U.S. patent application number 17/525793 was filed with the patent office on 2022-06-09 for angio-3 for treatment of retinal angiogenic diseases.
The applicant listed for this patent is NATIONAL UNIVERSITY OF SINGAPORE, SINGAPORE HEALTH SERVICES PTE LTD.. Invention is credited to Michael BELKIN, Gemmy Chui Ming CHEUNG, RUOWEN Ge, Manjunatha R. KINI, LAKSHMINARAYANAN Rajamani, BARATH Amutha Veluchamy, Tien Yin WONG.
Application Number | 20220175876 17/525793 |
Document ID | / |
Family ID | 1000006153293 |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220175876 |
Kind Code |
A1 |
BELKIN; Michael ; et
al. |
June 9, 2022 |
ANGIO-3 FOR TREATMENT OF RETINAL ANGIOGENIC DISEASES
Abstract
This disclosure provides methods of a method of treating a
retinal angiogenic disease in a subject comprising administering an
effective amount of an Angio-3 peptide.
Inventors: |
BELKIN; Michael; (Givat
Shumuel, IL) ; Veluchamy; BARATH Amutha; (Singapore,
SG) ; Rajamani; LAKSHMINARAYANAN; (Singapore, SG)
; KINI; Manjunatha R.; (Singapore, SG) ; Ge;
RUOWEN; (Singapore, SG) ; WONG; Tien Yin;
(Singapore, SG) ; CHEUNG; Gemmy Chui Ming;
(Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SINGAPORE HEALTH SERVICES PTE LTD.
NATIONAL UNIVERSITY OF SINGAPORE |
SINGAPORE
SINGAPORE |
|
SG
SG |
|
|
Family ID: |
1000006153293 |
Appl. No.: |
17/525793 |
Filed: |
November 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16641978 |
Feb 25, 2020 |
11266710 |
|
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PCT/IB2018/056685 |
Aug 31, 2018 |
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17525793 |
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62553051 |
Aug 31, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0048 20130101;
A61P 27/02 20180101; A61K 38/08 20130101 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61P 27/02 20060101 A61P027/02; A61K 9/00 20060101
A61K009/00 |
Claims
1.-24. (canceled)
25. A method of treating a retinal angiogenic disease in a subject
comprising: (a) selecting a subject with retinal angiogenic disease
not responsive to anti-VEGF therapy; and (b) administering to the
subject a composition comprising a peptide consisting of the
sequence Thr Pro His Thr His Gin Xaa Thr Pro Glu (SEQ ID NO:4)
wherein administration treats the retinal angiogenic disease in the
subject.
26. The method of claim 25, wherein the composition comprises 2 to
50 mg/kg Bwt of the peptide and is administered by intravenous
injection.
27. The method of claim 25, wherein the composition comprises 0.1
.mu.g/kg to 2 mg/kg Bwt of the peptide and is administered by
intravitreal injection.
28. The method of claim 25, wherein the composition comprises 2 to
10 mg/kg Bwt of the peptide and is administered orally.
29. The method of claim 25, wherein the composition is administered
via either intravenous (IV) or intravitreal (IVT) route at least
once every 4 to 10 weeks.
30. The method of claim 25, wherein the composition is administered
orally at least once daily for 1 to 2 weeks at intervals of 6
months.
31. The method of claim 25, wherein the anti-VEGF therapy is an
anti-VEGF antibody.
32. The method of claim 25, wherein the subject has age-related
macular degeneration, retinopathy, or vascular occlusion.
33. The method of claim 25, wherein the subject has diabetic
retinopathy, diabetic macular edema, central retinal vein
occlusion, branch retinal vein occlusion, or corneal
neovascularization.
34. The method of claim 25, wherein the subject is a human.
35. The method of claim 25, wherein the composition is formulated
for intravenous administration.
36. The method of claim 25, wherein the composition is formulated
for intravitreal injection.
37. The method of claim 25, wherein the composition is formulated
for oral administration.
Description
CROSS-SECTION TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/641,978, filed Aug. 31, 2018, which is a
U.S. National Phase of International Application No.
PCT/M2018/056685, filed Aug. 31, 2018, which claims priority to
U.S. Patent Application No. 62/553,051, filed Aug. 31, 2017, the
disclosure of which is hereby incorporated by reference in its
entirety for purposes.
BACKGROUND
[0002] The Sequence Listing written in file
101286_1283483_SEQ_LST.txt created on Nov. 9, 2021, containing
1,371 bytes, machine format IBM-PC, MS-Windows operating system, is
hereby incorporated by reference in its entirety.
[0003] Retinal angiogenic diseases include age-related macular
degeneration, retinopathy, vascular occlusion, diabetic
retinopathy, diabetic macular edema, central retinal vein
occlusion, branch retinal vein occlusion, and corneal
neovascularization. Retinal angiogenic disease, e.g., age-related
macular degeneration (AMD), is the most frequent cause of legal
blindness in the elderly in industrialized countries (Van Leeuwen
et al. (2003), European Journal of Epidemiology 18: 845-854). It is
a heterogeneous disease, which is characterized by progressive loss
of central, high acuity vision. For the patient it dramatically
compromises quality of life, as they lose their ability to read, to
recognize faces, and day-to-day tasks become major obstacles.
According to the World Health Organization (WHO) a total of 30-50
million individuals are affected and about 14 million people are
blind or severe visual impairment due to AMD (Gehrs et al., (2006)
Annals of Medicine 38:450-471).
[0004] The pathological process responsible for retinal angiogenic
diseases is the formation of chaotically oriented and
physiologically deficient new blood vessels under the retina, known
as choroidal neovascularization (CNV). Although aging, oxidative
stress, genetics and inflammation have all been described to
contribute to the pathogenesis of CNV; angiogenesis is currently
believed to be responsible for the final common pathway.
[0005] Current treatment options for AMD include laser therapy,
surgery to remove or destroy the abnormal blood vessels, and
anti-angiogenic therapies, e.g., anti-vascular endothelial growth
factor ("VEGF"), i.e., anti-VEGF therapies. These anti-angiogenic
medications are typically injected into the vitreous body of the
eye, which cause great discomfort and inconvenience to the
patients. In addition, some patients have developed resistance to
anti-VEGF therapy and are in need of other treatment options. Yang
et al., Drug. Des. Devel. Ther. 2016: 10:1857-1867.
BRIEF SUMMARY
[0006] Provided herein is a method of treating a retinal angiogenic
disease in a subject. The methods include administering to the
subject a pharmaceutically effective amount of a composition
comprising a peptide having the sequence Thr Pro His Thr His Asn
Arg Thr Pro Glu (SEQ ID NO:1). The composition can be administered
to the subject orally, by intravenous injection, or by intravitreal
injection, or by sublingual delivery wherein administration treats
the retinal angiogenic disease in the subject.
[0007] Optionally, the composition comprises 2 to 50 mg/kg body
weight (Bwt) of the peptide and is administered by intravenous
injection. Optionally, the composition comprises 0.1 .mu.g/kg to 5
mg/kg Bwt of the peptide and is administered by intravitreal
injection. Optionally, the composition comprises 2 to 10 mg/kg Bwt
of the peptide and is administered orally.
[0008] Optionally, the composition is administered via either
intravenous (IV) or intravitreal (IVT) route at least once every 4
to 24 weeks. Optionally, the composition is administered orally at
least once daily for 1 to 2 weeks at intervals of 6 months. In this
treatment protocol, the composition is not administered during the
6 month interval. Optionally, for the subject not responsive to
anti-VEGF therapy, for example, a VEGF antibody.
[0009] Optionally, the subject has age-related macular
degeneration, retinopathy, or vascular occlusion. Optionally, the
subject has diabetic retinopathy, diabetic macular edema, central
retinal vein occlusion, branch retinal vein occlusion, or corneal
neovascularization. Optionally, the subject is a human.
[0010] Also provided herein is a method of treating a retinal
angiogenic disease in a subject. The methods include selecting a
subject with retinal angiogenic disease not responsive to an
anti-angiogenesis therapy, and administering to the subject a
composition comprising a peptide having the sequence SEQ ID NO:1,
wherein administration treats the retinal angiogenic disease in the
subject. Optionally, the subject is not responsive to an anti-VEGF
therapy.
[0011] Optionally, the composition disclosed herein is formulated
for intravenous administration, for intravitreal injection, or for
oral administration. Optionally, the subject has age-related
macular degeneration, retinopathy, or vascular occlusion.
Optionally, the subject has diabetic retinopathy, diabetic macular
edema, central retinal vein occlusion, branch retinal vein
occlusion, or corneal neovascularization. Optionally, the subject
is not responsive to anti-angiogenesis therapy. Optionally, the
subject is not responsive to anti-VEGF therapy, e.g., a VEGF
antibody. Optionally, the subject is a human.
[0012] Also provided is a method of treating a retinal angiogenic
disease in a subject. The method includes administering to the
subject a pharmaceutically effective amount of a composition
comprising a peptide N having the sequence Thr Pro His Thr His Asn
Xaa Thr Pro Glu wherein Xaa is homoarginine (SEQ ID NO:3). The
composition can be administered to the subject orally, by
intravenous injection, or by intravitreal injection, wherein
administration treats the retinal angiogenic disease in the
subject.
[0013] Also provided is a method of treating a retinal angiogenic
disease in a subject. The method includes administering to the
subject a pharmaceutically effective amount of a composition
comprising a peptide Q having the sequence Thr Pro His Thr His Gin
Xaa Thr Pro Glu wherein Xaa is homoarginine (SEQ ID NO:4), wherein
the composition is administered to the subject orally, by
intravenous injection, or by intravitreal injection, wherein
administration treats the retinal angiogenic disease in the
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A, 1B, 1C and 1D show the efficacy of a single IV
dose of angio-3 in attenuating the retinal angiogenesis in KIMBA
mice, which have over expression of human VEGF (KIMBA-hVEGF
Transgenic). FIG. 1A is a schematic of the experimental design.
[0015] FIG. 1B are images showing Fundus Fluorescein Angiography
(FFA) images of baseline and weekly after post treatment. Digital
color fundus photographs were taken using a MICRON IV comprehensive
system for rodent retinal imaging (Phoenix Research Labs,
Pleasanton, Calif.) after pupil dilatation with topical
administration of 1% tropicamide (Alcon Laboratories, Inc., Fort
Worth, Tex.) and 2.5% phenylephrine (Bausch and Lomb
Pharmaceuticals, Inc., Tampa, Fla.) ophthalmic solutions. For FFA,
mice were injected intraperitoneally with 10% sodium fluorescein
dye at a dose of 0.01 mL/5-6 g BWt and fundus images were obtained
using MICRON IV. FIG. 1C is a graph showing regression of leakage
in Kimba mice by each eye and by batch. IV single injection of 100
.mu.L of Angio-3 was given at week 4 and followed for further 4
weeks. (25 mg/Kg Bwt). Readout is the leakage of retinal
vascularization post-administration of the drug. n=6 eyes per
batch; n=3 batches; Values are expressed as means.+-.s.e.m.,
**P<0.01, *P<0.05, student's t-test. FIG. 1D are images
showing Isolectin Retinal staining. Control and Angio-3 treated
eyes were enucleated and fixed in 4% paraformaldehyde (PFA) in PBS
for 15 minutes at room temperature. The eyes were then transferred
to cold 1.times. PBS on ice for 5-10 minutes. The neural retina and
choroid/RPE were dissected separately and placed in cold
(-70.degree. C.) ethanol. Retinas were then rinsed in PBS and
blocked in 1% Triton-X/PBS for 30 min. The whole mounts were then
incubated with Isolectin GS-IB4 from Griffonia Simplicofolia, Alexa
Fluor 594 Conjugate (Molecular Probes, 121413 1:100) overnight at
4.degree. C. Stained whole mounts were flat-mounted with Prolong
Gold (Invitrogen) and left overnight. All imaging was performed
with a laser-scanning confocal fluorescence microscope.
[0016] FIGS. 2A, 2B, 2C and 2D show the efficacy of a single IVT
dose of angio-3 and Eylea (anti-VEGF), positive control in
attenuating the retinal angiogenesis in KIMBA mice. FIG. 2A is a
schematic of the experimental design. FIG. 2B are images showing
Fundus Fluorescein Angiography (FFA) images of baseline and weekly
after post treatment. FIG. 2C is a graph showing regression of
leakage in Kimba mice by each eye. FIG. 2D are graphs showing
regression of leakage in Kimba mice by batch. IVT single injection
of 1 .mu.g in 1 .mu.L of Angio-3 scramble; Angio-3 peptide and
Eylea was given at week 7 and followed for further 16 weeks.
Readout is the leakage of retinal vascularization
post-administration of the drug. n=10 eyes per batch; n=2 batches;
Values are expressed as means.+-.s.e.m., **P<0.01, *P<0.05,
student's t-test. The results showed that treating with Eylea
(positive control) is effective in attenuating retinal angiogenesis
for 4 weeks post treatment and treating the mice with angio-3 is
effective in attenuating retinal angiogenesis for 16 weeks post
treatment.
[0017] FIG. 3A is a schematic of the experimental design on the
efficacy of Angio-3 in a laser-induced choroidal neovascularization
(CNV) model in mice (B6J WT mice). FIG. 3B are FFA images of
baseline and at different time points post treatment. IV single
injection of 100 .mu.L of Angio-3 (25 mg/Kg Bwt) was given at Day 0
(prevention mode) and 4 days after laser (regression mode) and
followed for 10 weeks. FIG. 3C is a graph showing the area of
leakage by each eye for various groups. FIG. 3D are graphs of the
mean area of leakage of all batches for each group. Single dose of
Angio-3 decreased the lesions in a laser-induced mice CNV model in
both prevention and regression mode via IV route till 10 weeks and
this was very significant in prevention mode. The attenuation
retinal vascularization was better than scrambled peptide. n=6 eyes
per batch; n=3 batches. Values are expressed as means.+-.s.e.m.,
**P<0.01, *P<0.05, student's t-test. FIG. 3E is a graph of
the result of the Retinal Function Test that measures ERG changes
in Angio-3 treated and control. The a- and b-wave amplitude of the
Angio-3 treated group, however, was similar to that of baseline
values after weak 1.
[0018] FIG. 4A is a schematic of the experimental design on the
efficacy of Angio-3 by IVT administration. FIG. 4B are graphs of
the area of leakage by each eye for various groups and FIG. 4C
shows the mean area of leakage of all batches for each group. This
figure illustrates that a single dose Angio-3 significantly reduced
angiogenesis as compared to Eylea (anti-VEGF) and scrambled peptide
in a laser-induced mice CNV model via IVT route. n=6 eyes per
batch; n=3 batches; Values are expressed as means.+-.s.e.m.,
**P<0.01, *P<0.05, student's t-test.
[0019] FIG. 5A is a schematic of the experimental design on the
efficacy of Angio-3 in a laser-induced CNV model by sub-lingual
route. FIG. 5B are images showing Angio-3 significantly reduced
angiogenesis than murine anti-VEGF or scrambled peptide in
laser-induced mice CNV model via sub-lingual route. FIG. 5C is a
graph of the mean area of leakage by groups and FIG. 5D shows the
mean area of leakage of all batches for each group. n=6 eyes per
batch; n=3 batches. Values are expressed as means.+-.s.e.m.,
**P<0.01, *P<0.05, student's t-test.
[0020] FIG. 6A is a schematic of the experimental design on the
efficacy of Angio-3 in a laser-induced CNV model by IVT route in a
prevention mode. FIG. 6B are graphs showing a single dose Angio-3
significantly reduced angiogenesis as compared to Eylea (anti-VEGF)
in laser-induced rat CNV model via IVT route. n=10 rats per group;
Values are expressed as means.+-.s.e.m., n=3 independent
experiment, **P<0.01, *P<0.05, student's t-test.
[0021] FIG. 7A is a schematic of the study design on the efficacy
of Angio-3 in a monkey model of laser-induced CNV. FIG. 7B are
images of fundus fluorescein angiography ("FFA") of the eye in
monkeys treated with a single dose (2 mg) of Angio-3, anti-VEGF
(Eylea and Lucentis) in laser induced monkey CNV model via IVT
route. FIG. 7C is a graph showing regression of leakage was plotted
by each eye in each group before and after treatment. n=2 monkeys
per group; Values are expressed as means.+-.s.e.m., ***P<0.001,
*P<0.05, student's t-test.
[0022] FIG. 8 is a graph showing severity of grade and percentage
of grades in monkeys treated as described in FIG. 7. Following a
single IVT dose of Angio-3, Eylea and Lucentis.RTM., the test
article formulations were well tolerated and all animals appeared
generally healthy. A few animals developed intraocular
inflammation, mostly short-lived, in the study eye after
intravitreal injection in all groups. In this intervention study,
no change in lesion severity was observed for the vehicle control
and the change in lesion severity was significantly different for
all treatments compared with vehicle control.
[0023] FIG. 9 is a graph showing results of laser spot area
thickness and volume in monkeys before and after administration of
Eylea or Angio-3. The amount of Angio-3 or Eyelea is the same as
described in FIG. 7. The laser spot area was significantly reduced
after treatment as compared to pre-treatment in both groups.
[0024] FIG. 10 are images of araldite retina sections stained with
toluidine blue (Magnification 20.times.). The eyes were removed,
postfixed for 2 to 3 days in half-strength Karnovsky fixative, and
then stored in formalin until processed. Strips of tissue
containing 1 or 2 lesion sites were embedded in plastic. Sections 2
.mu.m thick were taken at 30-.mu.m steps through the middle of each
lesion. The sections were stained with toluidine blue, and the
sample with the most robust lesion was designated as the central
cut. This section was then evaluated by an observer (R.R.D.) masked
to the treatment condition. A tissue proliferation score were
calculated for each lesion based on 3 criteria: the size of the
spindle cell proliferative lesion, the extent of new blood vessel
proliferation in the subretinal space, and the elevation of the
retina above the choriocapillaris. Vehicle treated (control)
sections are thicker and more vascular compared with the
drug-treated eyes.
[0025] Control sections showed more choroidal fibroplasia,
increased retinal thickness, more choroidal neovascularization,
multiple vessels extending once or twice the retinal thickness and
retinal elevation as compared to drug-treated eyes.
[0026] FIG. 11 A are FFA images of baseline and at different time
points post treatment. Mice received single IVT injection of
Angio-3 (at a dose of 1 .mu.g in 1 .mu.l or 10 ng in 1 .mu.l), and
chemically modified Angio-3 peptides Pep-N (at doses of 5 .mu.g in
.mu.l; 1 .mu.g in .mu.l; and 100 ng in 1 .mu.l, respectively) and
Pep-Q peptides (at doses of 5 .mu.g in .mu.l; 1 .mu.g in .mu.l; and
100 ng in 1 .mu.l, respectively). FIG. 11 B is a graph of the area
of leakage by each eye. Single dose attenuates retinal angiogenesis
in laser-induced mice CNV model via IVT route till 4 weeks. All
three peptides were able to significantly attenuate the choroidal
angiogenesis in laser-induced CNV mouse model. Eylea is the
positive control. n=18 eyes per group; Values are expressed as
means.+-.s.e.m. n=3 independent experiment, **P<0.01,
*P<0.05, student's t-test.
[0027] FIGS. 12A and 12B illustrates effect of Angio-3 on VEGF
induced cell proliferation of HRMECs. FIG. 12A is a graph showing
the effect of Angio-3 in combination of 10 ng/ml VEGF. FIG. 12B is
a graph showing the effect of Angio-3 in combination of 50 ng/ml
VEGF.
[0028] FIGS. 13A and 13B show effect of Angio-3 on HRMEC cells
migration in the presence of: 1) in EBM-2 alone, 2) EBM-2
supplemented with 50 ng/ml VEGF, 3) EBM-2 supplemented with 300
ug/ml Avastin plus 50 g/ml VEGF, 4) EBM-2 supplemented with 300
ug/ml Angio3 plus 50 g/ml VEGF, and 5) EBM-2 supplemented with 600
ug/ml Angio3 plus 50 g/ml VEGF. FIG. 13B are FFA images and FIG.
13A is a graph of the number of cells in microscopic fields under
100.times. objective based on the images of FIG. 13B.
[0029] FIGS. 14A and 14B show effect of Angio-3 on VEGF induced
HRMECs tube formation in the presence of: 1) in EBM-2 alone, 2)
EBM-2 supplemented with 50 ng/ml VEGF, 3) EBM-2 supplemented with
300 ug/ml Avastin plus 50 g/ml VEGF, 4) EBM-2 supplemented with 300
ug/ml Angio3 plus 50 g/ml VEGF, and 5) EBM-2 supplemented with 600
ug/ml Angio3 plus 50 g/ml VEGF. FIG. 14B are FFA images of cells.
FIG. 14A is a graph of quantifications of total tube length, number
of junctions, and total number of loops based on the images in FIG.
14B. **P<0.01, *P<0.05. Unless explicitly noted otherwise,
the VEGF peptide disclosed in this application refers to
VEGF.sub.165, a subtype of human VEGF that has the most potent
biological activity and is the most abundantly present in vivo.
[0030] FIGS. 15A and 15B are graphs showing that Angio-3 induced
human umbilical vein endothelial cells (HUVECs) apoptosis in the
presence of either VEGF or bFGF. FIG. 15A shows Angio-3 induced
HUVEC apoptosis in the presence of 20 ng/ml VEGF in a
dose-dependent manner. FIG. 15B shows that Angio-3 induced HUVEC
apoptosis in the presence of 20 ng/ml bFGF in a dose-dependent
manner. * represents p<0.05, n=3.
[0031] FIGS. 16A and 16B are graphs showing Angio-3 inhibited HUVEC
proliferation stimulated by VEGF and bFGF. FIG. 16A shows Angio-3
suppressed HUVEC proliferation induced by 20 ng/ml VEGF in a
dose-dependent manner. FIG. 16B shows Angio-3 suppresses HUVEC
proliferation induced by 20 ng/ml bFGF in a dose-dependent manner.
* represents p<0.05, n=3.
[0032] FIGS. 17A, 17B, 17C and 17D show that Angio-3 inhibited VEGF
and bFGF-induced EC migration and inhibits capillary network
formation. FIG. 17 A are images and 17 C is a graph showing Angio-3
suppressed HUVEC chemotactic migration induced by 20 ng/ml VEGF in
a dose-dependent manner. Migrated cells were stained with Hoechst,
imaged and counted. FIG. 17 B are images and 17 D is a graph
showing Angio-3 suppressed HUVEC chemotactic migration that was
induced by 20 ng/ml bFGF in a dose-dependent manner. Migrated cells
were stained with Hoechst, imaged and counted. n=3; * represents
significant reduction compared to control at P<0.05 by one-way
ANOVA.
[0033] FIGS. 18A and 18B show that Angio-3 inhibited HUVEC
capillary network formation on Matrigel. FIG. 18A are
representative images of HUVEC tube formation on Matrigel. HUVECs
were pre-incubated with increasing doses of Angio-3 for 30 min
prior to seeding on Matrigel. FIG. 18B is a graph of percentage
area covered by HUVEC tubes. n=3; * represents significant
reduction compared to control at P<0.05 by one-way ANOVA.
[0034] FIGS. 19A, 19B and 19C are graphs showing that Angio-3 is a
novel anti-permeability agent that can inhibit VEGF-induced
vascular permeability (VP) with multiple endothelial cell types. In
FIG. 19A, post-confluent HUVEC monolayers were treated with
increasing concentrations of Angio-3 or medium alone. The results
show that Angio-3 inhibited VEGF-induced permeability across
confluent HUVECs in a dose-dependent manner without affecting the
basal level permeability. In these experiments, post-confluent
HUVEC monolayers were pre-treated with Angio-3 for 30 minutes prior
to stimulation with 100 ng/ml VEGF. In FIG. 19B, Post-confluent
HMVEC monolayers were treated with increasing concentrations of
Angio-3 or medium alone. The results show that Angio-3 inhibited
VEGF-induced permeability across confluent human dermal
microvascular endothelial cells (HMVECs) in a dose-dependent manner
without affecting the basal level permeability. In FIG. 19C,
post-confluent HREC monolayers were treated with Angio-3 and VEGF
for 3 h. The results show that Angio-3 inhibited VEGF-induced
permeability across confluent human retinal endothelial cells
(HRECs) in a dose-dependent manner without affecting the basal
level permeability. * represents p<0.05, n=3.
[0035] FIGS. 20A and 20B are images and a graph, respectively,
showing that Angio-3 inhibited local VEGF-induced dermal vascular
permeability in mice. In FIG. 20A, Angio-3 was administered via
intradermal injection to mice and the results show that Angio-3
inhibited VEGF-induced dermal permeability in a dose-dependent
manner within 15 min. The dermal permeability was visualized by
Evans blue dye extravasation. In FIG. 20B, dye extravasation was
quantified by formamide extraction of the dye and measuring OD 610.
n=5 animals per group, * represent significantly increased as
compared with the simultaneous control at p<0.05.
[0036] FIGS. 21A, 21B, 21C and 21D are images showing Angio-3
prevented VEGF-induced dissociation of Vascular endothelial
("VE")-cadherin from Adherens junctions (AJs) on HUVECs. Angio-3
protected VE-cadherin from VEGF-induced dissociation from cell-cell
AJ. Confluent HUVEC monolayers were pre-treated with 100 .mu.M
Angio-3 for 30 min following which the monolayers were stimulated
with 100 ng/ml VEGF for 20 min. Cells were then fixed,
permeabilized and probed for VE-cadherin. FIGS. 21A-D shows control
cells, cells treated with 100 ng/ml VEGF, cells treated with 100 uM
Angio-3, and cells treated with 100 ng/ml VEGF plus 100 uM Angio-3,
respectively.
[0037] FIGS. 22A and 22B are images showing Angio-3 suppressed
VEGF-induced dissociation of tight junction (TJ) proteins ZO-1 and
ZO-2 from TJs in HUVECs. Cells were treated under control, 100
ng/ml VEGF, 100 .mu.M Angio-3, and 100 ng/ml VEGF plus 100 .mu.M
Angio-3, respectively.
[0038] FIGS. 23A, 23B, 23C and 23D are images showing Angio-3
suppressed VEGF-induced actin stress fiber formation in HUVECs.
However, in the absence of VEGF, Angio-3 promoted cortical actin
fiber formation. The red color represents action staining and the
blue represent DAPI staining.
[0039] FIG. 24 are graphs showing the results of retinal function
tests recorded via electroretinogram (ERG). Untreated mice has no
response of both a and b-waves after 12 weeks of age. Angio-3
increased the a and b-wave responses for 6 weeks post treatment and
then again increased the response for 20 weeks with second dose.
This result shows that Angio-3 is rescuing the retinal function in
KIMBA mice. Data is represented as mean.+-.S.D. **=P<0.05;
***=P<0.01.
[0040] FIGS. 25A, 25B, 25C, 25D, 25E, and 25F show the results of
testing in a laser-induced chorodial neovascularization (CNV) model
in Cynomolgus moneys developed as an experimental model of wet AMD.
FIG. 25A is a schematic of the study design. FIG. 25B are
representative fundus fluorescein angiography (FFA) images of all
groups. FIG. 25C is a graph showing change in mean lesion grade of
the eye in monkeys treated with a single dose 2 mg of Angio-3, dose
2 mg of anti-VEGF (Eylea), 2 mg of peptide (Q2) and 4 mg of peptide
(Q4) and control. FIG. 25D is a graph of percentage of all grades
pre and post treatment of Angio-3, PepQ-low dose (2 mg); PepQ-high
dose (4 mg), Eylea and control eyes that was tested in laser
induced choroidal neo-vascularization non-human primate model. FIG.
25E is a graph of laser area quantified from the FFA images by
ImageJ software. FIG. 25F are graphs of laser volume quantified
from PS-OCT images by ImageJ software. All 4 treated groups
significantly reduces leakage and neovessel area as compared to
vehicle control. Eylea shows the superior efficacy as compared to
test peptides. However, Pep-Q shows dose dependent efficacy and
higher dose efficacy is as close to Eylea.
[0041] FIGS. 26A and 26B are images showing progression of corneal
neovascularization seven days after alkali-burn injury. FIG. 26A
are representative image of a vehicle treated eye. FIG. 26B are
representative image of an eye treated three times per day with our
compound of interest (PeptideQ). White arrow indicates the
difference in corneal opacity and neovascularization.
[0042] FIG. 27 is a graph of the area of corneal neovascularization
(NV) seven days after alkali-burn injury was quantified by ImageJ
software. Peptide Q treated eyes were significantly reduced corneal
NV area as compared to vehicle control eyes.
[0043] FIGS. 28A and 28B are images and a graph showing results of
testing wound healing in a mouse model. FIG. 28A are representative
slitlamp biomicroscopy images of murine cornea following the
removal of corneal epithelium from Angio-3 treated and control
wild-type mice and topical fluorescein staining of the epithelial
defect (green). FIG. 28B is a graph of the percentage of wound
defect remaining (vertical axis) over time (horizontal axis) in
Angio-3 treated and control wild-type mice, n=6 for each group from
2 independent experiments. Both groups are statistically
significant. Angio-3 didn't affect the normal wound healing
process. Data is represented as mean.+-.S.D.
DETAILED DESCRIPTION
[0044] Provided herein are compositions and methods for treating
subjects having a retinal angiogenic disease by administering a
composition comprising Angio-3 peptides. The Angio-3 peptide acts
to block the formation of new vessels in patients having retinal
angiogenic disease and is particularly useful for patients who are
not responsive to anti-VEGF therapies. The therapy can be
conveniently delivered orally or by intravenous injection, or by
intravitreal injection.
[0045] The term "about" when used in conjunction with a value means
any value that is reasonably close to the value, i.e., within the
range of +10% of the value. In particular, it would include the
value itself. For example, both a value of 45 mg/kg and a value of
55 mg/kg are deemed to be "about 50 mg/kg".
[0046] The terms "subject", "patient" or "individual" are used
herein interchangeably to refer to a human or animal. For example,
the animal subject may be a mammal, a primate (e.g., a monkey), a
livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat),
a companion animal (e.g., a dog, a cat), a laboratory test animal
(e.g., a mouse, a rat, a guinea pig, a bird), an animal of
veterinary significance, or an animal of economic significance.
[0047] As used herein, the terms "not responsive," and
"non-responsive" to a treatment are used herein interchangeably to
refer to a condition in which a patient or subject does not respond
to a particular treatment or does not obtain a desired benefit
after treatment for a particular disease. The terms "not
responsive" and "non-responsive" include conditions in which a
subject receives a treatment but does not experience a reduction in
at least one symptom associated with the disease in the absence of
the treatment. By way of example, a subject is not responsive to an
anti-VEGF therapy if new blood vessels under the retinal of the
patient continue to form despite receiving an anti-VEGF therapy or
if a new blood vessel continues to grow while receiving the
treatment.
[0048] As used herein, the term "non-responder" refers to a subject
that is administered a therapeutic treatment for a particular
disease but does not respond to or obtain benefit from the therapy.
The term refers to subjects that do not experience a reduction in
at least one symptom associates with the disease, e.g., a retinal
angiogenic disease.
[0049] As used herein, "treating" or "treatment of" a condition,
disease or disorder or symptoms associated with a condition,
disease or disorder refers to an approach for obtaining beneficial
or desired results, including clinical results. Beneficial or
desired clinical results can include, but are not limited to,
alleviation or amelioration of one or more symptoms or conditions,
diminishment of extent of condition, disorder or disease,
stabilisation of the state of condition, disorder or disease,
prevention of development of condition, disorder or disease,
prevention of spread of condition, disorder or disease, delay or
slowing of condition, disorder or disease progression, delay or
slowing of condition, disorder or disease onset, amelioration or
palliation of the condition, disorder or disease state, and
remission, whether partial or total. "Treating" can also mean
prolonging survival of a subject beyond that expected in the
absence of treatment. "Treating" can also mean inhibiting the
progression of the condition, disorder or disease, slowing the
progression of the condition, disorder or disease temporarily,
although in some instances, it involves halting the progression of
the condition, disorder or disease permanently. As used herein the
terms treatment, treat, or treating refers to a method of reducing
the effects of one or more symptoms of a disease or condition
characterized by expression of the protease or symptom of the
disease or condition characterized by expression of the protease.
Thus in the disclosed method, treatment can refer to a 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the
severity of an established disease, condition, or symptom of the
disease or condition. For example, a method for treating a disease
is considered to be a treatment if there is a 10% reduction in one
or more symptoms of the disease in a subject as compared to a
control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, or any percent reduction in between 10% and
100% as compared to native or control levels. It is understood that
treatment does not necessarily refer to a cure or complete ablation
of the disease, condition, or symptoms of the disease or condition.
Further, as used herein, references to decreasing, reducing, or
inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or greater as compared to a control level and such terms
can include but do not necessarily include complete
elimination.
[0050] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to include a polymer of amino acid residues.
The terms apply to amino acid polymers in which one or more amino
acid residue is an artificial chemical mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers and non-naturally occurring amino acid
polymers. As used herein, the terms encompass amino acid chains of
any length, including full-length proteins (i.e., antigens),
wherein the amino acid residues are linked by covalent peptide
bonds.
[0051] The term "amino acid" includes naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs include compounds that have the
same basic chemical structure as a naturally occurring amino acid,
i.e., an .alpha. carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. "Amino acid mimetics" include
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0052] Amino acids may be referred to herein by either the commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
[0053] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions and/or additions to a nucleic
acid, peptide, polypeptide, or protein sequence which alters, adds
or deletes a single amino acid or a small percentage of amino acids
in the encoded sequence is a "conservatively modified variant"
where the alteration results in the substitution of an amino acid
with a chemically similar amino acid. Conservative substitution
tables providing functionally similar amino acids are well known in
the art. Such conservatively modified variants are in addition to
and do not exclude polymorphic variants, interspecies homologs,
and/or alleles.
[0054] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0055] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
[0056] (see, e.g., Creighton, Proteins (1984)).
[0057] The term "therapeutically effective amount" or "effective
mount" includes an amount or quantity effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0058] The term "administering" includes oral administration,
topical contact, administration as a suppository, intravenous,
intraperitoneal, intramuscular, intralesional, intrathecal,
intranasal, or subcutaneous administration, or the implantation of
a slow-release device, e.g., a mini-osmotic pump, to a subject.
Administration is by any route, including parenteral and
transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal,
vaginal, rectal, or transdermal). Parenteral administration
includes, e.g., intravenous, intramuscular, intra-arteriole,
intradermal, subcutaneous, intraperitoneal, intraventricular, and
intracranial. Other modes of delivery include, but are not limited
to, the use of liposomal formulations, intravenous infusion,
transdermal patches, etc. One skilled in the art will know of
additional methods for administering a therapeutically effective
amount of a fusion protein described herein.
[0059] Described herein are methods to treat subjects suffering
from a number of retinal angiogenic diseases, e.g., age-related
macular degeneration, retinopathy, or vascular occlusion.
Retinopathy refers to a disease of the retina that results in
impairment or loss of vision. Age-related macular degeneration
("AMD") is characterized by the invasion of new blood vessels into
different-structures of the eye such as macular and retinal pigment
epithelium. Vascular occlusion is a blockage in the retinal blood
vessels, arteries or veins. Optionally, the subject has diabetic
retinopathy, diabetic macular edema, central retinal vein
occlusion, branch retinal vein occlusion, or corneal
neovascularization. Subjects having one of these conditions may
experience one or more of the following symptoms, visual field
defects, difficulty to see textures and subtle changes in the
environment, and difficulty to adjust for changing light levels,
and impaired depth perception. Retinal angiogenic diseases can be
diagnosed by a trained optometrist or other medical professional
using methods well known in the art to examine the blood vessel
formation under the retina, such as Dilated Eye Exam,
Autofluorescence, Fundus Photography, Fluorescein Angiography,
Optical Coherence Tomography (OCT), Tonometry, Fundoscopy or
Ophthalmoscopy
[0060] Optionally, the subject treated with the Angio-3 peptide was
previously determined to be non-responsive to anti-angiogenesis
therapies, e.g., anti-VEGF therapies. As used herein,
anti-angiogenesis therapy refers to a therapy that blocks
angiogenesis. As used herein, an anti-VEGF therapy refers to a
therapy that blocks one or more VEGF function. Non-limiting
examples of anti-angiogenesis therapies include pegaptanib
(Macugen.TM.. by Pfizer), ranibizumab (Lucentis.TM. by Genentech)
bevacizumab (Avastin.TM. by Genentech), carboxyamidotriazole,
TNP-470, CM101, IFN-.alpha., IL-12, platelet factor 4, suramin,
SU5416, thrombospondin, VEGFR antagonists, angiostatic
steroids+heparin, cartilage-derived angiogenesis inhibitory factor,
matrix metallopreteinase inhibitors, angiostatin, endostatin,
2-methoxyestradiol, tecogalan, prolactin, alpha.sub.V.beta.sub.3
inhibitors, linomide, VEGF-Trap (by Regeneron Pharmaceuticals),
Aminosterols (Evizion.RTM. by Genera Corp.), Cortisen (Retaane.RTM.
by Alcon), tyrosine kinase inhibitors, anti-angiogenic siRNA,
inhibitors of the complement system, gentherapeutic therapies (e.g.
AdPEDF.11 by Genvec (Gaithersburg, Md.). Optionally, the anti-VEGF
therapy is an anti-VEGF antibody, for example bevacizumab that are
commercially available.
[0061] Optionally, the method of treating a retinal angiogenic
disease comprises selecting a subject that has been diagnosed with
a retinal angiogenic disease but is not responsive to anti-VEGF
therapy. Administering the composition comprises an Angio-3 peptide
disclosed herein, e.g., any one of SEQ ID Nos 1-4, or any
combination thereof.
[0062] Angiostatin is a fragment of plasmin, which is a fragment of
plasminogen. Plasminogen (UniProt No. P00747) contains five
homologous repeats that form looped "kringle" structures held
together by disulfide bonds. Plasminogen binds to fibrin through
lysine binding sites located on the five kringle domains (k1
through k5) (Folkman et al, Nature Medicine, vol. 1, No. 1, pp.
27-31 1999). Each kringle domain is about 80 amino acid residues in
length and different kringle domains are highly homologous to each
other in amino acid sequences. Angio-3, is derived from the kringle
domain k4 of plasminogen and is an angiogenesis inhibitor, i.e., it
blocks the growth of new blood vessels, and has
anti-inflammatory/anti-angiogenic activity. Angio-3 signaling
pathway mediates the switch between a quiescent and an activated
(i.e., angiogenic) endothelium. Unlike Vascular Endothelial Growth
Factor (VEGF), which appears to uniformly promote angiogenesis,
Angio-3 appears to have differing functions depending on
endothelial cell context, with both cell-to-cell and cell-to-matrix
contacts modulating the resulting signals.
[0063] It is believed that in many cases, patients having retinal
angiogenic disease are not responsive to VEGF inhibitor therapy or
become non-responsive after a period of time of treatment resulting
in protection of VEGF-dependent endothelium. As described herein,
delivering Angio-3 can disrupt the formation of new vessels and
patients may be able to overcome resistance to anti-VEGF therapies.
In particular, administering Angio-3 peptides can attenuate the
retinal and/or choroidal angiogenesis and/or reduce lesion area of
leakage in the eye caused by retinal angiogenesis.
[0064] The Angio-3 peptide that can be used to treat a retinal
angiogenic disease can be the native Angio-3 peptide, which has a
sequence of Thr Pro His Thr His Asn Arg Thr Pro Glu (SEQ ID NO: 1).
The Angio-3 peptide can also be a peptide that contains
modifications from SEQ ID NO:1 and yet retains the function of
blocking angiogenesis. Some exemplar modifications to the peptide
include sequence modifications and chemical modifications.
[0065] The polynucleotide sequences may encode Angio-3 polypeptides
including those sequences with deletions, insertions, or
substitutions of different nucleotides, which result in a
polynucleotide encoding a polypeptide with at least one functional
characteristic of the instant polypeptides, as described herein.
Included within this definition are polymorphisms which may or may
not be readily detectable using a particular oligonucleotide probe
of the polynucleotide encoding the instant polypeptides, and
improper or unexpected hybridization to allelic variants, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding the instant polypeptides.
[0066] Sequence alterations that do not change the amino acid
sequence encoded by the polynucleotide are termed "silent"
variations. Due to the degeneracy of the genetic code, with the
exception of the codons ATG and TGG, encoding methionine and
tryptophan, respectively, any of the possible codons for the same
amino acid can be substituted by a variety of techniques, for
example, site-directed mutagenesis, available in the art.
Accordingly, any and all such variations of a sequence selected
from the Sequence Listing are a feature of the instant
disclosure.
[0067] In addition to silent variations, other conservative
variations that alter one or a few amino acid residues in the
Angio-3 peptide, can be made without altering the function of the
polypeptide. For example, substitutions, deletions and insertions
introduced into the sequences provided herein are also envisioned.
Amino acid substitutions are typically of single residues;
insertions usually will be on the order of about from 1 to 10 amino
acid residues; and deletions will range about from 1 to 30
residues. In preferred embodiments, deletions or insertions are
made in adjacent pairs, for example, a deletion of two residues or
insertion of two residues. Substitutions, deletions, insertions or
any combination thereof can be combined to arrive at a sequence.
The mutations that are made in the polynucleotide encoding the
peptide should not place the sequence out of the reading frame and
should not create complementary regions that could produce
secondary mRNA structure. Preferably, the polypeptide encoded by
the DNA performs the desired function.
[0068] Conservative substitutions are those in which at least one
residue in the amino acid sequence has been removed and a different
residue inserted in its place. Such substitutions generally are
made when it is desired to maintain the activity of the protein.
Although all conservative amino acid substitutions (e.g., one basic
amino acid substituted for another basic amino acid) in a
polypeptide will not necessarily result in the polypeptide
retaining the same activity as the native polypeptide, it is
expected that many of these conservative mutations would result in
the polypeptide retaining its activity.
[0069] Sequence variants of the Angio-3 peptides can be produced by
modifying the respective wild-type sequences according to methods
well-known to the skilled in the art. Such methods include, but not
limited to, mutagenesis by PCR, which uses primers designed to
contain desired changes; nested primers to mutate a target region;
and inverse PCR, which amplifies a region of unknown sequence using
primers orientated in the reverse direction. Many other mutation
and evolution methods are also available and expected to be within
the skill of a person of ordinary skill in the relevant art.
Sequence variants of the Angio-3 peptide (SEQ ID NO: 1), as well as
the Angio-3 peptide itself, can also be synthesized in the
laboratory using methods well known in the art for peptide
synthesis.
[0070] Accordingly, the disclosure also provides a scrambled
Angio-3 peptide that has a sequence of Asn Thr Thr Glu Thr Pro His
Pro His Arg (SEQ ID NO:2), which is used as a negative control for
some of the studies disclosed herein.
[0071] Chemical or enzymatic alterations of expressed nucleic acids
and polypeptides can be performed by standard methods. For example,
sequences can be modified by the addition of lipids, sugars,
peptides, organic or inorganic compounds, by the inclusion of
modified nucleotides or amino acids, or the like. These methods can
be used to modify any given sequence, or to modify any sequence
produced by the various mutation and artificial evolution
modification methods described herein and known to those of skill
in the art.
[0072] The Angio-3 peptides disclosed herein may include natural
amino acids, and, optionally, post-translational modifications
thereof. However, in vitro peptide synthesis permits the use of
modified and/or non-natural amino acids. A table of exemplary, but
not limiting, modified and/or non-natural amino acids is provided
herein below.
TABLE-US-00001 TABLE 1 Modified Amino Acids Abbr. Amino Acid Abbr.
Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad
3-Aminoadipic acid Hyl Hydroxylsine BAla beta-alanine,
beta-Amino-propionic AHyl allo-Hydroxylysine acid Abu
2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid,
piperidinic 4Hyp 4-Hydroxyproline acid Acp 6-Aminocaproic acid Ide
Isodesmosine Ahe 2-Aminoheptanoic acid Aile allo-Isoleucine Aib
2-Aminoisobutyric acid MeGly N-Methylglycine, sarcosine BAib
3-Aminoisobutyric acid Melle N-Methylisoleucine Apm 2-Aminopimelic
acid MeLys 6-N-Methylly Dbu 2,4-Diaminobutyric acid MeVal
N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2'-Diaminopimelic
acid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine
EtGly N-Ethylglycine
[0073] Accordingly, the present disclosure provides for
modifications of any given nucleic acid by mutation, chemical or
enzymatic modification, or other available methods, as well as for
the products produced by practicing such methods, e.g., using the
sequences herein as a starting substrate for the various
modification approaches.
[0074] Optionally, the modification to the native Angio-3 is to
substitute arginine with a homoarginine. Homoarginine is the
methylene homologue of L-arginine (Arg). It is an amino acid
derivative and may increase nitric oxide availability and enhance
endothelial function. Thus, optionally, the Angio-3 peptide that
can be used to treat the retinal angiogenic diseases is Pep-N,
which has a sequence of Thr Pro His Thr His Asn Xaa Thr Pro Glu,
wherein Xaa is homoarginine (SEQ ID NO:3). Optionally, the Angio-3
peptide that can be used to treat the retinal angiogenic disease is
Pep-Q, which has a sequence of Thr Pro His Thr His Gin Xaa Thr Pro
Glu, wherein Xaa is homoarginine (SEQ ID NO:4).
[0075] The Angio-3 peptide may be prepared by methods known in the
art. These methods include synthetic peptide chemistry, recombinant
expression of the peptides of the disclosure using appropriate
prokaryotic or eukaryotic host cells and expression systems or
recombinant expression of the peptide as a feature of somatic gene
transfer, i.e., expression as part of the administration regimen at
the site of treatment.
[0076] Optionally, the peptide can be synthesized chemically using
standard peptide synthesis techniques, e.g., solid-phase or
solution-phase peptide synthesis. That is, the peptides disclosed
as SEQ ID NOs:1-4 may be chemically synthesized, for example, on a
solid support or in solution using compositions and methods well
known in the art, see, e.g., Fields, G. B. (1997) Solid-Phase
Peptide Synthesis. Academic Press, San Diego, incorporated by
reference in its entirety herein. Such standard peptide-preparation
techniques include, for example, solution synthesis or
Merrifield-type solid phase synthesis, Boc (tert.butyloxycarbonyl),
and the Fmoc (9-fluorenylmethyloxycarbonyl) strategies. Optionally,
the peptides are synthesized by solid phase Fmoc chemistry using
methods well known in the art (Ajikumar P K, Lakshminarayanan R,
Ong B T, Valiyaveettil S, Kini R M. Biomacromolecules. 2003
September-October; 4(5):1321-6).
[0077] Provided herein is a pharmaceutical composition including a
pharmaceutically acceptable excipient and an Angio-3 peptide. Also
provided is a method of administering the composition for treating
retinal angiogenic diseases, especially for subjects who are not
responsive to anti-angiogenesis therapies, e.g. anti-VEGF
therapies.
[0078] Pharmaceutical compositions or medicaments can be formulated
by standard techniques using one or more physiologically acceptable
carriers or excipients. Suitable pharmaceutical carriers are
described herein and in, e.g., "Remington: The Science and Practice
of Pharmacy, Twenty-First Edition" by E. W. Martin. The Angio-3
peptides and their physiologically acceptable salts and solvates
can be formulated for administration by any suitable route,
including, but not limited to, orally, topically, nasally,
rectally, parenterally (e.g., intravenously, subcutaneously,
intramuscularly, etc.), and combinations thereof. Optionally, the
therapeutic agent is dissolved in a liquid, for example, water.
[0079] For oral administration, a pharmaceutical composition or a
medicament disclosed herein can take the form of, e.g., a tablet or
a capsule prepared by conventional means with a pharmaceutically
acceptable excipient. Preferred are tablets and gelatin capsules
comprising the active ingredient(s), together with (a) diluents or
fillers, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,
cellulose (e.g., ethyl cellulose, microcrystalline cellulose),
glycine, pectin, polyacrylates and/or calcium hydrogen phosphate,
calcium sulfate, (b) lubricants, e.g., silica, anhydrous colloidal
silica, talcum, stearic acid, its magnesium or calcium salt (e.g.,
magnesium stearate or calcium stearate), metallic stearates,
colloidal silicon dioxide, hydrogenated vegetable oil, corn starch,
sodium benzoate, sodium acetate and/or polyethyleneglycol; for
tablets also (c) binders, e.g., magnesium aluminum silicate, starch
paste, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropyl
methylcellulose; if desired (d) disintegrants, e.g., starches
(e.g., potato starch or sodium starch), glycolate, agar, alginic
acid or its sodium salt, or effervescent mixtures; (e) wetting
agents, e.g., sodium lauryl sulfate, and/or (f) absorbents,
colorants, flavors and sweeteners. Optionally, the tablet contains
a mixture of hydroxypropyl methylcellulose, polyethyleneglycol 6000
and titatium dioxide. Tablets may be either film coated or enteric
coated according to methods known in the art.
[0080] Liquid preparations for oral administration can take the
form of, for example, solutions, syrups, or suspensions, or they
can be presented as a dry product for constitution with water or
other suitable vehicle before use. Such liquid preparations can be
prepared by conventional means with pharmaceutically acceptable
additives, for example, suspending agents, for example, sorbitol
syrup, cellulose derivatives, or hydrogenated edible fats;
emulsifying agents, for example, lecithin or acacia; non-aqueous
vehicles, for example, almond oil, oily esters, ethyl alcohol, or
fractionated vegetable oils; and preservatives, for example, methyl
or propyl-p-hydroxybenzoates or sorbic acid. The preparations can
also contain buffer salts, flavoring, coloring, and/or sweetening
agents as appropriate. If desired, preparations for oral
administration can be suitably formulated to give controlled
release of the active compound.
[0081] For topical administration, the compositions of the present
disclosure can be in the form of emulsions, lotions, gels, creams,
jellies, solutions, suspensions, ointments, and transdermal
patches. For delivery by inhalation, the composition can be
delivered as a dry powder or in liquid form via a nebulizer. For
parenteral administration, the compositions can be in the form of
sterile injectable solutions and sterile packaged powders.
Preferably, injectable solutions are formulated at a pH of about
4.5 to about 7.5.
[0082] The compositions can also be provided in a lyophilized form.
Such compositions may include a buffer, e.g., bicarbonate, for
reconstitution prior to administration, or the buffer may be
included in the lyophilized composition for reconstitution with,
e.g., water. The lyophilized composition may further comprise a
suitable vasoconstrictor, e.g., epinephrine. The lyophilized
composition can be provided in a syringe, optionally packaged in
combination with the buffer for reconstitution, such that the
reconstituted composition can be immediately administered to a
patient.
[0083] The compounds can be encapsulated in a controlled
drug-delivery system such as a pressure controlled delivery
capsule, a colon targeted delivery system, a osmotic controlled
drug delivery system, and the like. The pressure controlled
delivery capsule can contain an ethylcellulose membrane. The colon
target delivery system can contain a tablet core containing
lactulose which is over coated with an acid soluble material, e.g.,
Eudragit E.RTM., and then overcoated with an enteric material,
e.g., Eudragit L.RTM.. The osmotic controlled drug delivery system
can be a single or more osmotic unit encapsulated with a hard
gelatin capsule (e.g., capsule osmotic pump; commercially available
from, e.g., Alzet, Cupertino, Calif.). Typically, the osmotic unit
contains an osmotic push layer and a drug layer, both surrounded by
a semipermeable membrane.
[0084] Pharmaceutical compositions or medicaments can be
administered to a subject at a therapeutically effective dose to
treat a retinal angiogenic disease as described herein. Optionally,
the pharmaceutical composition or medicament is administered to a
subject in an amount sufficient to elicit an effective therapeutic
response in the subject.
[0085] Typically, a dosage of the active compounds is a dosage that
is sufficient to achieve the desired effect. Optimal dosing
schedules can be calculated from measurements of agent accumulation
in the body of a subject. Generally, administered dosages can vary
depending on a number of factors, including, but not limited to,
the subject's body weight, age, individual condition, surface area
or volume of the area to be contacted, and/or on the routes of
administration. The size of the dose will also be determined by the
existence, nature, and extent of any adverse effects that accompany
the administration of a particular compound in a particular
subject. Preferably, the smallest dose and concentration required
to produce the desired result should be used. Dosage should be
appropriately adjusted for children, the elderly, debilitated
patients, and patients with cardiac and/or liver disease. Further
guidance can be obtained from studies known in the art using
experimental animal models for evaluating dosage.
[0086] Optionally, the composition is administered by intravenous
injection. A unit dosage for intravenous administration to an
individual (e.g., human) may contain 2-50 mg of active ingredient
per 1 kg of body weight, which is also referred to as 2-50 mg/kg
Bwt. For example, for a patient of about 50 kg, the unit dosage may
contain 100 mg-2,500 mg of the active ingredient of Angio-3
peptide. Optionally, the unit dosage is 2-50 mg/kg Bwt., e.g.,
10-50 mg/kg Bwt., 25-45 mg/kg Bwt., or 20 to 40 mg/kg Bwt. The
volume of the unit dosage varies, Optionally, the volume is within
a range of 10-200 .mu.l, e.g., 40-150 .mu.l, 100-200 .mu.l, 120-150
.mu.l, or 50-160 .mu.l.
[0087] Optionally, the composition is administered by intravitreal
injection. Typically, a unit dosage for intravitreal administration
may contain 0.1 .mu.g/kg Bwt.-2 mg/kg Bwt., e.g., 0.5 .mu.g/kg
Bwt.-2 mg/kg Bwt., 1-2 mg/kg Bwt., or 1 to 1.5 mg/kg of the angio-3
peptide. The volume of the unit dosage may vary, for example, it
may be a volume within the range of 10-80 .mu.l, e.g., 20-60 .mu.l,
or 30-50 .mu.l.
[0088] Optionally, the composition is administered orally.
Typically, a unit dosage for oral administration may contain 2
mg/kg Bwt.-10 mg/kg Bwt., e.g., 2 .mu.g/kg Bwt.-8 mg/kg Bwt., 5-10
mg/kg Bwt., or 4 to 6 mg/kg of the Angio-3 peptide.
[0089] The dosage of a composition can be monitored and adjusted
throughout administration period, depending on severity of
symptoms, frequency of recurrence, and/or the physiological
response to the therapeutic regimen. Those of skill in the art
commonly engage in such adjustments in therapeutic regimens.
[0090] To achieve the desired therapeutic effect, the compositions
may be administered for multiple days at the therapeutically
effective dose. Thus, therapeutically effective administration of
the compositions of the disclosure to treat a pertinent condition
or disease described herein in a subject requires periodic (e.g.,
daily) administration that continues for a period ranging from four
weeks or two years or longer. A therapeutically beneficial effect
can be achieved if the agents are administered daily, or at a
frequency that is enough to maintain a therapeutically effective
concentration of the agents in the subject. For example, one can
administer the agents every day, every other day, or, if higher
dose ranges are employed and tolerated by the subject, e.g., twice
a week.
[0091] The duration of treatment with Angio-3 peptides to treat
patients vary according to severity of the condition in a subject
and the subject's response to Angio-3. Treatment with the Angio-3
in accordance with the disclosure thus may last for as long as
five, six, eight, ten weeks or even longer. Optionally, the
composition can be administered for a period of about 4 weeks to 2
years, more typically about 6 weeks to about 1 year, most typically
about 6 months to 1 year. Suitable periods of administration also
include about 18 weeks to 1 year, 9 to 16 weeks, 16 to 24 weeks, 16
to 32 weeks, 24 to 32 weeks, 24 to 48 weeks, 32 to 48 weeks, 32 to
52 weeks, 48 to 52 weeks, 48 to 64 weeks, 52 to 64 weeks, 52 to 72
weeks, 64 to 72 weeks, 64 to 80 weeks, 72 to 80 weeks, 72 to 88
weeks, 80 to 88 weeks, 80 to 96 weeks, 88 to 96 weeks, and 96 to
104 weeks. Suitable periods of administration also include 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 30, 32,
35, 40, 45, 48, and 50 weeks. Generally administration of the
composition should be continued until clinically significant
improvement of the condition is observed.
[0092] Optionally, administration of the composition comprising the
Angio-3 peptide is not continuous and can be stopped for one or
more periods of time, followed by one or more periods of time where
administration resumes. Suitable periods where administration stops
include 1 to 9 months, 1 to 6 months, 9 to 16 weeks, 16 to 24
weeks, 2 to 32 weeks, 24 to 32 weeks, 24 to 48 weeks, 32 to 48
days, 32 to 52 days, 48 to 52 days, 48 to 64 days, 52 to 64 days,
52 to 72 days, 64 to 72 days, 64 to 80 days, 72 to 80 days, 72 to
88 days, 80 to 88 days, 80 to 96 days, 88 to 96 days, and 96 to 100
days. Suitable periods where administration stops also include 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 30,
32, 35, 40, 45, 48 50, 52, 55, 60, 64, 65, 68, 70, 72, 75, 80, 85,
88 90, 95, 96, and 100 days.
[0093] Optionally, the composition is administered orally to
patients once, twice, or more times per day. Optionally, the daily
oral administration of the composition comprising the Angio-3
peptide is administered for at least two (2) weeks, at least three
(3) weeks, or for 1-2 weeks during a time interval. In some cases,
the interval is 3, 4, 6, 7, or 8 months. In general, administration
of Angio-3 is continued until the desired therapeutic benefit is
achieved. The entire treatment period, from the delivering the
first dose to the delivery of the last dose, may be 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 30, 32 months.
Optionally, the entire treatment period lasts 6 to 10 months, 12 to
24 months, 6 to 32 weeks, 24 to 32 weeks, 24 to 48 weeks, 32 to 48
months, or 32 to 52 months.
[0094] Optionally, the composition is delivered either
intravenously or intravitreally at least once, twice, or more every
24 weeks, every 20 weeks, every 15 weeks, every 12 weeks, every 10
weeks, or every 4 weeks. Optionally, the composition is delivered
either intravenously or intravitreally once every 4 to 10
weeks.
[0095] The Angio-3 composition disclosed herein can be used in
combination with other active agents known to be useful for
treating a retinal angiogenic disease, or with adjunctive agents
that may not be effective alone, but may contribute to the efficacy
of atropine. The Angio-3 composition can also be used in
conjunction with laser therapy or surgery to treat a retinal
angiogenic disease.
[0096] An Angio-3 composition disclosed herein can be placed in an
appropriate container, such as bottles or droppers, and labeled for
treatment of an indicated condition. For administration of the
atropine composition, such labeling would include, e.g.,
instructions concerning the amount, frequency and method of
administration.
[0097] Patients are monitored by eye examinations at the beginning
of the treatment and during periodic examinations during and/or
after treatment. In some cases, the patients are monitored, e.g.,
every four, five, six months, seven, or eight months during and/or
after the treatment period. Methods for determining the progression
of retinal angiogenic disease are well known, for example through
Colour Fundus photography ("CFP"), Fundus Fluorescein Angiography
("FFA"), Optical coherence tomography ("OCT") an visual acuity
test. The response to treatment is assessed by both qualitative and
quantitative analysis via clinical scoring system.
EXAMPLES
[0098] The following examples are for illustrative purposes only
and should not be interpreted as limitations. There are a variety
of alternative techniques and procedures available to those of
skill in the art, which would similarly permit one to successfully
perform the provided methods.
Example 1. Efficacy of Angio-3 in Non-Human Primate (NHP)
Laser-Induced CNV
[0099] This Pilot efficacy study was conducted on NHP laser induced
choroidal neovascularization ("CNV") model. Four monkeys were used
for this pilot study and each animal was undergone eye examination
procedures. Animals were sedated with ketamine intramuscularly
(10-20 mg/kg) and Medetomidine (0.02 mg/kg; IM). Topical
anaesthesia (1-2 drops of 1% xylocaine) was applied to reduce the
discomfort of the eyes. Pupils of non-human primates were dilated
with 2.5% phenylephrine hydrochloride and 1% tropicamide drops for
the ocular imaging procedures.
Laser Photocoagulation (Laser-Induced CNV):
[0100] Laser photocoagulation was performed on both eyes of the
animals to create a model for CNV as previously described (Lai C M
et al., 2005). Briefly, nine laser burns was delivered around the
macula of each eye in the manner of a grid with a protocol of
500-800 mW power intensity, 50-micron spot size, and 0.1-s
duration. The distance from each laser spot to the central fovea
was maintained at 0.5 to 1 disc diameter size. Care was taken to
avoid damaging the fovea.
[0101] Animals was observed twice daily for signs of potential
adverse events, and once daily for qualitative assessment of food
consumption. The body weights of the animals were recorded on the
day of transfer, at the time of animal selection for laser injury
and on the day of dose administration, and every week throughout
the remainder of the study.
Intravitreal Injection:
[0102] After the animal being anesthetized, the eyes were locally
anesthetized by putting a drop of xylocaine in the conjunctival
sac. A 5% povidone iodine solution was placed in the conjunctival
sac. A self-retaining eyelid speculum was placed in the eye.
Caliper was used to measure and mark a location at 2 mm behind the
limbus. Forceps was also be used to stabilize the eye and the
intravitreal injection was performed using a 30-gauge needle. The
tested compounds (2 mg in 50 .mu.l) were administered single IVT
injection into both eyes on day 15 (14 days after laser injury).
Daily cage-side observations were performed on all animals to
monitor for clinical signs of poor health, including any ocular
abnormalities.
Colour Fundus Photography (CFP) and Fundus Fluorescein Angiography
(FFA):
[0103] Fundus photography was carried out on both eyes at pre-laser
and day 14 post-laser and day 29 post laser (2 weeks after
treatment). For photography the pupils were dilated as mentioned
above and imaged with a fundus camera (TopCon Corp., Tokyo, Japan).
The fundus photographs were used to detect any changes in the
retina such as inflammation and pigmentation.
[0104] FFA was performed on both eyes by intravenous injection of
10% sodium fluorescein dye (0.1 ml/kg body wt). The fundus images
were taken between 10 s and 15 min after dye injection. FFA images
were assessed and graded according to standardized system: [0105]
Grade 1--No hyperfluorescence--No leak [0106] Grade
2--Hyperfluorescence--No leak [0107] Grade
3--Hyperfluorescence--Late leak [0108] Grade 4--Bright
Hyperfluorescence with Late Leak Beyond Spot
Optical Coherence Tomography (OCT):
[0109] OCT was carried out on both eyes at pre-laser and day 14
post-laser and day 29 post laser (2 weeks after treatment).
Euthanasia and Tissue Collection:
[0110] Animals were sacrificed on Day 30. Eye, blood, ocular fluids
and internal organs were collected for further analysis.
[0111] Animals were sacrificed on the proposed day (day 30) and the
upper body was perfused through the aorta (descending clamped) with
half-strength Karnovsky fixative. The eyes were removed, postfixes
for 2 to 3 days in half-strength Karnovsky fixative, and then
stored in formalin until processed. Strips of tissue containing 1
or 2 lesion sites were embedded in plastic. Sections 2 .mu.m thick
were taken at 30-.mu.m steps through the middle of each lesion. The
sections were stained with toluidine blue, and the sample with the
most robust lesion was designated as the central cut. This section
was then evaluated by an observer masked to the treatment
condition.
[0112] A tissue proliferation score was calculated for each lesion
based on 3 criteria: the size of the spindle cell proliferative
lesion, the extent of new blood vessel proliferation in the
subretinal space, and the elevation of the retina above the
choriocapillaris. Each measure was graded from 0 to 3, with 0
indicating not present. The total tissue proliferation score
comprises the sum of each of the described measures for each laser
lesion site.
Results
[0113] Our study result showed that Angio-3 peptide significantly
attenuates the retinal angiogenesis in KIMBA mice (FIG. 1A-D).
Eylea (positive control) is only effective for 4 weeks post
treatment when commenced treatment at 6 weeks of old Kimba mice via
IVT. However, Angio-3 (test peptide) is effective for up to 16
weeks post treatment (FIG. 2A-D). This data indicates the long
duration of Angio-3 efficacy in mice model Laser-induced mice model
study demonstrated that Angio-3 peptide significantly attenuates
the retinal and choroidal angiogenesis via IN route by single dose
till week 10 (FIGS. 3A-C). The systemic route did not affect the
natural wound healing process, menstrual cycle and behavioural
change, which shows that Angio-3 is safe to given via IV route.
Angio-3 also significantly attenuates the retinal and choroidal
angiogenesis than anti-VEGF via IVT route (FIGS. 4A-B). Angio-3
also shows significantly attenuates the retinal and choroidal
angiog than anti-VEGF via sub-lingual route (FIG. 5A-C). In
addition, our study shows that Angio-3 could be a potential long
acting anti-angiogenic for retinal and choroidal angiogenesis
diseases (FIGS. 3 and 4). In addition, we also determine the
retinal function with and without Angio-3 treatment. There was no
significant difference in retinal function noticed in all groups.
However, a and b-wave response was lower than baseline at week 26
that was due to normal ageing process (FIG. 3D). The optimal dose
was found in rat CNV model study. Angio-3 significantly attenuates
the retinal and choroidal angiogenesis than anti-VEGF via IVT route
at 5 .mu.g dose (FIGS. 6A-B).
[0114] This NHP pilot study shows that 2 mg in 50 .mu.l dose of
SIPRAD-0276 (Angio-3) was well tolerated via IVT and no signs of
inflammation found. This dose showed that same efficacy as compared
to 2 mg in 50 .mu.l of Eylea (FIGS. 7A-C). However, this was not
superior to Lucentis. Severity of lesion grade was significantly
reduced as compared to vehicle control (FIG. 8). Lesion area of
volume and thickness was significantly reduced post treatment (FIG.
9).
[0115] Based on histopathological analysis (FIG. 10), vehicle
treated (control) sections are thicker and more vascular compared
with the drug-treated eyes. Control sections showed more choroidal
fibroplasia (red arrows), increased retinal thickness, more
choroidal neovascularization (white arrows), multiple vessels
extending once or twice the retinal thickness and retinal elevation
as compared to drug-treated eyes. In this figure, the degree of
vessel leakiness was associated with morphological changes in the
retina at the site of laser injury. A distorted retinal
architecture was apparent if a laser spot was strongly leaky at the
day 29 time point, as revealed by thickening of the retina, massive
fibrosis, and edematous vacuoles. Control sections are thicker and
more vascular compared with the drug-treated eyes.
[0116] Angio-3 peptide also suppresses VEGF-induced endothelial
cell vascular permeability (VP) in vitro including VEGF-induced VP
of human retinal endothelial cells (HRECs). It also inhibits dermal
vascular permeability in mice. It is envisioned that Angio-3 would
also inhibit VP in the eye and this function contribute to
Angio-3's function to suppress vascular leakage in the Kimba mice
eye.
CONCLUSIONS
[0117] Laser-induced CNV in NHP is the gold-standard for drug
discovery and development of RAD. This pilot study result confirms
that Angio-3 shows efficacy as close to Eylea and this peptide
would be benefit to anti-angiogenesis therapy non-responders.
[0118] This peptide can be administered as intravenous or oral or
via IVT application for prevention or treatment of retinal
angiogenic diseases.
[0119] The animal model study result confirms that Angio-3 has a
long-term anti-VEGF effect, therefore has potentially greater
benefit than current drug on the market.
[0120] As compared to current drugs for the same disease condition,
we can produce even higher concentration (will find out tolerable
highest dose from next study) for low cost as this peptide has only
10 amino acid residues.
Example 2. Efficacy of Angio-3 Peptide Against Comparator Murine
Anti-VEGF on Retinal Angiogenic Diseases
Animals:
[0121] Aim of this study was to evaluate the efficacy of Angio-3
peptide against comparator murine anti-VEGF on retinal angiogenic
diseases. C57BL/6J wild type (WT) mice were purchased from InVivos
(Singapore). Kimba transgenic mice breeders were purchased from The
Lions Eye Institute, Perth, Australia. Brown Norway rats were
purchased from Charles & River Laboratories. In our facility,
KIMBA mice breeding colony was maintained and mice were bred for
the present study. Animals were housed on a 12 h light/12 h dark
cycle with food and water provided ad libitum. Handling and care of
all animals were performed according to the guidelines approved by
SingHealth Institutional Animal Care and Use Committee (IACUC],
Singapore, and is conducted in accordance with the Association for
Research in Vision and Ophthalmology (ARVO) recommendations for
animal experimentation.
Animal Model
[0122] The Kimba mouse (n=39) is a transgenic mouse model for
retinal neovascularisation, generated through
photoreceptor-specific over expression of human vascular
endothelial growth factor (hVEGF) protein. The retinal neovascular
changes include increased permeability, pericyte and endothelial
cell loss, vessel tortuosity, leukostasis and capillary blockage,
dropout and haemorrhage. The Kimba mouse model is particularly
suitable for testing anti-angiogeneic molecules designed to target
hVEGF.
[0123] Laser induced choridal neo-vascularization (CNV) in C57/BL6J
(B6J) wild type mice (n=90).
[0124] Laser induced choridal neo-vascularization (CNV) in
Brown-Norway rats (n=30).
Treatment Mode
[0125] Kimba mice received 25 mg/kg Bwt Angio-3 via intravenous
(IV) route, single injection of 100 .mu.l volume.
Angio-3 was also injected via intravitreal (IVT) route in Kimba
mice, single injection of 1 .mu.l volume at 1 .mu.g dose.
[0126] In B6J mice, single injection of 100 .mu.l volume at 25
mg/kg Bwt concentration Angio-3 was injected via intravenous (i/v)
route and this was tested in both Preventive mode (before laser)
and Regression mode (4 days after laser).
[0127] In B6J mice, 10 .mu.l volume at 3 mg/kg Bwt concentration
Angio-3; 10 .mu.l volume at 3 mg/kg Bwt concentration Angio-3
scramble and 10 ul volume at 0.1 mg/kg Bwt concentration anti-VEGF
was given via sub-lingual (oral) route and this was tested in
Regression mode (4 days after laser) and continued for 5 days
(single dose per day).
[0128] Angio-3 was also injected via intravitreal (IVT) route,
single injection of 1 .mu.l volume at 2 different doses; 1 .mu.g
(High dose) and 100 ng (Low dose).
[0129] This was further evaluated in Brown-Norway rats to identify
the optimal dose of Angio-3. Rats received 5 .mu.g (High dose) and
1 .mu.g (Low dose) of Angio-3 via IVT route, single injection of 3
.mu.l volume.
Laser Induced CNV:
[0130] 4 laser photocoagulation sites were placed concentrically
around the optic disc of both eyes to induce CNVs. A diode laser
(810 nm) was used with a relative potency scale of 120 mW for mice
and 250 mW for rats, an exposure time of 0.05 s, and a spot size of
50 .mu.m. Laser spots were focused with crystal covers to avoid
laser beam dispersion. Bubble formation was confirmed the rupture
of Bruch's membrane.
[0131] Fundus photography and fundus fluorescein angiography (FFA)
was imaged using a MICRON IV fundus camera (Phoenix Laboratories
USA). For FFA, animals were intraperitoneally injected with 10%
sodium fluorescein at a dose of 0.01 ml per 5-6 .mu.m body
weight.
[0132] The whole procedure took about 10-15 min per animal. At end
post treatment day 28 (Kimba and IVT mice; rat group) and week 25
(IV mice group)], animals were euthanized by overdose of
pentobarbital for blood and tissue collection.
Electroretinography (ERG)
[0133] Animals were dark-adapted overnight (12 h), and the
preparations for recordings were carried out under dim red light.
Anesthesia and pupil dilation were induced as described. Animals
were lightly secured to a stage with fastener strips across the
upper and lower back to ensure a stable, reproducible position for
ERG recordings. Body temperature was maintained between 37.degree.
C. and 38.degree. C. with a pumped water heating pad (TP500 T/Pump;
Gaymar Industries, Orchard Park, N.Y.) fixed to the top of the
stage. ERGs were recorded (Espion; Diagnosis LLC, Redwood City,
Calif.) with corneal monopolar electrodes (Mayo, Aichi, Japan). A
gold-cup electrode (Grass-Telefactor, West Warwick, R.I.) was
placed in the mouth to serve as the reference electrode, and a
silver-silver chloride electrode (Grass-Telefactor, West Warwick,
R.I.) was placed in the tail to serve as the ground electrode.
Recordings were performed at a wide range of stimulus intensities
(3.3 to 1.0 log cd*s/m2 in 0.3-log unit increments) in dark-adapted
(scotopic) condition. The response at each intensity was an average
of at least five trials. Signals were band-pass filtered from 1 to
100 Hz and were acquired at 1 kHz. The duration of the ERG
recording session was approximately 30 minutes for each animal.
Isolectin Staining
[0134] The flattened retinas were made permeable in ice-cold 70%
vol/vol ethanol for 20 minutes and then in PBS/1% Triton X-100 for
30 minutes. Retinas were incubated with AlexaFluor 568-conjugated
Griffonia simplicifolia isolectin B4 (5 .mu.g/mL;
Invitrogen-Molecular Probes, Eugene, Oreg.) in 1.times.PBS
overnight at 4.degree. C. for staining of the vasculature. Then
retinas were rinsed three times in 1.times.PBS for 10 min each and
mounted in antifade medium (Prolong Antifade Kit (P7481);
Invitrogen-Molecular Probes) and was sealed with the coverslip.
Images of retinal vasculature were captured with fluorescence
imaging and confocal microscopy (Live Cell TIRF System and MR
confocal microscope; Singapore Bio Imaging Centre-Nikon Imaging
Centre, Singapore).
Results
[0135] Our study result showed that Angio-3 peptide significantly
attenuates the retinal angiogenesis in KIMBA mice (FIG. 1A-D).
Eylea (positive control) is only effective for 4 weeks post
treatment when commenced treatment at 6 weeks of old Kimba mice via
IVT. However, Angio-3 (test peptide) is effective for up to 16
weeks post treatment (FIG. 2A-D). This data indicates the long
duration of Angio-3 efficacy in mice model Laser-induced mice model
study demonstrated that Angio-3 peptide significantly attenuates
the retinal and choroidal angiogenesis via IN route by single dose
till week 10 (FIG. 3A-C). The systemic route did not affect the
natural wound healing process, menstrual cycle and behavioural
change, which shows that Angio-3 is safe to given via IV route.
Angio-3 also significantly attenuates the retinal and choroidal
angiogenesis than anti-VEGF via IVT route (FIGS. 4A-B). Angio-3
also shows significantly attenuates the retinal and choroidal
angiog than anti-VEGF via sub-lingual route (FIGS. 5A-C). In
addition, our study shows that Angio-3 could be a potential long
acting anti-angiogenic for retinal and choroidal angiogenesis
diseases (FIGS. 2 and 3).
[0136] This peptide can be administered as intravenous or via IVT
application for prevention or treatment of retinal angiogenic
diseases. Our animal model study result confirms that Angio-3 is a
long-acting anti-VEGF, which has potential benefit than current
drug in the market.
Example 3. Efficacy of Modified Angio-3 Peptides on Retinal
Angiogenic Diseases
[0137] In B6J mice, single intravitreous (IVT) injection of 100 ng,
1 .mu.g, 5 .mu.g PEP-Q, PEP-N (modified Angio-3), Angio-3 and Eylea
were executed and this was tested in Prevention mode (before laser)
Method of Llser induced CNV model and injection was described as
above.
[0138] The study result showed that chemical modified angio-3
peptides: PEP-N and PEP-Q peptide significantly attenuates the
choroidal angiogenesis in laser-induced CNV mice via single
intravitreal injection at dose 100 ng, 1 .mu.g, and 5 .mu.g. (FIGS.
11A-B). The original Angio-3 (100 ng and 1 .mu.g) and positive
control Eylea.RTM. at the same dose (100 ng, 1 .mu.g, and 5 .mu.g)
also shows superior efficacy that is similar to PEP-Q and PEP-N
(FIG. 11; Table 2). PEP-Q and PEP-N could be a potential long
acting anti-angiogenic for retinal and choroidal angiogenic
diseases.
TABLE-US-00002 TABLE 2 Summary of PEP-N, PEP-Q, Angio-3 and Eylea
.RTM. on Area of Leakage Area of Leakage (Mean) Groups Week 1 Week
4 % Reduction Vehicle control 14738.20 9110.67 0% PEP-N 100 ng
11287.40 2237.13 42.00% 1 .mu.g 12808.95 2402.54 43.06% 5 .mu.g
8251.43 749.71 52.73% PEP-Q 100 ng 9987.09 1283.52 48.96% 1 .mu.g
13444.41 1271.30 52.36% 5 .mu.g 12839.27 742.03 56.04% Eylea 100 ng
10273.37 3433.52 28.40% 1 .mu.g 12502.09 3005.85 37.77% 5 .mu.g
11724.39 1834.08 46.17% Angio-3 100 ng 7404.61 3334.89 50.03% 1
.mu.g 6214.08 2867.88 48.92%
[0139] This two Angio-3 peptides, PEP-Q and PEP-N, can be
administered as intravitreal injection for prevention or treatment
of retinal angiogenic diseases. The animal model study result
confirmed that chemically-modified Angio-3 peptides, PEP-Q and
PEP-N, are novel and has potentially greater benefit than current
drugs on the market.
Example 4. Mechanistic Study of Angio-3
Effect of Angio-3 on VEGF Induced Proliferation of HRMEC Cells
[0140] 2000 HRMEC cells were seeded in 96-well plates in 100 .mu.l
of media and placed in a CO.sub.2 incubator at 37.degree. C. To
evaluate the effect the compounds have on VEGF-induced HRMEC
proliferation, cells were treated with 10 ng/ml VEGF.sub.165 or 50
ng/ml VEGF.sub.165 in the presence of different concentrations of
Angio-3 after serum starved in EBM-2 supplemented with 0.5% FBS
overnight. 300 .mu.g/ml Avastin was used as a positive control.
Alamar-Blue assay was performed after 3 days of treatment.
[0141] HRMEC cells were incubated with different concentrations of
compounds in the presence or absence of VEGF.sub.165 for 3 days.
Results showed that HRMEC proliferation was induced by 10 ng/ml and
50 ng/ml of VEGF.sub.165. 300 .mu.g/ml Avastin was sufficient to
inhibit VEGF induce proliferation. However, Angio-3 showed no
effect on VEGF induced proliferation at all three concentrations
tested (FIGS. 12A and B).
Effect of Angio-3 on HRMEC Cells Migration
[0142] Transwell migration assays were performed in a 24-well cells
culture plate containing 8.0 .mu.m pore size inserts. HRMEC cells
were serum starved overnight prior of assay. The lower chamber were
filled with EBM-2, EBM-2 supplemented with 50 ng/ml VEGF.sub.165
plus different concentrations of inhibitors. 1.times.10.sup.5 Cells
in EBM-2 were seeded in the upper chamber. Cells were allowed to
migrate to the other side of membrane for overnight at 37.degree.
C. Cells on top of the membrane were wiped off and cells migrated
to the other side were fixed and stained with 0.4% crystal violet.
Cell numbers were counted under a 10.times. objective for 5
randomly selected fields.
[0143] The results showed that HRMEC cells migration was strongly
induce by VEGF.sub.165. Migration stimulated by VEGF was completely
blocked by 300 .mu.g/ml Avastin 300 .mu.g/ml. 300 .mu.g/ml and 600
.mu.g/ml Angio-3 also showed a moderate inhibitory effect on VEGF
induced migration (FIGS. 13A-B).
Effect of Angio-3 on HRMECs Tube Formation
[0144] Cold 50 .mu.l matrigel GFR (BD Biosciences) was added into
96-well plates, and the plates were incubated at 37.degree. C. for
1 h to allow gel formation. HRMEC (1.times.10.sup.5/well) in EBM-2,
EBM-2 supplemented with 50 ng/ml VEGF.sub.165 with or without
different concentrations of Angio-3 were then plated on the
matrigel, 300 .mu.g/ml Avastin was used as a positive control.
After overnight incubation, 5 randomly selected fields of the
network growth area of the cells were photographed using an
inverted phase contrast photomicroscope. Tube networks were
quantified using Image J Angiogenesis Analyzer.
[0145] Effect of Angio-3 on VEGF induced HRMEC cells tube formation
was examined and quantified in term of total tube length, number of
junctions and number of loops. FIG. 14 showed that VEGF induced
endothelial cells tube formation network was strongly inhibited by
all drug tested.
[0146] FIGS. 15A and 15B show that Angio-3 induced human umbilical
vein endothelial cells (HUVECs) apoptosis in the presence of both
VEGF and bFGF. FIG. 15A showed Angio-3 induced HUVEC apoptosis in
the presence of 20 ng/ml VEGF in a dose-dependent manner. FIG. 15B
showed that Angio-3 induced HUVEC apoptosis in the presence of 20
ng/ml bFGF in a dose-dependent manner. * represents p<0.05,
n=3.
[0147] FIGS. 16A and 16B show that Angio-3 inhibited HUVEC
proliferation stimulated by VEGF and bFGF. FIG. 16A shows Angio-3
suppressed HUVEC proliferation induced by 20 ng/ml VEGF in a
dose-dependent manner. FIG. 16B shows Angio-3 suppressed HUVEC
proliferation induced by 20 ng/ml bFGF in a dose-dependent manner.
* represents p<0.05, n=3.
[0148] FIGS. 17 A-D show Angio-3 inhibited VEGF and bFGF-induced EC
migration and inhibits capillary network formation. FIGS. 17 A and
22 C show Angio-3 suppressed HUVEC chemotactic migration induced by
20 ng/ml VEGF in a dose-dependent manner. Migrated cells were
stained with Hoechst, imaged and counted. FIGS. 17 B and 22 D show
Angio-3 suppressed HUVEC chemotactic migration that was induced by
20 ng/ml bFGF in a dose-dependent manner. Migrated cells were
stained with Hoechst, imaged and counted.
[0149] FIG. 18 shows Angio-3 inhibited HUVEC capillary network
formation on Matrigel. HUVECs were pre-incubated with increasing
doses of Angio-3 for 30 min prior to seeding onto Matrigel and
cultured under complete EC growth media. HUVEC tube formation was
imaged after 6 h of incubation. Percentage area covered by HUVEC
tubes were quantified as the level of tube formation. n=3; *
represents significant reduction compared to control at P<0.05
by one-way ANOVA.
[0150] FIGS. 19A, 19B and 19C show Angio-3 is a novel
anti-permeability agent that can inhibit VEGF-induced vascular
permeability (VP) with multiple endothelial cell types. In FIG.
19A, post-confluent HUVEC monolayers were treated with increasing
concentrations of Angio-3 or medium alone. The results show that
Angio-3 inhibited VEGF-induced permeability across confluent HUVECs
in a dose-dependent manner without affecting the basal level
permeability. In these experiments, post-confluent HUVEC monolayers
were pre-treated with Angio-3 for 30 minutes prior to stimulation
with 100 ng/ml VEGF. In FIG. 19B, Post-confluent HMVEC monolayers
were treated with increasing concentrations of Angio-3 or medium
alone. The results show that Angio-3 inhibited VEGF-induced
permeability across confluent human dermal microvascular
endothelial cells (HMVECs) in a dose-dependent manner without
affecting the basal level permeability. In FIG. 19C, post-confluent
HREC monolayers were treated with Angio-3 and VEGF for 3 h. The
results show that Angio-3 inhibited VEGF-induced permeability
across confluent human retinal endothelial cells (HRECs) in a
dose-dependent manner without affecting the basal level
permeability. * represents p<0.05, n=3.
[0151] FIGS. 20A and 20B show Angio-3 inhibited local VEGF-induced
dermal vascular permeability in mice. In FIG. 20A, Angio-3 was
administered via intradermal injection to mice and the results show
that Angio-3 inhibited VEGF-induced dermal permeability in a
dose-dependent manner within 15 min. The dermal permeability was
visualized by Evans blue dye extravasation. In FIG. 20B, dye
extravasation was quantified by formamide extraction of the dye and
measuring OD 610. n=5 animals per group, * represent significantly
increased as compared with the simultaneous control at
p<0.05.
[0152] FIG. 21 shows Angio-3 prevented VEGF-induced dissociation of
Vascular endothelial ("VE")-cadherin from Adherens junctions (AJs)
on HUVECs. Confluent HUVECs were stimulated with 100 ng/ml VEGF to
induce VE-cadherin dissociation from the AJs. The HUVECs confluent
monlayes were pre-treated by 100 .mu.M Angio-3 prior to VEGF
stimulation. VE-cadherin is stained by antibody and DAPI was used
to counter-stain the nucleus. Angio-3 can interferes with the
ability of VEGF to induce VE-cadherin dissociation from AJs.
[0153] FIG. 22 shows Angio-3 suppressed VEGF-induced dissociation
of tight junction (TJ) proteins ZO-1 and ZO-2 from TJs in HUVECs.
HUVEC cells Cells were treated as described above and stained with
an ZO-1 antibody or a ZO-2 antibody, and counter-stained with DAPI
to show the nucleus. The results show that VEGF induces the TJ
protein ZO-1 and ZO-2 to dissociate from the TJs of confluent HUVEC
monolayers. Pre-treatment of Angio-3 (100 .mu.M) can suppress this
VEGF function.
[0154] FIG. 23 shows Angio-3 suppressed VEGF-induced actin stress
fiber formation in HUVECs. However, in the absence of VEGF, Angio-3
promoted cortical actin fiber formation.
Example 4. Retinal Function Tests in KIMBA Mice Overexpressing
hVEGF
[0155] Eight-week-old KIMBA mice that overexpress hVEGF were
dark-adapted overnight (for at least 12 hours) and all the
procedures were carried out under dim red light. Mice were
anesthetized with a combination of ketamine (20 mg/kg body weight)
and xylazine (2 mg/kg body weight). Pupils were dilated with a
topical administration of 1% tropicamide (Alcon Laboratories, Inc.,
Fort Worth, Tenn. USA) and 2.5% phenylephrine (Bausch and Lomb
Pharmaceuticals, Inc., Tampa, Fla., USA) ophthalmic solutions.
[0156] Animals were placed in ERG recording table with body
temperature controller for ERG recordings. Electroretinograms were
recorded (Espion; Diagnosys LLC, Lowell, Mass., USA) with corneal
monopolar electrodes. A goldcup electrode was placed in the mouth
to serve as the reference electrode, and a silver-silver chloride
electrode (Grass-Telefactor, West Warwick, R.I., USA) was placed in
the tail to serve as the ground electrode. Recordings were
performed at a wide range of stimulus intensity (-3.0 to 1.0 log
cds/m2) in dark-adapted (scotopic) condition. The response ateach
flash intensity was an average of at least five trials. Signals
were band-pass filtered from 1 to 100 Hz and were acquired at 1
kHz.
[0157] ERG was recorded from 8 weeks old to 24 weeks old of mice in
untreated group (naive group). In Angio-3 IVT treated group, ERG
was recorded at 8 weeks old as baseline (BL) and then 6 and 12
weeks post treatment. 2nd IVT was given at 12 weeks after 1st
treatment and then followed for 8 weeks post treatment. Student's
t-test was used to compare data between two groups; P<0.05 was
considered to be significant. The results are shown in FIG. 24. The
untreated mice has no response of both a and b-waves after 12 weeks
of age. Angio-3 increased the a and b-wave responses for 6 weeks
post treatment and then again increased the response for 20 weeks
with second dose. This result shows that Angio-3 is rescuing the
retinal function in KIMBA mice.
Example 5. Testing Angio-3 in a Laser-Induced Chorodial
Neovascularization (CNV) Model Developed in Cynomolgus Monkeys as
an Experimental Model of Wet AMD
[0158] A laser-induced choroidal neovascularization (CNV) model was
developed and validated in Cynomolgus monkeys as experimental model
of wet AMD. The aim was to differentiate efficacy and dose response
of test peptide from Eylea (clinical compound). 5 groups of 5 male
Cynomolgus monkeys were used in this study. 3 groups were dosed IVT
with 2 mg Angio-3; PeptideQ (SEQ ID NO:4) and Eylea.RTM. (dose
volume 50 .mu.L) respectively. 1 group of 5 monkeys received 4 mg
of PeptideQ. 1 group was served as laser control without any drug
treatment. Both eyes were injected by a single IVT injection of the
drugs at Day 14 after application of laser. The development of
active CNV was assessed by fluorescein angiography, at Day 14 the
baseline degree of neovascularization and leakiness were measured
and the final fluorescein angiography assessment was performed on
Day 28. Leakage area was quantified by ImageJ software. The laser
volume was quantified by Spectralis-Heidelberg software using
SD-OCT images. The results are shown in FIGS. 25A-F.
[0159] In this intervention study, no change in lesion severity was
observed for the laser control and the change in lesion severity
and laser area was significantly different for all drug treated
groups as compared with control (analysis of variance [ANOVA]
followed by Tukey's multiple comparison, @=p<0.05 compared with
Control). Eylea shows the superior efficacy as compared to test
peptides. However, Pep-Q shows dose dependent efficacy and higher
dose efficacy close to Eylea.
Example 6. Effects of Angio-3 Peptides after Alkali-Burn Injury
[0160] Mice were anesthetized with the combination of 80 mg/kg
ketamine and 5 mg/kg xylazine. One drop of 1% xylocaine was applied
to the corneal surface for local analgesia. Round piece of filter
paper, approximately 2 mm in diameter was soaked in a solution of 1
M NaOH. A piece of NaOH soaked filter paper was picked by sterile
forceps. NAOH soaked filter paper was placed on the central cornea
under microscope to ensure properly placing the filter paper. Left
it for 30 sec to generate an acute alkali-burn of approximately
2.times.2 mm.sup.2 in area. Filter paper was removed and then
gently flushed the eye with 10 ml of 1.times.PBS twice to wash away
residual 1 M NaOH. Only one eye of the mouse was injured and the
other serving as a control. A drop of 1.times. Peptide Q (1%
solution as test compound) or a vehicle control consisting of PBS
was topically applied to the cornea. Repeated application occurred
3.times./day for 7 days. At end point, clinical assessment was done
as stated below and eyes were enucleated for corneal flat
mount.
[0161] A daily examination of the mice in a blinded fashion was
performed under a surgical microscope and score corneal
neo-vascularization (NV) was based on corneal opacity, NV and
vessel size. Two observers were scored and record a final score
that was the average of the two. Score corneal opacity on a scale
of 0-4. 0=completely clear; 1=slightly hazy, iris and pupil easily
visible; 2=slightly opaque, iris and pupil still detectable;
3=opaque, pupils hardly detectable; and 4=completely opaque with no
view of the pupil. Score NV on a scale of 0-3. 0=no neovessels;
1=neovessels at the corneal limbus; 2=neovessels spanning the
corneal limbus and approaching the corneal center; 3=neovessels
spanning the corneal center. Score vessel size on a scale of 0-3.
0=no neovessels; 1=neovessels detectable under surgical microscope;
2=neovessels easily seen under surgical microscope; 3=neovessels
easily seen without the microscope. Images were captured using
surgical microscope camera and slitlamp biomicroscopy. The results
are shown in FIGS. 26 and 27. Peptide Q treated eyes had
significantly reduced corneal NV area as compared to vehicle
control eyes.
Example 7. Wound Healing in a Mouse Model
[0162] Two (2)-mm trephine was used to mark the wound size.
Epithelium, basement membrane and some stromal layers were removed
by peeling using forceps. Fluorescein staining to the wounded area
of cornea was done from day 0 to Day 5 (n=6 mice per group from 2
independent experiments). Gentle and meticulous approach when
wounding and handling mouse cornea, fluorescein was diluted, slit
lamp images had to be taken within a few seconds after staining.
The wound area was quantified by ImageJ software. Student's t-test
was used to compare data between two groups; P<0.05 was
considered to be significant. The results are shown in FIGS. 28A
and 28B. Angio-3 didn't adversely affect the normal wound healing
process.
[0163] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, sequence accession numbers, patents, and patent
applications cited herein are hereby incorporated by reference in
their entirety for all purposes.
Sequence CWU 1
1
4110PRTArtificial SequenceSynthetic construct 1Thr Pro His Thr His
Asn Arg Thr Pro Glu1 5 10210PRTArtificial SequenceSynthetic
construct 2Asn Thr Thr Glu Thr Pro His Pro His Arg1 5
10310PRTArtificial SequenceSynthetic
constructmisc_feature(7)..(7)Xaa can be any naturally occurring
amino acid 3Thr Pro His Thr His Asn Xaa Thr Pro Glu1 5
10410PRTArtificial SequenceSynthetic
constructmisc_feature(7)..(7)Xaa can be any naturally occurring
amino acid 4Thr Pro His Thr His Gln Xaa Thr Pro Glu1 5 10
* * * * *