U.S. patent application number 15/753674 was filed with the patent office on 2019-01-03 for an eye drop solution for enhanced hyaluronic acid retention and delivery.
The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY. Invention is credited to Jennifer H. Elisseeff, David Lee, Nicole Lu, Anirudha Singh.
Application Number | 20190000983 15/753674 |
Document ID | / |
Family ID | 58051345 |
Filed Date | 2019-01-03 |
United States Patent
Application |
20190000983 |
Kind Code |
A1 |
Singh; Anirudha ; et
al. |
January 3, 2019 |
AN EYE DROP SOLUTION FOR ENHANCED HYALURONIC ACID RETENTION AND
DELIVERY
Abstract
The present invention provides biomaterial compositions that can
immobilize HA to the ocular surfaces through an HABpep and
transmembrane mucins and collagen of ocular tissues can act as
anchoring sites. These biomaterial compositions provide prolonged
HA binding and retention in both ex vivo and in vivo animal models.
HA eye drop solutions with these the inventive biomaterials can
prolong the biological and physical benefits to the ocular surface
and potentially be more effective in treating eye disorders
including dry eye than standard HA-eye drop presently available.
Methods of making and use of the compositions are also
provided.
Inventors: |
Singh; Anirudha; (Baltimore,
MD) ; Elisseeff; Jennifer H.; (Baltimore, MD)
; Lee; David; (Baltimore, MD) ; Lu; Nicole;
(Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY |
Baltimore |
MD |
US |
|
|
Family ID: |
58051345 |
Appl. No.: |
15/753674 |
Filed: |
August 17, 2016 |
PCT Filed: |
August 17, 2016 |
PCT NO: |
PCT/US2016/047248 |
371 Date: |
February 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62207417 |
Aug 20, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/08 20130101;
H01L 2224/75745 20130101; A61P 27/04 20180101; H01L 21/67092
20130101; H01L 24/00 20130101; A61K 47/60 20170801; H01L 2224/75821
20130101; H01L 21/67132 20130101; A61K 31/728 20130101; H01L 24/75
20130101 |
International
Class: |
A61K 47/60 20060101
A61K047/60; A61K 38/08 20060101 A61K038/08; A61K 31/728 20060101
A61K031/728; A61P 27/04 20060101 A61P027/04 |
Claims
1. A biomaterial comprising at least one biologically compatible
polymer having one or more HA binding peptides (HABPep) covalently
linked to the biologically compatible polymer, and one or more
sialic acid binding peptides (SABPep) covalently linked to the
biologically compatible polymer.
2. (canceled)
3. A biomaterial comprising at least one biologically compatible
polymer having one or more HA binding peptides (HABPep) covalently
linked to the biologically compatible polymer, and one or more
sialic acid binding peptides (SABPep) and one or more collagen
binding peptides (ColBPep) covalently linked to the biologically
compatible polymer.
4. The biomaterial of claim 1, wherein HA is mixed into the
biologically compatible polymer.
5. The biomaterial of claim 1, further comprising HA bound to the
HABPep.
6. The biomaterial of claim 5, wherein the HA has a molecular
weight of between 5000 Da and 20,000,000 Da.
7. The biomaterial of claim 6, wherein the HA is crosslinked.
8. The biomaterial of claim 1, wherein the HABPep is a peptide
comprising the following amino acid sequence: TABLE-US-00002 (SEQ
ID NO: 1) RRDDGAHWQFNALTVR, (SEQ ID NO: 2) CRRDDGAHWQFNALTVR, (SEQ
ID NO: 3) GAAWQFNALTVR, (SEQ ID NO: 4) GAHWQFAALTVR, (SEQ ID NO: 5)
GAHWQFNALTVA, (SEQ ID NO: 6) GAHWQFNALTVR, (SEQ ID NO: 7)
STMMSRSHKTRSHHV, (SEQ ID NO: 8) RYPISRPRKRC, (SEQ ID NO: 9)
TAGHGRRWS, or (SEQ ID NO: 10) LKQKIKHVVKLKVVVKLRSQLVKRKQN.
9. The biomaterial of claim 1, wherein the ColBpep is selected from
the group consisting of RRANAALKAGELYKSILYGC (SEQ ID NO: 11),
SYIRIADTNIT (SEQ ID NO: 12), YSFYSDESLQ (SEQ ID NO: 13) and WYRGRL
(SEQ ID NO: 14).
10. The biomaterial of claim 1, wherein the SABpep is selected from
the group consisting of GGSPYGRC (SEQ ID NO: 15), and
GGPQEQITQHGSPYGRC (SEQ ID NO: 16).
11. The biomaterial of claim 1, wherein the biocompatible polymer
is hydrophilic.
12. The biomaterial of claim 11, wherein the biocompatible polymer
is selected from the group consisting of: Poly(ethylene glycol),
Poly(propylene glycol), Poly(methyl vinyl ether), Oligoethylene,
Poly(isobutylene) Poly(tetrahydrofuran) Poly(oxytrimethylene),
Poly(dimethylsiloxsane), Poly(dimethylsilane), Nylon 6, Nylon 11,
Poly(acrylonitrile), Squalane, Poly(1,3-dioxolane),
Poly(iminooligomethylene), Poly(l-lysine), Polyethyleneimine,
Poly(adipate), Poly(l-caprolactone), Poly(L-lactic acid), or
derivatives thereof.
13. The biomaterial of claim 11, wherein the one or more
biocompatible polymers are mono, disubstituted, or multisubstituted
with at least one functional group.
14. The biomaterial of claim 1, comprising SABpep linked to
Poly(ethylene glycol) linked to HABpep (SABpep-PEG-HABpep).
15. The biomaterial of claim 1, comprising ColBpep linked to
Poly(ethylene glycol) linked to HABpep (ColBpep-PEG-HABpep).
16. A biomaterial comprising at least one biologically compatible
polymer having HA conjugated to a linker molecule covalently linked
to the biologically compatible polymer, and one or more sialic acid
binding peptides (SABPep) covalently linked to the biologically
compatible polymer.
17. The biomaterial of claim 16, wherein HA is mixed into the
biologically compatible polymer.
18. The biomaterial of claim 16, wherein HA is conjugated to a
linker molecule and conjugated to a detectable moiety which is one
or more sialic acid binding peptides (SABPep) covalently linked to
the biologically compatible polymer.
19. The biomaterial of claim 18, wherein the detectable moiety is
selected from the group consisting of: radioactive isotopes,
magnetic beads, metallic beads, colloidal particles, fluorescent
dyes, electron-dense reagents, enzymes, biotin, digoxigenin, or
haptens.
20. The biomaterial of claim 19, wherein the detectable moiety is
fluorescein.
21. A pharmaceutical composition comprising the biomaterial of
claim 1, and a pharmaceutically acceptable carrier.
22. The pharmaceutical composition of claim 21, wherein the
pharmaceutically acceptable carrier is suitable for ophthalmic
use.
23. The pharmaceutical composition of either of claim 22, further
comprising an additional therapeutic agent.
24. A method for treatment of a disease or condition of the eye of
a subject in need of treatment, comprising administering to the
subject an effective amount of the biomaterial composition of claim
1, or the pharmaceutical composition of claim 21.
25. The method of claim 24, wherein the disease or condition is Dry
Eye.
26. A method for treating eye diseases by means of an eye surgery
treatment in a subject in need of treatment comprising
administering to the subject an effective amount of the biomaterial
composition of claim 1 or the pharmaceutical composition of claim
21.
27. The method of claim 26, wherein the surgery treatment is
selected from the group consisting of corneal transplantation,
cataract surgery, glaucoma surgery, and surgery to repair retinal
detachment.
28. The biomaterial of claim 3 wherein HA is mixed into the
biologically compatible polymer.
29. The biomaterial of claim 3, further comprising HA bound to the
HABPep.
30. The biomaterial of claim 29, wherein the HA has a molecular
weight of between 5000 Da and 20,000,000 Da.
31. The biomaterial of claim 30, wherein the HA is crosslinked.
32. The biomaterial of claim 3, wherein the HABPep is a peptide
comprising the following amino acid sequence: RRDDGAHWQFNALTVR (SEQ
ID NO: 1), CRRDDGAHWQFNALTVR (SEQ ID NO: 2), GAAWQFNALTVR (SEQ ID
NO: 3), GAHWQFAALTVR (SEQ ID NO: 4), GAHWQFNALTVA (SEQ ID NO: 5),
GAHWQFNALTVR (SEQ ID NO: 6), STMMSRSHKTRSHHV (SEQ ID NO: 7),
RYPISRPRKRC (SEQ ID NO: 8), TAGHGRRWS (SEQ ID NO: 9), or
LKQKIKHVVKLKVVVKLRSQLVKRKQN (SEQ ID NO: 10).
33. The biomaterial of claim 3, wherein the ColBpep is selected
from the group consisting of RRANAALKAGELYKSILYGC (SEQ ID NO: 11),
SYIRIADTNIT (SEQ ID NO: 12), YSFYSDESLQ (SEQ ID NO: 13) and WYRGRL
(SEQ ID NO: 14).
34. The biomaterial of claim 3, wherein the SABpep is selected from
the group consisting of GGSPYGRC (SEQ ID NO: 15), and
GGPQEQITQHGSPYGRC (SEQ ID NO: 16).
35. The biomaterial of claim 3, wherein the biocompatible polymer
is hydrophilic.
36. The biomaterial of claim 35, wherein the biocompatible polymer
is selected from the group consisting of: Poly(ethylene glycol),
Poly(propylene glycol), Poly(methyl vinyl ether), Oligoethylene,
Poly(isobutylene) Poly(tetrahydrofuran) Poly(oxytrimethylene),
Poly(dimethylsiloxsane), Poly(dimethylsilane), Nylon 6, Nylon 11,
Poly(acrylonitrile), Squalane, Poly(1,3-dioxolane),
Poly(iminooligomethylene), Poly(l-lysine), Polyethyleneimine,
Poly(adipate), Poly(l-caprolactone), Poly(L-lactic acid), or
derivatives thereof.
37. The biomaterial of claim 35, wherein the one or more
biocompatible polymers are mono, disubstituted, or multisubstituted
with at least one functional group.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of International Patent
Application No. 62/207,419, filed Aug. 20, 2015, which is hereby
incorporated by reference for all purposes as if fully set forth
herein.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 12, 2016, is named P13662-02_ST25.txt and is 4,807 bytes in
size.
BACKGROUND OF THE INVENTION
[0003] Dry eye is a prevalent ophthalmic condition especially to
elderly population, affecting over 15% of the US population (Ding
and Sullivan 2012). Symptoms include discomfort, dryness or lack of
hydration, visual disturbances and pain in the eye, which can
hinder the performance of everyday activities (Abetz, Rajagopalan
et al. 2011; Pouyeh, Viteri et al. 2012; Pili, Kastelan et al.
2014). Although dry eye is a multifactorial disease, it occurs
mostly due to any alterations in the tear film composition and
stability with potential damage to the ocular surface. There are
three tear film layers that have unique functions. All together,
they prevent tear evaporation, protect the eye from foreign
objects, lubricate and hydrate the ocular surface (Gipson 2007)
(FIG. 1). Tear film-related dry eye disease is further categorized
into two major conditions: aqueous-deficient and evaporative.
Aqueous-deficient dry eye is caused by lacrimal gland's inability
to produce sufficient tear fluid, whereas evaporative dry eye is
caused by increased tear film evaporation rate due to a compromised
protective lipid layer (Horwath-Winter, Berghold et al. 2003;
Sharma and Hindman 2014). To treat dry eye disorders, many
different approaches have been employed (Young, Veys et al. 2002;
Johnson, Murphy et al. 2006; Ali and Byrne 2009; Papas, Ciolino et
al. 2013); however, these strategies are temporary, involving the
application of a saline or an emulsion-based eye drop to reduce
discomfort and increase water retention at the ocular surface
(Young, Veys et al. 2002; Johnson, Murphy et al. 2006; Prabhasawat,
Tesavibul et al. 2007; Ali and Byrne 2009; Papas, Ciolino et al.
2013; Labetoulle 2015; Simmons, Carlisle-Wilcox et al. 2015). These
artificial tears must be reapplied frequently owing to their short
residence time in the eye.
[0004] Currently, commercially available eye drops containing
0.1-0.2% hyaluronic acid (HA) solutions, such as Opticalm.TM.,
Scope.TM., and Oxyal.TM. are commonly used as an active ingredient
in Europe to relieve dry eye symptoms, while in USA as an inactive
ingredient of the eye drop formulation (e.g. in Blink.TM.).
[0005] HA is an anionic glycosaminoglycan that is present in many
tissues and organs as a major component of the extracellular
matrix, has been shown to ameliorate dry eye because it retains
water through hydrogen bonding (Hargittai and Hargittai 2008),
stabilizes the tear film and lubricates the surface (Tonge, Jones
et al. 2001; Read, Morgan et al. 2009). HA has also been shown to
slow tear removal and permit uninterrupted blinking (Tsubota and
Yamada 1992; Nakamura, Hikida et al. 1993; Hamano, Horimoto et al.
1996). In addition, HA has several desirable therapeutic
properties; for example, it encourages corneal wound healing by
promoting epithelial cell migration (Stuart and Linn 1985;
Shimmura, Ono et al. 1995; Gomes, Amankwah et al. 2004; Johnson,
Murphy et al. 2006; Rah 2011), reduces inflammation (Pauloin, Dutot
et al.) and protects cells from free-radical damage (Presti and
Scott). Recent studies (Bray, J. Theor. Biol, 2001; Georgiev et
al., Soft Matter, 2013; Cerretani et al., Adv. Colloid Interface
Sci. 2013) further indicate that polyanionic polysaccharide
moieties, including HA can enhance the spreading of the tear film
lipid layer, which may be an added clinical benefit of HA for
ocular application. HA eye drop solutions effectively wet and
lubricate the contact lens and ocular surfaces, however similar to
saline, poly(vinyl alcohol)/PVA, methyl cellulose (MC) and
hydroxypropyl methylcellulose (HPMC)-based eye drops, they suffer
from low ocular residence time (<10 min) due to limited
adherence to ocular surface (Snibson, Greaves et al. 1992;
Mochizuki, Yamada et al. 2008; Papas, Ciolino et al. 2013).
Conversely, highly concentrated HA-based eye drops are too viscous
and interfere with vision and blinking (Marner, Mooller et al.
1996; Reddy, Grad et al. 2004; Ali and Byrne 2009; Papas, Tilia et
al. 2014).
[0006] There still exists, however a need to develop an improved HA
binding eye drop technology that prolongs HA retention and delivery
on ocular surfaces providing biological and physical benefits of
HA.
SUMMARY OF THE INVENTION
[0007] The present invention provides biomaterial compositions
developed to enhance the availability of HA that can retain a thin
film of moisture to mimic the smooth, hydrated and lubricated
ocular surface.
[0008] In accordance with one or more embodiments, the present
inventors have accomplished this by, in an embodiment, immobilizing
HA through a HA binding peptide that localizes HA to the ocular
surfaces. In accordance with another embodiment, the HA is
immobilized through conjugation of the HA to a hydrophilic linker
which is also conjugated to a binding peptide which binds
transmembrane mucins of the epithelium that contain sialic acid and
collagen of ocular matrices and act as anchoring sites for HA
immobilization.
[0009] In accordance with a further embodiment, the present
invention comprises, in an embodiment, an eye drop technology based
on a heterobifunctional HA-binding peptide (HABpep) polymer-peptide
system that binds and retains HA, from tear fluid or exogenous
application, for longer periods of time at the surface of the eye.
The key is to anchor HABpep polymer-peptide system on either sialic
acid-containing glycosylated transmembrane molecules, such as
mucins, the peripheral extracellular ocular epithelium protecting
and lubricating surface, or the ocular tissue matrix enriched in
collagen type I.
[0010] In some embodiments the HA can be provided within the
biologically compatible polymer matrix, or added separately.
[0011] In accordance with an embodiment, the present invention
provides a biomaterial comprising at least one biologically
compatible polymer having one or more HA binding peptides (HABPep)
covalently linked to the biologically compatible polymer, and one
or more sialic acid binding peptides (SABPep) covalently linked to
the biologically compatible polymer.
[0012] In accordance with another embodiment, the present invention
provides a biomaterial comprising at least one biologically
compatible polymer having one or more HA binding peptides (HABPep)
covalently linked to the biologically compatible polymer, and one
or more collagen binding peptides (ColBPep) covalently linked to
the biologically compatible polymer.
[0013] In accordance with a further embodiment, the present
invention provides a biomaterial comprising at least one
biologically compatible polymer having one or more HA binding
peptides (HABPep) covalently linked to the biologically compatible
polymer, and one or more sialic acid binding peptides (SABPep) and
one or more collagen binding peptides (ColBPep) covalently linked
to the biologically compatible polymer.
[0014] In accordance with an embodiment, the present invention
provides a biomaterial comprising SABpep linked to Poly(ethylene
glycol) linked to HABpep (SABpep-PEG-HABpep).
[0015] In accordance with another embodiment, the present invention
provides a biomaterial comprising ColBpep linked to Poly(ethylene
glycol) linked to HABpep (ColBpep-PEG-HABpep).
[0016] In accordance with an embodiment, the present invention
provides a biomaterial comprising HA covalently linked to one or
more sialic acid binding peptides (SABPep) via a linker.
[0017] In accordance with another embodiment, the present invention
provides a biomaterial comprising HA covalently linked to a
detectable moiety which is covalently linked to one or more sialic
acid binding peptides (SABPep) (for example, with a fluorescent
moiety (FL), HA-FL-SABPep) via a linker.
[0018] In accordance with an embodiment, the present invention
provides a biomaterial a pharmaceutical composition comprising the
biomaterial as described above, and pharmaceutically acceptable
carrier.
[0019] In accordance with another embodiment, the present invention
provides a biomaterial a pharmaceutical composition comprising the
biomaterial as described above, an additional therapeutic agent,
and pharmaceutically acceptable carrier.
[0020] In accordance with an embodiment, the present invention
provides a method of treatment of an ophthalmic disease or
condition of the eye comprising administering to the eye of a
subject an effective amount of the biomaterial described above or a
pharmaceutical composition comprising the biomaterial.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A-1B: Schematic of an HA binding eye drop technology.
1A) Exterior ocular surface has three layers: lipid, aqueous and
mucin layers. 1B) On application of HA binding eye drop, HABpep
binds HA through an anchoring peptide on the ocular surface.
Specifically, sialic acid present in extracellular domains, such as
transmembrane mucins is targeted (magnified); alternatively,
collagen of the ocular tissue present in conjunctiva or cornea
tissues can also be targeted to immobilize HA.
[0022] FIGS. 2A-2C: Selecting ocular surface binding peptides:
targeting sialic acid and collagen as sites for binding SABpep and
ColBpep on the conjunctival tissue ex vivo. 2A) A HABA assay
quantifying the amount of peptide bound to conjunctiva tissue
(normalized to the dry weight) indicated that out of the six
peptides tested (as listed in Table 1), peptide 3 (ColBpep) and
peptide 6 (ColBpep) bound the most to conjunctiva tissue compared
to control (p<0.5). 2B) Immunohistochemistry showed that both
SABpep and anti-mucin 1 antibody stained positively for the
transmembrane mucin-1 on the epithelial surface, while ColBpep
binds on ocular matrices throughout cellular and non-cellular
regions (scale bar=100 .mu.m). 2C) A magnified immunostaining image
showing that SABpep specifically binds to the cellular periphery
throughout the epithelium (scale bar=100 .mu.m).
[0023] FIGS. 3A-3D QCM-D measurements of in vitro binding of SABpep
and HABpep to sialic acid and HA, respectively. QCM-D curves
indicate successful deposition of substrates and binding peptides:
3A) Sialic acid-containing mucin adsorbed onto gold substrate
followed by attachment of SABpep. 3B) Thiolated HA is covalently
immobilized onto pristine gold, subsequent HABpep biding to HA. 3C)
Deposition of the substrate layers of mucin and HA on pristine gold
QCM-D surfaces quantified by the visco-elastic Voigt model,
respectively. 3D) Attachment of binding peptides to mucin and HA
substrates quantified by Sauerbrey relationship (overtone n=5).
Conditions: All concentrations cw=0.1 mg/ml, T=37.degree. C., flow
rate=25 .mu.L/min in PBS buffer (pH=7.4) (n=3).
[0024] FIGS. 4A-4C: HA binding through HABpep and overtime HA
release profile. SABpep-PEG-HABpep treated eyes SABpep-PEG-HABpep
and ColBpep-PEG-HABpep bind HA ex vivo. 4A) SABpep-PEG-HABpep
treated rabbit eyes (corneas were dissected and imaged after
treatments) (i) showed intense fluorescence compared to the eyes
treated with only HA (ii) and PBS (iii). Similarly,
ColBpep-PEG-HABpep treated rabbit eyes (iv) showed a higher
fluorescence compared to the eyes treated with only HA (v) and PBS
(vi). Scale bar 100 .mu.m. 4B) Short-time HA release profile for
SABpep-PEG-HABpep (-/+HA) treated eyes showed a higher amount of HA
was releasing at each time point compared to HA without the
peptide. SABpep-PEG-HABpep treated eyes bind to .about.1.8 times
more HA in the beginning as indicated by the greater initial
fluorescence value and retain HA longer, showing signal
significantly greater (.about.x1.6) than the control after 25
minutes. 4C) Long-term HA release profile for SABpep-PEG-HABpep
(+/-HA) treated eyes showed that compared to the controls, a higher
amount of HA was bound to the ocular surface even after 15 h.
[0025] FIG. 5: Friction measurement of rabbit ocular tissues
treated with HA-only compared with tissues treated with
SABpep-PEG-HABpep then HA and a non-HA-binding PEG control peptide.
Kinetic friction values (mean.+-.SEM) for three sequential times
(repeat 1, 2, 3) at the rabbit eyelid-cornea biointerface in a PBS
bath showed enhanced lubrication for tissues treated with peptide
and HA compared to HA-only (Cohen's d effect size, d=1.25, 2.13,
and 2.10) and the PEG control peptide (d=2.02, 2.70 and 1.34) as
indicated by the strong effect sizes in all three repeats in both
controls, respectively.
[0026] FIGS. 6A-6B: HA retention on in vivo mice eyes. 6A)
Harvested mice eyes applied with SABpep-PEG-HABpep+HA eye drops
(top row) retained HA for at least three fold time period longer
than those applied with control eye drops (only HA, bottom row)
(n=2). Even after 15 minutes, HA was present on the samples treated
with the binding peptide. 6B) Magnified images showed that both at
beginning and after 15 min, SABpep-PEG-HABpep+HA (i- only eye,
i'-magnified image of i) was bound homogenously to the ocular
epithelial cells, while samples treated with only HA showed
relatively less homogenous distribution. (Scale bar=100 .mu.m)
[0027] FIG. 7: HA binding on ocular surfaces through SABpep and
ColBpep. Images from FIG. 4A were analyzed using ImageJ to quantify
the relative amount of fluorescence between SABpep+HA and
ColBpep+HA treated samples. Background signal from the PBS controls
were subtracted from the images.
[0028] FIGS. 8A-8B: Estimated HA release over time by area under
curve calculation (AUC). Area under curve (AUC) was calculated for
each experimental group. The background from PBS control was then
subtracted from each AUC to quantify total amount of HA retained
for each experimental condition. A standard curve of fluorescence
versus HA was used to convert AUC values into HA (.mu.g). AUC for
release time of A) 25 minutes, and B) 16 h.
[0029] FIGS. 9A-9B: HA binding through covalently conjugated SABpep
and overtime bound HA profile ex vivo. 9A) Overtime bound HA
profile for tissues treated with SABpep-conjugated HA-FL showed a
higher amount of retained HA at each time point compared to HA
without the peptide. It is more obvious in samples treated with 3.0
mg/mL or 0.3% w/v HA-FL, probably due to the higher overall amount
of SABpep that bound to the tissue. At beginning, tissues treated
with SABpep conjugated to HA-FL showed similar fluorescence values
as for HA-FL with no peptide. This is due to the same amount of
FITC concentration in starting solutions. However, the rate of drop
of fluorescence (%) was slower in samples treated with HA-SABpep
compared to no peptide. As expected, the rates of drop in
fluorescence values remain similar for HA-FL at concentrations of 1
mg/mL and 3 mg/mL. The initial drop in fluorescence rate after 1st
wash (within 30 min) is .about.24% vs .about.4% while after 2nd
wash (within 60 min) is -28% vs 8% for tissues treated with HA-FL
but no peptide vs SABpep conjugated HA-FL. After 6 h, fluorescence
values for HA-FL-SABpep treated tissue samples were .about.1.7
times more than that of tissues treated with HA-FL but no peptide.
9B) Similarly, bound HA for SABpep-HA treated eyes showed that
compared to the control, a higher amount of HA (.about.1.7 times
for 3.0 mg/mL solution) was still bound to the ocular surface even
at 24 h time point.
DETAILED DESCRIPTION OF THE INVENTION
[0030] A current challenge in HA-based eye drop technology is
finding a way for HA to remain on the ocular surface for a
prolonged time. Increased HA retention can benefit dry eye patients
by reducing symptoms of dry eye and by increasing corneal
wettability and reducing tear evaporation. Recently, an in vitro
study demonstrated that HA is superior to two other common
lubricants, carboxymethylcellulose (CMC) and hydroxypropyl
methylcelluolose (HPMC) tested for its water retention ability and
its protective effectiveness of human corneal epithelial cells
against dehydration (Cornea 2013; 32:1260-4). Moreover, HA is
demonstrated to be beneficial for both, patients with aqueous tear-
and lipid-deficient dry eye because it increases tear volume and
tear film stability (Br J Ophthalmol 2007; 91:47-50). The present
inventors have been able to concentrate and retain HA using the
inventive compositions and methods in order to provide many
physical and biological benefits to parts of the ocular surfaces,
such as cornea and conjunctiva.
[0031] The goal of the present invention is to enhance the binding
of HA to the eye surface, which would retain a thin film of
moisture to mimic the smooth, hydrated and lubricated ocular
surface. The present invention utilizes a HA binding peptide
(HABpep) (Tolg, Hamilton et al. 2012; Amemiya, Nakatani et al.
2005) which can localize HA to the ocular surface through anchoring
sites on the epithelium. Sialic acid is usually found at the
outermost of the glycan chains on the cell surface proteins and
lipids (Varki 2008), and it is widely distributed through different
types of tissues, including ocular epithelium. Membrane-bound
mucins (e.g. mucin-1, mucin-16 and mucin-20) known as the
glycocalyx contain sialic acid in 0-glycosylated extracellular
domains and are present in the peripheral extracellular epithelium
layer of the ocular tissues (FIG. 1). The inventors chose sialic
acid as the potential anchoring site for immobilizing HA on the
ocular tissue surface. Moreover, in case of severe dry eye,
sight-threatening complications, such as persistent corneal
epithelial defect or sterile stromal ulceration, allow the stromal
tissue to be exposed to the external environment (Pflugfelder
2004). At the same time, the ocular surface epithelium is damaged
and membrane-bound mucins in the glycocalyx decrease significantly
(Corrales, Narayanan et al. 2011). Therefore, in an alternative
embodiment, the present inventors also utilize collagen as an
alternative anchoring site, as stromal collagen will be exposed
when the epithelium is absent.
[0032] In accordance with some embodiments, the present inventors
developed an eye drop composition comprising a HABpep that
noncovalently binds HA. The inventors have also developed a
chemical spacer or linker with HABpep to facilitate interactions of
HABpep with HA. In some embodiments the linker is poly(ethylene
glycol) (PEG). The other end of the PEG linker is modified by a
peptide that can bind to tissues or proteins on the ocular surface.
A number of candidate peptides that were capable of binding to the
ocular surface through different mechanisms were screened to
determine which ocular binding-peptides were the most effective
(Table 1).
TABLE-US-00001 TABLE 1 Peptides Screened Peptide Sequence (#1-6)
Description Reference K-K(Palm)-K-K-K-K(Palm) Cell membrane binding
(Matsuda, Hatanaka et al. (SEQ ID NO: 18) 2014) GWQPPRARI
Epithelial binding- (Mooradian, McCarthy et al. (SEQ ID NO: 19)
Fibronectin derived 1993) GGSPYGR Sialic acid binding (Heerze,
Chong et al. 1992) (SEQ ID NO: 15) GGPQEQITQHGSPYGRC Sialic acid
binding (Heerze, Smith et al. 1995) (SEQ ID NO: 20)
RRANAALKAGELYKSILYGC Collagen I binding peptide (Paderi, Sistiabudi
et al.) (SEQ ID NO: 11) derived from platelet rich plasma
SYIRIADTNIT Collagen I binding peptide (Kalamajski, Aspberg et al.
(SEQ ID NO: 12) derived from decorin 2007)
[0033] Out of these peptides, the inventors found that SABpep
GGSPYGRC (SEQ ID NO: 15) and ColBpep SYIRIADTNIT (SEQ ID NO: 12)
were the most promising candidates (FIG. 2A). SABpep is especially
promising, because it can directly target epithelial cells, which
consist of sialic-acid rich Mucin-1. Transmembrane mucins are the
first layer on the epithelial layer that can be targeted to
localize HA, therefore, we investigated SABpep for HA binding in
depth.
[0034] The inventors later confirmed by immunochemistry and
fluorescent cell imaging that SABpep-biotin binds selectively to
epithelial lining of conjunctiva while ColBpep-biotin binds to any
collagen I on the tissue (FIG. 2A). This selectivity can be of
importance when treating dry eye since the disease can be caused by
abnormality of any of the tear film's three layers (lipid, aqueous,
and mucin). Therefore, different ocular surface-binding peptides
can be used for different dry eye causes. Damage to a certain
region of the eye, such as where epithelium is removed, can be
selectively treated for by using ColBpep-PEG-HABpep, while in
conditions where epithelium is still partially or fully present can
be treated with SABpep.
[0035] The polymer-peptide system with collagen-binding peptide is
most useful for treating conditions related to severe dry eye that
can lead to blindness, such as Sjogren syndrome, in which the
stromal collagen is exposed as the epithelium is missing
(Pflugfelder 2004). In conditions where epithelium is still
partially or fully present (Gipson 2004), the eyes can be treated
with the polymer-peptide system containing SABpep. The binding
sites of SABpep was specifically localized on the cell membrane
throughout the ocular epithelium (FIG. 2C). It is well known that
transmembrane mucins, such as mucin-1, mucin-4 and mucin-16 are
localized primarily within apical cell membranes of the stratified
corneal and conjunctival epithelia, while transmembrane mucin-20 is
uniquely distributed throughout the conjunctival and corneal
epithelia (Gipson 2014, Woodard 2014). Since, mucins are rich in
sialic acid, SABpep should bind to these molecules.
[0036] In accordance with an embodiment, the present invention
provides novel biomaterial compositions which bind to tissue
surfaces, such as the eye, and bind HA. The embodiments of the
present invention provide biomaterials that allow tissue
modification with HA binding polymer coatings, and utilize a
synthetic peptide to target and locally concentrate hyaluronic acid
to those tissue surfaces.
[0037] The HA-binding biomaterials of the present invention can be
powerful tools for effective modification of the lubrication
environment of the eye and for other tissues where lubrication or
the presence of HA is critical for homeostasis and health.
[0038] In accordance with an embodiment, the present invention
provides a biomaterial comprising at least one biologically
compatible polymer having one or more HA binding peptides (HABPep)
covalently linked to the biologically compatible polymer, and one
or more sialic acid binding peptides (SABPep) covalently linked to
the biologically compatible polymer.
[0039] In accordance with another embodiment, the present invention
provides a biomaterial comprising at least one biologically
compatible polymer having one or more HA binding peptides (HABPep)
covalently linked to the biologically compatible polymer, and one
or more collagen binding peptides (ColBPep) covalently linked to
the biologically compatible polymer.
[0040] In accordance with a further embodiment the present
invention provides a biomaterial comprising at least one
biologically compatible polymer having one or more HA binding
peptides (HABPep) covalently linked to the biologically compatible
polymer, and one or more sialic acid binding peptides (SABPep) and
one or more collagen binding peptides (ColBPep) covalently linked
to the biologically compatible polymer.
[0041] In accordance with an embodiment, the present invention
provides a biomaterial comprising SABpep linked to Poly(ethylene
glycol) linked to HABpep (SABpep-PEG-HABpep).
[0042] In accordance with another embodiment, the present invention
provides a biomaterial comprising ColBpep linked to Poly(ethylene
glycol) linked to HABpep (ColBpep-PEG-HABpep).
[0043] In accordance with an embodiment, the present invention
provides a biomaterial comprising HA covalently linked to one or
more sialic acid binding peptides (SABPep) via a linker.
[0044] In accordance with another embodiment, the present invention
provides a biomaterial comprising HA covalently linked to a
detectable moiety (such as a fluorescent dye FL) and to one or more
sialic acid binding peptides (SABPep) (HA-FL-SABPep) via a
linker.
[0045] The HA is linked to the polymer and/or the detectable moiety
by a linker molecule. For instance linking groups having alkyl,
aryl, combination of alkyl and aryl, or alkyl and aryl groups
having heteroatoms may be present. For example, the linker can be a
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl, C.sub.1-C.sub.20 hydroxyalkyl, C.sub.1-C.sub.20 alkoxy,
C.sub.1-C.sub.20 alkoxy C.sub.1-C.sub.20 alkyl, C.sub.1-C.sub.20
alkylamino, di-C.sub.1-C.sub.20 alkylamino, C.sub.1-C.sub.20
dialkylamino C.sub.1-C.sub.20 alkyl, C.sub.1-C.sub.20 thioalkyl,
C.sub.2-C.sub.20 thioalkenyl, C.sub.2-C.sub.20 thioalkynyl,
C.sub.6-C.sub.22 acyloxy, C.sub.6-C.sub.22 arylamino
C.sub.2-C.sub.20 acyloxy, C.sub.2-C.sub.20 thioacyl,
C.sub.1-C.sub.20 amido, and C.sub.1-C.sub.20 sulphonamido.
[0046] In some embodiments the HA can be provided within the
biologically compatible polymer matrix, or added separately.
[0047] As used herein, the term "biocompatible biomaterial" means
materials that can be used for binding to tissues, and which are
acceptable for use in a mammal, preferably in a human subject.
[0048] A biologically compatible polymer refers to a polymer which
is functionalized to serve as a composition for applying to a
biological surface. The polymer is one that is a naturally
occurring polymer or one that is not toxic to the host. The polymer
can, e.g., contain at least an imide. The polymer may be a
homopolymer where all monomers are the same or a hetereopolymer
containing two or more kinds of monomers. The terms "biocompatible
polymer," "biocompatible cross-linked polymer matrix" and
"biocompatibility" when used in relation to the instant polymers
are art-recognized are considered equivalent to one another,
including to biologically compatible polymer. For example,
biocompatible polymers include polymers that are neither toxic to
the host (e.g., an animal or human), nor degrade (if the polymer
degrades at a rate that produces monomeric or oligomeric subunits
or other byproducts at toxic concentrations in the host).
[0049] As used herein, the term "crosslinked" refers to a
composition containing intermolecular links and, optionally,
intramolecular links, arising from the formation of covalent bonds.
Covalent bonding between two crosslinkable components may be
direct, in which case, an atom in one component is directly bound
to an atom in the other component, or it may be indirect, that is,
for example, through a linking group. A crosslinked gel or polymer
matrix may, in addition to covalent bonds, also include
intermolecular and/or intramolecular noncovalent bonds such as
hydrogen bonds and electrostatic (ionic) bonds.
[0050] By "detectable label(s) or moieties" is meant a composition
that when linked to a molecule of interest renders the latter
detectable, via spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include radioactive isotopes, magnetic beads, metallic beads,
colloidal particles, fluorescent dyes, electron-dense reagents,
enzymes (for example, as commonly used in an ELISA), biotin,
digoxigenin, or haptens. Specific radioactive labels include most
common commercially available isotopes including, for example,
.sup.3H, .sup.11C, .sup.13C, .sup.15N, .sup.18F, .sup.19F,
.sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.86Y, .sup.89Zr,
.sup.111In, .sup.94mTc, .sup.99mTc, .sup.64Cu and .sup.68Ga.
Suitable dyes include any commercially available dyes such as, for
example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye
800CW, ruthenium polypyridyl dyes, and the like.
[0051] Gel refers to a state of matter between liquid and solid,
and is generally defined as a polymer network swollen in a liquid
medium. Typically, a gel is a two-phase colloidal dispersion
containing both solid and liquid, wherein the amount of solid is
greater than that in the two-phase colloidal dispersion referred to
as a "sol." As such, a "gel" has some of the properties of a liquid
(i.e., the shape is resilient and deformable) and some of the
properties of a solid (i.e., the shape is discrete enough to
maintain three dimensions on a two-dimensional surface). "Gelation
time," also referred to herein as "gel time," refers to the time it
takes for a composition to become non-flowable under modest stress.
This is generally exhibited as reaching a physical state in which
the elastic modulus, G', equals or exceeds the viscous modulus,
G'', i.e., when tan(A) becomes 1 (as may be determined using
conventional rheological techniques).
[0052] The term "polymer" is used to refer to molecules composed of
repeating monomer units, including homopolymers, block copolymers,
heteropolymers, random copolymers, graft copolymers and so on.
"Polymers" also include linear polymers as well as branched
polymers, with branched polymers including highly branched,
dendritic, and star polymers.
[0053] In accordance with a further embodiment, the biocompatible
polymer can be hydrophilic.
[0054] A hydrogel is a water-swellable polymeric matrix that can
absorb water to form elastic gels. Hydrogels consist of hydrophilic
polymers crosslinked to from a water-swollen, insoluble polymer
network. Crosslinking can be initiated by many physical or chemical
mechanisms, for example, such as, a light-induced reaction.
[0055] A "matrix" is a three-dimensional network of macromolecules
held together by covalent or noncovalent crosslinks. On placement
in an aqueous environment, dry hydrogels swell to the extent
allowed by the viscosity, the gel state and/or degree of
crosslinking in the polymer or network. A matrix can be a
network.
[0056] Hydrogels are semi-interpenetrating networks that promote
cell, tissue and organ repair, and in some instances, discourage
scar formation. Hydrogels can be derivatized to contain a reactive
group to facilitate polymerization and linking. Hydrogels can also
carry a reactive group or a functional group reactive with a
biological surface, an artificial surface and/or a second polymer
or network. The latter form of reactivity also can anchor a gel of
interest at and to a site of interest. Hydrogels of interest also
are configured to have a viscosity that will enable the gelled
hydrogel to remain or reside in place for longer periods of time.
Viscosity can be controlled by the monomers and polymers used, the
degree of crosslinking, by the level of water trapped in the
hydrogel and by incorporated thickeners, such as biopolymers, such
as proteins, lipids, saccharides and the like. An example of such a
hydrogel is HA, whether crosslinked or not.
[0057] The term "functionalized" as used herein, refers to a
modification of an existing molecular segment to generate or
introduce a new reactive or more reactive group (e.g., an amine,
ester or imide group) that is capable of undergoing reaction with
another molecule, polymer or functional group (e.g., an amine, an
ester or a carboxyl group) to form a covalent bond. For example,
carboxylic acid groups can be functionalized by reaction with a
carbodiimide and an imide reagent using known procedures to provide
a new reactive functional group in the form of an imide group
substituting for the hydrogen in the hydroxyl group of the carboxyl
function.
[0058] The terms "substituted," "functional group" and "reactive
group" are contemplated to include all permissible substituents of
organic compounds on the monomers, polymers and networks of
interest. In a broad aspect, the permissible substituents include
acyclic and cyclic, branched and unbranched, carbocyclic and
heterocyclic, aromatic and nonaromatic substituents of organic
compounds. Illustrative substituents include, for example, carboxy
groups, amine groups, amide groups, hydroxyl groups and so on, as
known in the art. The permissible substituents may be one or more
and the same or different for appropriate organic compounds.
[0059] A functional group or a moiety capable of mediating
formation of a polymer or network can be added to a naturally
occurring molecule or a synthetic molecule practicing methods known
in the art. Functional groups include the various radicals and
chemical entities taught herein, and include alkenyl moieties such
as acrylates, methacrylates, dimethacrylates, oligoacrylates,
oligomethacrylates, ethacrylates, itaconates or acrylamides.
[0060] Further functional groups include aldehydes. Other
functional groups may include ethylenically unsaturated monomers
including, for example, alkyl esters of acrylic or methacrylic acid
such as methyl methacrylate, ethyl methacrylate, butyl
methacrylate, ethyl acrylate, butyl acrylate, hexyl acrylate,
n-octyl acrylate, lauryl methacrylate, 2-ethylhexyl methacrylate,
nonyl acrylate, benzyl methacrylate, the hydroxyalkyl esters of the
same acids such as 2-hydroxyethyl acrylate, 2-hydroxyethyl
methacrylate, and 2-hydroxypropyl methacrylate, the nitrite and
amides of the same acids such as acrylonitrile, methacrylonitrile,
and methacrylamide, vinyl acetate, vinyl propionate, vinylidene
chloride, vinyl chloride, and vinyl aromatic compounds such as
styrene, t-butyl styrene and vinyl toluene, dialkyl maleates,
dialkyl itaconates, dialkyl methylene-malonates, isoprene and
butadiene. Suitable ethylenically unsaturated monomers containing
carboxylic acid groups include acrylic monomers such as acrylic
acid, methacrylic acid, ethacrylic acid, itaconic acid, maleic
acid, fumaric acid, monoalkyl itaconate including monomethyl
itaconate, monoethyl itaconate, and monobutyl itaconate, monoalkyl
maleate including monomethyl maleate, monoethyl maleate, and
monobutyl maleate, citraconic acid and styrene carboxylic acid.
Suitable polyethylenically unsaturated monomers include butadiene,
isoprene, allylmethacrylate, diacrylates of alkyl dials such as
butanediol diacrylate and hexanediol diacrylate, divinyl benzene
and the like.
[0061] In some embodiments, other suitable hydrophilic polymers
which can serve as the biocompatible polymer include synthetic
polymers such as poly(ethylene glycol), poly(ethylene oxide),
partially or fully hydrolyzed poly(vinyl alcohol),
poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene
oxide)-co-poly(propylene oxide) block copolymers (poloxamers and
meroxapols), poloxamines, carboxymethyl cellulose, and
hydroxyalkylated celluloses such as hydroxyethyl cellulose and
methylhydroxypropyl cellulose, and natural polymers, such as,
polysaccharides or carbohydrates such as Ficoll.TM. polysucrose,
dextran, heparan sulfate, chondroitin sulfate or alginate, and
polypeptides or proteins such as gelatin, collagen, albumin or
ovalbumin, or copolymers or blends thereof.
[0062] In accordance with still another embodiment, the
biocompatible polymers are selected from the group consisting of:
Poly(ethylene glycol), Poly(propylene glycol), Poly(methyl vinyl
ether), Oligoethylene, Poly(isobutylene) Poly(tetrahydrofuran)
Poly(oxytrimethylene), Poly(dimethylsiloxsane),
Poly(dimethylsilane), Nylon 6, Nylon 11, Poly(acrylonitrile),
Squalane, Poly(1,3-dioxolane), Poly(iminooligomethylene),
Poly(1-lysine), Polyethyleneimine, Poly(adipate),
Poly(1-caprolactone), Poly(L-lactic acid), or derivatives
thereof.
[0063] By way of example, and not limitation, and in particular
embodiments, the polymer can comprise synthetic reactants and
comprises poly(ethylene glycol) (PEG) or a derivative thereof.
[0064] Polysaccharides that are very viscous liquids or that are
thixotropic, and form a gel over time by the slow evolution of
structure, are also useful. For example, HA, which can form an
injectable gel with a consistency like a hair gel, may be utilized.
Modified hyaluronic acid can also be useful. As used herein, the
term "modified hyaluronic acids" refers to chemically modified
hyaluronic acids. Modified hyaluronic acids may be designed and
synthesized with preselected chemical modifications to adjust the
rate and degree of linking and biodegradation. For example,
modified hyaluronic acids may be designed and synthesized to be
esterified with a relatively hydrophobic group such as propionic
acid or benzylic acid to render the polymer more hydrophobic and
gel-forming, or which are grafted with amines to promote
electrostatic self-assembly. Modified hyaluronic acids thus, may be
synthesized which are injectable, to flow under stress, but
maintain a gel-like structure when not under stress. Hyaluronic
acid and hyaluronic derivatives are available from Genzyme,
Cambridge, Mass. and Fidia, Italy. Other commercially available HA
useful in the invention are Restylane, comprising a crosslinked HA,
and Juvederm (Allergan) comprising HA.
[0065] HA is a polymer of disaccharides, themselves composed of
D-glucuronic acid and D-N-acetylglucosamine, linked via alternating
(3-1,4 and (3-1,3 glycosidic bonds. HA can have 25,000 disaccharide
repeats in length. Polymers of HA can range in size from 5,000 to
20,000,000 Da in vivo.
[0066] In accordance with one or more embodiments, the HA that can
be used with the inventive compositions and methods can have
molecular weights in the range of 5 kDa to about 20.times.10.sup.4
kDa, in some embodiments, HA can have a molecular weight of between
about 10 kDa to about 2.times.10.sup.4 kDa.
[0067] In accordance with one or more embodiments, the HA that can
be used with the inventive compositions and methods can be
crosslinked.
[0068] Photopolymerization is a method to covalently crosslink
polymer chains, whereby a photoinitiator and polymer solution
(termed "pre-gel" or monomer solution) are exposed to a light
source specific to the photoinitiator. On activation, the
photoinitiator reacts with specific functional groups in the
polymer chains, linking the functional groups to form the hydrogel.
The reaction generally is rapid (3-5 minutes) and can proceed at
room or body temperature. Photoinduced gelation enables spatial and
temporal control of gel formation, permitting shape manipulation
after injection and during gelation in vivo. Cells and bioactive
factors can be incorporated into the hydrogel scaffold by simply
mixing same in and with the polymer solution prior to gelation
[0069] In accordance with an embodiment, the HABPep is a peptide
which is capable of specifically binding HA. Many such HA binding
peptides are known in the art. See for example WO/2006/130974,
which describes many such peptides which have at least one
repetition of the ammo acid residue sequence B.sub.rX.sub.7-B.sub.2
where B is any basic amino acid residue and X.sub.7 are any 7
non-acidic amino acid residues. The binding of the peptide to HA
may be enhanced by the addition of basic ammo acid residues between
B1 and B2 or flanking either end of motif (non-conservative
substitutions).
[0070] The HABP52 family of HA binding peptides includes peptides
with an amino acid sequence selected from the group consisting of
i) (RRDDGAHWQFNALTVR) (SEQ ID NO: 1) or (CRRDDGAHWQFNALTVR) (SEQ ID
NO: 2) or a conservative amino acid substitution thereof at a
residue position other than 4, 5, 6, 9, 10 or 11, ii) GAAWQFNALTVR,
(SEQ ID NO: 3) or a conservative amino acid substitution thereof at
a residue position other than 4, 5, 6, 9, 10 or 11, iii)
GAHWQFAALTVR, (SEQ ID NO: 4) or a conservative amino acid
substitution thereof at a residue position other than 4, 5, 6, 9,
10 or 11, and iv) GAHWQFNALTVA (SEQ ID NO: 5) or a conservative
amino acid substitution thereof at a residue position other than 4,
5, 6, 9, 10 or 11.
[0071] In accordance with some embodiments the HABpep comprises the
peptide GAHWQFNALTVR (SEQ ID NO: 6) or a conservative amino acid
substitution thereof at a residue position other than 4, 5, 6, 9,
10 or 11.
[0072] In accordance with some embodiments the HABpep comprises the
peptide STMMSRSHKTRSHHV (SEQ ID NO: 7); or RYPISRPRKRC (SEQ ID NO:
8); or TAGHGRRWS (SEQ ID NO: 9); or LKQKIKHVVKLKVVVKLRSQLVKRKQN
(SEQ ID NO: 10) or a peptide derivative thereof with conservative
amino acid substitutions.
[0073] As used herein, the term "collagen binding peptide
(ColBPep)" means a protein, peptide or fragment which is capable of
specifically binding extracellular matrix proteins, such as
collagen I or collagen II. In accordance with one or more
embodiments, the ColBpep can comprise a peptide having the
following sequences: RRANAALKAGELYKSILYGC (SEQ ID NO: 11),
SYIRIADTNIT (SEQ ID NO: 12), YSFYSDESLQ (SEQ ID NO: 13) and WYRGRL
(SEQ ID NO: 14) or a peptide derivative thereof with conservative
amino acid substitutions.
[0074] As used herein, the term "sialic acid binding peptide
(SABPep)" means a protein, peptide or fragment which is capable of
specifically binding a glycoprotein having sialic acid sugar
moieties. In accordance with one or more embodiments, the SABpep
can comprise a peptide having the following sequences: GGSPYGRC
(SEQ ID NO: 15), and GGPQEQITQHGSPYGRC (SEQ ID NO: 16) or a peptide
derivative thereof with conservative amino acid substitutions.
[0075] It will be understood by those of ordinary skill in the art
that any known conjugation method which can be used to attach both
peptides, HABpep, ColBpep or SABpep to the PEG spacer or linker, or
any biocompatible spacer through functional reactive groups can be
used in the compositions and methods of the present invention.
[0076] In accordance with an embodiment, the present invention
provides pharmaceutical compositions comprising the biomaterials
described herein, and a pharmaceutically acceptable carrier.
[0077] Therapeutic formulations of the product may be prepared for
storage as lyophilized formulations or aqueous solutions by mixing
the biomaterials described herein, having the desired degree of
purity with optional pharmaceutically acceptable carriers,
diluents, excipients or stabilizers typically employed in the art,
i.e., buffering agents, stabilizing agents, preservatives,
isotonifiers, non-ionic detergents, antioxidants and other
miscellaneous additives, see Remington's Pharmaceutical Sciences,
16th ed., Osol, ed. (1980). Such additives are generally nontoxic
to the recipients at the dosages and concentrations employed,
hence, the excipients, diluents, carriers and so on are
pharmaceutically acceptable.
[0078] The compositions can take the form of solutions,
suspensions, emulsions, powders, sustained-release formulations,
depots and the like. Examples of suitable carriers are described in
"Remington's Pharmaceutical Sciences," Martin. Such compositions
will contain an effective amount of the biopolymer of interest,
preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
patient. As known in the art, the formulation will be constructed
to suit the mode of administration.
[0079] Buffering agents help to maintain the pH in the range which
approximates physiological conditions. Buffers are preferably
present at a concentration ranging from about 2 mM to about 50 mM.
Suitable buffering agents for use with the instant invention
include both organic and inorganic acids, and salts thereof, such
as citrate buffers (e.g., monosodium citrate-disodium citrate
mixture, citric acid-trisodium citrate mixture, citric
acid-monosodium citrate mixture etc.), succinate buffers (e.g.,
succinic acid monosodium succinate mixture, succinic acid-sodium
hydroxide mixture, succinic acid-disodium succinate mixture etc.),
tartrate buffers (e.g., tartaric acid-sodium tartrate mixture,
tartaric acid-potassium tartrate mixture, tartaric acid-sodium
hydroxide mixture etc.), fumarate buffers (e.g., fumaric
acid-monosodium fumarate mixture, fumaric acid-disodium fumarate
mixture, monosodium fumarate-disodium fumarate mixture etc.),
gluconate buffers (e.g., gluconic acid-sodium glyconate mixture,
gluconic acid-sodium hydroxide mixture, gluconic acid-potassium
gluconate mixture etc.), oxalate buffers (e.g., oxalic acid-sodium
oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic
acid-potassium oxalate mixture etc.), lactate buffers (e.g., lactic
acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture,
lactic acid-potassium lactate mixture etc.) and acetate buffers
(e.g., acetic acid-sodium acetate mixture, acetic acid-sodium
hydroxide mixture etc.). Phosphate buffers, carbonate buffers,
histidine buffers, trimethylamine salts, such as Tris, HEPES and
other such known buffers can be used.
[0080] Preservatives may be added to retard microbial growth, and
may be added in amounts ranging from 0.2%-1% (w/v). Suitable
preservatives for use with the present invention include phenol,
benzyl alcohol, m-cresol, octadecyldimethylbenzyl ammonium
chloride, benzyaconium halides (e.g., chloride, bromide and
iodide), hexamethonium chloride, alkyl parabens, such as, methyl or
propyl paraben, catechol, resorcinol, cyclohexanol and
3-pentanol.
[0081] Isotonicifiers are present to ensure physiological
isotonicity of liquid compositions of the instant invention and
include polhydric sugar alcohols, preferably trihydric or higher
sugar alcohols, such as glycerin, erythritol, arabitol, xylitol,
sorbitol and mannitol. Polyhydric alcohols can be present in an
amount of between about 0.1% to about 25%, by weight, preferably 1%
to 5% taking into account the relative amounts of the other
ingredients.
[0082] Stabilizers refer to a broad category of excipients which
can range in function from a bulking agent to an additive which
solubilizes the therapeutic agent or helps to prevent denaturation
or adherence to the container wall. Typical stabilizers can be
polyhydric sugar alcohols; amino acids, such as arginine, lysine,
glycine, glutamine, asparagine, histidine, alanine, ornithine,
L-leucine, 2-phenylalanine, glutamic acid, threonine etc.; organic
sugars or sugar alcohols, such as lactose, trehalose, stachyose,
arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol,
myoinisitol, galactitol, glycerol and the like, including cyclitols
such as inositol; polyethylene glycol; amino acid polymers; sulfur
containing reducing agents, such as urea, glutathione, thioctic
acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and
sodium thiosulfate; low molecular weight polypeptides (i.e., <10
residues); proteins, such as human serum albumin, bovine serum
albumin, gelatin or immunoglobulins; hydrophilic polymers, such as
polyvinylpyrrolidone, saccharides, monosaccharides, such as xylose,
mannose, fructose or glucose; disaccharides, such as lactose,
maltose and sucrose; trisaccharides, such as raffinose;
polysaccharides, such as, dextran and so on. Stabilizers can be
present in the range from 0.1 to 10,000 w/w per part of
biopolymer.
[0083] Additional miscellaneous excipients include bulking agents,
(e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g.,
ascorbic acid, methionine or vitamin E) and cosolvents.
[0084] Non-ionic surfactants or detergents (also known as "wetting
agents") may be added to help solubilize the therapeutic agent, as
well as to protect the therapeutic protein against
agitation-induced aggregation, which also permits the formulation
to be exposed to shear surface stresses without causing
denaturation of the protein. Suitable non-ionic surfactants include
polysorbates (20, 80 etc.), polyoxamers (184, 188 etc.),
Pluronic.RTM. polyols and polyoxyethylene sorbitan monoethers
(TWEEN-20.RTM., TWEEN-80.RTM. etc.). Non-ionic surfactants may be
present in a range of about 0.05 mg/ml to about 1.0 mg/ml,
preferably about 0.07 mg/ml to about 0.2 mg/ml.
[0085] The present invention provides liquid formulations of a
biopolymer having a pH ranging from about 5.0 to about 7.0, or
about 5.5 to about 6.5, or about 5.8 to about 6.2, or about 6.0, or
about 6.0 to about 7.5, or about 6.5 to about 7.0.
[0086] The instant invention encompasses formulations, such as,
liquid formulations having stability at temperatures found in a
commercial refrigerator and freezer found in the office of a
physician or laboratory, such as from about 20.degree. C. to about
5.degree. C., said stability assessed, for example, by microscopic
analysis, for storage purposes, such as for about 60 days, for
about 120 days, for about 180 days, for about a year, for about 2
years or more. The liquid formulations of the present invention
also exhibit stability, as assessed, for example, by particle
analysis, at room temperatures, for at least a few hours, such as
one hour, two hours or about three hours prior to use.
[0087] Examples of diluents include a phosphate buffered saline,
buffer for buffering against gastric acid in the bladder, such as
citrate buffer (pH 7.4) containing sucrose, bicarbonate buffer (pH
7.4) alone, or bicarbonate buffer (pH 7.4) containing ascorbic
acid, lactose, or aspartame. Examples of carriers include proteins,
e.g., as found in skim milk, sugars, e.g., sucrose, or
polyvinylpyrrolidone. Typically these carriers would be used at a
concentration of about 0.1-90% (w/v) but preferably at a range of
1-10%.
[0088] The formulations to be used for in vivo administration must
be sterile. That can be accomplished, for example, by filtration
through sterile filtration membranes. For example, the formulations
of the present invention may be sterilized by filtration.
[0089] The biomaterial compositions of the present invention will
be formulated, dosed and administered in a manner consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The "therapeutically effective amount" of the biomaterial to be
administered will be governed by such considerations, and can be
the minimum amount necessary to prevent, ameliorate or treat a
disorder of interest. As used herein, the term "effective amount"
is an equivalent phrase refers to the amount of a therapy (e.g., a
prophylactic or therapeutic agent), which is sufficient to reduce
the severity and/or duration of a disease, ameliorate one or more
symptoms thereof, prevent the advancement of a disease or cause
regression of a disease, or which is sufficient to result in the
prevention of the development, recurrence, onset, or progression of
a disease or one or more symptoms thereof, or enhance or improve
the prophylactic and/or therapeutic effect(s) of another therapy
(e.g., another therapeutic agent) useful for treating a
disease.
[0090] Generally, the ingredients are supplied either separately or
mixed together in unit dosage form, for example, as a dry
lyophilized powder or water-free concentrate in a sealed container,
such as an ampule or sachet indicating the quantity of active
agent. Where the composition is to be administered by ophthalmic
solution, it can be dispensed with a bottle containing sterile
pharmaceutical grade water or saline.
[0091] An "active agent" and a "biologically active agent" are
phrases used interchangeably herein to refer a chemical or
biological compound that induces a desired pharmacological or
physiological effect, wherein the effect may be prophylactic or
therapeutic. The terms also encompass pharmaceutically acceptable,
pharmacologically active derivatives of those active agents
specifically mentioned herein, including, but not limited to,
salts, esters, amides, prodrugs, active metabolites, analogs and
the like.
[0092] When the terms "active agent," "pharmacologically active
agent" and "drug" are used, it is to be understood that the
invention includes the active agent per se, as well as
pharmaceutically acceptable, pharmacologically active salts,
esters, amides, prodrugs, metabolites, analogs etc. The active
agent can be a biological entity, such as a virus or cell, whether
naturally occurring or manipulated, such as transformed.
[0093] In accordance with an embodiment, the present invention
provides the use of the biomaterial compositions disclosed herein,
for treating eye diseases by means of an eye surgery treatment in a
subject, characterized in that an effective amount of the
biomaterial composition is administered to the tissue of the
subject in need of treatment.
[0094] In accordance with an embodiment, the present invention
provides a therapeutic method for the treatment of eye diseases by
means of an eye surgery treatment, comprising applying to the eye
of a subject in need of such treatment a therapeutically effective
amount of the biomaterial compositions described herein. Such
surgical procedures include, but are not limited to, corneal
transplantation, cataract surgery, glaucoma surgery, and surgery to
repair retinal detachment.
[0095] In accordance with another embodiment, the present invention
provides a therapeutic method for the treatment of dry eye or
keratoconjunctivitis sicca (KCS) which can be the result of a
number of disorders, including, for example, Sjogren's syndrome.
The inventive methods comprise applying an effective amount of the
biomaterial compositions of the present invention on the cornea of
the eye, and which may include other therapeutic agents, such as
estrogens, or cyclosporine.
[0096] In accordance with an embodiment, the present invention
provides the use of the biomaterial compositions disclosed herein,
for treating dry eye in a subject, characterized in that an
effective amount of the biomaterial composition is applied to the
eye of a subject in need of such treatment.
[0097] In accordance with an embodiment, the present invention
provides the use of a biomaterial comprising HA covalently linked
to a detectable moiety (such as, for example, a fluorescent dye FL)
and to one or more sialic acid binding peptides (SABPep)
(HA-FL-SABPep) via a linker for diagnosis or confirmation or
grading the severity of dry eye or keratoconjunctivitis sicca (KCS)
in a subject, comprising administering to the eye of the subject
suspected of having dry eye or KCS an effective amount of the
biomaterial and detecting the amount of binding of the biomaterial
to the cornea of the eye. If the amount of binding of the
composition is less than the amount of binding in a control eye,
the subject is diagnosed or confirmed as having dry eye or KCS, or
in the case of comparing the binding amount to the amounts in cases
of known severity, the severity of the disease can be graded or
assessed.
[0098] One of ordinary skill in the art would understand that the
type of detectable moiety can be other than a fluorescent moiety,
including for example, colloidal particles, fluorescent dyes,
electron-dense reagents, enzymes, and dyes such as, for example,
5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW. It
will be understood that the uses provided herein can also include
administration of at least one additional therapeutic agent with
the compositions disclosed herein.
[0099] Examples of biologically active agents include, without
limitation, enzymes, receptor antagonists or agonists, hormones,
growth factors, antibiotics, antimicrobial agents, and antibodies.
The term "biologically active agent" is also intended to encompass
various cell types and genes that can be incorporated into the
compositions of the invention.
[0100] In certain embodiments, the subject compositions comprise
about 1% to about 75% or more by weight of the total composition,
alternatively about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70%,
of a biologically active agent.
[0101] Non-limiting examples of biologically active agents include
following: adrenergic blocking agents, androgenic steroids,
anti-allergenic materials, anti-cholinergics and sympathomimetics,
anti-infective agents, anti-inflammatory agents such as steroids,
non-steroidal anti-inflammatory agents, anti-pyretic and analgesic
agents, antihistamines, biologicals, decongestants, estrogens, ion
exchange resins, growth factors, neuromuscular drugs, nutritional
substances, peripheral vasodilators, progestational agents,
prostaglandins, vitamins, antigenic materials, and prodrugs.
[0102] In accordance with one or more embodiments, there is
provided ophthalmic formulations comprising the inventive
biomaterials, wherein the formulation is suitable for
administration to the eye of a subject. The ophthalmic formulation
may have a pH between 5.5 and 7. In some embodiments the ophthalmic
formulation is an aqueous formulation. In some embodiments the
ophthalmic formulation is in the form of a single dose unit. In
some embodiments the ophthalmic formulation does not comprise a
preservative. The ophthalmic formulation may further comprise one
or more additional therapeutic agents, such as antioxidants. The
ophthalmic formulation may further comprise one or more tear
substitutes. In some embodiments, at least one of the tear
substitutes contains an ophthalmic lubricant (e.g.,
hydroxypropylmethylcellulose).
[0103] A variety of tear substitutes are known in the art and
include, but are not limited to: monomeric polyols, such as,
glycerol, propylene glycol, and ethylene glycol; polymeric polyols
such as polyethylene glycol; cellulose esters such
hydroxypropylmethyl cellulose, carboxy methylcellulose sodium and
hydroxy propylcellulose; dextrans such as dextran 70; water soluble
proteins such as gelatin; vinyl polymers, such as polyvinyl
alcohol, polyvinylpyrrolidone, and povidone; and carbomers, such as
carbomer 934P, carbomer 941, carbomer 940 and carbomer 974P. Many
such tear substitutes are commercially available, which include,
but are not limited to cellulose esters such as Bion Tears.RTM.,
Celluvisc.RTM., Genteal.RTM., OccuCoat.RTM., Refresh.RTM., Teargen
II.RTM., Tears Naturale.RTM., Tears Natural II.RTM., Tears Naturale
Free.RTM., and TheraTears.RTM.; and polyvinyl alcohols such as Akwa
Tears.RTM., HypoTears.RTM., Moisture Eyes.RTM., Murine
Lubricating.RTM., and Visine Tears.RTM.. Tear substitutes may also
be comprised of paraffins, such as the commercially available
Lacri-Lube.RTM. ointments. Other commercially available ointments
that are used as tear substitutes include Lubrifresh PM.RTM.,
Moisture Eyes PM.RTM. and Refresh PM.RTM.. Preservatives and other
additives may also be present such as, for example, antimicrobials,
antioxidants, chelating agents, and inert gases and the like.
[0104] It will be understood by those of ordinary skill that a
dosing regimen used in the inventive compositions and methods can
be any length of time sufficient to provide enhanced HA
concentration in the eyes of the subject. The term "chronic" as
used herein, means that the length of time of the dosage regimen
can be hours, days, weeks, months, or possibly years. In accordance
with an embodiment, the composition is administered for at least 48
hours.
[0105] Typically, an attending physician will decide the dosage of
the composition with which to treat each individual subject, taking
into consideration a variety of factors, such as age, body weight,
general health, diet, sex, compound to be administered, route of
administration, and the severity of the condition being treated. By
way of example, and not intending to limit the invention, the dose
of the compositions of the present invention can be at a
concentration from about 0.1 mg/ml of biomaterial composition to
about 100 mg/ml, preferably from about 1 mg/ml to about 10 mg/ml.
In some embodiments, solutions comprising HA can have HA in
concentrations of between about 0.01% to 1% by weight, preferably
between about 0.1% to about 0.5% by weight of solution.
[0106] In accordance with an embodiment, the present invention
provides a method for making the biomaterial composition as
described herein, comprising: a) obtaining a sufficient amount of
one or more biocompatible polymers conjugated to at least one or
more N-succinimide groups and one or more maleimide groups in a
suitable solution; b) adding to the solution of a) a sufficient
amount of one or more ColBPep or SABpep and allowing it to react
with the one or more N-succinimide groups to produce one or more
biocompatible polymers having one or more ColBPep or SABpep which
are covalently linked to the biocompatible polymers; c) obtaining a
sufficient amount of having one or more thiolated HA binding
peptides (C-HABPep) in a suitable solution; d) adding the solution
of b) to the solution of c) and mixing for a sufficient period of
time to produce one or more biocompatible polymers having one or
more HA binding peptides (HABPep) which are covalently linked to
the biocompatible polymers which are covalently linked to one or
more ColBPep or SABpep; e) adding to the solution of d) a
sufficient amount of hyaluronic acid (HA) in a suitable solvent for
a sufficient time to allow HA to bind to the HABPep in the
solution; f) removing the unreacted reagents of b), c) and e) and
purifying the remaining product.
[0107] It will be understood by those of ordinary skill in the art
that any biocompatible polymer can be used in the inventive
methods. In a preferred embodiment, the biocompatible polymer is
poly(ethylene glycol).
[0108] In accordance with an embodiment, the present invention
provides a bifunctional biopolymer composition comprising a
biologically compatible polymer having at least one amine reactive
moiety and at least one thiol reactive moiety.
[0109] The term "bifunctional biopolymer composition" means a
biocompatible polymer which has been chemically modified to have at
least one amine reactive moiety and at least one thiol reactive
moiety covalently linked to the polymer either directly or via a
linking moiety.
[0110] In one or more embodiments, the amine reactive moiety can
include N-hydroxysuccinimide or N-hydroxysulfosuccinimide. Other
bifunctional biopolymer compositions can include, for example,
maleimide-PEG-N-hydroxysuccinimide;
iodoacetamide-PEG-hydroxysuccinimide/sulfsuccinimide; and
acrylate-PEG-N-hydroxysuccinimide. Other molecules such as
azlactones, imidoesters, epoxides, fluorophenyl ester, anhydride,
caronate, acyl azide, isothiocyanate, isocyanate, aldehyde, etc.
can also be used.
[0111] In one or more embodiments, the thiol reactive moiety can
include maleimide or iodoacetamide.
[0112] One of ordinary skill in the art will understand that the
chemical modifications to the biopolymers to incorporate the amine
reactive moieties and thiol reactive moieties are known in the art
and can be accomplished using known methods.
[0113] In some embodiments, the biomaterial composition described
herein can be administered to the site in the subject along with HA
in a one-step process. In other embodiments the biomaterial
composition described herein can be administered to the site either
before or after administering HA to the site.
[0114] Therefore, in an embodiment, the inventive SABpep-PEG-HABpep
and/or ColBpep-PEG-HABpep compositions can be applied to existing
eye drop technology by simply mixing the composition to a standard
HA-eye drop solution. The advantages of this technology are that it
is easy to prepare and does not require any modifications to
standard HA eye drop solution--the peptide construct can simply be
added to the HA-eye drop solution. The practical implication is
that HABpep technology may overcome frequent HA-containing eye drop
instillation and problems associated with dry eyes by localizing HA
on ocular surface that retains water for prolonged time and
enhances wettability.
EXAMPLES
[0115] Materials
[0116] Six different binding peptide candidates that could
potentially bind ocular tissues were custom synthesized (NeoBioLab,
MA, USA) (Table 1) (36-41). The peptides were biotinylated with a
linker, N-hydroxysuccinimde (NHS)-PEG-biotin (Life Technologies,
NY) with a standard method as recommended by the manufacturer's
protocol. HA, HA-biotin and HA-fluorescein (HA-FL) (each with Mw
800 kDa) with a degree of substitution .about.5 mol %, were
purchased from Creative PEGWorks (Chapel Hill, N.C.).
Heterofunctional poly(ethylene glycol) (PEG, Mw 1000 Da) with
SABpep (GGSPYGRC) (SEQ ID NO: 15) and HABpep (STMMSRSHKTRSHHVGC)
(SEQ ID NO: 17) (Amemiya, Nakatani et al. 2005; Tolg, Hamilton et
al. 2012) were custom synthesized from SynPeptide (China).
[0117] Screening of ocular surface-binding peptides.
[0118] Conjunctival tissue from the ocular surface was targeted to
test the binding affinity of the multiple peptides. Conjunctiva was
harvested from rabbit eyes (Pel-Freez Biologicals, Rogers, Ariz.)
with the underlying tenon's capsule removed, and then cut into 6 mm
pieces with a biopsy punch. The conjunctival epithelium was kept
intact for testing SABpep, while the epithelium was scrapped off
with a cell scraper for testing type I collagen-binding peptide
(ColBpep). The tissue pieces were merged in the biotin-conjugated
peptide solutions (1.0 mg/mL in PBS, pH 7.4; Life Technologies, NY)
for 20 minutes and then washed three times with PBS to remove any
unbound peptides. A HABA (4'-hydroxyazobenzene-2-carboxylic acid)
assay (Life Technologies, NY) was used to measure the amount of
bound peptide (biotin), per manufacturer's instructions. An aqueous
solution of biotin (1.0 mg/mL) without peptides was used as a
negative control. Peptides with the highest binding to conjunctiva
corresponding to their fluorescence values were chosen for further
experiments.
[0119] Immunohistochemistry.
[0120] To visualize binding of SABpep and ColBpep to the ocular
surface, we performed immunohistochemistry with biotin-conjugated
peptides on tissue section of rabbit conjunctiva. Conjunctival
tissue dissected from rabbit eyes were fixed with 4%
paraformaldehyde (PFA) solution (Electron Microscopy Sciences,
Hatfield, Pa.), embedded and sectioned (thickness-5 .mu.m) in
paraffin. The sections were first incubated with biotin-conjugated
peptides (1.0 mg/ml in PBS pH 7.4) for 1 h at room temperature,
washed thrice with PBS and then stained with streptavidin-Texas Red
conjugate (Vector Laboratories, CA) in accordance to manufacturer's
protocol. The samples were then washed again three times, mounted
with DAPI mounting medium (Vector Laboratories) and imaged with
Zeiss Axio Imager 2 microscope (Carl Zeiss, Jena, Germany).
Sections stained with only secondary antibody served as negative
control. Mouse anti-mucin-1 primary antibody (Novus Biologicals,
Littleton, Colo.) and mouse anti-collagen type I antibody (Abcam,
Cambridge, Mass.) were used as replacements to SABpep and ColBpep
in the positive controls, respectively, and stained with goat
anti-mouse IgG secondary antibody (Life Technologies, NY).
[0121] Quartz Crystal microbalance with dissipation (QCM-D) binding
studies.
[0122] QCM-D studies were conducted using a Qsense E4 system and
gold-coated sensor crystals QSX 301 (Biolin Scientific, Linthicum
Heights, Md.) to directly evaluate the binding polymer-peptide
constructs of SABpep and HABpep to mucin and HA, respectively. At a
flow rate of 25 .mu.l/min in PBS (pH=7.4), mucin from the bovine
submaxillary glands (type I-S, Sigma-Aldrich, St. Louis, Mo.) or
HA-thiol (Creative PEGWorks, Chapel Hill, N.C.) were applied on
pristine surfaces at cw=0.1 mg/mL in PBS at 37.degree. C. After a
PBS rinse, either SABpep (0.1 mg/mL) or SABPep-PEG-HABpep (Mw
3698.43 Da, 0.1 mg/mL) was applied to the respective substrates.
Changes of frequency (.DELTA.f) and dissipation (.DELTA.D) were
recorded, and the mass per surface area (.DELTA.m) for immobilized
SABpep and HABpep was calculated using the Sauerbrey relationship
on the fifth overtone (.DELTA.m/C=-.DELTA.f, C=17.7 ng/cm.sup.2)
and for HA-thiol and Mucin substrates using the visco-elastic Voigt
model (Reviakine, Johannsmann et al. 2011; Majd, Kuijer et al.
2014).
[0123] Ex vivo imaging for studying HA binding.
[0124] SABpep-PEG-HABpep and ColBpep-PEG-HABpep (1.0 mg/mL)
solutions were applied on the corneal surface of the rabbit eyes
for 20 minutes. The epithelium was scrapped off with a cell scraper
for testing ColBpep-PEG-HABpep. After washing twice with PBS, 10
.mu.g/ml of HA-FL was applied to the same area for 20 minutes.
Positive controls received HA-FL with no peptides and negative
controls were treated with only PBS. For imaging, the eyes were
washed again with PBS and corneas were excised and imaged under
Axio Imager 2 microscope (Carl Zeiss). ImageJ was used to process
and quantify the amount fluorescence in each image. We further
performed overtime HA release studies on SABpep-PEG-HABpep treated
samples by measuring fluorescence values with full area scan
periodically every 5 minutes for a total of 25 minutes and also 2,
4 and 16 hours after the initial fluorescence measurement. The
value of fluorescence was compared to positive and negative
controls. All groups were performed in triplicate.
[0125] Friction Measurement
[0126] Ocular biomechanical friction tests were conducted on
freshly excised rabbit ocular tissues mounted on a Bose ELF3200
instrument using a custom eyelid-cornea friction testing procedure
that was previously reported (Morrison, Sullivan et al. 2012;
Schmidt, Sullivan et al. 2013; Samsom, Chan et al. 2015). To assess
the lubricating ability of eye drops, the tissues were soaked in
either an HA-only solution (1.0 mg/mL), or a PEG non-HA-binding
peptide control solution (SABpep-PEG, 1.0 mg/mL), or an
SABpep-PEG-HABpep solution (1.0 mg/mL) followed by a 1.0 mg/mL HA
solution for 1 h in each solution and washed three times in saline
for two minutes each between peptide and HA solutions and before
mounting the samples for friction testing. During the friction
testing, the tissues were articulated against each other at an
effective sliding velocity of 0.3 mm per second and under three
different normal loads of approximately 4-20 kPa, mimicking the
blinking of a human eye. Each test sequence was repeated 3 times in
a 0.25 mL saline lubricant bath.
[0127] HA release profile studies for SABpep-PEG-HABpep treated
eyes.
[0128] Rabbit eyes (n=3) were placed in a 24-well plate and were
incubated in 0.5 mg/ml SABpep-PEG-HABpep solution for 20 minutes.
They were then washed with PBS three times and emerged in HA-FITC
(10 .mu.g/ml) solution. Similar to the ex vivo method, positive
controls were treated with only HA-FITC and negative controls were
treated with only PBS. After twenty minutes, the eyes were washed
with PBS three times and fluorescence was measured using an area
scan from a microwell plate reader. Change in fluorescence was
recorded periodically every 5 minutes for a total of 25 minutes and
also after 2 hours, 4 hours and 16 hours after the initial
fluorescence measurement.
[0129] In Vivo Studies.
[0130] In vivo studies were performed on mice treated with an HA
eye drop solution containing SABpep-PEG-HABpep. SABpep eye drop
solution (total volume 5 .mu.l) consisting of SABpep-PEG-HABpep and
HA-FL was prepared by combining a 1:1 volume ratio of 5 mg/ml
SABpep-PEG-HABpep and 1 mg/ml HA-FL solution (corresponds to 0.1%
HA eye drop solution). Five mg/ml HA-FL solution mixed with an
equal volume of PBS served as a control eye drop. Mice (C57BL/6,
Charles River) were anesthetized with isoflurane (Baxter, Ill.) and
SABpep-PEG-HABpep/HA eye drop solution was applied to the right eye
while the control eye drop was applied to the left one. Isoflurane
gas was then removed and the mice were allowed to regain
consciousness. Mice were sacrificed approximately 5, 10 or 15
minutes after regaining consciousness. Whole mice were imaged using
a fluorescent dissecting microscope (Nikon, Melville, N.Y.),
allowing us to take images of the eye without harvesting the
tissues. However, we also harvested eyes and imaged with Zeiss Axio
Imager 2 microscope to obtain magnified images.
[0131] Live/dead staining and AlamarBlue assay for the cytotoxicity
assessment of SABpep and ColBpep.
[0132] Rabbit conjunctival primary epithelial cells were isolated
as described previously in the main text. Cells were seeded in 96
well microplates for AlamarBlue assays (Life Technologies, NY) and
live/dead staining at a density of 10,000 cells/cm.sup.2. Cells
were allowed to grow to about 90% confluence before experiments
were carried out. Three different concentrations of HA-FL were
tested: 1.0 mg/ml, 100 .mu.g/ml and 10 .mu.g/ml. SABpep and ColBpep
were added to the medium for 24 h with a concentration of 1.0
mg/ml. Calcein AM and ethidium homodimer-1 (EthD-1; Life
Technologies, NY) were used as live/dead staining agents according
manufacture's manual, and cells were imaged using Zeiss Axio Imager
2 microscope. AlamarBlue assay was performed with quadruplets for
every group. After 2 h incubation, the absorbance values at 570 nm
on a Synergy 2 microplate reader were recorded.
[0133] Binding specificity of SABpep.
[0134] To examine if SABpep binds specifically to sialic acid
segment on the oligosaccharide chains, paraffin embedded and 4% PFA
fixed rabbit conjunctiva tissue sections were stained with three
different groups were selected (SABpep, sialidase treated+SABpep,
SA+SABpep). Sections in the first group were stained with SABpep as
described in the main text; sections in the second group were
treated with sialidase (.alpha.2-3,6,8 Neuraminidase, 60 U/ml; New
England Biolabs, Ipswich, Mass.) first for one hour at 37.degree.
C. before the addition of SABpep; and finally, sections in the
third group were stained with SABpep pre-incubated with sialic acid
(N-Acetylneuraminic acid, 100 mM; Carbosynth, Compton, Berkshire,
UK) for one hour at room temperature. The staining procedure was
the same in all three groups (streptavidin-fluorescein was used for
detecting biotin conjugated SABpep; DAPI was used as counterstain
for nuclei). The sections were imaged using Zeiss Axio Imager 2
microscope.
[0135] Statistical Analysis
[0136] One-way ANOVA was performed among groups to determine any
statistically significance in mean values of HA retention on ex
vivo rabbit ocular tissues (statistically significant values with
p.ltoreq.0.05 was marked as *, p<0.0001 marked as ****,
p<0.002 marked as ***, and p<0.005 marked as **). The
measured kinetic coefficient of friction values between the
treatment groups for each repeat of the test were analyzed by
Cohen's d effect size. Cohen's d measure is defined as the
difference between two means divided by the pooled standard
deviation for the data and classifies effect sizes as small
(d=0.2), medium (d=0.5), and large (d.gtoreq.0.8) (Cohen 1988). The
effect size was calculated instead of significance as it allowed
for the determination the magnitude of the difference between the
two treatment groups independent from sample size (n=3).
[0137] In the following examples, SABpep-PEG-HABpep of the present
invention was investigated for the amount and duration of HA bound
on ocular surfaces and found that SABpep-PEG-HABpep bound HA that
is immobilized to the ocular surfaces with the sialic acid as the
anchoring sites prolongs HA retention in both ex vivo and in vivo
animal models. In the ex vivo rabbit eye model, HA was bound 1.8
times more in the beginning and .about.1.2 times more at 24 h
through SABpep-PEG-HABpep compared to control. In addition, the
lubricating ability of the eye drops was assessed which showed a
strong magnitude of difference in kinetic coefficient friction
values for rabbit ocular tissues treated with SABpep-PEG-HABpep and
HA solution compared to the tissues treated with HA-only solution
(.about.20% in repeat 1 and .about.30% in repeat 2 and 3). The
kinetic coefficient of friction measurements suggest that
SABpep-PEG-HABpep was effective in immobilizing HA onto the ocular
surface, and the surface bound HA without binding peptide sheared
off from the tissue to a greater extent, which is consistent with
the longer retention time of HA bound with SABpep-PEG-HABpep.
Kinetic friction coefficient results from the SABpep-PEG-HABpep and
HA solution showed a strong magnitude of difference compared to the
tissues treated with a non-HA-binding PEG control peptide. This
suggests that the PEG present in the SABpep-PEG-HABpep solution
does not play a significant role in the kinetic coefficient of
friction measurements obtained.
[0138] While these results were performed on a rabbit model and not
on human tissue, the rabbit animal model has often been used in
ophthalmic research due to their similarities to human ocular
anatomy. Although rabbit ocular tissues have differences in tear
film stability and sialic acid-containing membrane-bound mucin
structures compared to human ocular tissue (Tsonis 2011), the
rabbit model is a better alternative than testing against synthetic
materials. Lastly, while increased in vitro friction of commercial
contact lenses has been correlated with increased discomfort of
wears, there are no `clinically relevant changes in tear friction`
to reference from the literature. As such, the reported friction
values are simply an in vitro measurement yet still provide a
valuable information related to lubricating function of the peptide
studied here.
[0139] In an in vivo mouse model, HA immobilized through the
peptide was observed even at 15 min compared to 5 min for the
control HA that was not bound with the peptide. Although HA bound
was not quantified in vivo, the amount of HA-FL decreased more
dramatically over the course of 30 min. However, this difference
between in vivo and ex vivo retention times is expected because in
a live animal the tear film is active and is constantly flushing
fluid.
[0140] From a combination of in vivo, ex vivo, and in vitro studies
we posit using animal models that peptide-binding agents included
in HA eye-drops can prolong the physical and biological benefits of
HA. Although glycan compositions on the ocular surface are
different across species, human ocular surface has a higher
occurrence of sialic acid than other species such as rabbits and
canine, and we expect that SABpep eye drop technology will be
extrapolated to human tissue too.
Example 1
[0141] Peptide screening: selecting peptide that binds to the
ocular tissues.
[0142] We screened multiple peptides that are supposed to bind
different sites of the ocular tissues and cells (data not shown).
Out of the 6 screened peptides, a sialic acid binding peptide
GGSPYGRC (SEQ ID NO: 15) hereafter respectively referred to as
SABpep, and a collagen binding peptide SYIRIADTNIT (SEQ ID NO: 12)
hereafter respectively referred to as ColBpep were chosen for
further experiments (FIG. 2A). The chosen SABpep and ColBpep
exhibited little to no cytotoxicity to epithelial cells as well
(data not shown)
Example 2
[0143] SABpep and ColBpep binding on conjunctiva tissue.
[0144] Rabbit conjunctival sections bound SABpep specifically on
the epithelial lining of the conjunctiva (FIG. 2B), indicating the
strong presence of sialic acid on mucin-1 as compared to mucin-1
antibody staining. A higher magnification confirmed that SABpep
staining was localized to only on cell membranes (FIG. 2C).
Conversely, as expected, ColBpep staining was localized on
conjunctiva tissue more indiscriminately, which is similar to type
I collagen antibody staining (FIG. 2B). The binding specificity of
SABpep was further proven by sialidase digestion and pre-incubation
with sialic acid. SABpep could not bind to conjunctival epithelium
if the section was digested by sialiadase before staining. However,
SABpep still stained the epithelium even after preincubation with
sialic acid solution (data not shown).
Example 3
[0145] Mucin and HA binding studies by Quartz Crystal Microbalance
with Dissipation (QCM-D) monitoring.
[0146] Surface binding of polymer-peptide conjugates to mucin and
HA was directly measured using QCM-D. First, sialic acid bearing
mucin was adsorbed on gold substrates to mimic the interface at the
corneal epithelium (FIG. 3A), as native mucin from bovine
submaxillary glands is abundant with O-linked oligosaccharide
chains, and can be used as a source of sialic acids. A
visco-elastic layer of highly hydrated mucin was formed as shown by
a frequency change of .DELTA.f=-32 Hz and dissipation change of
.DELTA.D=7. Then SABpep-PEG-HABpep was applied to the mucin layer
and specific binding was indicated with a frequency change of
.DELTA.f=-5 Hz. In turn, SABpep-PEG-HABpep was immobilized onto HA
substrates with a .DELTA.f=-3 Hz (FIG. 3B). The Voigt mass
quantified the consistent formation of visco-elastic substrates
Mucin and HA (FIG. 3C). In turn, HApep binding showed a frequency
but not a dissipation change. Considering the relatively small
molecular weight of SABpep-PEG-HABpep (Mw=3698.43 Da) we observed a
considerable binding to mucin and HA with Sauerbrey mass of
.DELTA.m=68.+-.28 and 54.+-.5 ng/cm.sup.2. For example, the drop in
frequency directly corresponds to binding of the polymer-peptide to
mucin and can be attributed to sialic acid chains branching out
from the proteoglycan (Gipson 2004) with binding sites available in
a 3-dimensional network.
Example 4
[0147] Ex vivo HA binding and release profile.
[0148] To confirm the binding of HA on SABpep-PEG-HABpep and
ColBpep-PEG-HABpep treated ocular tissue samples, we applied HA-FL
solution and monitored the fluorescence overtime. The peptide
treated samples fluoresced with higher intensity compared to both
positive (only HA) and negative controls (PBS) (FIG. 4A). The
positive control had minimal fluorescence, and it was significantly
lower than the peptide-treated groups, indicating that the ocular
surface is poor at retaining HA without an aid from the peptide
constructs. Fluorescent HA release studies performed on
SABpep-PEG-HABpep treated samples showed .about.1.8-fold more bound
HA in within the first 25 min (FIG. 4B), and .about.1.3-fold more
after incubating overnight compared with HA only-treated controls
(FIG. 4C). Although a detailed stoichiometric and control study to
compare the binding ability of SABpep vs. ColBpep is required for
an accurate comparison, our initial experiments with conjunctiva
(physically abraded to expose collagen) showed a greater relative
fluorescence value (only as an estimate) for SABpep+HA treated
samples than ColBpep+HA treated samples (Fig. S3 add fig). We
further calculated Area under curve (AUC) for HA release overtime
(Fig. S4 add fig) that showed the total released amount of HA on
ocular surface bound through SABpep was significantly higher than
that for samples with only HA after 25 minutes and after 16 h.
Example 5
[0149] Lubrication of ocular tissues with eye drop solutions.
[0150] Rabbit ocular tissues treated with SABpep-PEG-HABpep and HA
solution had reduced coefficient of friction values, and therefore
improved tissue lubrication, compared to HA-only treated tissues as
control. Kinetic coefficient of friction values of
<.mu..sub.kinetic, N.sub.eq>, expressed as the
mean.+-.standard error of the mean (SEM), decreased from
0.05.+-.0.01, to 0.04.+-.0.01 (d=1.25), 0.06.+-.0.01 to
0.04.+-.0.01 (d=2.13) and 0.07.+-.0.00 to 0.05.+-.0.01 (d=2.10)
during the first, second and third repeats of the HA treated
tissues compared to the SABpep-PEG-HABpep followed by HA treated
tissues, respectively (FIG. 5). A strong effect size was observed
in all three repeats, indicating that there is a strong magnitude
of difference between the SABpep-PEG-HABpep+HA group compared to
the HA control group. Kinetic friction coefficient values for
SABpep-PEG-HABpep followed by HA treated tissues were also compared
to tissues treated with a PEG non-HA-binding peptide (SABpep-PEG)
solution, which acted as a polymer control. <.mu..sub.kinetic,
N.sub.eq> values during the first, second and third repeats of
the PEG non-HA-binding peptide solution were 0.08.+-.0.01,
0.07.+-.0.01 and 0.07.+-.0.01, exhibiting strong effect sizes
(d=2.02, 2.70 and 1.34) when compared to the kinetic friction
coefficient values of the tissues treated with SABpep-PEG-HABpep+HA
(FIG. 5).
Example 6
[0151] In Vivo HA Retention Studies
[0152] An eye drop formulation with equal volume mixture of
SABpep-PEG-HABpep and 0.1% HA (as in commercial HA eye drops) was
applied in vivo to one of the mice eyes, while the other eye was
applied with only 0.1% HA, and monitored overtime till 30 min. Eyes
treated with SABpep eye drops retained HA longer than eyes treated
with regular HA eye drops (15 minutes versus 10 minutes,
respectively) (FIGS. 6A&B). The fluorescence from eyes treated
with only HA was quickly vanishing and lasted till 10 min, while
the fluorescence from peptide and HA treated eyes was still intense
at 10 min and lasted even at 15 min (FIG. 6C). We further harvested
mice eyes and imaged them at a higher magnification, which, as
expected, showed more bound HA on ocular surface at epithelial cell
level at 5 min and lasted till 15 min (FIGS. 6B&C). Initial and
over time fluorescence of standard HA-FL eye drop solution with and
without peptide solutions remained approximately the same that
ruled out the difference in fluorescence values due to the
pipetting errors (data not shown).
Example 7
[0153] SABpep with thiol end group was conjugated to HA (750K Da)
with Maleimide functional group and FITC (fluorescent tag) in a
standard Maleimide-thiol Michael addition chemistry. This HA with
SABpep was used as an eye drop for the ex vivo applications in FIG.
9.
[0154] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0155] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0156] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
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Sequence CWU 1
1
20116PRTArtificial Sequencesynthetic sequence 1Arg Arg Asp Asp Gly
Ala His Trp Gln Phe Asn Ala Leu Thr Val Arg 1 5 10 15
217PRTArtificial Sequencesynthetic sequence 2Cys Arg Arg Asp Asp
Gly Ala His Trp Gln Phe Asn Ala Leu Thr Val 1 5 10 15 Arg
312PRTArtificial Sequencesynthetic sequence 3Gly Ala Ala Trp Gln
Phe Asn Ala Leu Thr Val Arg 1 5 10 412PRTArtificial
Sequencesynthetic sequence 4Gly Ala His Trp Gln Phe Ala Ala Leu Thr
Val Arg 1 5 10 512PRTArtificial Sequencesynthetic sequence 5Gly Ala
His Trp Gln Phe Asn Ala Leu Thr Val Ala 1 5 10 612PRTArtificial
Sequencesynthetic sequence 6Gly Ala His Trp Gln Phe Asn Ala Leu Thr
Val Arg 1 5 10 715PRTArtificial Sequencesynthetic sequence 7Ser Thr
Met Met Ser Arg Ser His Lys Thr Arg Ser His His Val 1 5 10 15
811PRTArtificial Sequencesynthetic sequence 8Arg Tyr Pro Ile Ser
Arg Pro Arg Lys Arg Cys 1 5 10 99PRTArtificial Sequencesynthetic
sequence 9Thr Ala Gly His Gly Arg Arg Trp Ser 1 5 1027PRTArtificial
Sequencesynthetic sequence 10Leu Lys Gln Lys Ile Lys His Val Val
Lys Leu Lys Val Val Val Lys 1 5 10 15 Leu Arg Ser Gln Leu Val Lys
Arg Lys Gln Asn 20 25 1120PRTArtificial Sequencesynthetic sequence
11Arg Arg Ala Asn Ala Ala Leu Lys Ala Gly Glu Leu Tyr Lys Ser Ile 1
5 10 15 Leu Tyr Gly Cys 20 1211PRTArtificial Sequencesynthetic
sequence 12Ser Tyr Ile Arg Ile Ala Asp Thr Asn Ile Thr 1 5 10
1310PRTArtificial Sequencesynthetic sequence 13Tyr Ser Phe Tyr Ser
Asp Glu Ser Leu Gln 1 5 10 146PRTArtificial Sequencesynthetic
sequence 14Trp Tyr Arg Gly Arg Leu 1 5 158PRTArtificial
Sequencesynthetic sequence 15Gly Gly Ser Pro Tyr Gly Arg Cys 1 5
1617PRTArtificial Sequencesynthetic sequence 16Gly Gly Pro Gln Glu
Gln Ile Thr Gln His Gly Ser Pro Tyr Gly Arg 1 5 10 15 Cys
1717PRTArtificial Sequencesynthetic sequence 17Ser Thr Met Met Ser
Arg Ser His Lys Thr Arg Ser His His Val Gly 1 5 10 15 Cys
186PRTArtificial Sequencesynthetic sequence 18Lys Lys Lys Lys Lys
Lys 1 5 199PRTArtificial Sequencesynthetic sequence 19Gly Trp Gln
Pro Pro Arg Ala Arg Ile 1 5 2017PRTArtificial Sequencesynthetic
sequence 20Gly Gly Pro Gln Glu Gln Ile Thr Gln His Gly Ser Pro Tyr
Gly Arg 1 5 10 15 Cys
* * * * *