic treatments in patients have been minimal at
best.
[0016] Many factors may require consideration before angiostatic
therapies ultimately become successful. Naturally occurring
compensatory mechanisms may ultimately render angiogenic
monotherapies obsolete. Angiostatic drugs generally target a single
cytokine or intracellular angiogenic pathway. In vivo, angiogenesis
is likely to be initiated by the combined signaling of multiple
pathways. Thus, blocking a single pathway may be insufficient to
prevent angiogenesis during the treatment of neovascular diseases.
Further complicating matters, it is also likely that blocking a
single pathway induces compensation and increased roles of other
angiogenic pathways.
[0017] It has now been discovered that a concurrent administration
of a combination of angiostatic compounds that target different
pathways enhances angiostatic potency and also interferes with
natural compensatory mechanisms.
SUMMARY OF THE INVENTION
[0018] The present invention provides compositions and a method of
treating a neovascular disease, such as a retinal neovascular
disease, by administering to a mammal suffering from a neovascular
disease an amount of a combination of angiogenesis suppressing
drugs sufficient to inhibit new blood vessel formation. These drugs
can be a combination of an angiostatic fragment of
tryptophanyl-tRNA synthetase (TrpRS) and a therapeutic agent.
Preferably, the therapeutic agent is a VEGF signaling inhibitor, an
integrin signaling inhibitor, or a combination thereof.
Additionally, the therapeutic agent can comprise an angiostatic
steroid, an anti-neoplastic agent, an anti-bacterial agent, an
anti-viral agent, and an anti-inflammatory agent, and the like.
Preferably the mammal is a human.
[0019] A particularly preferred method embodiment comprises
administering to a mammal suffering from a neovascular disease a
vascular development inhibiting amount of an admixture of drugs
comprising an angiostatic fragment of TrpRS (e.g., the T1 fragment,
the T2 fragment, or the mini TrpRS fragment described herein) and
at least one compound selected from a VEGF signaling inhibitor and
an integrin signaling inhibitor. Another preferred embodiment for
this purpose is the triple combination of T2-TrpRS angiostatic
fragment, aVEGF signaling inhibitor such as a VEGF aptamer, and an
integrin signaling inhibitor such as an .alpha..sub.v.beta..sub.3
and .alpha..sub.v.beta..sub.5 integrin signaling inhibitor. A
particularly preferred triple combination comprises the T2 fragment
of human TrpRS, a VEGF aptamer specific for VEGF-165 (e.g.,
pegaptanib sodium), and a peptidomimetic .alpha..sub.v.beta..sub.3
and .alpha..sub.v.beta..sub.5 integrin signaling inhibitor (e.g.,
Compound (1) described herein). This particular triple combination
exhibits a strong synergistic effect on the inhibition of
neovascularization in the mammalian eye.
[0020] Neovascular diseases treatable by the methods of the present
invention include, without limitation, ocular diseases such as
retinal degenerative diseases, retinal vascular degenerative
diseases, ischemic retinopathies, vascular hemorrhages, vascular
leakage, and choroidopathies in neonatal, juvenile, or fully mature
mammals. The methods of the present invention can also be utilized
to treat neovascular diseases such as solid tumor cancers (e.g.,
lung cancer, breast cancer, and prostate cancer) and rheumatoid
arthritis, for example.
[0021] A therapeutic composition useful for inhibition of
angiogenesis, and thus for the treatment of neovascular diseases,
comprises an admixture of an angiostatic fragment of
tryptophanyl-tRNA synthetase (TrpRS), a VEGF signaling inhibitor,
and an integrin signaling inhibitor, together with a
pharmaceutically acceptable excipient and a carrier therefor.
Optionally the present therapeutic compositions can also include
one or more of an angiostatic steroid, an anti-neoplastic agent, an
anti-bacterial agent, an anti-viral agent, an anti-inflammatory
agent, and the like therapeutic agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts the amino acid sequences of an angiostatic
fragments of tryptophanyl-tRNA synthetase designated as T2-TrpRS,
SEQ ID NO: 1 and T2-TrpRS-GD, SEQ ID NO: 2 (a mutant thereof).
[0023] FIG. 2 depicts the amino acid sequence of an angiostatic
fragments of tryptophanyl-tRNA synthetase designated as mini-TrpRS,
SEQ ID NO: 3 and T1-TrpRS, SEQ ID NO: 4.
[0024] FIG. 3 depicts the amino acid sequence of full length TrpRS
(SEQ ID NO: 5) and indicates the position of the T1, T2 and Mini
fragments thereof.
[0025] FIG. 4 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 1
intravitreally injected with PBS.
[0026] FIG. 5 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 1 intravitreally
injected with (A) 0.5.times.concentration (10 mg/ml) of
peptidomimetic integrin signaling inhibitor Compound (1); (B) a
1.times.concentration (2 mg/ml) of VEGF aptamer Compound (2); and
(C) a combination of integrin signaling inhibitor Compound (1) and
VEGF aptamer Compound (2).
[0027] FIG. 6 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 2
intravitreally injected with phosphate buffered saline (PBS).
[0028] FIG. 7 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 2 intravitreally
injected with 0.1.times.concentration (0.05 mg/ml) of T2-TrpRS.
[0029] FIG. 8 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 2 intravitreally
injected with 0.1.times.concentration of VEGF aptamer Compound
(2).
[0030] FIG. 9 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 2 intravitreally
injected with a combination of 0.1.times.concentration of T2-TrpRS
and 0.1.times.concentration of VEGF aptamer Compound (2).
[0031] FIG. 10 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 3
intravitreally injected with PBS.
[0032] FIG. 11 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 3 intravitreally
injected with a 1.times.concentration of T2-TrpRS.
[0033] FIG. 12 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 3 intravitreally
injected with a 1.times.concentration of VEGF aptamer Compound
(2).
[0034] FIG. 13 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 3 intravitreally
injected with a 1.times.concentration of T2-TrpRS and a
1.times.concentration of VEGF aptamer Compound (2).
[0035] FIG. 14 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 4
intravitreally injected with phosphate buffered saline (PBS).
[0036] FIG. 15 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 4 intravitreally
injected with a 1.times.concentration of T2-TrpRS.
[0037] FIG. 16 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 4 intravitreally
injected with a 0.5.times.concentration of VEGF aptamer Compound
(2).
[0038] FIG. 17 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 4 intravitreally
injected with a combination of a 1.times.concentration of T2-TrpRS
and 0.5.times.concentration of VEGF aptamer Compound (2).
[0039] FIG. 18 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 5
intravitreally injected with PBS.
[0040] FIG. 19 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 5 intravitreally
injected with a 1.times.concentration of T2-TrpRS.
[0041] FIG. 20 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 5 intravitreally
injected with a 0.5.times.concentration of peptidomimetic integrin
signaling inhibitor Compound (1).
[0042] FIG. 21 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 5 intravitreally
injected with a combination of a 1.times.concentration of T2-TrpRS
and a 0.5.times.concentration of peptidomimetic integrin signaling
inhibitor Compound (1).
[0043] FIG. 22 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 6
intravitreally injected with PBS.
[0044] FIG. 23 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 6 intravitreally
injected with a 1.times.concentration of T2-TrpRS.
[0045] FIG. 24 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 6 intravitreally
injected with a 1.times.concentration of VEGF aptamer Compound
(2).
[0046] FIG. 25 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 6 intravitreally
injected with a 0.5.times.concentration of peptidomimetic integrin
signaling inhibitor Compound (1).
[0047] FIG. 26 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 6 intravitreally
injected with a combination of a 1.times.concentration of T2-TrpRS
and a 1.times.concentration of VEGF aptamer Compound (2).
[0048] FIG. 27 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 6 intravitreally
injected with a combination of a 1.times.concentration of T2-TrpRS
and a 0.5.times.concentration of peptidomimetic integrin signaling
inhibitor Compound (1).
[0049] FIG. 28 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 6 intravitreally
injected with a combination of a 1.times.concentration of T2-TrpRS,
a 0.5.times.concentration of peptidomimetic integrin signaling
inhibitor Compound (1), and a normal concentration of VEGF aptamer
Compound (2).
[0050] FIG. 29 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 7
intravitreally injected with PBS.
[0051] FIG. 30 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 7 intravitreally
injected with a 1.times.concentration of T2-TrpRS.
[0052] FIG. 31 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 7 intravitreally
injected with a 1.times.concentration of VEGF aptamer Compound
(2).
[0053] FIG. 32 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 7 intravitreally
injected with a 0.5.times.concentration of peptidomimetic integrin
signaling inhibitor Compound (1).
[0054] FIG. 33 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 7 intravitreally
injected with a combination of a 0.5.times.concentration of
peptidomimetic integrin signaling inhibitor Compound (1) and a
1.times.concentration of VEGF aptamer Compound (2).
[0055] FIG. 34 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 7 intravitreally
injected with a combination of a 1.times.concentration of T2-TrpRS,
a 0.5.times.concentration of peptidomimetic integrin signaling
inhibitor Compound (1), and a 1.times.concentration of VEGF aptamer
Compound (2).
[0056] FIG. 35 depicts photomicrographs of primary and secondary
vascular layers of retinas of control mice from Example 9
intravitreally injected with PBS.
[0057] FIG. 36 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 9 intravitreally
injected with a 1.times.concentration of peptidomimetic integrin
signaling inhibitor Compound (1).
[0058] FIG. 37 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 9 intravitreally
injected with a 1.times.concentration of VEGF aptamer Compound
(2).
[0059] FIG. 38 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 9 intravitreally
injected with a 1.times.concentration of T2-TrpRS.
[0060] FIG. 39 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 9 intravitreally
injected with a 1.times.concentration of each of T2-TrpRS and
peptidomimetic integrin signaling inhibitor Compound (1).
[0061] FIG. 40 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 9 intravitreally
injected with a 1.times.concentration of each of peptidomimetic
integrin signaling inhibitor Compound (1) and VEGF aptamer Compound
(2).
[0062] FIG. 41 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 9 intravitreally
injected with a combination of a 1.times.concentration of each of
T2-TrpRS and VEGF aptamer Compound (2).
[0063] FIG. 42 depicts photomicrographs of primary and secondary
vascular layers of retinas of mice from Example 9 intravitreally
injected with a combination of a 1.times.concentration of each of
the inhibitors T2-TrpRS, peptidomimetic integrin signaling
inhibitor Compound (1), and VEGF aptamer Compound (2).
[0064] FIG. 43 is a graphical representation of data from a
T2-TrpRS dosing experiment;
[0065] FIG. 44 is a graphical representation of data from a
VEGF-aptamer dosing experiment;
[0066] FIG. 45 is a graphical representation of data showing
inhibition of deep vascular plexus formation as a function of
administered optimal concentration of VEGF aptamer and T2-TrpRS
compound, alone and in combination;
[0067] FIG. 46 is a graphical representation of data from
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrin
antagonist dosing experiment.
[0068] FIG. 47 is a graphical representation of data showing
inhibition of deep vascular plexus formation as a function of
administered optimal concentration of a small molecule
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrin
antagonist and T2-TrpRS, alone and in combination;
[0069] FIG. 48 is a graphical summary of data showing inhibition of
deep vascular plexus formation at various combinations of
therapies;
[0070] FIG. 49 is a graphical representation of data showing degree
of inhibition of deep vascular plexus formation at various
combinations of therapies;
[0071] FIG. 50 is a series of photomicrographs of primary and deep
(secondary) vascular layers at various therapies and combinations
thereof at dosing levels shown in FIG. 49;
[0072] FIG. 51 is a graphical representation of levels of
inhibition of vascular plexus formation with triple combination
therapy at various dosage levels;
[0073] FIG. 52 is a graphical representation similar to FIG. 51 but
showing inhibition of >75%, >90% and 100%;
[0074] FIG. 53 is a graphical representation of levels of
inhibition of vascular plexus formation comparing monotherapies
versus combination therapies at various dosage levels;
[0075] FIG. 54 is a graphical representation of >75%, >90%
and 100% levels of inhibition of vascular plexus formation
comparing monotherapies versus combination therapies at various
dosage levels;
[0076] FIG. 55 is a graphical representation of data showing an
area of neovascular tufts as a function of various monotherapies as
well as a triple combination therapy utilizing a single injection
of a therapeutic agent or agents;
[0077] FIG. 56 is similar to FIG. 55, but shows data from a dual
injection of a therapeutic agent or agents;
[0078] FIG. 57 is a graphical representation of data showing areas
of neovascular tufts as a function of various monotherapies, dual
therapies and a triple therapy;
[0079] FIG. 58 is a series of photomicrographs showing mouse
retinas treated with angiostatic compounds singly and in
combination;
[0080] FIG. 59 shows the structure of VEGF aptamer Compound (2)
(SEQ ID NO: 6), pegaptanib sodium; and
[0081] FIG. 60 shows a graph of survival for tumor bearing rats
treated with a composition of the invention (squares) versus
control rats treated only with PBS (triangles).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0082] A composition suitable for treating a neovascular disease
comprises an angiostatic fragment of tryptophanyl-tRNA synthetase
(TrpRS) and a therapeutic agent. Preferably the therapeutic agent
comprises at least one anti-angiogenic agent selected from the
group consisting of a VEGF signaling inhibitor (E.g, a VEGF
aptamer) and an integrin signaling inhibitor (e.g., angiostatic
integrin antagonist).
[0083] Preferred angiostatic fragments of TrpRS include a 43 kDa
fragment (e.g., the T2 fragment, "T2-TrpRS", SEQ ID NO: 1; a mutant
of T2-TrpRS, "T2-TrpRS-GD", SEQ ID NO: 2; both of which are shown
in FIG. 1), a 48 kDa fragment such as the truncated TrpRS known as
mini-TrpRS (SEQ ID NO: 3, shown in FIG. 2), and a 46 kDa fragment
such as the truncated TrpRS known as T1-TrpRS (SEQ ID NO: 4, shown
in FIG. 2). The amino acid residue sequence of T2-TrpRS-GD (SEQ ID
NO: 2), differs from SEQ ID NO: 1 by two amino acid residue
substitutions (i.e., S121G and Y122D). The amino acid residue
sequence of full length human TrpRS (SEQ ID NO: 5) is shown in FIG.
3, along with an indication of the position of the T1, T2 and mini
fragments thereof. Without being bound by theory, it is believed
that the angistatic fragments of TrpRS can form non-covalent dimers
(see e.g., Yu et al. J. Biol. Chem. 2004, 279: 8378-8388), which
may contribute to the biological activity of the fragments.
Accordingly, any reference herein an in the appended claims to an
angiostatic fragment of TrpRS (e.g., T1-TRpRS, T2-TrpRS,
mini-TrpRS) is to be construed as a reference to the monomer form,
the dimer form, or a mixture thereof.
[0084] Preferred integrin signaling inhibitors are
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5
antagonists, including RGD peptides, such as those described in
U.S. Pat. No. 5,693,612, U.S. Pat. No. 5,766,591, U.S. Pat. No.
5,767,071, U.S. Pat. No. 5,780,426, and U.S. Pat. No. 6,610,826,
the relevant disclosures of which are incorporated herein by
reference, and peptidomimetic integrin antagonists such as those
described in U.S. Pat. No. 5,614,531, U.S. Pat. No. 5,614,535, U.S.
Pat. No. 6,326,403, U.S. Pat. No. 6,455,529, U.S. Pat. No.
6,521,646, U.S. Pat. No. 6,559,144, U.S. Pat. No. 6576,637, U.S.
Pat. No. 6,602,876, U.S. Pat. No. 6,645,991, and U.S. Pat. No.
6,649,613, the relevant disclosures of which are incorporated
herein by reference. A particularly preferred peptidomimetic
integrin signaling inhibitor is a compound having the formula of
Compound (1), available from Merck KGaA (Darmstadt, Germany) as EMD
472523.
##STR00001##
[0085] Preferred VEGF signaling inhibitors include VEGF-selective
aptamers (protein binding oligonucleotides), preferably nuclease
resistant aptamers that bind to VEGF- 165, such as the
2'-fluoropyrimidine RNA-based aptamers that bind to VEGF-165
described by Ruckman et al. J. Biol. Chem. 1998, 273: 20556-20567
(the relevant disclosure of which is incorporated herein by
reference), and the like; anti-VEGF antibodies and fragments
thereof that bind to VEGF, such as the Rhu antibody available from
Genentech (San Francisco, Calif.) and an Fab fragment thereof
(RhuFab V2); soluble VEGF receptors such as soluble VEGFR1; and
small interfering RNAs (siRNA) that target VEGF or its receptors,
such as the siRNA described by Reich et al. Mol. Vis. 2003;
9:210-216 (the relevant disclosure of which is incorporated herein
by reference). Preferred VEGF signaling inhibitors are nuclease
resistant VEGF aptamers, more preferably 2'-fluoropyrimidine
RNA-based aptamers specific for VEGF-165, such as pegaptanib sodium
(Compound (2)), which is a polyethoxylated oligonucleotide having
the following formula (SEQ ID NO: 6; FIG. 59, R in FIG. 59 is a 40
kiloDalton polyethylene glycol (PEG) chain):
TABLE-US-00001 5'-40K PEG-C5
aminolinker-CfGmGmArArUfCfAmGmUfGmAmAmUf
GmCfUfUfAmUfAmCfAmUfCfCfGm3'-3'dT wherein Cf = 2'fluoro C Ar = 2'
OH (ribo) A Uf = 2'fluoro U 3'-3'dT = inverted deoxyT Am = 2'OMe A
C5 aminolinker = pentyl amino linker Gm = 2'OMe G 40K PEG = 40K
polyethelene glycol amide.
[0086] A polyethoxylated oligonucleotide of SEQ ID NO: 6 is sold
commercially under the trademark MACUGEN.RTM. by from Eyetech
Pharmaceuticals, Inc., and is also known as NX1838 or pegaptanib
sodium.
[0087] In one embodiment the combination of drugs also includes at
least one additional therapeutic agent such as an angiostatic
steroid, an anti-neoplastic agent, an anti-bacterial agent, an
anti-viral agent, an anti-inflammatory agent, and the like.
[0088] Examples of suitable angiostatic steroids include anecortave
acetate and triamcinolone acetonide.
[0089] Examples of suitable anti-neoplastic agents include
Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin;
Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;
Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;
Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin;
Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride;
Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar
Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;
Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin
Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin ;
Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide;
Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride;
Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate;
Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;
Droloxifene; Droloxifene Citrate; Dromostanolone Propionate;
Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin;
Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride;
Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine
Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide;
Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;
Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;
Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine;
Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin
Hydrochloride; Ifosfamide; Imofosine; Interferon Alfa-2a;
Interferon Alfa-2b ; Interferon Alfa-n1; Interferon Alfa-n3;
Interferon Beta-Ia; Interferon Gamma-Ib; Iproplatin; Irinotecan
Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate
Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone
Hydrochloride; Masoprocol; Maytansine; Mechlorethamine
Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;
Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;
Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;
Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone
Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;
Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;
Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman;
Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine
Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin;
Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;
Semustine; Simtrazene; Sparfosate Sodium; Sparsomycinl,
Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;
Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;
Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur;
Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone;
Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;
Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate;
Trestolone Acetate; Triciribine Phosphate; Trimetrexate;
Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride;
Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine
Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate;
Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate;
Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate;
Vorozole; Zeniplatin; and Zinostatin; Zorubicin Hydrochloride.
[0090] Examples of suitable anti-bacterial agents include, but are
not limited to, penicillins, aminoglycosides, macrolides,
monobactams, rifamycins, tetracyclines, chloramphenicol,
clindamycin, lincomycin, imipenem, fusidic acid, novobiocin,
fosfomycin, fusidate sodium, neomycin, polymyxin, capreomycin,
colistimethate, colistin, gramicidin, minocycline, doxycycline,
vanomycin, bacitracin, kanamycin, gentamycin, erythromicin and
cephalosporins.
[0091] Examples of suitable anti-inflammatory agents include, but
are not limited to, aspirin (acetylsalicylic acid), indomethacin,
sodium indomethacin trihydrate, salicylamide, naproxen, colchicine,
fenoprofen, sulindac, diflunisal, diclofenac, indoprofen and sodium
salicylamide.
[0092] Examples of suitable anti-viral agents include, but are not
limited to, alpha-methyl-P-adamantane methylamine,
1-D-ribofuranosyl-1,2,4-triazole-3 carboxamide,
9-(2-hydroxy-ethoxy)methylguanine, adamantanamine,
5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine
arabinoside, CD4, 3'-azido-3'-deoxythymidine (AZT),
9-(2-hydroxyethoxymethyl)-guanine (acyclovir), phosphonoformic
acid, 1-adamantanamine, peptide T, and 2',3'dideoxycytidine.
[0093] A particularly preferred method of treating neovascular
diseases comprises administering to a mammal suffering from a
neovascular disease a vascular development inhibiting amount of a
combination of drugs comprising T2-TrpRS, at least one VEGF-165
signaling inhibitor, and optionally, at least one
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrin
signaling inhibitor.
[0094] Neovascular diseases treatable by the methods of the present
invention include, without limitation, neovascular diseases of the
eye (e.g., retinal and choroidal neovascular diseases), rubeotic
glaucoma, pterygia, solid tumor cancers (e.g., lung cancer, breast
cancer, and prostate cancer), osteoarthritis, rheumatoid arthritis,
vascular anomalies and malformations (e.g., hemangiomas,
lymphangiomas, and the like), and psoriasis.
[0095] The present method of treating retinal neovascular diseases
in a mammal preferably comprises intravitreally injecting into the
eye of a mammal suffering from a neovascular disease a vascular
development inhibiting amount of a combination of antiangiogenic
and angiostatic compositions that provide an angiostatic fragment
of TrpRS, a VEGF signaling inhibitor, and an integrin signaling
inhibitor.
[0096] This method can be utilized to treat ocular diseases such as
vascular degenerative diseases, ischemic retinopathies, vascular
hemorrhages, vascular leakage, and choroidopathies in neonatal,
juvenile or fully mature mammals. Examples of such diseases include
age related macular degeneration, diabetic retinopathy, presumed
ocular histoplasmosis, retinopathy of prematurity, sickle cell
anemia, hemangioma, pterygia, ischemic central retinal vein
occlusion, blanch retinal vein occlusion, ocular melanoma, retinal
blastoma, and retinitis pigmentosa, as well as retinal
injuries.
[0097] Another aspect of the present invention is a therapeutic
composition useful for the treatment of neovascular diseases, which
comprises an angiostatic fragment of TrpRS, a VEGF signaling
inhibitor, and an integrin signaling inhibitor, together with one
or more pharmaceutically acceptable excipient.
[0098] In a preferred embodiment, the composition further comprises
at least one additional therapeutic agent such as an angiostatic
steroid, an anti-neoplastic agent, an anti-bacterial agent, an
anti-viral agent, an anti-inflammatory agent, and the like.
[0099] A method of treating a neovascular disease comprises
administering to a mammal suffering from a neovascular disease a
vascular development inhibiting amount of a combination of drugs
comprising an angiostatic fragment of tryptophanyl-tRNA synthetase
(TrpRS) and at least one compound selected from the group
consisting of a vascular endothelial growth factor (VEGF) signaling
inhibitor and an integrin signaling inhibitor.
[0100] Generally, a vascular development inhibiting amount of a
composition of the present invention is at least about 10 .mu.g/kg
body weight and, in most cases, not in excess of about 8 mg/kg body
weight per day for systemic treatments. Preferably the dosage is in
the range of about 10 .mu.g/kg body weight to about 1 mg/kg body
weight daily. For ocular, intravitreal treatment of human patients,
the preferred dosage is in the range of about 0.1 to about 5
milligrams per eye for a given treatment. The compositions may be
administered in a single dose or in multiple doses over time. One
of ordinary skill in the medical arts would be capable of
determining the optimum effective therapeutic dosage of a
composition of the present invention, taking into account the
particular patient, the drugs present in the composition, the
disease state, and other factors that are well known in the medical
arts.
[0101] The therapeutic compositions of this invention can be
embodied in a variety of physical forms. These forms include, for
example, solid, semi-solid, and liquid dosage forms, such as
tablets, pills, powders, liquid solutions or suspensions, aerosols,
liposomes, suppositories, injectable and infusible solutions and
sustained release forms. One of ordinary skill in the medical arts
will choose a suitable dosage form depending on the intended mode
of administration and on the disease to be treated, using
pharmacological principles well known in the art.
[0102] A therapeutic composition according to this invention can be
administered by conventional routes of administration, such as
parenteral, subcutaneous, intravenous, intramuscular,
intralesional, intrastemal, intravitreal, intracranial, or aerosol
routes. Topical routes of administration can also be used, with
application of the compositions locally to a particular part of the
body (e.g., eye, skin, lower intestinal tract, vagina, rectum)
where appropriate. The therapeutic compositions also include
conventional pharmaceutically acceptable carriers and excipients
that are known to those of skill in the art.
[0103] Generally, the therapeutic compositions of the present
invention can be formulated and administered using methods and
compositions similar to those used for the individual classes of
active ingredients present in the compositions. It will be
understood by those of skill in the art that conventional doses
will vary depending upon the particular active ingredients in the
composition, as well as the patient's health, weight, age, sex, the
condition or disease, and the desired mode of administration.
[0104] The therapeutic compositions of this invention include
pharmacologically appropriate, pharmaceutically acceptable
carriers, excipients and vehicles. In general, these carriers
include aqueous or alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media such as phosphate
buffered saline (PBS). Parenteral vehicles can include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. In addition, intravenous vehicles
can include fluid and nutrient replenishers, and electrolyte
replenishers, such as those based on Ringer's dextrose. Excipients
such as preservatives and other additives can also be present, such
as, for example, antimicrobials, antioxidants, chelating agents,
and inert gases. Suitable formulation aids, carriers, other
excipients, and methods of formulating pharmaceutical compositions
are disclosed in Remington's Pharmaceutical Sciences, 14th Ed.,
Mack Publishing Co., 1970, particularly Part VIII, "Pharmaceutical
Preparations and Their Manufacture", pages 1461-1762, the relevant
disclosures of which are incorporated herein by reference.
[0105] The therapeutic compositions of the present invention can be
packaged in suitably sterilized bottles or vials, either in
multi-dose or in unit dose forms. The containers are preferably
hermetically sealed after being filled with a composition of the
invention. Preferably, the compositions are packaged in a container
having a label affixed thereto, which label identifies the drugs
present in the composition, and bears a notice in a form prescribed
by a government agency such as the United States Food and Drug
Administration, reflecting approval of the composition under
appropriate laws, dosage information, and the like. The label
preferably contains information about the composition that is
useful to a health care professional administering the composition
to a patient. The package also preferably contains printed
informational materials relating to the administration of the
composition, instructions, indications, and any necessary required
warnings.
METHODS
Neonatal Mouse Retinal Angiogenesis Model
[0106] Description of the Model. Immediately after birth (postnatal
day zero; "P0"), retinal vasculature is virtually absent in the
mouse. By four weeks after birth (P28) the retina has attained an
adult pattern of retinal vessels coincident with the onset of
vision. Physiological neovascularization of the retina occurs
during this period via a stereotypical, biphasic developmental
pattern of angiogenesis. During the primary phase of retinal
vascular development, spoke-like peripapillary vessels grow
radially from the central retinal artery and vein, becoming
progressively interconnected by a capillary plexus that forms
between them. This "inner retinal plexus" grows in area, volume and
complexity, centrifugally, as a monolayer within the nerve fiber
layer during the first seven to ten days following birth.
[0107] The second phase of retinal vessel formation begins between
postnatal days 7 (P7) and 10 (P10) when collateral branches sprout
from capillaries of the superficial plexus and penetrate into the
retina where their tips branch and anastamose laterally to form a
planar "deep vascular plexus". While the deep vascular plexus is in
place by P14, it undergoes extensive remodeling from P14 to P21. It
is of interest to note that the formation of these vascular
networks in the neonatal mouse are strikingly similar to the events
occurring in the third trimester human fetus.
Advantages and Quantiftcation of the Model
[0108] The reproducibility of the murine retinal development
process and its easy accessibility in neonatal animals provide an
opportunity to assess the efficacy of antiangiogenic compounds in a
physiologically relevant model of angiogenesis. Additional
advantages of the neonatal mouse model are the ability to
qualitatively and quantitatively evaluate the angiostatic effect of
putative antagonists of angiogenesis. Angiostatic activity was
evaluated based upon the degree of angiogenesis in the deep, outer
retinal vascular layer (secondary layer) that develops between P8
and P12. The appearance of the inner blood vessel network (primary
layer) was evaluated for normal development and signs of toxicity.
No abnormalities in the inner vascular layer were observed in any
of the assays performed and described herein. Qualitative
evaluation of the secondary layer vascularization can be performed
by microscopically photographing appropriately stained superficial
and deep layers of excised retinas and determining the percentage
of eyes in which formation of the deep vascular layer is completely
or partially inhibited. All data presented herein are based on
qualitative analysis of the percentage of eyes that demonstrated a
75 to 100% inhibition of deep retinal vascular network formation
after treatment. In most cases, the percentage of mice that
exhibited >95% and 100% inhibition of deep retinal vascular
network formation are also provided.
Preparation of Compositions
[0109] Peptidomimetic integrin signaling inhibitor Compound (1) is
solubilized in PBS at a concentration of about 20 mg/ml in PBS
(1.times.concentration). T2-TrpRS is solubilized in PBS at a
concentration of about 0.5 mg/ml (1.times.concentration). VEGF
aptamer (pegaptanib sodium; Compound (2)) is solubilized in PBS at
a concentration of about 2 mg/ml (1.times.concentration) to achieve
an injection of approximately 1 .mu.g/eye at 1.times.concentration.
For all combination assays the compounds were prepared at 2 or 3
times the 1.times.concentration of each material and then combined
to produce a final solution containing each individual compound at
the same concentration as was used alone (e.g., 1.times.,
0.5.times., 0.25.times. or 0.1.times., as the case may be). For all
assays, a single injection of 0.5 .mu.l of PBS solutions of the
drugs was administered intravitreally regardless of the number of
compounds being injected. As used herein, the term "0.1.times."
refers to one tenth of the 1.times.concentration of a given
material, "0.25.times." refers to one quarter of the
1.times.concentration of a given material, "0.5.times." refers to
one half of the 1.times.concentration of a given material, and so
forth for similar designations.
[0110] Mouse model of Oxygen Induced Retinopathy (OIR). This model
is described by Smith, L., Invest. Ophthalmol. Vis. Sci. 35,
101-111 (1994). Mice are placed in hyperoxia (75% O.sub.2) from
P7-P12, followed by return to normoxia. While under hyperoxia, the
central retinal vessels obliterate, and deep vasculature fails to
form. Upon return to normoxia, the retina becomes hypoxic and
pathological neovascularization results.
[0111] Quantification of neovascularization in the OIR model
involved quantification of enovascular tuft formation, as well as
quantification of obliteration. Retinal whole mounts are prepared
and blood vessels thereof are stained with isolectin GS-IB.sub.4.
Confocal imaging, focusing just above the superficial vascular
plexus, is carried out and a montage of four quadrants is made.
Neovascular tufts are identified (Adobe PHOTOSHOP.RTM.) and the
area of pixelation is quantified. In addition, areas of
obliteration are traced (Adobe PHOTOSHOP.RTM.), and the area of
pixelation is quantified. Conversion factor based on image
acquisition (resolution, size etc.) is then applied to obtain a
value in .mu.m.sup.2.
General Angiogenesis Assay Procedure
[0112] An in vivo angiogenesis assay in the neonatal mouse (Balb/C,
The Jackson Laboratory, Bar Harbor, Me.) was used to evaluate the
angiostatic activity of integrin signaling inhibitor Compound (1),
T2-TrpRS, and VEGF aptamer Compound (2). Intravitreous injection
and retina isolation was performed with a dissecting microscope
(SMZ 645, Nikon, Japan). An eyelid fissure was created at postnatal
day 7 (P7) with a fine blade to expose the globe for injection. The
samples (0.5 .mu.l) were injected with a Hamilton syringe fitted
with a 32-gauge needle (Hamilton Company, Reno, Nev.). The
injection was made between the equator and the corneal limbus.
During injection, the location of the needle tip was monitored by
direct visualization to determine that it was in the vitreal
cavity. Eyes with needle-induced lens or retinal damage were
excluded from the study. After the injection, the eyelids were
repositioned to close the fissure.
[0113] On postnatal day 12 (P12), animals were euthanized and the
eyes were enucleated. After about 10 minutes in 4% paraformaldehyde
(PFA) the cornea, lens, sclera, and vitreous were excised through a
limbal incision. The isolated retina was prepared for staining by
soaking in methanol for about 10 minutes on ice, followed by
blocking in 50% fetal bovine serum (Gibco, Grand Island, N.Y.) with
20% normal goat serum (The Jackson Laboratory, Bar Harbor, Me.) in
PBS for about one hour on ice. The blood vessels were specifically
visualized by staining the retina for about 18 hours at about
4.degree. C. with a rabbit anti-mouse collagen IV antibody
(Chemicon, Temecula, Calif.) diluted 1:200 in blocking buffer or
with a fluorescent conjugated isolectin (Griffonia simplicifolia,
Molecular Probes). An ALEXA FLUOR.RTM. (Alexa) 594-conjugated goat
anti-rabbit IgG antibody (Molecular probes, Eugene, Oreg.) (1:200
dilution in blocking buffer) was incubated with the retina for
about 2 hours at about 4.degree. C. The retinas were then mounted
for microscopic evaluation with slow-fade mounting media (Molecular
Probes, Eugene, Oreg.).
Example 1
Treatment Of Neonatal Mouse Eyes With A Combination Of A
Peptidomimetic Integrin Signaling Inhibitor And A VEGF Signaling
Inhibitor
[0114] Following the General Angiogenesis Assay Procedure ("General
Procedure") described hereinabove, the eyes of neonatal Balb/C mice
were intravitreally injected on postnatal day 8 (P8) with either a
0.25.times.concentration of integrin signaling inhibitor Compound
(1) (five mice), a 0.5.times.concentration of VEGF aptamer Compound
(2) (five mice), or a combination of a 0.25.times.concentration
Compound (1) and a 0.5.times.concentration of Compound (2) (six
mice). As a control, another group of six mice received only an
intravitreal injection of PBS. At P12, the mice were euthanized and
the retinas were removed from the injected eyes, stained, mounted
and microscopically evaluated as described in the General
Procedure. The vascularity of the secondary (outer retinal
vascular) layer was evaluated based on the percentage of
vascularization compared to the control eyes. The results are shown
in Table 1 and in FIGS. 4 and 5.
TABLE-US-00002 TABLE 1 % inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% PBS 100 0 0 0 0 0.5.times. Compound (2) 20 20 20 20 40
0.1.times. Compound (1) 40 0.5 20 20 20 Combination 0 17 0 0 83
Example 2
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS And A VEGF Signaling Inhibitor
[0115] Following the General Procedure, the eyes of neonatal Balb/C
mice were intravitreally injected on P4 with either a
0.1.times.concentration of T2-TrpRS (eight mice), a
0.1.times.concentration of VEGF aptamer Compound (2) (eight mice),
or a combination of a 0.1.times.concentration T2-TrpRS and a
0.1.times.concentration of Compound (2) (ten mice). As a control,
another group of eight mice received only an intravitreal injection
of PBS. At P12, the mice were euthanized and the retinas were
removed from the injected eyes, stained, mounted and
microscopically evaluated as described in the General Procedure.
The vascularity of the secondary (outer retinal vascular) layer was
evaluated based on the percentage of vascularization compared to
the control eyes. The results are shown in Table 2 and in FIGS. 6,
7, 8, and 9.
TABLE-US-00003 TABLE 2 % inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 100.0 0 0 0 0 0 0 0.1.times. 87.5 12.5 0 0
0 0 0 Comp. (2) 0.1.times. 50 37.5 12.5 0 0 0 0 T2- TrpRS Combi- 10
30 20 20 20 0 0 nation
Example 3
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS And A VEGF Signaling Inhibitor
[0116] Following the General Procedure, the eyes of neonatal Balb/C
mice were intravitreally injected on P4 with either a
1.times.concentration of T2-TrpRS (eight mice), a
1.times.concentration of VEGF aptamer Compound (2) (eight mice), or
a combination of a 1.times.concentration T2-TrpRS and a
1.times.concentration of Compound (2) (ten mice). As a control,
another group of six mice received only an intravitreal injection
of PBS. At P12, the mice were euthanized and the retinas were
removed from the injected eyes, stained, mounted and
microscopically evaluated as described in the General Procedure.
The vascularity of the secondary (outer retinal vascular) layer was
evaluated based on the percentage of vascularization compared to
the control eyes. The results are shown in Table 3 and in FIGS. 10,
11, 12, and 13.
TABLE-US-00004 TABLE 3 % inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 100 0 0 0 0 0 0 1.times. 37.5 12.5 12.5
12.5 25 0 0 Comp. (2) 1.times. 12.5 12.5 0 12.5 62.5 12.5 12.5 T2-
TrpRS Combi- 0 0 10 10 80 40 20 nation
Example 4
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS And A VEGF Signaling Inhibitor
[0117] Following the General Procedure, the eyes of neonatal Balb/C
mice were intravitreally injected on P4 with either a
1.times.concentration of T2-TrpRS (eight mice), a
0.5.times.concentration of VEGF aptamer Compound (2) (ten mice) or
a combination of a 1.times.concentration T2-TrpRS and a
0.5.times.concentration of Compound (2) (ten mice). As a control,
another group of six mice received only an intravitreal injection
of PBS. At P12, the mice were euthanized and the retinas were
removed from the injected eyes, stained, mounted and
microscopically evaluated as described in the General Procedure.
The vascularity of the secondary (outer retinal vascular) layer was
evaluated based on the percentage of vascularization compared to
the control eyes. The results are shown in Table 4 and in FIGS. 14,
15, 16, and 17.
TABLE-US-00005 TABLE 4 % inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 66.6 16.7 16.7 0 0 0 0 0.5.times. 60 30 0
0 10 0 0 Comp. (2) 1.times. T2- 12.5 37.5 12.5 12.5 25 0 0 TrpRS
Combi- 0 0 10 10 70 30 20 nation
Example 5
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS And An Integrin Signaling
Inhibitor
[0118] Following the General Procedure, the eyes of neonatal Balb/C
mice were intravitreally injected on P4 with either a
1.times.concentration of T2-TrpRS, a 0.5.times.concentration of
integrin signaling inhibitor Compound (1) or a combination of a
1.times.concentration T2-TrpRS and a 0.5.times.concentration of
Compound (1), in groups of six mice for each treatment regimen. As
a control, another group of four mice received only an intravitreal
injection of PBS. At P12, the mice were euthanized and the retinas
were removed from the injected eyes, stained, mounted and
microscopically evaluated as described in the General Procedure.
The vascularity of the secondary (outer retinal vascular) layer was
evaluated based on the percentage of vascularization compared to
the control eyes. The results are shown in Table 5 and in FIGS. 18,
19, 20, and 21.
TABLE-US-00006 TABLE 5 % inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 50 25 25 0 0 0 0 0.5.times. 50 33.3 16.7 0
0 0 0 Comp. (1) 1.times. T2- 33.3 16.7 33.3 0 16.7 16.7 0 TrpRS
Combi- 0 33.3 16.7 0 50 16.7 0 nation
Example 6
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS, A VEGF Signaling Inhibitor, And An
Integrin Signaling Inhibitor
[0119] Following the General Procedure, the eyes of neonatal Balb/C
mice were intravitreally injected on P4 with either a
1.times.concentration of T2-TrpRS, a 0.5.times.concentration of
integrin signaling inhibitor Compound (1), a 1.times.concentration
of VEGF aptamer Compound (2), a combination of a
1.times.concentration T2-TrpRS and a 0.5.times.concentration of
Compound (1), a combination of a 1.times.concentration T2-TrpRS and
a 1.times.concentration of Compound (2), or a combination of a
1.times.concentration T2-TrpRS, a 0.5.times.concentration of
Compound (1), and a 1.times.concentration of Compound (2), in
groups of eight mice for each treatment regimen. As a control,
another group of eight mice received only an intravitreal injection
of PBS. At P12, the mice were euthanized and the retinas were
removed from the injected eyes, stained, mounted and
microscopically evaluated as described in the General Procedure.
The vascularity of the secondary (outer retinal vascular) layer was
evaluated based on the percentage of vascularization compared to
the control eyes. The results are shown in Table 6 and in FIGS. 22,
23, 24, 25, 26, 27, and 28.
TABLE-US-00007 TABLE 6 % Inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 100 0 0 0 0 0 0 0.5.times. Comp. (1) 50
12.5 12.5 12.5 12.5 0 0 1.times. Comp. (2) 37.5 25 12.5 25 0 0 0
1.times. T2-TrpRS 50 25 12.5 12.5 0 0 0 T2-TrpRS + Comp. (2) 25 25
25 0 25 25 12.5 T2-TrpRS + Comp. (1) 50 0 0 0 50 37.5 25 T2-TrpRS +
(1) and (2) 0 0 0 0 100 87.5 75
Example 7
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS, A VEGF Signaling Inhibitor, And An
Integrin Signaling Inhibitor
[0120] Following the General Procedure, the eyes of neonatal Balb/C
mice were intravitreally injected on P4 with either a
1.times.concentration of T2-TrpRS, a 0.5.times.concentration of
integrin signaling inhibitor Compound (1), a 1.times.concentration
of VEGF aptamer Compound (2), a combination of a
0.5.times.concentration Compound (1) and a 1.times.concentration of
Compound (2), or a combination of a 1.times.concentration T2-TrpRS,
a 0.5.times.concentration of Compound (1), and a
1.times.concentration of Compound (2), in groups of eight mice for
each treatment regimen. As a control, another group of six mice
received only an intravitreal injection of PBS. At P12, the mice
were euthanized and the retinas were removed from the injected
eyes, stained, mounted and microscopically evaluated as described
in the General Procedure. The vascularity of the secondary (outer
retinal vascular) layer was evaluated based on the percentage of
vascularization compared to the control eyes. The results are shown
in Table 7 and in FIGS. 29, 30, 31, 32, 33, and 34.
TABLE-US-00008 TABLE 7 % Inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 100 0 0 0 0 0 0 0.5.times. Comp. (1) 12.5
25 12.5 12.5 37.5 0 0 1.times. Comp. (2) 25 25 0 12.5 37.5 0 0
1.times. T2-TrpRS 25 12.5 12.5 0 50 12.5 0 Comp. (1) + (2) 0 12.5
12.5 25 50 25 25 T2-TrpRS + (1) and (2) 0 0 0 0 100 62.5 50
Example 8
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS And A VEGF Signaling Inhibitor
[0121] Following the General Procedure, the eyes of neonatal Balb/C
mice were intravitreally injected on P4 with either a
1.times.concentration of T2-TrpRS (ten mice), a
0.25.times.concentration of VEGF aptamer Compound (2) (eleven
mice), or a combination of a 1.times.concentration T2-TrpRS and a
0.25.times.concentration of Compound (2) (eleven mice). As a
control, another group of eight mice received only an intravitreal
injection of PBS. At P12, the mice were euthanized and the retinas
were removed from the injected eyes, stained, mounted and
microscopically evaluated as described in the General Procedure.
The vascularity of the secondary (outer retinal vascular) layer was
evaluated based on the percentage of vascularization compared to
the control eyes. The results are shown in Table 8.
TABLE-US-00009 TABLE 8 % inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 100 0 0 0 0 0 0 0.25.times. 54.5 36.4 0 0
9.1 0 0 Comp. (2) 1.times. T2- 30 30 20 10 10 0 0 TrpRS Combi- 18.2
18.2 9.1 18.2 27.3 0 0 nation
Example 9
Treatment Of Neonatal Mouse Eyes With A Combination Of An
Angiostatic Fragment Of TrpRS, A VEGF Signaling Inhibitor, And An
Integrin Signaling Inhibitor
[0122] Following the General Procedure, the eyes of neonatal Balb/C
mice (in groups of eight mice each) were intravitreally injected on
P4 with either a 1.times.concentration of T2-TrpRS, a
1.times.concentration of integrin signaling inhibitor Compound (1),
a 1.times.concentration of VEGF aptamer Compound (2), a combination
of a 1.times.concentration T2-TrpRS and a 1.times.concentration of
Compound (1), a combination of a 1.times.concentration Compound (1)
and a 1.times.concentration of Compound (2), or a combination of a
1.times.concentration T2-TrpRS, a 1.times.concentration of Compound
(2), and a 1.times.concentration of Compound (2). As a control,
another group of eight mice received only an intravitreal injection
of PBS. At P12, the mice were euthanized and the retinas were
removed from the injected eyes, stained, mounted and
microscopically evaluated as described in the General Procedure.
The vascularity of the secondary (outer retinal vascular) layer was
evaluated based on the percentage of vascularization compared to
the control eyes. The results are shown in Table 9 and FIGS.
35-42.
TABLE-US-00010 TABLE 9 % Inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% >95% 100% PBS 75 25 0 0 0 0 0 1.times. Comp. (1) 25 12.5
12.5 37.5 12.5 0 0 1.times. Comp. (2) 25 0 12.5 12.5 50 25 12.5
1.times. T2-TrpRS 12.5 0 25 12.5 50 12.5 0 Comp. (1) + T2-TrpRS 0
12.5 12.5 25 50 25 12.5 Comp. (1) + (2) 12.5 12.5 0 12.5 62.5 62.5
50 T2-TrpRS + Comp. (2) 0 12.5 12.5 12.5 62.5 37.5 25 T2-TrpRS +
(1) + (2) 0 25 0 0 75 75 62.5
Example 10
Treatment Of Neonatal Mouse Eyes With Varying Doses Of An
Angiostatic Fragment Of TrpRS
[0123] Following the General Procedure, the eyes of neonatal Balb/C
mice (in groups of eight to twelve mice each) were intravitreally
injected on P4 with T2-TrpRS, at concentrations of 0.1.times.(8
mice), 0.3.times.(12 mice), 1.times.(12 mice), 2.times.(12 mice),
and 3.times.(12 mice). As a control, another group of 10 mice
received only an intravitreal injection of PBS. At P12, the mice
were euthanized and the retinas were removed from the injected
eyes, stained, mounted and microscopically evaluated as described
in the General Procedure. The vascularity of the secondary (outer
retinal vascular) layer was evaluated based on the percentage of
vascularization compared to the control eyes. The results are shown
in Table 10.
TABLE-US-00011 TABLE 10 % Inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% PBS 100 0 0 0 0 0.1.times. T2-TrpRS 75 25 0 0 0 0.3.times.
T2-TrpRS 33.3 16.7 8.3 16.7 25 1.times. T2-TrpRS 8.3 16.7 25 8.7
41.7 2.times. T2-TrpRS 8.3 25 0 16.7 41.7 3.times. T2-TrpRS 66.7 25
8.7 0 0
Example 11
Treatment Of Neonatal Mouse Eyes With Varying Doses Of An
Angiostatic Fragment Of TrpRS
[0124] Following the General Procedure, the eyes of neonatal Balb/C
mice (in groups of six to fourteen mice each) were intravitreally
injected on P4 with T2-TrpRS, at concentrations of 0.3.times.(6
mice), 1.times.(14 mice), 2.times.(8 mice), 3.times.(7 mice), and
5.times.(6 mice). As a control, another group of 10 mice received
only an intravitreal injection of PBS. At P12, the mice were
euthanized and the retinas were removed from the injected eyes,
stained, mounted and microscopically evaluated as described in the
General Procedure. The vascularity of the secondary (outer retinal
vascular) layer was evaluated based on the percentage of
vascularization compared to the control eyes. The results are shown
in Table 11.
TABLE-US-00012 TABLE 11 % Inhibition: 0-10% 10-25% 25-50% 50-75%
75-100% PBS 60 40 0 0 0 0.3.times. T2-TrpRS 33.3 16.7 0 0 50
1.times. T2-TrpRS 14.3 14.3 7.1 14.3 50 2.times. T2-TrpRS 12.5 12.5
12.5 12.5 50 3.times. T2-TrpRS 14.3 14.3 14.3 28.5 28.5 5.times.
T2-TrpRS 16.6 33.3 33.3 16.6 0
[0125] The data for mice exhibiting >95% and 100% inhibition in
the foregoing Examples demonstrates that even compositions
comprising at least two materials selected from the group
consisting of an angiostatic fragment of TrpRS, a VEGF signaling
inhibitor, and an integrin signaling inhibitor afford unexpectedly
greater efficacy for inhibition of neovascularization in the
neonatal mouse eye model than the expected levels of inhibition
from the simple additive effects of the combination of individual
components. This is also evident when the results of the various
examples are combined as in Table 12, which compiles the results
from the Examples at concentrations of 1.times. to 2.times. of
integrin inhibitor Compound (1), 0.5.times. to 1.times. of VEGF
aptamer Compound (2), and 1.times. of T2-TrpRS, as well as
compositions of the present invention (inhibition values in bold
type in Table 12) comprising combinations of at least two of
Compound (1), Compound (2) and T2-TrpRS. The number of mice in each
group is indicated in parenthesis for each grouping. The data in
Table 12 clearly shows an unexpectedly higher level of inhibition
of blood vessel formation in the deep vascular layer for mouse eyes
treated with the compositions of the present invention compared to
the inhibition levels of the treatments with the individual
inhibitors by themselves or the numerical sum thereof.
TABLE-US-00013 TABLE 12 Composite Data from the Examples.. %
Inhibition: 0-10% 10-25% 25-50% 50-75% 75-100% >95% 100% PBS (38
mice) 84.2 10.5 5.3 0 0 0 0 1.times.-2.times. Comp. (1) 33.3 20
13.3 16.7 16.7 0 0 (30 mice) 1.times. Comp. (2) (30 mice) 39.1 20
7.6 11.9 21.4 4.7 2.4 1.times. T2-TrpRS (46 mice) 23.9 17.4 15.2
8.7 34.8 8.7 2.2 Comp. (1) + T2-TrpRS 18.2 13.6 9.1 9.1 50 27.3
13.6 (22 mice) Comp. (1) + (2) (16 6.3 12.5 6.3 18.8 56.3 43.7 37.5
mice) T2-TrpRS + Comp. (2) 5.6 11.1 13.9 8.3 61.1 33.3 19.4 (36
mice) T2-TrpRS + (1) + (2) 0 8.3 0 0 91.7 79.2 62.5 (24 mice)
[0126] The results from Examples 10 and 11 (Tables 10 and 11) show
that the efficacy of T2-TrpRS reached a maximum at about 1.times.
to 2.times.concentration and did not provide any more than 50% of
mice having 75-100% inhibition in the deep vascular layer. The
efficacy began to fall off at 2.times. and greater concentration.
Thus even with increasing dosages, T2 did not provide greater than
about 50% efficacy at the 95-100% inhibition level.
Example 12
Synergistic Effects Of The Administration Of T2-TrpRS Angiostatic
Fragment, VEGF Aptamer And Peptidominetic .alpha..sub.v.beta..sub.3
And .alpha..sub.v.beta..sub.5 Integrin Signaling Inhibitor
[0127] To study the effect of combining different angiostatic
molecules, three angiostatic compounds known to target critical,
but separate, angiogenic pathways were utilized: a small molecule
integrin .alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5
antagonist (Compound (1); "EMD 472523" obtained from Merck KGaA,
Darmstadt, Germany), a VEGF.sub.165 antagonist (Compound (2);
pegaptanib sodium), and a truncated form of Tryptophan tRNA
synthetase (T2-TrpRS, obtained from Angiosyn Inc., La Jolla,
Calif.). Although the exact mechanism of action for T2-TrpRS has
not been fully elucidated, its mechanism of action is not directly
linked to VEGF or integrin antagonism.
[0128] The neonatal mouse retinal angiogenesis model was used to
test the efficacy of each monotherapy, and various combinations of
these individual compounds. As stated hereinabove, mice are born
without a retinal vasculature. During the first three weeks after
birth an adult-like vasculature develops. The retinal vasculature
forms three distinct planar plexuses with the superficial vascular
plexus developing during the first post-natal week. At post-natal
day 8 (P8), the vessels of the superficial plexus branch and
migrate toward the deep plexus at the outer edge of the inner
nuclear layer. To test the angiostatic properties of each
monotherapy or combination solution, intravitreal injections were
performed at P7, when the formation of the superficial network is
nearing completion, but before the formation of the deep plexuses
has begun. The effects on formation of the deep vascular plexus
were then analyzed five days later (P 12). The degree of inhibition
for each injected retina was scored as 0-10%, 10-25%, 25-50%,
50-75%, or 75-100% (FIG. 48) with the 75-100% inhibition group
further separated into >90% and 100% inhibition levels (FIG.
49). The appearance of the previously formed primary vascular
plexus, as well as the overall retinal morphology, were evaluated
for signs of toxicity.
[0129] Sample preparation. The .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 integrin antagonist (Compound (1)) was
stored as lyophilized powder in a desiccator at room temperature
(I.A.) or -20.degree. C. (V.A.), and solubilized in sterile,
RNAse-free 1.times. PBS immediately before use. The VEGF aptamer
(Compound (1)) was synthesized as a 40 kDa PEG conjugated compound
(Transgenomic Inc, Boulder, Colo.) based on published information,
Bridonneau, et al., J. Chromatogr. B. Diomed. Sci. App. 726: 237-47
(1999). The compound was determined to be pure by reverse phase
liquid chromatography. Concentrations reported herein refer to the
final concentration of the active VEGF aptamer rather than the
total concentration of the PEG conjugated compound and were
determined by spectrophotometric analysis at 260/280 nm. T2-TrpRS
peptide was made as a recombinant compound as described in Otani,
et al., Proc. Nat'l. Acad. Sci., USA 99: 178-183 (2002) and U.S.
Provisional Application for Patent Serial No. 60/598,019 filed Aug.
2, 2004, which are incorporated herein by reference in their
entireties. Purified product was stored in 50% glycerol at
-20.degree. C., and dialyzed into sterile 1.times.PBS immediately
before use. Combination solutions were prepared by initially
creating 3.times.stock solutions of each individual compound. The
compounds were then mixed together, and with PBS where appropriate,
to make a final solution containing each desired compound at a
concentration equivalent to each corresponding monotherapy
concentration.
[0130] Intravitreal injections. All animal work adhered to strict
protocol guidelines for the humane care and use of animals.
Intravitreal injections were performed, the retinas were dissected,
and the vasculature was visualized. OIR was induced according to
the protocol described by Smith, et al., Invest. Ophthalmol. Vis.
Sci., 35: 101-111 (1994), by exposing post-natal day 7 (P7) pups
and their mothers to an environment of 75% oxygen (hyperoxia) for 5
days, followed by a return to room air (normoxia). Intravitreal
injections were performed at P12, immediately following return to
normoxia and the retinas were analyzed at P17. Blood vessels were
stained using isolectin GS-IB.sub.4 from Griffonia simplicifolia
(lectin GS), conjugated to Alexa Fluor 594 (Molecular Probes, 1:150
dilution in PBS). Confocal images were taken using a
4.times.objective lens, carefully focusing just above the inner
limiting membrane of the retina, making the pre-laminar neovascular
tufts prominent. Four overlapping images were acquired from each
retina and each individual image was converted to a 2.times.2 inch
size with 300 pixels per inch. The neovascular tuft areas were
quantified by masked individuals using retinal whole mounts. Tufts
were specifically selected, based on their characteristic
appearance and the higher intensity of isolectin staining, using
the magic wand tool in Adobe PHOTOSHOP.RTM. software. The total
area in pixels was then determined. The areas of neovascular tuft
formation were normalized to PBS-injected control OIR retinas.
[0131] Dosing. Dosing experiments were first performed to determine
the maximum efficacy dose for each individual compound. Each
compound was found to have a bell-shaped efficacy curve with
optimal effective doses of 5-10 .mu.g (10-20 nmoles) per eye for
the integrin antagonist (FIG. 46), 1.0-2.0 .mu.g (108-215 pmoles)
per eye for the VEGF aptamer (FIG. 44), and 0.25-0.5 .mu.g
(5.2-10.4 pmoles) per eye for T2-TrpRS (FIG. 43). Single injections
of each monotherapy at the optimal dose, and solutions containing
appropriate combinations of each compound at equivalent doses were
then performed to compare the angiostatic activities. At the
maximum individual doses, around 35% of the retinas were unaffected
by injection of either the integrin antagonist or the VEGF aptamer.
The other 67% of the retinas basically fell evenly within the
10-25%, 25-50%, 50-75%, or 75%-100% range (Table 13A below).
Inhibition of the deep vascular network with T2-TrpRS peptide was
slightly better. 24% of the T2-TrpRS peptide injected retinas
developed a normal, complete deep vascular plexus while 35% of the
T2-TrpRS peptide injected retinas exhibited over 75% inhibition
compared to 17% and 21% for the integrin antagonist and the VEGF
aptamer respectively (Table 13A below). When the angiostatic
compounds were injected in combination, the angiostatic effects on
neovascularization were striking. Each dual combination, integrin
antagonist +T2-TrpRS, integrin antagonist +VEGF aptamer, and
T2-TrpRS +VEGF aptamer, demonstrated significant improvement of
angiostatic activity over the monotherapies. Significantly fewer
retinas were resistant to angiostatic treatment using the
combination therapies. Neovascularization was inhibited by over 75%
in a majority of the retinas treated with any of the dual
combinations (Table 13A below).
[0132] When all three compounds were injected together at the same
optimal doses as the corresponding monotherapy injections
(1.times.triple combination), over 90% of the retinas had >75%
inhibition. Unlike the monotherapy or double combination-treated
retinas, all retinas treated with the triple combination exhibited
some degree of neovascular inhibition. In addition, only 8% of the
injected retinas still had any significant levels of
neovascularization at all. Nearly complete inhibition of
angiogenesis was observed in the other 92% of the retinas injected
with the triple combination compound (Table 13A below; FIG. 48).
The differences in angistatic efficacy became even more pronounced
when the >75% inhibition category was subcategorized into
>90% inhibition and 100% inhibition levels (FIG. 49). Over 80%
of the retinas injected with the triple combination had greater
than 90% inhibition of deep vascular plexus formation and 63% had
100% inhibition of neovascularization where not even a single
neovascular sprout could be observed. This is a substantial
improvement over both the monotherapies which demonstrated 100%
inhibition in <5% of the treated retinas and the dual
combination therapies. In addition, the superficial vascular plexus
of many of the triple combination-treated retinas resembled that of
a normal P7 retina rather than P12 retinas, indicating that further
vascular growth within the superficial plexus had also been
prevented by the triple combination immediately following
injection. Inhibition of superficial plexus growth was not observed
in any mono- or dual-therapy treated retina. The more central
vessels of the superficial vascular plexus that had already formed
prior to injection remained normal, indicating negligible levels of
toxicity to pre-existent vasculature. In addition, no signs of
neuronal toxicity were observed, and the retinal morphology was
unaltered, indicating that no observable negative side effects had
occurred by injection of the triple combination solution. Images of
the superficial and deep vascular plexuses from one complete
representative experiment are shown in FIG. 50.
[0133] To analyze synergism, and to test if potent angiostatic
activity could be maintained using lower doses of the triple
combination, serial dilutions were tested. The triple combination
was still highly effective at inhibiting angiogenesis when diluted
up to 100-fold (0.01.times. triple combination) (Table 13B below;
FIGS. 52 and 53). When the triple combination was made by combining
the individual compounds at one-tenth their optimal dose, nearly
80% of the treated retinas still exhibited >75% inhibition, and
50% of the retinas exhibited complete (100%) inhibition of
neovascularization. At the 0.1.times.concentrations (1 .mu.g/eye
integrin antagonist, 0.2 .mu.g/eye VEGF aptamer, and 0.025
.mu.g/eye T2-TrpRS), inhibition of neovascularization by the
individual angiostatic compounds was negligible (Table 13C below;
FIG. 54). Some efficacy was observed after injection of the double
0.1.times.T2-TrpRS and 0.1.times.VEGF aptamer combination. However,
despite the fact that this combination was the most effective
angiostatic of all the double combinations tested, the angiostatic
activity was still minimal compared to the inhibition levels
observed by injection of the 0.1.times.triple combination.
TABLE-US-00014 TABLE 13 N 0-10% 10-25% 25-50% 50-75% >75%
>90% 100% 13A. Neonatal mouse angiogenesis model combination
experiment Percentage of retinas with the indicated levels of
neovascular inhibition Injection PBS 38 84.2 10.5 5.3 0.0 0.0 0.0
0.0 5-10 .mu.g integrin 30 33.3 20.0 13.3 16.7 16.7 0.0 0.0
antagonist 1-2 .mu.g VEGF 42 39.1 20.0 7.6 11.9 21.4 4.7 2.4
aptamer 0.25 .mu.g T2-TrpRS 46 23.9 17.4 15.2 8.7 34.8 8.7 2.2
T2-TrpRS + 22 18.2 13.6 9.1 9.1 50.0 27.3 13.6 integrin ant.
Integrin ant. + 21 6.3 12.5 6.3 18.8 56.3 43.7 28.5 VEGF apt.
T2-TrpRS + VEFG 36 5.6 11.1 13.9 8.3 61.1 38.3 19.4 apt. Triple
combination 24 0 8.3 0 0 91.7 83.2 62.6 13B. Triple combination
serial dilution experiment Injection PBS 14 71.4 14.3 14.3 0.0 0.0
0.0 0.0 1.times. Triple 16 0.0 0.0 0.0 7.1 92.8 71.4 57.1
Combination 0.5.times. Triple 16 0.0 0.0 0.0 0.5 100.0 87.5 50.0
Combination 0.25.times. Triple 16 0.0 0.0 0.0 0.5 100.0 68.8 43.8
Combination 0.1.times. Triple 18 0.0 5.5 11.1 5.5 77.8 61.1 44.4
Combination. 0.05.times. Triple 10 0.0 10.0 20.0 20.0 50.0 30.0
20.0 Combination 0.01.times. Triple 10 10.0 10.0 30.0 30.0 20.0
20.0 10.0 Combination 13C. Low dosing monotherapy vs. combination
experiment Inhibition Levels: PBS 8 100 0 0 0 0 0 0 0.1.times.
integrin ant. (1.0 .mu.g) 10 90 10 0 0 0 0 0 0.1.times. VEGF apt.
(0.20 .mu.g) 8 75 25 0 0 0 0 0 0.1.times. T2-TrpRS (0.025 .mu.g) 10
50 37.5 12.5 0 0 0 0 0.1.times. T2-TrpRS + VEFG 10 10 30 20.0 20 20
0 0 apt. 0.1.times. Triple Combination 18 0 5.5 11.1 5.5 77.8 61.1
44.4
Example 13
Synergistic Effects Of A "Triple Therapy"
[0134] The mouse model of oxygen-induced retinopathy (OIR)
described hereinabove is a well-accepted model of hypoxia-induced
neovascularization in the retina. The associated vascular changes
are consistent, reproducible and quantifiable. In recent years the
use of this model has been extended to the general study of
disease-related ischemic vasculopathies and related anti-angiogenic
interventions. To study the synergistic properties of these
angiostatic compounds in a more pathological model of angiogenesis,
the effects of monotherapies and combination therapies on the
formation of pathological neovascularization were tested in the
mouse OIR model. For initial experiments, the optimal doses
obtained from the neonatal angiogenesis model were used. In each
case, combination therapies demonstrated improved angiostatic
activities compared to the monotherapies. However, due to the
angiostatic activities of each monotherapy, it was difficult to
determine if the results of combining the various compounds were
synergistic or simply additive. Thus, based on the results observed
using the neonatal mouse retinal angiogenesis model which
demonstrated equivalent efficacies of the combination therapies at
relatively low doses, each monotherapy and the various combination
therapies were tested at one-tenth of the optimal doses. Again, the
concentration of each compound in the combination solutions was
equivalent to the corresponding monotherapy concentration. At the
lower concentrations, no significant inhibition of pathological
neovascular tuft formation was observed following monotherapy
treatments. However, significant reductions in neovascular tuft
formations were observed using each double combination (FIG. 57).
When the integrin antagonist was combined with T2-TrpRS peptide,
pathological tuft formation was reduced by >50%. Combining the
integrin antagonist with the VEFG aptamer reduced tuft formation by
>40%. When T2-TrpRS peptide was combined with the VEGF aptamer,
pathological neovascularization was reduced by nearly 80% compared
to control-treated retinas. Many of the retinas treated with the
double T2-TrpRS/VEGF aptamer combination looked nearly normal with
virtually no pathological neovascularization evident (FIG. 58).
[0135] A dramatic increase in angiostatic activity by combining
multiple angiostatic compounds that target distinct angiogenesis
pathways has been demonstrated. Strong angiostatic activities were
observed in both a developmental and a pathological model of
angiogenesis even after combining the compounds at doses which have
no monotherapeutic activity. This suggests a synergistic effect
rather than simply an additive effect. This data also suggests that
targeting multiple pathways may be required for effective clinical
anti-angiogenic therapy and may provide a new paradigm for the
treatment of neovascular diseases. Preferably, at least two
anti-angiogenic therapies are combined (e.g., a combination of VEGF
signaling inhibitor, such as a VEGF aptamer, combined with an
angiostatic fragment of TrpRS, such as the T2 fragment of TrpRS,
and optionally and integrin antagonist.
[0136] By targeting and inhibiting three separate angiogenic
pathways, nearly complete inhibition of angiogenesis was obtained
in two separate models of angiogenesis. The complete inhibition of
neovascularization may be important for the effective treatment of
angiogenesis-related diseases using angiostatic therapies. In our
models, even the best results from monotherapy injections generally
only blocked 50-75% of new vessel growth. This means that in most
cases, a significant amount of neovascularization still developed.
In contrast, two-thirds of the neonatal mouse retinas injected with
the triple combination therapy had complete 100% inhibition of
neovascular formation (Table 13). Similarly, in the OIR model of
pathological angiogenesis, a large portion of the treated mice
demonstrated little or no pathological neovascular tuft formation
(FIG. 58). During cancer treatment, high levels of angiogenesis
inhibition may be required to cause the complete starvation of
tumor cells and prevent further tumor growth. Monotherapies that
only inhibit 50% of neovascular growth are only likely to reduce,
rather than eliminate, the oxygen and nutrients available to the
rapidly growing tumor cells. Although this may initially slow
growth, it may not be sufficient to prevent further tumor growth.
In these instances, combination therapies that can achieve complete
inhibition of neovascularization would greatly improve the results
of anti-angiogenic therapies during cancer treatments. In addition,
by using relatively low doses while maintaining strong angiostatic
potential, the possibility of adverse side effects generated by
angiostatic treatments can be minimized. Together, the foregoing
data demonstrate the beneficial utility of combining different
angiostatic molecules for the treatment of neovascularization
associated with disease.
[0137] The compositions of the present invention, comprising an
angiostatic fragment of tryptophanyl-tRNA synthetase (TrpRS), a
vascular endothelial growth factor (VEGF) signaling inhibitor, and
an integrin signaling inhibitor, and the methods of use thereof,
provide a new and surprisingly efficacious treatment regimen for
neovascular diseases, particularly for neovascular diseases of the
eye.
Example 14
Treatment Of A Tumor
[0138] Glioblastoma multiform is an incurable malignant brain
tumor, usually fatal within one year of diagnosis. The 9L rat
gliosarcoma cell line is used as a model for malignant gliomas. In
both forms of glioma, the tumor is highly vascularized and
infiltrates into normal brain tissue. Untreated animal receiving a
bolus of 9L cells intracerebrally have a survival tie of
approximately 3 weeks, once the tumor cells have been implanted. At
day 0, intracerebral 9L tumors were established by stereotactic
inoculation of about 50,000 cells in about 2 .mu.l of Delbecco's
Modified Eagles's Medium (DMEM; Life Technologies, Gaithersburg,
Md.) into the right frontal lobe of CD 344 Fisher rats that had
previously been anesthetized with ketamine and xylazine.
[0139] At day 6, a 10 .mu.l bolus of a composition of the invention
(4.5 mg/l of T2-TrpRS, 30 mg/l of Compound (1), and 6 mg/l of
Compound (2); pegaptanib sodium) was injected stereotactically over
the course of about 2 minutes into the same region of the brain as
the 9L cells were implanted. Follwing injection of the bolus, a
pump was inserted into a subcutaneous pocket between the scapulae.
A catheter connected via tubing to the pump was inserted into the
same burr hole made for intorduction of the 9L cells, and was fixed
in place. Each pump had a flow rate of about 8 .mu.l per hour. An
additional quantity of the composition of the invention was
continuously pumped into the brain of each animal for about 24
hours. The continuous pumping distributed the composition
throughout the entire hemisphere of the brain in which the tumor
cells had been implanted. Nine rats received the composition of the
invention and nine rats received straight PBS as a control
group.
[0140] At day 13, an incision was made between the scapulae, and
the pump was removed and replaced with an fresh pump. Treatment
with the composition of the invention or PBS was resumed for an
additional 24 hours with the new pump at the same pumping flow
rates. There was a 21 percent increase in survival for rats treated
with the composition of the invention compared to the PBS treated
control group (See FIG. 60).
[0141] Numerous variations and modifications of the embodiments
described above may be effected without departing from the spirit
and scope of the novel features of the invention. No limitations
with respect to the specific embodiments illustrated herein are
intended or should be inferred.
Sequence CWU 1
1
61379PRTHOMO SAPIENS 1Met Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu
Ile Val Arg Phe Gly1 5 10 15Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn
Arg Ile Glu Arg Ala Thr 20 25 30Gly Gln Arg Pro His His Phe Leu Arg
Arg Gly Ile Phe Phe Ser His 35 40 45Arg Asp Met Asn Gln Val Leu Asp
Ala Tyr Glu Asn Lys Lys Pro Phe 50 55 60Tyr Leu Tyr Thr Gly Arg Gly
Pro Ser Ser Glu Ala Met His Val Gly65 70 75 80His Leu Ile Pro Phe
Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn 85 90 95Val Pro Leu Val
Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys 100 105 110Asp Leu
Thr Leu Asp Gln Ala Tyr Ser Tyr Ala Val Glu Asn Ala Lys 115 120
125Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser
130 135 140Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn
Val Val145 150 155 160Lys Ile Gln Lys His Val Thr Phe Asn Gln Val
Lys Gly Ile Phe Gly 165 170 175Phe Thr Asp Ser Asp Cys Ile Gly Lys
Ile Ser Phe Pro Ala Ile Gln 180 185 190Ala Ala Pro Ser Phe Ser Asn
Ser Phe Pro Gln Ile Phe Arg Asp Arg 195 200 205Thr Asp Ile Gln Cys
Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr 210 215 220Phe Arg Met
Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro225 230 235
240Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr
245 250 255Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr
Asp Thr 260 265 270Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His Ala
Phe Ser Gly Gly 275 280 285Arg Asp Thr Ile Glu Glu His Arg Gln Phe
Gly Gly Asn Cys Asp Val 290 295 300Asp Val Ser Phe Met Tyr Leu Thr
Phe Phe Leu Glu Asp Asp Asp Lys305 310 315 320Leu Glu Gln Ile Arg
Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly 325 330 335Glu Leu Lys
Lys Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu 340 345 350His
Gln Ala Arg Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe 355 360
365Met Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln 370 3752379PRTHOMO
SAPIENS 2Met Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg
Phe Gly1 5 10 15Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu
Arg Ala Thr 20 25 30Gly Gln Arg Pro His His Phe Leu Arg Arg Gly Ile
Phe Phe Ser His 35 40 45Arg Asp Met Asn Gln Val Leu Asp Ala Tyr Glu
Asn Lys Lys Pro Phe 50 55 60Tyr Leu Tyr Thr Gly Arg Gly Pro Ser Ser
Glu Ala Met His Val Gly65 70 75 80His Leu Ile Pro Phe Ile Phe Thr
Lys Trp Leu Gln Asp Val Phe Asn 85 90 95Val Pro Leu Val Ile Gln Met
Thr Asp Asp Glu Lys Tyr Leu Trp Lys 100 105 110Asp Leu Thr Leu Asp
Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys 115 120 125Asp Ile Ile
Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser 130 135 140Asp
Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val145 150
155 160Lys Ile Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe
Gly 165 170 175Phe Thr Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro
Ala Ile Gln 180 185 190Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln
Ile Phe Arg Asp Arg 195 200 205Thr Asp Ile Gln Cys Leu Ile Pro Cys
Ala Ile Asp Gln Asp Pro Tyr 210 215 220Phe Arg Met Thr Arg Asp Val
Ala Pro Arg Ile Gly Tyr Pro Lys Pro225 230 235 240Ala Leu Leu His
Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr 245 250 255Lys Met
Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr 260 265
270Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly
275 280 285Arg Asp Thr Ile Glu Glu His Arg Gln Phe Gly Gly Asn Cys
Asp Val 290 295 300Asp Val Ser Phe Met Tyr Leu Thr Phe Phe Leu Glu
Asp Asp Asp Lys305 310 315 320Leu Glu Gln Ile Arg Lys Asp Tyr Thr
Ser Gly Ala Met Leu Thr Gly 325 330 335Glu Leu Lys Lys Ala Leu Ile
Glu Val Leu Gln Pro Leu Ile Ala Glu 340 345 350His Gln Ala Arg Arg
Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe 355 360 365Met Thr Pro
Arg Lys Leu Ser Phe Asp Phe Gln 370 3753424PRTHOMO SAPIENS 3Met Ser
Tyr Lys Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp Cys Pro1 5 10 15Pro
Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr Glu 20 25
30Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala
35 40 45Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser
Lys 50 55 60Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly
Gln Arg65 70 75 80Pro His His Phe Leu Arg Arg Gly Ile Phe Phe Ser
His Arg Asp Met 85 90 95Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys
Pro Phe Tyr Leu Tyr 100 105 110Thr Gly Arg Gly Pro Ser Ser Glu Ala
Met His Val Gly His Leu Ile 115 120 125Pro Phe Ile Phe Thr Lys Trp
Leu Gln Asp Val Phe Asn Val Pro Leu 130 135 140Val Ile Gln Met Thr
Asp Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr145 150 155 160Leu Asp
Gln Ala Tyr Ser Tyr Ala Val Glu Asn Ala Lys Asp Ile Ile 165 170
175Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp
180 185 190Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val Lys
Ile Gln 195 200 205Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe
Gly Phe Thr Asp 210 215 220Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro
Ala Ile Gln Ala Ala Pro225 230 235 240Ser Phe Ser Asn Ser Phe Pro
Gln Ile Phe Arg Asp Arg Thr Asp Ile 245 250 255Gln Cys Leu Ile Pro
Cys Ala Ile Asp Gln Asp Pro Tyr Phe Arg Met 260 265 270Thr Arg Asp
Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu 275 280 285His
Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser 290 295
300Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr Ala Lys
Gln305 310 315 320Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly
Gly Arg Asp Thr 325 330 335Ile Glu Glu His Arg Gln Phe Gly Gly Asn
Cys Asp Val Asp Val Ser 340 345 350Phe Met Tyr Leu Thr Phe Phe Leu
Glu Asp Asp Asp Lys Leu Glu Gln 355 360 365Ile Arg Lys Asp Tyr Thr
Ser Gly Ala Met Leu Thr Gly Glu Leu Lys 370 375 380Lys Ala Leu Ile
Glu Val Leu Gln Pro Leu Ile Ala Glu His Gln Ala385 390 395 400Arg
Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe Met Thr Pro 405 410
415Arg Lys Leu Ser Phe Asp Phe Gln 4204401PRTHOMO SAPIENS 4Ser Asn
His Gly Pro Asp Ala Thr Glu Ala Glu Glu Asp Phe Val Asp1 5 10 15Pro
Trp Thr Val Gln Thr Ser Ser Ala Lys Gly Ile Asp Tyr Asp Lys 20 25
30Leu Ile Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn
35 40 45Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro His His Phe Leu Arg
Arg 50 55 60Gly Ile Phe Phe Ser His Arg Asp Met Asn Gln Val Leu Asp
Ala Tyr65 70 75 80Glu Asn Lys Lys Pro Phe Tyr Leu Tyr Thr Gly Arg
Gly Pro Ser Ser 85 90 95Glu Ala Met His Val Gly His Leu Ile Pro Phe
Ile Phe Thr Lys Trp 100 105 110Leu Gln Asp Val Phe Asn Val Pro Leu
Val Ile Gln Met Thr Asp Asp 115 120 125Glu Lys Tyr Leu Trp Lys Asp
Leu Thr Leu Asp Gln Ala Tyr Ser Tyr 130 135 140Ala Val Glu Asn Ala
Lys Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn145 150 155 160Lys Thr
Phe Ile Phe Ser Asp Leu Asp Tyr Met Gly Met Ser Ser Gly 165 170
175Phe Tyr Lys Asn Val Val Lys Ile Gln Lys His Val Thr Phe Asn Gln
180 185 190Val Lys Gly Ile Phe Gly Phe Thr Asp Ser Asp Cys Ile Gly
Lys Ile 195 200 205Ser Phe Pro Ala Ile Gln Ala Ala Pro Ser Phe Ser
Asn Ser Phe Pro 210 215 220Gln Ile Phe Arg Asp Arg Thr Asp Ile Gln
Cys Leu Ile Pro Cys Ala225 230 235 240Ile Asp Gln Asp Pro Tyr Phe
Arg Met Thr Arg Asp Val Ala Pro Arg 245 250 255Ile Gly Tyr Pro Lys
Pro Ala Leu Leu His Ser Thr Phe Phe Pro Ala 260 265 270Leu Gln Gly
Ala Gln Thr Lys Met Ser Ala Ser Asp Pro Asn Ser Ser 275 280 285Ile
Phe Leu Thr Asp Thr Ala Lys Gln Ile Lys Thr Lys Val Asn Lys 290 295
300His Ala Phe Ser Gly Gly Arg Asp Thr Ile Glu Glu His Arg Gln
Phe305 310 315 320Gly Gly Asn Cys Asp Val Asp Val Ser Phe Met Tyr
Leu Thr Phe Phe 325 330 335Leu Glu Asp Asp Asp Lys Leu Glu Gln Ile
Arg Lys Asp Tyr Thr Ser 340 345 350Gly Ala Met Leu Thr Gly Glu Leu
Lys Lys Ala Leu Ile Glu Val Leu 355 360 365Gln Pro Leu Ile Ala Glu
His Gln Ala Arg Arg Lys Glu Val Thr Asp 370 375 380Glu Ile Val Lys
Glu Phe Met Thr Pro Arg Lys Leu Ser Phe Asp Phe385 390 395
400Gln5471PRTHOMO SAPIENS 5Met Pro Asn Ser Glu Pro Ala Ser Leu Leu
Glu Leu Phe Asn Ser Ile1 5 10 15Ala Thr Gln Gly Glu Leu Val Arg Ser
Leu Lys Ala Gly Asn Ala Ser 20 25 30Lys Asp Glu Ile Asp Ser Ala Val
Lys Met Leu Val Ser Leu Lys Met 35 40 45Ser Tyr Lys Ala Ala Ala Gly
Glu Asp Tyr Lys Ala Asp Cys Pro Pro 50 55 60Gly Asn Pro Ala Pro Thr
Ser Asn His Gly Pro Asp Ala Thr Glu Ala65 70 75 80Glu Glu Asp Phe
Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys 85 90 95Gly Ile Asp
Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile 100 105 110Asp
Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro 115 120
125His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His Arg Asp Met Asn
130 135 140Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe Tyr Leu
Tyr Thr145 150 155 160Gly Arg Gly Pro Ser Ser Glu Ala Met His Val
Gly His Leu Ile Pro 165 170 175Phe Ile Phe Thr Lys Trp Leu Gln Asp
Val Phe Asn Val Pro Leu Val 180 185 190Ile Gln Met Thr Asp Asp Glu
Lys Tyr Leu Trp Lys Asp Leu Thr Leu 195 200 205Asp Gln Ala Tyr Ser
Tyr Ala Val Glu Asn Ala Lys Asp Ile Ile Ala 210 215 220Cys Gly Phe
Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp Tyr225 230 235
240Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val Lys Ile Gln Lys
245 250 255His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly Phe Thr
Asp Ser 260 265 270Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln
Ala Ala Pro Ser 275 280 285Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg
Asp Arg Thr Asp Ile Gln 290 295 300Cys Leu Ile Pro Cys Ala Ile Asp
Gln Asp Pro Tyr Phe Arg Met Thr305 310 315 320Arg Asp Val Ala Pro
Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu His 325 330 335Ser Thr Phe
Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser Ala 340 345 350Ser
Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr Ala Lys Gln Ile 355 360
365Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp Thr Ile
370 375 380Glu Glu His Arg Gln Phe Gly Gly Asn Cys Asp Val Asp Val
Ser Phe385 390 395 400Met Tyr Leu Thr Phe Phe Leu Glu Asp Asp Asp
Lys Leu Glu Gln Ile 405 410 415Arg Lys Asp Tyr Thr Ser Gly Ala Met
Leu Thr Gly Glu Leu Lys Lys 420 425 430Ala Leu Ile Glu Val Leu Gln
Pro Leu Ile Ala Glu His Gln Ala Arg 435 440 445Arg Lys Glu Val Thr
Asp Glu Ile Val Lys Glu Phe Met Thr Pro Arg 450 455 460Lys Leu Ser
Phe Asp Phe Gln465 470628DNAArtificial SequenceChemically
synthesized mixed DNA RNA sequence 6cggaaucagu gaaugcuuau acauccgn
28
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