U.S. patent number 8,329,673 [Application Number 13/304,292] was granted by the patent office on 2012-12-11 for alkylated semi synthetic glycosaminoglycosan ethers, and methods for making and using thereof.
This patent grant is currently assigned to University of Utah Research Foundation. Invention is credited to Thomas P. Kennedy, Glenn D. Prestwich, Narayanam V. Rao, Jianxing Zhang.
United States Patent |
8,329,673 |
Prestwich , et al. |
December 11, 2012 |
**Please see images for:
( Certificate of Correction ) ** |
Alkylated semi synthetic glycosaminoglycosan ethers, and methods
for making and using thereof
Abstract
Described herein are alkylated and semi-synthetic
glycosaminoglycosan ethers, referred to herein as "SAGEs." The
synthesis of sulfated and alkylated SAGEs is also described. The
compounds described herein are useful in a number of applications
including use for ocular or ophthalmic treatment.
Inventors: |
Prestwich; Glenn D. (Eastsound,
WA), Zhang; Jianxing (Salt Lake City, UT), Kennedy;
Thomas P. (Charlotte, NC), Rao; Narayanam V. (Salt Lake
City, UT) |
Assignee: |
University of Utah Research
Foundation (Salt Lake City, UT)
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Family
ID: |
41055226 |
Appl.
No.: |
13/304,292 |
Filed: |
November 23, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120058161 A1 |
Mar 8, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12870774 |
Aug 27, 2010 |
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PCT/US2009/039498 |
Apr 3, 2009 |
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61042310 |
Apr 4, 2008 |
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Current U.S.
Class: |
514/54; 424/427;
424/400; 536/55.1; 514/62 |
Current CPC
Class: |
A61P
27/04 (20180101); C08B 37/0072 (20130101); A61P
17/08 (20180101); A61P 17/00 (20180101); A61P
15/00 (20180101); A61P 17/02 (20180101); A61P
25/02 (20180101); A61K 31/728 (20130101); A61P
17/06 (20180101); A61P 9/00 (20180101); A61P
17/14 (20180101); A61P 13/00 (20180101); A61P
11/02 (20180101); A61P 13/12 (20180101); A61P
27/02 (20180101); A61P 1/04 (20180101); A61P
11/00 (20180101); A61P 19/02 (20180101); C08L
5/08 (20130101); A61K 45/06 (20130101); A61P
13/10 (20180101); A61P 17/10 (20180101); A61P
7/06 (20180101); A61P 11/06 (20180101); A61P
25/00 (20180101); A61P 43/00 (20180101); A61P
25/28 (20180101); A61P 35/00 (20180101); A61P
17/12 (20180101); A61P 37/02 (20180101); A61P
1/02 (20180101); A61P 29/00 (20180101); A61P
1/00 (20180101); Y02A 50/30 (20180101); Y02A
50/387 (20180101) |
Current International
Class: |
A61K
31/728 (20060101); A61K 9/00 (20060101); A61P
27/04 (20060101); A61P 27/02 (20060101); C08B
37/08 (20060101) |
References Cited
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WO |
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WO 2010/130468 |
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Nov 2010 |
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WO |
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Primary Examiner: Baek; Bong-Sook
Attorney, Agent or Firm: Whitlock; Ted
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part application of U.S. patent
application Ser. No. 12/870,774, filed Aug. 27, 2010, which is a
continuation-in-part of International Application No.
PCT/US09/039498, filed Apr. 3, 2009, which claims the benefit of
U.S. provisional application Ser. No. 61/042,310, filed Apr. 4,
2008. These applications are hereby incorporated by reference in
their entirety for all of their teachings.
Claims
The invention claimed is:
1. A method of treating an ocular or ophthalmic condition selected
from the group consisting of ocular rosacea, ocular inflammatory
disease, blepharitis, conjunctivitis or allergic conjunctivitis,
corneal inflammation, corneal scarring, corneal ulceration, corneal
damage or trauma, conjunctiva damage or trauma, diabetic
retinopathy, dry eye or dry eye syndrome, iritis, keratitis,
uveitis, and age-related macular degeneration in a subject, said
method comprising administering an effective amount of a compound
which is a modified hyaluronan or a pharmaceutically acceptable
salt or ester thereof comprising at least one sulfate group and the
primary C-6 hydroxyl proton of at least one N-acetyl-glucosamine
residue substituted with an unsubstituted C.sub.1-C.sub.10 alkyl
group to a subject symptomatic of or predisposed to at least one of
said ocular or ophthalmic conditions.
2. The method of claim 1 wherein said unsubstituted
C.sub.1-C.sub.10 alkyl group is methyl.
3. The method of claim 1, wherein said compound is sulfated at the
C-4 hydroxyl position of a N-acetyl glucosamine residue, the C-2
position of a glucuronic acid residue, the C-3 position of a
glucuronic acid residue, or any combination thereof.
4. The method of claim 1, wherein said compound has a molecular
weight of about 1 kDa to about 100 kDa.
5. The method of claim 1, wherein said compound has a molecular
weight of about 2 kDa to about 10 kDa.
6. The method of claim 1 comprising topically administering said
compound to ocular or ophthalmic tissue.
7. The method of claim 1 comprising intraocularly administering
said compound to the subject.
8. A pharmaceutical composition for the treatment of an ocular or
ophthalmic condition selected from the group consisting of ocular
rosacea, ocular inflammatory disease, blepharitis, conjunctivitis
or allergic conjunctivitis, corneal inflammation, corneal scarring,
corneal ulceration, corneal damage or trauma, conjunctiva damage or
trauma, diabetic retinopathy, dry eye or dry eye syndrome, iritis,
keratitis, uveitis, and age-related macular degeneration, said
composition comprising a compound, which is a modified hyaluronan
or a pharmaceutically acceptable salt or ester thereof comprising
at least one sulfate group and the primary C-6 hydroxyl proton of
at least one N-acetyl-glucosamine residue substituted with an
unsubstituted C.sub.1-C.sub.10 alkyl group, and one or more
pharmaceutically acceptable carriers, wherein the pharmaceutical
composition is formulated to be applied directly to the eye and/or
surrounding tissue of the eye.
9. The pharmaceutical composition of claim 8, wherein the
unsubstituted C.sub.1-C.sub.10 alkyl group is methyl.
10. The pharmaceutical composition of claim 8, wherein the
composition is a topical composition, wherein the compound is
present from 0.01% to 20% by weight of the composition.
11. The pharmaceutical composition of claim 8, wherein said
composition is formulated for intraocular administration.
12. A compound for the treatment of an ocular or ophthalmic
condition selected from the group consisting of ocular rosacea,
ocular inflammatory disease, blepharitis, conjunctivitis or
allergic conjunctivitis, corneal inflammation, corneal scarring,
corneal ulceration, corneal damage or trauma, conjunctiva damage or
trauma, diabetic retinopathy, dry eye or dry eye syndrome, iritis,
keratitis, uveitis, and age-related macular degeneration, or a
symptom associated with said ocular or ophthalmic condition, said
compound being a modified hyaluronan or a pharmaceutically
acceptable salt or ester thereof, wherein said compound comprises a
sulfate group at the C-4 hydroxyl position of at least one N-acetyl
glucosamine residue, the C-2 position of at least one glucuronic
acid residue, the C-3 position of at least one glucuronic acid
residue, or any combination thereof; and said compound is
substituted with an unsubstituted C.sub.1-C.sub.10 alkyl group at
the primary C-6 hydroxyl position of at least one
N-acetyl-glucosamine residue.
13. The compound of claim 12 wherein the unsubstituted
C.sub.1-C.sub.10 alkyl group is methyl.
14. The compound of claim 12 having a degree of sulfation from 0.5
to 3.5 per disaccharide unit.
15. The compound of claim 12 having a molecular weight of 1 kDa to
100 kDa.
16. A drug delivery device comprising a compound of claim 12 or a
composition comprising said compound of claim 12.
Description
BACKGROUND
Heparin and its derivatives have shown promise in treating
inflammatory diseases. Heparin and its derivatives disrupt at least
three important events in inflammatory cascades. First, heparin
attaches to and blocks the leukocyte integrins P- and L-selectin.
Second, heparin and its derivatives reduce the inflammatory cascade
by binding to and inhibiting the cationic PMN protease human
leukocyte elastase and cathepsin G, which reduces proteolytic
tissue injury by PMNs that escape the first heparin barrier of
selectin inhibition. Third, heparin and its derivatives potentially
inhibit the interaction of the receptor for advanced glycation
end-products (RAGE) with its ligands. However, treatments using
heparin and its derivatives exhibiting anticoagulant activity have
several major drawbacks. First, heparin and its derivatives are
porcine-derived; thus leading to concerns of cross-species transfer
of viruses. Second, because of their anticoagulant properties,
diabetics treated with this compound are at risk of excessive
bleeding. Third, heparin or some of its derivatives may induce
thrombocytopenia in certain individuals who produce an antibody to
the complex of heparin with the cationic protein platelet factor-4
(PF-4), resulting in catastrophic platelet aggregation and
generalized paradoxical arterial and venous clotting. Thus, there
exists an important unmet need for compounds which may be used to
treat inflammatory diseases while avoiding the myriad of side
effects seen from treatments using heparin or heparin-like
agents.
Inflammatory diseases such as psoriasis, dermatitis, acne, rosacea,
photo-dermal ageing, and numerous diseases linked to RAGE-mediated
signaling, plague people worldwide. To put these diseases into
perspective, the National Psoriasis Foundation reports that
psoriasis alone afflicts 2-3% of the world's population or
approximately 125 million people. These inflammatory conditions can
be aesthetically unpleasing and can create serious health issues if
left untreated. Conventionally accepted treatments of these
conditions may involve UV phototherapy, corticosteroids and
glucocorticoids, acitretin, cyclosporine, and methotrexate.
However, each of these treatments may cause serious side effects
ranging from immune suppression and liver disease to thinning skin
and causing birth defects. Due to partial or complete
ineffectiveness, these treatments often leave patients unsatisfied
with their results.
Inflammatory responses may also contribute to certain ocular or
ophthalmic conditions, including age-related macular degeneration,
diabetic retinopathy, dry eye syndrome and other inflammatory
conjunctivitis, iritis, uveitis, allergic conjunctivitis,
anti-inflammatory aid in cataract surgery, or in the prevention of
corneal inflammation and scarring.
Dry eye disease is a multi-factorial disease of the tears and
ocular surface that results in symptoms of discomfort, visual
disturbance, and tear film instability with potential damage to the
ocular surface. Dry eye results from decreased production,
increased evaporation, or decreased clearance of tears and if left
untreated, can lead to pain, blurred and fluctuating vision and an
increased risk of sight-threatening corneal infection and
ulceration, scars on the cornea, and some loss of vision.
Dry eye is a common condition that affects over 20 million
individuals in the US or 10% of the population between the ages of
30 and 60 years, with increasing prevalence to 15% of the
population aged more than 65 years. There are numerous causes of
dry eye including various systemic diseases, systemic medications,
hormonal changes, neural alterations, and environmental influences.
Treatment options may follow different approaches, depending on the
cause of the disease and include treatment of the underlying
associated conditions. Due to the many different causes and
pathophysiologies, dry eye treatment continues to present a
substantial challenge to the clinician.
Over-the-counter ocular lubricants remain the mainstay of treatment
and hyaluronic acid based products are key therapeutic alternatives
in Japan and the EU. A formulation of 0.05% cyclosporine A
(Restasis.RTM.) is the only Food and Drug Administration (FDA)
approved ophthalmic solution for dry eye. Cyclosporine A is an
immunomodulator whose mechanism of action appears to be associated
with suppression of inflammation associated with
keratoconjunctivitis sicca, however, it has limited efficacy and a
significant number of users experience side effects. Only 15% of
Restasis.RTM. ophthalmic emulsion treated patients, versus 5% of
vehicle treated patients had a significant increase in tear
production, as defined in clinical studies leading to drug
approval. The most commonly reported adverse event following the
use of Restasis.RTM. is ocular burning, present in approximately
17% of users. Other events reported in 1% to 5% of patients
included conjunctival hyperemia, discharge, epiphora, eye pain,
foreign body sensation, pruritus, stinging and visual disturbance
(most often blurring).
There is a clear unmet medical need for more effective, safer and
convenient treatment options for ocular or ophthalmic conditions,
including dry eye disease. A product that targets these conditions,
e.g., one or more of the multiple aspects of the complex dry eye
physiopathology, can have significant clinical benefit.
SUMMARY OF THE INVENTION
Described herein are semisynthetic glycosaminoglycosan ethers,
referred to herein as "SAGEs," and more particularly, SAGEs that
are both sulfated and alkylated, and the synthesis of these
compounds. The compounds described herein are useful in a number of
therapeutic and cosmetic applications and the treatment of a number
of inflammatory diseases and/or skin disorders and have particular
utility in treating ocular or ophthalmic conditions in patients in
need of such treatment. The advantages of the invention will be set
forth in part in the description which follows, and in part will be
clear from the description, or may be learned by practice of the
aspects described herein. The advantages of the invention are
realized and attained by means of the elements and combinations
particularly pointed out in the specification and the appended
claims. It is to be understood that the general and detailed
descriptions provided in this specification are exemplary and
explanatory only and are not restrictive.
Specifically described and claimed are compounds for the treatment,
prevention, or inhibition of an ocular, ophthalmic, or
dermatological conditions or symptoms associated with said ocular,
ophthalmic, or dermatological conditions, said compounds comprising
modified hyaluronans, or the pharmaceutically acceptable salts or
esters thereof, comprising at least one sulfate group and having at
least one primary C-6 hydroxyl position of an N-acetyl-glucosamine
residue comprising a C.sub.1-C.sub.10 alkyl group.
The alkylated and sulfated compounds of the subject invention
preferably have an alkyl group selected from the group consisting
of methyl, ethyl, propyl and butyl, and a more preferred compound
comprises a methyl group as the alkyl group. In addition, a
preferred compound of the subject invention comprises a sulfate
group at the C-2 or C-3 hydroxyl position of a glucuronic acid
moiety, and more preferably, at the C-2 and C-3 positions. An
additional embodiment can comprise a sulfate at the C-4 hydroxyl
position of the N-acetyl glucosamine moiety or any combination of
sulfation at the C-2, C-3 positions of the glucuronic acid moiety,
or C-4 hydroxyl position of the N-acetyl glucosamine moiety of the
compound. The sulfated compounds can have a degree of sulfation
from 0.5 to 3.5 per disaccharide unit, and a molecular weight of 2
kDa to 10 kDa.
The compounds of the subject invention can further be admixed with
or otherwise prepared using pharmaceutically acceptable excipients
to form a pharmaceutical composition comprising an alkylated and/or
sulfated compound of the subject invention. These pharmaceutical
compositions can be formulated for topical administration,
including formulations optimized for administration to ocular
tissue of a patient, e.g., by injection or by topical or
transmucosal administration.
Methods of treating or preventing an ocular, ophthalmic, or
dermatological condition or symptom associated with said ocular,
ophthalmic, or dermatological condition using the disclosed
alkylated and sulfated compounds comprise the steps of (a)
providing a compound as described, and (b) administering an
effective amount of said compound or a composition comprising said
compound, to a patient suffering from these ocular, ophthalmic, or
dermatological conditions or symptom associated with these ocular,
ophthalmic, or dermatological conditions. For purposes of the
subject invention, the terms "ocular" and "ophthalmic" conditions
are intended to be alternative expressions for the same conditions.
Accordingly, the terms "ocular" and "ophthalmic" conditions are
used synonymously and interchangeably herein. The term "ocular or
ophthalmic condition" is defined herein as any disorder of
associated with an ocular or ophthalmic tissue. The term "ocular or
ophthalmic tissue" is defined herein as (1) any component of the
eye or (2) a tissue that is in contact or in close proximity to the
eye. Ocular or ophthalmic tissues that can be treated using a
compound or composition of the subject invention include, but are
not limited to, conjunctiva, cornea, lacrimal glands, lacrimal
ducts, eyelids, Meibomian glands, ciliary glands, sebaceous ciliary
glands, anterior chamber, posterior chamber, lens, vitreous, uvea,
and retina.
Examples of ocular or ophthalmic conditions that can be treated
using a compound or composition of the subject invention include,
but are not limited to, age-related macular degeneration, diabetic
retinopathy, dry eye syndrome, ocular rosacea, or other ocular or
ophthalmic inflammatory conditions, e.g., blepharitis,
conjunctivitis (including allergic conjunctivitis), iritis,
keratitis, retinitis, scleritis, uveitis, in the prevention or
restoration of corneal damage, and the prevention of corneal
inflammation and scarring. The compounds or compositions described
herein can also facilitate corneal or conjunctiva wound healing
following eye surgery or other trauma to the eye. Compounds or
compositions of the subject invention can also be used for
increased tear production or tear film stability when a subject is
in need thereof, e.g., a condition leading to decreased tear
production or tear quality (referred to herein as "tear production
or tear quality dysfunction").
In a preferred embodiment, a SAGE derivative from hyaluronic acid
(HA), and particularly, GM-1111, demonstrates activity that can
provide substantial clinical benefits for the treatment of dry eye
due to increased efficacy and advantageous safety profiles,
including applicability for chronic use, as compared to existing
therapies. GM-1111 can act as a protective barrier to the corneal
epithelial cells, increases moisture and normalize the hyperosmolar
eye environment, and limit local ocular inflammatory reactions.
By virtue of the poly-anionic charges present in this sulfated
polysaccharide, GM-1111 has reported in vitro anti-inflammatory
activities at nanomolar concentrations, including inhibition of
cationic polymorphonuclear (PMN) proteases, inhibition of platelet
adhesion receptor P-selectin and inhibition of the interaction of
the receptor for advance glycation end-products (RAGE), a newly
recognized important component of innate immunity mediating
long-lived inflammation. The RAGE and P-selectin inhibiting
activities of GM-1111 might also retard the influx of CD4+
lymphocytes into the ocular epithelial environment. In addition, as
sulfated polysaccharides, GM-1111 could counteract many of the Th1
heparin-binding cytokines active in dry eye. As an example, the
related sulfated polysaccharide heparin antagonizes the biological
and molecular actions of interferon-.gamma. (IFN.gamma.) and
IFN.gamma.-inducible cytokines such as IL-10.
With these benefits, GM-1111 can be used to treat, inhibit, or
prevent hyperosmolarity-epithelial surface damage-inflammation and
produce substantial clinical benefit in dry eye disease. The
subject method preferably comprises administering said compound, or
a composition comprising a compound of the invention, directly to
the eye, topically, transmucosally, or by injection.
Dermatologic conditions which can be treated with the compounds or
compositions of the invention include rosacea, psoriasis, acne
vulgaris, hair loss, atopic dermatitis, and actinic keratosis. The
subject method preferably comprises topically administering said
compound to the skin.
Compositions useful in accordance with the compounds of the subject
invention can be formulated using processes and ingredients that
are well known in the art. For example, a preferred composition may
be a formulation containing a compound of the subject invention
that can be topically applied, such as eye drops (solutions or
suspensions), ointments, creams, or gels or as a spray or mist. In
addition, formulations for use with ocular or ophthalmic devices
(e.g. punctual plugs, contact lenses or the like), or drug delivery
devices, e.g., implants, coated with a compound or composition of
the subject invention for immediate release or controlled release
of drugs to the eye or surrounding tissues of the eye are described
herein. The compound or composition of the invention may also be
parenterally administered, e.g., intraocularly, as an injectable
solution or suspension.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute
a part of this specification, illustrate several aspects described
below.
FIG. 1 shows a synthetic scheme for producing alkylated and
fluoroalkylated hyaluronan and sulfated derivatives thereof.
FIG. 2 shows the structures of several exemplary SAGEs.
FIG. 3 shows the inhibition of P-Selectin by partially O-sulfated
and methylated HA (P-OSMEHA, or GM-131201).
FIG. 4 shows the inhibition of human leukocyte elastase by sulfated
hyaluronan derivatives, including alkylated and non-alkylated
hyaluronans, which are also sulfated.
FIG. 5 shows the inhibition of amphoterin, also known as high
mobility group box protein-1 (HMGB-I) binding to immobilized RAGE
by P-OSMEHA
FIG. 6 shows the inhibition of S100b calgranulin binding to
immobilized RAGE by P-OSMEHA (GM-131201).
FIG. 7 shows the inhibition of carboxymethyl lysine-BSA (CML-BSA)
binding to immobilized RAGE by P-OSMEHA (GM-131201).
FIG. 8 shows the inhibition of keratinocyte proliferation by high
molecular weight hyaluronan derivatives, including alkylated and
non-alkylated hyaluronans that are also sulfated.
FIG. 9 shows the inhibition of keratinocyte proliferation by low
molecular weight hyaluronan derivatives, including alkylated and
non-alkylated hyaluronans, which are also sulfated, and by 2,3-O
desulfated heparin (ODSH).
FIG. 10 shows SAGE (GM-111101) co-injection with LL-37 rosacea
model, (a) Gross picture of LL-37 injected skin region and (b)
co-injection model of LL-37 and SAGE, (c) H&E-stained
cross-sectional view of a LL-37 injected skin sample, (d)
H&E-stained cross-sectional view of a LL-37 mixed with SAGE
injected skin region, (e) Polymorphonuclear leukocyte (PMN)
infiltration into skin as measured by activity of the PMN enzyme
myeloperoxidase (MPO) in skin biopsies from mice injected with
LL-37 only, LL-37 plus SAGE or SAGE only injection groups, (f) area
of erythema and (g) erythema score in mice injected with LL-37 only
or LL-37 plus SAGE.
FIG. 11 shows SAGE (GM-111101) topical treatment in the LL-37
rosacea model, (a) Gross picture of LL-37 injected skin region, (b)
SAGE treatment immediately after LL-37 injection and (c) SAGE
treatment 12 h after LL-37 injection, (d) H&E-stained
cross-sectional view of a LL-37 injected skin sample, (e)
H&E-stained cross-sectional view of SAGE immediate treatment in
LL-37 injected skin region, (f) H&E-stained cross-sectional
view of SAGE 12 h treatment in LL-37 injected skin region, (g) MPO
activity measurement of LL-37 injection model with different SAGE
treatment strategies, (h) area of erythema and (g) erythema score
demonstration of LL-37 rosacea model treated with topical
application of SAGE.
FIG. 12 shows the outer Skin under natural light (treated with 1
mg/ml fluorescently labeled SAGE) (Panel a) and outer Skin
fluorescent image (Panel b); Inner Skin under natural light (Panel
c) and inner skin under fluorescent condition (Panel d).
FIG. 13 shows the effects of HA derivatives on the proliferation of
nHDF cells (a) and effect on nHEK (b) Gross pictures of mice
treated with different concentrations of GM-111101 and GM-212101;
the intact area (c) and formic acid irritated area (d) were
compared with 0.1 mg/ml GM-111101 (e), 1 mg/ml GM-111101 (f), 10
mg/ml GM-111101 (g), 0.1 mg/ml GM-212101 (h), 1 mg/ml GM-212101 (i)
and 10 mg/ml GM-212101 (j).
FIG. 14 shows the results of a skin irritation test in mice: (a)
Erythema scoring of SAGE in abraded area, (b) Edema scoring of SAGE
in abraded area (c) Erythema scoring of SAGE in intact area, (d)
Edema scoring of SAGE in intact area.
FIG. 15 shows topical SAGE treatment using croton oil inflammatory
model. Four hours after croton oil treatment in control (CTL)
group.
FIG. 16 shows a comparison between Right (untreated) (Panel a) and
Left (croton oil painted) (Panel b) ears of the same mouse in CTL
group. H&E staining was done for negative control with PBS
painting (Panel c), positive control with croton oil (Panel d) and
croton oil followed by SAGE treatment (Panel e). Leukocytic
infiltration and edema was identified in the croton oil positive
control group. Myeloperoxidase activity (Panel f) is an index of
polymorphonuclear leukocyte activation and was measured in ear
punches after SAGE treatment. Panel g and Panel h are changes in
ear thickness (from edema) and ear redness (from irritation) after
SAGE treatment. (p<0.05) FIG. 16 shows (a) H&E-stained
cross-sectional view of a LL-37 injected skin sample, (b)
H&E-stained cross-sectional view of HA treatment in LL-37
injected skin region, (f) H&E-stained cross-sectional view of
SAGE (GM-111101) treatment in LL-37 injected skin region, (g) MPO
activity measurement of LL-37 injection model with HA and SAGE
treatment, (h) Area of erythema illustration and (g) erythema score
demonstration of LL-37 rosacea model with HA and SAGE
treatment.
FIG. 17 shows AGE-induced RAGE expression in ARPE-19 cells is
increased by growth on the AGE product carboxymethyl lysine-bovine
serum albumin (CML-BSA) and prevented by modified heparin.
FIG. 18 shows that compared to control (a), the AGE product CML-BSA
induces apoptosis in ARPE-19 cells (b) that is inhibited by ODSH
(c) and almost eliminated by SAGE treatment (d).
FIG. 19 shows that LMW SAGEs do not activate Factor XII, unlike
heparin which activates Factor XII at a concentration of 0.4
.mu.g/ml, which is close to the therapeutic anticoagulating plasma
heparin concentration in humans.
FIG. 20 shows that concentration-dependent binding was observed for
GM-1111 measured by increased fluorescent intensity at 647 nm.
FIG. 21 shows confocal images of corneal epithelial cells exposed
to Alexa Fluor.RTM. 633-labeled GM-1111.
FIG. 22 shows a scopolamine- and ocular desiccation-induced dry eye
study comparing GM-1111 to Restasis.RTM. or vehicle control was
performed in mice. GM-1111 performed at least as well as
Restasis.RTM. in increasing tear production and tear film
stability.
DETAILED DESCRIPTION
Before the present compounds, compositions, and/or methods are
disclosed and described, it is to be understood that the aspects
described below are not limited to specific compounds, synthetic
methods, or uses as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular aspects only and is not intended to be
limiting.
In this specification and in the claims that follow, reference will
be made to a number of terms that shall be defined to have the
following meanings:
It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a pharmaceutical carrier" includes
mixtures of two or more such carriers, and the like. "Optional" or
"optionally" means that the subsequently described event or
circumstance can or cannot occur, and that the description includes
instances where the event or circumstance occurs and instances
where it does not. For example, the phrase "optionally substituted
lower alkyl" means that the lower alkyl group can or cannot be
substituted and that the description includes both unsubstituted
lower alkyl and lower alkyl where there is substitution.
References in the specification and concluding claims to parts by
weight of a particular element or component in a composition or
article, denotes the weight relationship between the element or
component and any other elements or components in the composition
or article for which a part by weight is expressed. Thus, in a
compound containing 2 parts by weight of component X and 5 parts by
weight component Y, X and Y are present at a weight ratio of 2:5,
and are present in such ratio regardless of whether additional
components are contained in the compound.
A weight percent of a component, unless specifically stated to the
contrary, is based on the total weight of the formulation or
composition in which the component is included.
A residue of a chemical species, as used in the specification and
concluding claims, refers to the moiety that is the resulting
product of the chemical species in a particular reaction scheme or
subsequent formulation or chemical product, regardless of whether
the moiety is actually obtained from the chemical species. For
example, hyaluronan that contains at least one --OH group can be
represented by the formula Y--OH, where Y is the remainder (i.e.,
residue) of the hyaluronan molecule.
The term "treat" as used herein is defined as maintaining or
reducing the symptoms of a pre-existing condition (i.e., someone
that is symptomatic of an ocular or ophthalmic condition). The term
"prevent" as used herein is defined as eliminating or reducing the
likelihood of the occurrence of one or more symptoms of a
condition, disease or disorder, or someone that is predisposed to
an ocular or ophthalmic condition. The term "inhibit" as used
herein is the ability of the compounds described herein to
completely eliminate the activity or reduce the activity when
compared to the same activity in the absence of the compound.
Described herein are alkylated hyaluronan or derivatives thereof.
In one aspect, at least one primary C-6 hydroxyl proton of the
N-acetyl-glucosamine residue of hyaluronan is substituted with an
alkyl group. The term "alkyl group" as used herein is a branched or
unbranched saturated hydrocarbon group of 1 to 24 carbon atoms. In
one aspect, the alkyl group is a C.sub.1-C.sub.10 branched or
straight chain alkyl group. The alkyl group can be unsubstituted or
substituted. The term "unsubstituted" with respect to the alkyl
group is a saturated hydrocarbon composed only of hydrogen and
carbon. Examples of unsubstituted alkyl groups include, but are not
limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
f-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, tetradecyl,
hexadecyl, eicosyl, tetracosyl and the like.
Described herein are methods for alkylating SAGEs. In one aspect,
the SAGEs are produced by (a) reacting the hyaluronan or a
derivative thereof with a sufficient amount of base to deprotonate
at least one primary C-6 hydroxyl proton of the
N-acetyl-glucosamine residue, and (b) reacting the deprotonated
hyaluronan or a derivative thereof with an alkylating agent for a
sufficient time and concentration to alkylate at least one
deprotonated primary C-6 hydroxyl group. It will be understood by
those skilled in the art that the basic conditions may also lead to
cleavage of the glycosidic linkage, leading to lower molecular
weight hyaluronan derivatives during the modification process. It
will also be understood that the basic conditions deprotonate the
acid to the carboxylate, and the secondary hydroxyl groups, and
that each of these nucleophilic moieties may participate in the
ensuing alkylation in proportion to their relative abundance at
equilibrium and the nucleophilicity of the anionic species. For
example, 2-O and/or 3-O hydroxyl protons of the glucuronic acid
moiety or the C-4 hydroxyl position of the N-acetyl glucosamine
moiety can be deprotonated and alkylated. An example of this is
depicted in FIG. 1, where R can be hydrogen, an alkyl group, or an
alkyl group. The hyaluronan starting material can exist as the free
acid or the salt thereof.
Derivatives of hyaluronan starting material can also be used
herein. The derivatives include any modification of the hyaluronan
prior to the alkylation step. A wide variety of molecular weight
hyaluronan can be used herein. In one aspect, the hyaluronan has a
molecular weight between about 1 kDa to about 25 kDa prior to
alkylation, preferably about 5 kDa to about 25 kDa, and more
preferably less than about 10 kDa. In one preferred embodiment, the
hyaluronan has an average molecular weight of about 6 kDa. In still
other aspects of the invention, the hyaluronan has about 25 kDa to
1,000 kDa, 100 kDa to 1,000 kDa, 25 kDa to 500 kDa, 25 kDa to 250
kDa, or 25 kDa to 100 kDa prior to alkylation. In certain aspects,
the hyaluronan starting material or a derivative thereof is not
derived from an animal source. In these aspects, the hyaluronan can
be derived from other sources such as bacteria. For example, a
recombinant B. subtilis expression system or Streptococcus strain
can be used to produce the hyaluronan starting material.
The hyaluronan starting material or derivative thereof is initially
reacted with a sufficient amount of base to deprotonate at least
one primary C-6 hydroxyl proton of the N-acetyl-glucosamine
residue. The selection of the base can vary. For example, an alkali
hydroxide such as sodium hydroxide or potassium hydroxide can be
used herein. The concentration or amount of base can vary depending
upon the desired degree of alkylation. In one aspect, the amount of
base is sufficient to deprotonate at least 0.001% of the primary
C-6 hydroxyl protons of the N-acetyl-glucosamine residue of the
hyaluronan starting material or derivative thereof. In other
aspects, the amount of base is sufficient to deprotonate from
0.001% to 50%, 1% to 50% 5% to 45%, 5% to 40%, 5% to 30%, 5% to
20%, 10% to 50%, 20% to 50%, or 30% to 50% of the primary C-6
hydroxyl protons of the N-acetyl-glucosamine residue of the
hyaluronan starting material or derivative thereof. It is
understood that the more basic the solution, the more likely are
chain cleavage reactions and the higher the degree of alkylation
that can be achieved. For example, other hydroxyl groups present on
hyaluronan (e.g., 2-OH and/or 3-OH can be alkylated). In one
aspect, all of the hydroxyl groups present on hyaluronan can be
alkylated. In other aspects, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or any range thereof
of hydroxyl protons present on hyaluronan can be deprotonated and
subsequently alkylated.
After the hyaluronan starting material or derivative thereof has
been treated with a base, the deprotonated hyaluronan is reacted
with an alkylating agent to produce the SAGE. Examples of
alkylating agents include, but are not limited to, an alkyl halide.
Alkyl bromides and iodides are particularly useful. Alkylating
agents commonly used in organic synthesis can be used herein.
An exemplary synthetic procedure for making alkylated SAGEs is
provided in FIG. 1. Referring to FIG. 1, hyaluronan (HA) is treated
with a base (e.g., NaOH) and an alkylating agent (e.g., CH.sub.3I)
to methylate a primary C-6 hydroxyl proton of the
N-acetyl-glucosamine residue of hyaluronan and produce methylated
hyaluronan (MHA). FIG. 1 also provides an exemplary synthetic
procedure for making a fluoroalkylated hyaluronan (FHA) using a
fluoroalkylating agent (e.g., CF.sub.3(CF.sub.2X.sub.1CH.sub.2Br),
which is a substituted alkyl group as defined herein.
In certain aspects, it is desirable to sulfate the alkylated SAGEs
described above. In one aspect, the alkylated SAGE is sulfated by
reacting the alkylated SAGE with a sulfating agent to produce a
sulfated product. The degree of sulfation can vary from partial
sulfation to complete sulfation. In general, free hydroxyl groups
present on the alkylated hyaluronan or a derivative thereof can be
sulfated. In one aspect, at least one C-2 hydroxyl proton and/or
C-3 hydroxyl proton is substituted with a sulfate group. An
additional embodiment can comprise a sulfate at the C-4 hydroxyl
position of the N-acetyl glucosamine moiety or any combination of
sulfation at the C-2, C-3 positions of the glucuronic acid moiety
and C-4 hydroxyl position of the N-acetyl glucosamine moiety of the
compound. The degree of sulfation can be from 0.5, 1.0, 1.5, 2.0,
2.5, 3.0, 3.5 or any range thereof per disaccharide unit of the
alkylated SAGE. In one aspect, the alkylated SAGE can be treated
with a base to deprotonate one or more hydroxyl protons followed by
the addition of the sulfating agent. The sulfating agent is any
compound that reacts with a hydroxyl group or deprotonated hydroxyl
group to produce a sulfate group. The molecular weight of the SAGE
can vary depending upon reaction conditions. In one aspect, the
molecular weight of the SAGE is from 2 kDa to 500 kDa, 2 kDa to 250
kDa, 2 kDa to 100 kDa, 2 kDa to 50 kDa, 2 kDa to 25 kDa, or from 2
kDa to 10 kDa. FIG. 1 depicts an exemplary synthesis of sulfated
alkylated SAGEs (SMHA and SFHA, respectively).
FIG. 2 provides the structures of several exemplary SAGEs. Each
SAGE is identified by the code GM-XYSTZZ, where: X=type of alkyl
group, where 1=methyl, 2=pentafluoropropyl, 3=heptafluorobutyl
Y=size of HA, where 1=low, 2=medium, 3=high; S=degree of sulfation,
where 1=partial, 2=full T=degree of alkylation, where 1=low,
2=high; ZZ=sequential lot number 01 or 02, where the 02 has been
made and has all the same properties as the 01 batch.
Table 1, below, provides a list of several SAGEs as defined by the
code system above.
TABLE-US-00001 TABLE 1 MW MW Alkylation SAGE # CHEMICAL NAME
(starting) (GPC) Alkylation SD Sulfation GM-211101
LMW--P--OSFHA-1(DS 1) 53K 6K Pentafluoropropyl (Pfp) 1 1.0-1.5
GM-311101 LMW--P--OSFHA-2(DS 1) 53K 5.8K Heptafluorobutyl (Hfb) 1
1.0-1.5 GM-111101 LMW--P--OSMeHA(DS 1) 53K 5.6k Methyl (Me) 1
1.0-1.5 GM-211201 LMW--P--OSFHA-1(DS 2) 53K 6K pentafluoropropyl 2
1.0-1.5 GM-311201 LMW--P--OSFHA-2(DS 2) 53K 5.6k heptafluorobutyl 2
1.0-1.5 GM-111201 LMW--P--OSMeHA(DS 2) 53K 5.5K methyl 2 1.0-1.5
GM-231101 P--OSFHA-1(DS 1) 950K 112k Pentafluoropropyl 1 1.0-1.5
GM-331101 P--OSFHA-2(DS 1) 950K 110k Heptafluorobutyl (Hfb) 1
1.0-1.5 GM-131101 P--OSMeHA(DS 1) 950K 123k methyl 1 1.0-1.5
GM-231201 P--OSFHA-1(DS 2) 950K 108k pentafluoropropyl 2 1.0-1.5
GM-331201 P--OSFHA-2(DS 2) 950K 130k heptafluorobutyl 2 1.0-1.5
GM-131201 P--OSMeHA(DS 2) 950K 120K methyl 2 1.0-1.5 GM-212101
LMW--F--OSFHA-1(DS 1) 53K 5k pentafluoropropyl 1 1.5-2.0 GM-312101
LMW--F--OSFHA-2(DS 1) 53K 4.8k heptafluorobutyl 1 1.5-2.0 GM-112101
LMW--F--OSMeHA(DS 1) 53K 5.6k methyl 1 1.5-2.0 GM-212201
LMW--F--OSFHA-1(DS 2) 53K 6K pentafluoropropyl 2 1.5-2.0 GM-312201
LMW--F--OSFHA-2(DS 2) 53K 6K heptafluorobutyl 2 1.5-2.0 GM-112201
LMW--F--OSMeHA(DS 2) 53K 5.4k methyl 2 1.5-2.0 GM-232101
F--OSFHA-1(DS 1) 950K 110k pentafluoropropyl 1 1.5-2.0 GM-332101
F--OSFHA-2(DS 1) 950K 105k heptafluorobutyl 1 1.5-2.0 GM-132101
F--OSMeHA(DS 1) 950K 112k Methyl 1 1.5-2.0 GM-232201 F--OSFHA-1(DS
2) 950K 120k pentafluoropropyl 2 1.5-2.0 GM-332201 F--OSFHA-2(DS 2)
950K 118k heptafluorobutyl 2 1.5-2.0 GM-132201 F--OSMeHA(DS 2) 950K
116K methyl 2 1.5-2.0 GM-431101 P--OSBGHA 950K 105k benzyl glycidyl
ether (BG) <1
In one aspect, the alkyl group of the SAGE is methyl and at least
one C-2 hydroxyl proton and/or C-3 hydroxyl proton of hyaluronan is
substituted with a sulfate group. In another aspect, the alkyl
group of the SAGE is methyl, at least one C-2 hydroxyl proton
and/or C-3 hydroxyl proton of hyaluronan is substituted with a
sulfate group, and the compound has a molecular weight of 2 kDa to
200 kDa after alkylation. An example of such a compound is
GM-111101 as shown in FIG. 2. An additional embodiment can comprise
a sulfate at the C-4 hydroxyl position of the N-acetyl glucosamine
moiety or any combination of sulfation at the C-2, C-3 positions of
the glucuronic acid moiety and C-4 hydroxyl position of the
N-acetyl glucosamine moiety of the compound.
The modified hyaluronans described herein can be prepared from
different sources of hyaluronic acid with different
polydispersities and initial average molecular weights. The in
vitro biochemical results, in vivo biological activities, and
alkylation/sulfation levels can vary based on the size and
solubility of the starting HA. For example, starting with poorly
soluble HA of size 60-70 kDa resulted in low levels of methylation,
sulfation, and dramatically reduced biological activities. In
contrast, starting with readily soluble HA of sizes 40-60 kDa
(whether obtained commercially at this size or prepared by partial
depolymerization of a higher molecular weight HA starting material)
resulted in reproducible levels of methylation, sulfation, and high
biological activity.
Any of the alkylated SAGEs described herein can be the
pharmaceutically acceptable salt or ester thereof. Pharmaceutically
acceptable salts are prepared by treating the free acid with an
appropriate amount of a pharmaceutically acceptable base.
Representative pharmaceutically acceptable bases are ammonium
hydroxide, sodium hydroxide, potassium hydroxide, lithium
hydroxide, calcium hydroxide, magnesium hydroxide, ferrous
hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide,
ferric hydroxide, isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine,
2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine,
histidine, and the like. In one aspect, the reaction is conducted
in water, alone or in combination with an inert, water-miscible
organic solvent, at a temperature of from about 0.degree. C. to
about 100.degree. C. such as at room temperature. The molar ratio
of compounds of structural formula I to base used are chosen to
provide the ratio desired for any particular salts. For preparing,
for example, the ammonium salts of the free acid starting material,
the starting material can be treated with approximately one
equivalent of pharmaceutically acceptable base to yield a neutral
salt.
Ester derivatives are typically prepared as precursors to the acid
form of the compounds, as illustrated in the examples below--and
accordingly can serve as prodrugs. Generally, these derivatives
will be lower alkyl esters such as methyl, ethyl, and the like.
Amide derivatives --(CO)NH.sub.2, --(CO)NHR and --(CO)NR.sub.2,
where R is an alkyl group defined above, can be prepared by
reaction of the carboxylic acid-containing compound with ammonia or
a substituted amine. Also, the esters can be fatty acid esters. For
example, the palmitic ester has been prepared and can be used as an
alternative esterase-activated prodrug.
The SAGEs described herein can be formulated in any excipient the
biological system or entity can tolerate to produce pharmaceutical
compositions. Examples of such excipients include, but are not
limited to water, aqueous hyaluronic acid, saline, Ringer's
solution, dextrose solution, Hank's solution, and other aqueous
physiologically balanced salt solutions. Nonaqueous vehicles, such
as fixed oils, vegetable oils such as olive oil and sesame oil,
triglycerides, propylene glycol, polyethylene glycol, and
injectable organic esters such as ethyl oleate can also be used.
Other useful formulations include suspensions containing viscosity
enhancing agents, such as sodium carboxymethylcellulose, sorbitol,
or dextran. Excipients can also contain minor amounts of additives,
such as substances that enhance isotonicity and chemical stability.
Examples of buffers include phosphate buffer, bicarbonate buffer
and Tris buffer, while examples of preservatives include
thimerosol, cresols, formalin and benzyl alcohol. In certain
aspects, the pH can be modified depending upon the mode of
administration. For example, the pH of the composition is from
about 5 to about 6, which is suitable for topical applications.
Additionally, the pharmaceutical compositions can include carriers,
thickeners, diluents, preservatives, surface active agents and the
like in addition to the compounds described herein.
The pharmaceutical compositions can also include one or more active
ingredients used in combination with the compounds described
herein. The resulting pharmaceutical composition can provide a
system for sustained, continuous delivery of drugs and other
biologically-active agents to tissues adjacent to or distant from
the application site. The biologically active agent is capable of
providing a local or systemic biological, physiological or
therapeutic effect in the biological system to which it is applied.
For example, the agent can act to control and/or prevent infection
or inflammation, restore corneal permeability, enhance cell growth
and tissue regeneration, control tumor growth, act as an analgesic,
promote anti-cell attachment, reduce alveolar bone and tooth loss,
inhibit degeneration of cartilage and weight bearing joints, and
enhance bone growth, among other functions. Additionally, any of
the compounds described herein can contain combinations of two or
more pharmaceutically-acceptable compounds.
Examples of such compounds include, but are not limited to,
antimicrobial agents, antiinflammatory agents, anesthetics,
immunomodulators, cell regeneration and anti-apoptotic compounds,
and the like. Methods for using these compositions as drug delivery
devices are described in detail below.
The pharmaceutical compositions can be prepared using techniques
known in the art. In one aspect, the composition is prepared by
admixing a SAGE described herein with a pharmaceutically-acceptable
compound and/or carrier. The term "admixing" is defined as mixing
the two components together so that there is no chemical reaction
or physical interaction. The term "admixing" also includes the
chemical reaction or physical interaction between the compound and
the pharmaceutically-acceptable compound. Covalent bonding to
reactive therapeutic drugs, e.g., those having nucleophilic groups,
can be undertaken on the compound. Second, non-covalent entrapment
of a pharmacologically active agent in a cross-linked
polysaccharide is also possible. Third, electrostatic or
hydrophobic interactions can facilitate retention of a
pharmaceutically-acceptable compound in the compounds described
herein.
It will be appreciated that the actual preferred amounts of SAGE in
a specified case will vary according to the specific compound being
utilized, the particular compositions formulated, the mode of
application, and the particular situs and subject being treated.
Dosages for a given host can be determined using conventional
considerations, e.g. by customary comparison of the differential
activities of the subject compounds and of a known agent, e.g., by
means of an appropriate conventional pharmacological protocol.
Physicians and formulators, skilled in the art of determining doses
of pharmaceutical compounds, will have no problems determining dose
according to standard recommendations (Physician's Desk Reference,
Barnhart Publishing (1999).
The pharmaceutical compositions described herein can be
administered in a number of ways depending on whether local or
systemic treatment is desired, and on the area to be treated.
Administration can be topically (including ophthalmically,
vaginally, rectally, intranasally, orally, or directly to the
skin). Formulations for topical administration can include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. In one aspect, the compounds described herein
can be formulated into a solution that can be applied directly to
the eye or surrounding tissues of the eye. In one aspect, the
compound is from 0.01% to 20%, 0.1% to 15%, or 0.5% to 10% by
weight of the composition. In another aspect, the compound can be
formulated to be isotonic (e.g., 300-310 mOsm/kg osmolarity).
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like can be necessary or desirable.
Administration can also be directly into the lung by inhalation of
an aerosol or dry micronized powder. Administration can also be by
direct injection into the inflamed or degenerating joint space. In
other aspects, the pharmaceutical composition can be formulated for
intraocular administration. For example, the compounds described
herein can be added to intra-ocular drug delivery rings and other
devices to provide a constant elution of the compound to the
intra-ocular environment for treatment of iritis and age-related
macular degeneration and diabetic retinopathy.
Preparations for administration include sterile aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Parenteral vehicles, if needed for collateral use of the disclosed
compositions and methods, include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's,
or fixed oils. Intravenous vehicles, if needed for collateral use
of the disclosed compositions and methods, include fluid and
nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives can also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like.
In other aspects, ocular or ophthalmic devices can be coated with
the compounds described herein. Examples of such devices include,
but are not limited to, punctual plugs, contact lenses, implants,
intraocular lenses, and the like. The device can be designed such
that the compound is immediately released from the device or the
rate of release of the compound is controlled. Methods for coating
ocular or ophthalmic devices with the compounds described herein
are well known in the art.
Dosing is dependent on severity and responsiveness of the condition
to be treated, but will normally be one or more doses per day, with
course of treatment lasting from several days to several months or
until one of ordinary skill in the art determines the delivery
should cease. Persons of ordinary skill can easily determine
optimum dosages, dosing methodologies and repetition rates.
The SAGEs, pharmaceutical compositions, and devices described
herein can be used in a variety of applications related to drug
delivery, small molecule delivery, wound healing, treatment of
inflammatory skin disorders, treatment of inflammatory dental
disorders, treatment of inflammatory respiratory disorders,
treatment of inflammatory eye disorders, burn injury healing, and
tissue regeneration/engineering. In one aspect, the SAGEs and
compositions described herein can improve wound healing in a
subject in need of such improvement. The SAGEs and pharmaceutical
compositions described herein can be placed directly in or on any
biological system without purification as it is composed of
biocompatible materials. Examples of sites the SAGEs can be placed
include, but are not limited to, soft tissue such as muscle or fat;
hard tissue such as bone or cartilage; areas of tissue
regeneration; a void space such as periodontal pocket; surgical
incision or other formed pocket or cavity; a natural cavity such as
the oral, vaginal, rectal or nasal cavities, the joint space, the
cul-de-sac of the eye, and the like; the peritoneal cavity and
organs contained within, and other sites into or onto which the
compounds can be placed including a skin surface defect such as a
cut, scrape or burn area. It is contemplated that the tissue can be
damaged due to injury or a degenerative condition or, in the
alternative, the SAGEs and compositions described herein can be
applied to undamaged tissue to prevent injury to the tissue.
In the case of inflammatory skin disorders such as psoriasis, acne,
atopic dermatitis, rosacea or UV light dependent photo-aging, the
SAGEs can be applied topically as part of an emollient to prevent
or treat the intended condition. In the case of respiratory
disorders such as asthma, chronic obstructive pulmonary disease,
acute lung injury or cystic fibrosis, the SAGEs can be dissolved in
a water-soluble isotonic vehicle compatible with airway lining
fluid and delivered to the lung or nasal passages as an inhaled
aerosol. Alternately, the SAGEs can be formulated into a micronized
powder and inhaled into the lung as a dry powder. In the case of
eye diseases, the SAGEs can be placed into an aqueous vehicle and
applied to the eye topically as drops, or injected directly into
the eye either by needle or using an implanted constant drug
delivery device. In the case of dental disorders such as
periodontal disease, the SAGEs can be added as a component of a
mouthwash or formulated into creams or gingival packing materials
to be applied directly to the gingival crevice.
The SAGEs can also be injected parenterally, either intravenously,
intramuscularly or subcutaneously, to treat or prevent systemic
inflammatory disorders such as diabetic vascular or renal disease
or inflammatory gastrointestinal diseases. Similarly, the SAGEs can
be injected intra-articularly to treat inflammatory and
degenerative arthritis. The SAGEs can also be administered orally
in capsules or formulated into an enema to be delivered
intra-rectally as treatment for inflammatory bowel diseases.
The SAGEs and compositions described herein can deliver at least
one pharmaceutically-acceptable compound to a patient in need of
such delivery, comprising contacting at least one tissue capable of
receiving the pharmaceutically-acceptable compound with one or more
compositions described herein. The SAGEs can be used as a carrier
for a wide variety of releasable biologically active substances
having curative or therapeutic value for human or non-human
animals. Many of these substances that can be carried by the SAGE
are discussed above. Included among biologically active materials
which are suitable for incorporation into the gels of the invention
are therapeutic drugs, e.g., anti-inflammatory agents, anti-pyretic
agents, steroidal and non-steroidal drugs for anti-inflammatory
use, hormones, growth factors, contraceptive agents, antivirals,
antibacterials, antifungals, analgesics, hypnotics, sedatives,
tranquilizers, anticonvulsants, muscle relaxants, local
anesthetics, antispasmodics, antiulcer drugs, peptidic agonists,
sympathimometic agents, cardiovascular agents, antitumor agents,
oligonucleotides and their analogues and so forth. A biologically
active substance is added in pharmaceutically active amounts.
In one aspect, the sulfated and alkylated SAGEs described herein
can inhibit the activity of the receptor for Advanced Glycation End
products (RAGE), P-selectin, or human leukocyte elastase. RAGE is
highly expressed in human skin, where it is present on dermal
fibroblasts, dendritic cells, keratinocytes, endothelial cells and
monocytes. RAGE is upregulated in sun-exposed skin by Advanced
Glycation End-Products (AGE) and by the cytokine tumor necrosis
factor-.alpha.. RAGE plays a prominent role in UV-induced
photo-ageing, where its ligation by AGE products such as UV-induced
carboxymethyl lysine (CML) promotes skin aging through stimulation
of extracellular matrix production by dermal fibroblasts. The role
of RAGE is likely to be even more prominent in psoriasis because
this disease is critically dependent on activated T-lymphocytes for
initiation of inflammation. T-lymphocytes may also be
mechanistically important in acne and atopic dermatitis. In the
case of acne, elevated dermal levels of CD3.sup.+ and CD4.sup.+
T-lymphocytes and macrophages stimulate hyper-proliferation of
keratinocytes in the ducts of follicles, producing the plugged
follicular ducts that lead to formation of the acne comedone. In
the case of atopic dermatitis, dermal antigens activate T.sub.H2
lymphocytes which secrete cytokines such as interleukin-4 (IL-4)
and interleukin-13 (IL-13), resulting in the recruitment of
eosinophils into skin. Eosinophils, in turn, release cationic
toxins such as major basic protein, which produces allergic skin
disease. Thus, the potent RAGE inhibiting activity of the compounds
described herein makes them useful in treating a variety of skin
disorders including, but not limited to, acne, eczema, atopic
dermatitis, psoriasis, or photo-dermal ageing.
In the adult state, RAGE is not always so entirely helpful to the
organism. Malignant tumors secrete amphoterin (or high mobility box
group protein-1, HMGB-1) as an autocrine factor and use the
interaction of amphoterin with RAGE to promote primary tumor growth
and metastasis. Blocking RAGE with a recombinant decoy (soluble
RAGE or s-RAGE) reduces tumor growth and inhibits metastasis.
During sepsis, monocytes and macrophages secrete amphoterin which
interacts with RAGE on blood vessels and other inflammatory cells
to enhance the severity of bacterial shock. Blocking this
interaction with antibodies against RAGE prevents organ damage in
severe sepsis. In the adult state, RAGE also functions as a
vascular adhesion receptor promoting the recruitment of PMNs,
monocytes and lymphocytes into areas of inflammation. Blocking RAGE
blunts inflammatory cell influx. This has been previously
demonstrated in animal models of multiple sclerosis, where
competitive blockade of vascular endothelial RAGE with s-RAGE
prevents the influx of activated encephalitogenic T-lymphocytes
into the central nervous system, and retards onset and progression
of neurologic inflammation and degeneration.
RAGE also interacts with a family of calcium binding proteins
called S100 calgranulins, which are secreted by PMNs, monocytes and
lymphocytes as potent inflammation-promoting factors. Elevated
levels of S 100 calgranulins are a prominent marker of PMN
inflammation in acute lung injury and in the airway secretions of
patients with cystic fibrosis. In the eye, the interaction of S 100
calgranulins with RAGE plays a prominent role leading to blindness
in age-related macular degeneration. RAGE also binds the
Alzheimer's .beta.-amyloid peptide and the .beta. sheets of amyloid
proteins. Through RAGE-related induction of neural cell death and
inflammation, RAGE-.beta. sheet fibrillar interactions mediate
Alzheimer's dementia and organ damage in systemic amyloidosis.
RAGE is also prominent in diabetes mellitus. When blood glucose is
elevated, the aldehyde group of glucose randomly attaches to the
amines of cellular proteins, covalent adducts. In the presence of
oxidants such as hypochlorous acid (HOCl), the oxidant produced by
PMNs, this glucose moiety can then become oxidized. These oxidized,
glycosylated proteins are known as Advanced Glycation End-Products,
(AGE). AGEs also bind the RAGE receptor avidly, and trigger
RAGE-mediated signaling. AGE-RAGE signaling accounts for the
vascular endothelial dysfunction, poor wound healing and
accelerated arterial atherosclerosis characteristic of poorly
controlled diabetes. In the eye, AGE-RAGE signaling produces the
proliferation of retinal microvessels that leads to diabetic
retinopathy and blindness. In the kidney, AGE-RAGE signaling
accounts for the initial renal hypertrophy and then fibrosis that
causes diabetic renal failure (diabetic nephropathy). AGE-RAGE
signaling likewise produces apoptosis of endothelium, inhibits
blood vessel growth and retards healing of cutaneous diabetic
ulcers. The ability of the SAGEs to block RAGE makes them
particularly valuable as therapeutic agents for inflammation. RAGE
functions in utero as a receptor binding the growth promoting
nuclear protein amphoterin, or high mobility box protein-1. There,
the amphoterin-RAGE interaction triggers growth signaling important
for nervous system development. In the adult state, RAGE is
expressed in the cells of vessel walls, neural tissues, cardiac
myocytes, monocytes and macrophages, T-lymphocytes, renal mesangial
cells, and in skin fibroblasts, dendrocytes and keratinocytes.
Thus, in one aspect, the SAGEs and compositions described herein
can be used to safely reduce or prevent inflammation in a subject
produced by a variety of different maladies attributed to
RAGE-related diseases including, but not limited to, cancer,
multiple sclerosis, osteoarthritis, cystic fibrosis, sickle cell
anemia, a cardiovascular inflammatory disorder, or a cardiovascular
inflammatory disorder, or diabetic complications.
In other aspects, the SAGEs and compositions, as negatively charged
entities, can also be administered to bind and inhibit cationic
skin peptides derived from cathelicidins, thereby treating or
preventing skin disorders. For example, the skin condition known as
acne rosacea, which is known to occur from excess skin expression
of active cathelicidin peptides, can be treated or prevented using
the SAGEs described herein. Examples of skin disorders that can be
treated or prevented using the SAGEs include, but are not limited
to, rosacea, atopic dermatitis (eczema), allergic contact
dermatitis, psoriasis, dermatitis herpetiformis, acne, diabetic
skin ulcers and other diabetic wounds, burns (including relieving
pain of thermal burns), sunburn (including relieving pain of
sunburn), prevention of scarring after plastic surgery, actinic
keratoses, inflammation from insect bites, poison ivy,
radiation-induced dermatitis/burn, facilitation of skin healing,
prevention and treatment of keloid scarring, or the treatment of
seborrheic dermatitis.
Due to the ability of the SAGEs to inhibit RAGE activity and other
biological mechanisms, the SAGEs have numerous therapeutic
applications in addition to treating or preventing skin disorders.
In one aspect, the SAGEs can be used in dental and oral surgery to
treat gingivitis (periodontal disease) and aphthous ulcers. In
other aspects, the SAGEs can be used in ophthalmological
applications such as, for example, in the treatment of age-related
macular degeneration, diabetic retinopathy, dry eye syndrome and
other inflammatory conjunctivitis, iritis, uveitis, allergic
conjunctivitis, anti-inflammatory aid in cataract surgery, or in
the prevention of corneal inflammation and scarring. In further
aspects, the SAGEs can be used in genitourinary applications (e.g.,
prevention of urinary tract infection, treatment of the
transitional cell cancer of the bladder and uroepithelial system;
treatment of interstitial cystitis; and use as a vaginal
lubricant/protective to prevent transmission of sexually
transmitted diseases).
In another aspect, the SAGEs can be used to treat a number of
respiratory disorders including cystic fibrosis, bronchiectasis,
rhinitis (both allergic and perennial), sinusitis, emphysema and
chronic bronchitis (COPD), acute lung injury/adult respiratory
distress syndrome, interstitial lung fibrosis, SARS, asthma, and
respiratory syncytial virus. In other aspects, the SAGEs can
prevent and treat snoring and obstructive sleep apnea, prevent
infection by common respiratory pathogens (Streptococcus
pneumoniae, Hemophilus influenzae, Staphylococcus, Mycoplasma
pneumoniae, Chlamydial pneumonia, Gram-negative enteric infections)
in immune suppressed hosts such as subjects who are HIV positive or
who have hematopoietic malignancies, or prevent and treat otitis
media.
The SAGEs can be used in cardiovascular applications (e.g.,
treating or preventing acute coronary syndrome or atherosclerosis);
hematological/oncological applications (e.g., prevention and
treatment of sickle cell anemia; prevention and treatment of
metastatic disease; and prevention of hypercoagulable state of
malignancy (Trousseau's syndrome)); treatment of infectious
diseases (e.g., cerebral vascular occlusive syndromes and nephritis
in Falciparum malaria, Yellow fever, Denge fever, systemic sepsis,
and adjunctive treatment of HIV to prevent viral fusion with and
infection of target cells); treatment of gastrointestinal diseases
(e.g., ulcerative colitis, Crohn's disease of the bowel,
Hemorrhoids, and the Prevention of stress ulceration of the stomach
and esophagus); treatment of rheumatological and immunological
diseases (e.g., prevention and treatment of osteoarthritis,
rheumatoid arthritis, systemic lupus erythematosis, prevention and
treatment of angioneurotic edema, Sjogren's syndrome, systemic
sclerosis, systemic amyloidosis, and systemic mastocytosis); renal
diseases (e.g., prevention and treatment of diabetic nephropathy
and glomerulonephritis); and neurologic diseases (e.g., multiple
sclerosis and Alzheimer's dementia). The SAGEs and compositions
described herein are safer than other related therapies. For
example, heparin and other sulfated polysaccharides can reduce
diabetic complications in both animal and clinical studies, and are
particularly effective against diabetic nephropathy. However,
heparins cannot be used in general clinical settings to prevent
diabetic complications because the anticoagulant properties present
an excessive risk of bleeding.
The SAGEs of the subject invention can also be used to treat,
inhibit, or prevent certain ocular or ophthalmic conditions. In
particular, GM-111101 can be used for dry eye or ocular rosacea by
acting as a protective barrier to the corneal epithelial cells,
increasing moisture and normalizing the hyperosmolar eye
environment, and limiting local ocular inflammatory reactions.
The SAGEs and compositions described herein possess low
anticoagulant activity, which is an important consideration for
long-term treatment, which is demonstrated below in the Examples.
Moreover, by virtue of the poly-anionic charges present in this
sulfated polysaccharide, GM-111101 has reported in vitro
anti-inflammatory activities at nanomolar concentrations, including
inhibition of cationic polymorphonuclear (PMN) proteases,
inhibition of platelet adhesion receptor P-selecting and inhibition
of the interaction of the receptor for advance glycation
end-products (RAGE), a newly recognized important component of
innate immunity mediating long-lived inflammation. The RAGE and
P-selectin inhibiting activities of GM-111101 might also retard the
influx of CD4+ lymphocytes into the ocular epithelial environment.
In addition, as sulfated polysaccharides, GM-111101 could
counteract many of the Th1 heparin-binding cytokines active in dry
eye. As an example, the related sulfated polysaccharide heparin
antagonizes the biological and molecular actions of
interferon-.gamma. (IFN.gamma.) and IFN.gamma.-inducible cytokines
such as IL-10. Additionally, the SAGEs have little to no toxicity,
which is also demonstrated in the Examples.
EXAMPLES
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, and methods
described and claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
scope of what the inventors regard as their invention. Efforts have
been made to ensure accuracy with respect to numbers (e.g.,
amounts, temperature, etc.) but some errors and deviations should
be accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C. or is at ambient temperature,
and pressure is at or near atmospheric. There are numerous
variations and combinations of reaction conditions, e.g., component
concentrations, desired solvents, solvent mixtures, temperatures,
pressures and other reaction ranges and conditions that can be used
to optimize the product purity and yield obtained from the
described process. Only reasonable and routine experimentation will
be required to optimize such process conditions. Compounds
described below are identified by code numbers as defined above and
referred to in FIG. 2.
Example I
Synthesis of Alkylated HA Derivatives
A. Preparation of Methyl HA (DS-2)
Hyaluronic acid (HA, Novozymes Biopolymers, 950 kDa) (2.0 g) was
dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the
mixture stirred for 2 h at room temperature (RT). The resulting
viscous liquid was transferred to a 400 mL beaker that contained
100 mL of isopropanol, and mixing was continued. To a stirred
mixture was added 10 mL of iodomethane, and the mixture was stirred
for 24 h at rt. The resulting suspension was filtered to collect
the crude methylated HA product. This crude MeHA was dissolved in
250 mL of distilled water, the solution was adjusted to pH -7.0,
and the solution was dialyzed against distilled water for 24 h,
changing the external water bath four times during this period. The
dialyzed MeHA product was lyophilized to afford 1.25 g of methyl HA
as a cottony mesh. .sup.1H NMR (D.sub.2O, .delta.): 1.85 (s, 3H,
NCH.sub.3), 3.20-3.80 (m, 1OH, OCH+OCH.sub.3). The substitution
degree (SD) was determined by .sup.1H NMR, SD=[(integration of
methyl HA at .delta. 3.20-3.8)-(integration of HA at .delta.
3.20-3.8)]/(integration of NCH.sub.3 at 1.85), and was estimated to
be SD=2, or an average of 2 methyl groups per disaccharide unit.
This suggests that both the primary hydroxyl and at least one
secondary hydroxyl group were modified by this process.
B. Preparation of Methyl HA (DS-I)
Hyaluronic acid (HA, Novozymes Biopolymers, 950 kDa) (2.0 g) was
dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the
mixture stirred for 2 h at room temperature (rt). The resulting
viscous liquid was transferred to a 400 mL beaker that contained
100 mL of isopropanol, and mixing was continued. To stirred mixture
was added 4 mL of iodomethane, and the mixture was stirred for 24 h
at rt. The resulting suspension was filtered to collect the crude
methylated HA product. This crude MeHA was dissolved in 250 mL of
distilled water, the solution was adjusted to pH -7.0, and the
solution was dialyzed against distilled water for 24 h, changing
the external water bath four times during this period. The dialyzed
MeHA product was lyophilized to afford 1.25 g of methyl HA as a
cottony mesh. .sup.1H NMR (D.sub.2O, .delta.): 1.8 S (s, 3H,
NCH.sub.3), 3.20-3.80 (m, 1OH, OCH+OCH.sub.3). The substitution
degree (SD) was determined by .sup.1H NMR, SD=[(integration of
methyl HA at .delta. 3.20-3.8)-(integration of HA at .delta.
3.20-3.8)]/(integration of NCH.sub.3 at 1.85), and was estimated to
be SD=1, or an average of 1 methyl groups per disaccharide unit.
This suggests that the primary hydroxyl and at least one secondary
hydroxyl group were modified by this process.
C. Preparation of FHA-2(DS-1)
To a 25 mL flask containing 1.0 g of
2,2,3,3,4,4-heptafluoro-l-butanol (5 mmol), 1.3 mL of phosphorus
tribromide (7.5 mmol) was added slowly. The mixture was stirred at
60.degree. C. for 30 minutes, and then saturated sodium bicarbonate
solution (15 mL) was slowly added to quench the reaction. The
aqueous solution was extracted with three 15-mL portions of
dichloromethane, the organic layer containing the heptafluorobutyl
bromide was concentrated, and the residue was used without
purification in the next step.
HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL
beaker and the mixture stirred for 2 h at room temperature (rt).
The resulting viscous liquid was transferred to a 400 mL beaker
that contained 100 mL of isopropanol, and mixing was continued. To
the stirred mixture was added the crude heptafluorobutyl bromide in
10 mL of isopropanol, and the mixture was stirred for 24 h at rt.
The resulting suspension was filtered to collect the crude FHA-2
product. This crude FHA-2 was dissolved in 250 mL of distilled
water, the solution was adjusted to pH -7.0, and the solution was
dialyzed against distilled water for 24 h, changing the external
water bath four times during this period. The dialyzed FHA-2
product was lyophilized to afford 1.5 g of FHA-2, designed as FHA-2
as cottony mesh. .sup.1H NfMR (D.sub.2O, .delta.): 1.82 (s, 3H,
NCH.sub.3), 3.15-3.80 (m, 8H, 0CH+0CH.sub.2). .sup.19F NMR
(D.sub.2O, .delta.): -115.3, -120.8. The substitution degree (SD)
was determined by .sup.1H NMR as 1.0. SD=[(integration of FHA-2 at
.delta. 3.15-3.80)-(integration of HA at .delta.
3.20-3.8)]/[(integration of NCH.sub.3 at 1.82).times.(2/3)].
D. Preparation of FHA-2(DS-2)
To a 25 mL flask containing 3.0 g of
2,2,3,3,4,4-heptafluoro-l-butanol (15 mmol), 2.5. mL of phosphorus
tribromide (16 mmol) was added slowly. The mixture was stirred at
60.degree. C. for 30 minutes, and then saturated sodium bicarbonate
solution (15 mL) was slowly added to quench the reaction. The
aqueous solution was extracted with three 15-mL portions of
dichloromethane, the organic layer containing the heptafluorobutyl
bromide was concentrated, and the residue was used without
purification in the next step.
HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL
beaker and the mixture stirred for 2 h at room temperature (rt).
The resulting viscous liquid was transferred to a 400 mL beaker
that contained 100 mL of isopropanol, and mixing was continued. To
the stirred mixture was added the crude heptafluorobutyl bromide in
10 mL of isopropanol, and the mixture was stirred for 24 h at rt.
The resulting suspension was filtered to collect the crude FHA-2
product. This crude FHA-2 was dissolved in 250 mL of distilled
water, the solution was adjusted to pH -7.0, and the solution was
dialyzed against distilled water for 24 h, changing the external
water bath four times during this period. The dialyzed FHA-2
product was lyophilized to afford 1.5 g of FHA-2, designed as FHA-2
as cottony mesh H NMR (D.sub.2O, .delta.): 1.82 (s, 3H, NCH.sub.3),
3.15-3.80 (m, 8H, OCH+OCH.sub.2). .sup.19F NMR (D.sub.2O, .delta.):
-1 15.3, -120.8. The substitution degree (SD) was determined by
.sup.1H NMR as 2.0. SD=[(integration of FHA-2 at .delta.
3.15-3.80)-(integration of HA at .delta. 3.20-3.8)]/[(integration
of NCH.sub.3 at 1.82).times.(2/3)].
E. Preparation of FHA-I(DS-I)
To a 25 mL flask containing 1.0 g of 2,2,3,3-pentafluoro-l-propanol
(5 mmol), 1.3 mL of phosphorus tribromide (7.5 mmol) was added
slowly. The mixture was stirred at 60.degree. C. for 30 minutes,
and then saturated sodium bicarbonate solution (15 mL) was slowly
added to quench the reaction. The aqueous solution was extracted
with three 15-mL portions of dichloromethane, the organic layer
containing the pentafluoropropyl bromide was concentrated, and the
residue was used without purification in the next step.
HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL
beaker and the mixture stirred for 2 h at room temperature (rt).
The resulting viscous liquid was transferred to a 400 mL beaker
that contained 100 mL of isopropanol, and mixing was continued. To
the stirred mixture was added the crude pentafluoropropyl bromide
in 10 mL of isopropanol, and the mixture was stirred for 24 h at
rt. The resulting suspension was filtered to collect the crude
FHA-I product. This crude FHA-I was dissolved in 250 mL of
distilled water, the solution was adjusted to pH -7.0, and the
solution was dialyzed against distilled water for 24 h, changing
the external water bath four times during this period. The dialyzed
FHA-I product was lyophilized to afford 1.5 g of FHA-I, designed as
FHA-I as cottony mesh. H NMR (D.sub.2O, .delta.): 1.80 (s, 3H,
NCH.sub.3), 3.15-3.80 (m, 8H, OCH+OCH.sub.2). .sup.19F NMR
(D.sub.2O, .delta.): -113.6, -118.0. The substitution degree (SD)
was determined by .sup.1H NMR as 1.0. SD=[(integration of FHA-I at
.delta. 3.15-3.80)-(integration of HA at .delta.
3.20-3.8)]/[(integration of NCH.sub.3 at 1.80).times.(2/3)].
F. Preparation of FHA-I (DS-2)
To a 25 mL flask containing 3.0 g of
2,2,3,3,-pentafluoro-l-propanol (15 mmol), 3 mL of phosphorus
tribromide (18 mmol) was added slowly. The mixture was stirred at
60 C for 30 minutes, and then saturated sodium bicarbonate solution
(15 mL) was slowly added to quench the reaction. The aqueous
solution was extracted with three 15-mL portions of
dichloromethane, the organic layer containing the pentafluoropropyl
bromide was concentrated, and the residue was used without
purification in the next step.
HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL
beaker and the mixture stirred for 2 h at room temperature (it).
The resulting viscous liquid was transferred to a 400 mL beaker
that contained 100 mL of isopropanol, and mixing was continued. To
the stirred mixture was added the crude pentafluoropropyl bromide
in 10 mL of isopropanol, and the mixture was stirred for 24 h at
it. The resulting suspension was filtered to collect the crude
FHA-I product. This crude FHA-I was dissolved in 250 mL of
distilled water, the solution was adjusted to pH -7.0, and the
solution was dialyzed against distilled water for 24 h, changing
the external water bath four times during this period. The dialyzed
FHA-I product was lyophilized to afford 1.5 g of FHA-I, designed as
FHA-I as cottony mesh. H NMR (D.sub.2O, .delta.): 1.80 (s, 3H,
NCH.sub.3), 3.15-3.80 (m, 8H, OCH+OCH.sub.2). .sup.19F NMR
(D.sub.2O, .delta.): -113.6, -118.0. The substitution degree (SD)
was determined by .sup.1H NMR as 2.0. SD=[(integration of FHA-I at
.delta. 3.15-3.80)-(integration of HA at .delta.
3.20-3.8)]/[(integration Of NCH.sub.3 at 1.80).times.(2/3)].
G. Preparation of Low Molecular Weight Methyl HA (DS-2)
Hyaluronic acid (HA, Novozymes Biopolymers, 53 kDa) (2.0 g) was
dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the
mixture stirred for 2 h at room temperature (it). The resulting
viscous liquid was transferred to a 400 mL beaker that contained
100 mL of isopropanol, and mixing was continued. To stirred mixture
was added 10 mL of iodomethane, and the mixture was stirred for 24
h at rt. The resulting suspension was filtered to collect the crude
Low molecule methylated HA product. This crude LMW MeHA was
dissolved in 250 mL of distilled water, the solution was adjusted
to pH -7.0, and the solution was dialyzed against distilled water
for 24 h, changing the external water bath four times during this
period. The dialyzed LMW MeHA product was lyophilized to afford 1.2
g of methyl HA as a cottony mesh. .sup.1H NMR (D.sub.2O, .delta.):
1.85 (s, 3H, NCH.sub.3), 3.20-3.80 (m, 1OH, OCH+OCH.sub.3). The
substitution degree (SD) was determined by .sup.1H NMR, as 2.
H. Preparation of Low Molecular Weight Methyl HA (DS-I)
Hyaluronic acid (HA, Novozymes Biopolymers, 53 kDa) (2.0 g) was
dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the
mixture stirred for 2 h at room temperature (rt). The resulting
viscous liquid was transferred to a 400 mL beaker that contained
100 mL of isopropanol, and mixing was continued. To stirred mixture
was added 4 mL of iodomethane, and the mixture was stirred for 24 h
at it. The resulting suspension was filtered to collect the crude
low molecule methylated HA product. This crude LMW MeHA was
dissolved in 250 mL of distilled water, the solution was adjusted
to pH .about.7.0, and the solution was dialyzed against distilled
water for 24 h, changing the external water bath four times during
this period. The dialyzed LMW MeHA product was lyophilized to
afford 1.2 g of LMW MeHA as a cottony mesh. .sup.1H NMR (D.sub.2O,
.delta.): 1.85 (s, 3H, NCH.sub.3), 3.20-3.80 (m, 1OH,
OCH+OCH.sub.3). The substitution degree (SD) was determined by
.sup.1H NMR as 1.
I. Preparation of Low Molecular Weight FHA-2(DS-1)
To a 25 mL flask containing 1.0 g of
2,2,3,3,4,4-heptafluoro-l-butanol (5 mmol), 1.3 mL of phosphorus
tribromide (7.5 mmol) was added slowly. The mixture was stirred at
60.degree. C. for 30 minutes, and then saturated sodium bicarbonate
solution (15 mL) was slowly added to quench the reaction. The
aqueous solution was extracted with three 15-mL portions of
dichloromethane, the organic layer containing the heptafluorobutyl
bromide was concentrated, and the residue was used without
purification in the next step.
HA (2.0 g, 53 kDa was dissolved in 20 mL of NaOH (40% w/v) in a 100
mL beaker and the mixture stirred for 2 h at room temperature (rt).
The resulting viscous liquid was transferred to a 400 mL beaker
that contained 100 mL of isopropanol, and mixing was continued. To
the stirred mixture was added the crude heptafluorobutyl bromide in
10 mL of isopropanol, and the mixture was stirred for 24 h at rt.
The resulting suspension was filtered to collect the crude LMW
FHA-2 product. This crude LMW FHA-2 was dissolved in 250 mL of
distilled water, the solution was adjusted to pH -7.0, and the
solution was dialyzed against distilled water for 24 h, changing
the external water bath four times during this period. The dialyzed
LMW FHA-2 product was lyophilized to afford 1.5 g of LMW FHA-2,
designed as LMW FHA-2 as cottony mesh. H NMR (D.sub.2O, .delta.):
1.82 (s, 3H, NCH.sub.3), 3.15-3.80 (m, 8H, OCH+OCHz). .sup.19F NMR
(D.sub.2O, .delta.): -115.3, -120.8. The substitution degree (SD)
was determined by .sup.1H NMR as 1.0.
J. Preparation of Low Molecular Weight FHA-2(DS-2)
To a 25 mL flask containing 3.0 g of
2,2,3,3,4,4-heptafluoro-l-butanol (15 mmol), 2.5. mL of phosphorus
tribromide (16 mmol) was added slowly. The mixture was stirred at
60.degree. C. for 30 minutes, and then saturated sodium bicarbonate
solution (15 mL) was slowly added to quench the reaction. The
aqueous solution was extracted with three 15-mL portions of
dichloromethane, the organic layer containing the heptafluorobutyl
bromide was concentrated, and the residue was used without
purification in the next step. HA (2.0 g, 53 kDa) was dissolved in
20 mL of NaOH (40% w/v) in a 100 mL beaker and the mixture stirred
for 2 h at room temperature (rt). The resulting viscous liquid was
transferred to a 400 mL beaker that contained 100 mL of
isopropanol, and mixing was continued. To the stirred mixture was
added the crude heptafluorobutyl bromide in 10 mL of isopropanol,
and the mixture was stirred for 24 h at rt. The resulting
suspension was filtered to collect the crude LMW FHA-2 product.
This crude LMW FHA-2 was dissolved in 250 mL of distilled water,
the solution was adjusted to pH -7.0, and the solution was dialyzed
against distilled water for 24 h, changing the external water bath
four times during this period. The dialyzed LMW FHA-2 product was
lyophilized to afford 1.5 g of LMW FHA-2, designed as LMW FHA-2 as
cottony mesh. H NMR (D.sub.2O, .delta.): 1.82 (s, 3H, NCH.sub.3),
3.15-3.80 (m, 8H, 0CH+0CH.sub.2). .sup.19F NMR (D.sub.2O, .delta.):
-115.3, -120.8. The substitution degree (SD) was determined by
.sup.1H NMR as 2.0.
K. Preparation of Low Molecular Weight FHA-I(DS-I)
To a 25 mL flask containing 1.0 g of 2,2,3,3-pentafluoro-l-propanol
(5 mmol), 1.3 mL of phosphorus tribromide (7.5 mmol) was added
slowly. The mixture was stirred at 60.degree. C. for 30 minutes,
and then saturated sodium bicarbonate solution (15 mL) was slowly
added to quench the reaction. The aqueous solution was extracted
with three 15-mL portions of dichloromethane, the organic layer
containing the pentafluoropropyl bromide was concentrated, and the
residue was used without purification in the next step.
HA (2.0 g, 53 kDa) was dissolved in 20 mL of NaOH (40% w/v) in a
100 mL beaker and the mixture stirred for 2 h at room temperature
(rt). The resulting viscous liquid was transferred to a 400 mL
beaker that contained 100 mL of isopropanol, and mixing was
continued. To the stirred mixture was added the crude
pentafluoropropyl bromide in 10 mL of isopropanol, and the mixture
was stirred for 24 h at rt. The resulting suspension was filtered
to collect the crude LMW FHA-I product. This crude LMW FHA-I was
dissolved in 250 mL of distilled water, the solution was adjusted
to pH -7.0, and the solution was dialyzed against distilled water
for 24 h, changing the external water bath four times during this
period. The dialyzed LMW FHA-I product was lyophilized to afford
1.5 g of LMW FHA-I, designed as LMW FHA-I as cottony mesh. H NMR
(D.sub.2O, .delta.): 1.80 (s, 3H, NCH.sub.3), 3.15-3.80 (m, 8H,
OCH+OCH.sub.2). .sup.19F NMR (D.sub.2O, .delta.): -113.6, -1 18.0.
The substitution degree (SD) was determined by .sup.1H NMR as
1.0.
L. Preparation of LMW FHA-I(DS-2)
To a 25 mL flask containing 3.0 g of
2,2,3,3,-pentafluoro-l-propanol (15 mmol), 3 mL of phosphorus
tribromide (18 mmol) was added slowly. The mixture was stirred at
60.degree. C. for 30 minutes, and then saturated sodium bicarbonate
solution (15 mL) was slowly added to quench the reaction. The
aqueous solution was extracted with three 15-mL portions of
dichloromethane, the organic layer containing the pentafluoropropyl
bromide was concentrated, and the residue was used without
purification in the next step.
HA (2.0 g, 53 kDa) was dissolved in 20 mL of NaOH (40% w/v) in a
100 mL beaker and the mixture stirred for 2 h at room temperature
(rt). The resulting viscous liquid was transferred to a 400 mL
beaker that contained 100 mL of isopropanol, and mixing was
continued. To the stirred mixture was added the crude
pentafluoropropyl bromide in 10 mL of isopropanol, and the mixture
was stirred for 24 h at rt. The resulting suspension was filtered
to collect the crude LMW FHA-I product. This crude LMW FHA-I was
dissolved in 250 mL of distilled water, the solution was adjusted
to pH .about.7.0, and the solution was dialyzed against distilled
water for 24 h, changing the external water bath four times during
this period. The dialyzed LMW FHA-I product was lyophilized to
afford 1.5 g of LMW FHA-I, designed as LMW FHA-I as cottony mesh.
.sup.1H NMR (D.sub.2O, .delta.): 1.80 (s, 3H, NCH.sub.3), 3.15-3.80
(m, 8H, OCH+OCH.sub.2). .sup.19F NMR (D.sub.2O, .delta.): -1 13.6,
-118.0. The substitution degree (SD) was determined by .sup.1H NMR
as 2.0.
Example II
Sulfation of Alkylated HA Derivatives
1. Preparation of LMW-P-OSFHA-I (DS-I) (GM-211101)
First, the tributylammonium (TBA) salt of LMW FHA-I(DS-I) was
prepared by the addition of 1 mL of tributylamine to LMW FHA-I (1.0
g) in 100 mL of deionized water which was adjusted to pH 3.0 with
IN HCl. The mixture was mixed vigorously, dried by lyophilization.
The resulting salt (FHA-I-TBA) was dissolved in 25 mL of DMF to
which the required excess (6 mol/equiv of available hydroxy group
in HA) of pyridine-sulfur trioxide complex (0.4 g) was added. After
stirring for 3 h at 40.degree. C., the reaction was quenched by
addition of 50 mL of water, and the crude material was precipitated
by adding 75 mL of cold ethanol saturated with anhydrous sodium
acetate, and then collected by filtration. The resulting crude
partially O-sulfated HA was dissolved in distilled water and
dialyzed against 100 mM of NaCl solution for two days, changing the
solution four times a day, and lyophilized to give the product (330
mg) in 75% yield and characterized by .sup.1H NMR, sulfation
SD=1.0. The substitution degree is estimated by comparing the NMR
shift of OCH to those in literature (Carbohydrate Research, 1998,
306, 35-43).
2. Preparation of LMW-P-OSFHA-2 (DS I) (GM-311101)
The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(LMW FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (6 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.4 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated LMW FHA-2 was dissolved in water, dialyzed, and
lyophilized as in 1 to give the product (300 mg) in 68% yield and
characterized by .sup.1H NMR, estimated sulfation SD=1.0.
3. Preparation of LMW-P-OSMeHA (DS-I) (GM-111101)
The TBA salt of LMW MeHA (DS-I) (from MW 53 kDa HA) was prepared
from 0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water
as in 1. The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL
of DMF to which the required excess (6 mol/equiv of available
hydroxy groups in MeHA) of pyridine-sulfur trioxide complex (0.8 g)
was added. After stirring for 3 h at 40.degree. C., the reaction
was quenched by adding 100 mL of water, and crude material was
precipitated by adding 150 mL of cold ethanol saturated with
anhydrous sodium acetate, and then collected by filtration. The
resulting crude partially O-sulfated MeHA was dissolved in water,
dialyzed, and lyophilized as in 1 to give the product (540 mg) in
62% yield, which was estimated by 1H NMR to have a sulfation
SD=1.0-1.5.
4. Preparation of LMW-P-OSFHA-I (DS-2) (GM-211201)
The TBA salt of LMW FHA-I (FHA-I from MW 53 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-I-TBA) was dissolved in 25 mL of DMF to which the required
excess (6 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.4 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated HA was dissolved in distilled water and dialyzed against
100 mM of NaCl solution for two days, changing the solution four
times a day, and lyophilized to give the product (300 mg) in 70%
yield and characterized by 1H NMR, estimated sulfation SD=1.0.
5. Preparation of LMW-P-OSFHA-2 (DS-2) (GM-311201)
The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(LMW FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (6 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.4 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated LMW FHA-2 was dissolved in water, dialyzed, and
lyophilized as in 1 to give the product (310 mg) in 70% yield and
characterized by 1H NMR, estimated sulfation SD=1.0.
6. Preparation of LMW-P-OSMeHA (DS-2) (GM-111201)
The TBA salt of LMW MeHA (DS-I) (from MW 53 kDa HA) was prepared
from 0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water
as in 1. The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL
of DMF to which the required excess (6 mol/equiv of available
hydroxy groups in MeHA) of pyridine-sulfur trioxide complex (0.8 g)
was added. After stirring for 3 h at 40.degree. C., the reaction
was quenched by adding 100 mL of water, and crude material was
precipitated by adding 150 mL of cold ethanol saturated with
anhydrous sodium acetate, and then collected by filtration. The
resulting crude partially (O-sulfated MeHA was dissolved in water,
dialyzed, and lyophilized as in 1 to give the product (560 mg) in
64% yield, which was shown by 1H NMR to have estimated sulfation
SD=1.0-1.5.
7. Preparation of P-OSFHA I (DS-I) (GM-231101)
The TBA salt of FHA-1 (FHA-1 from MW 950 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-I-TBA) was dissolved in 25 mL of DMF to which the required
excess (6 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.4 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated HA was dissolved in distilled water and dialyzed against
100 mM of NaCl solution for two days, changing the solution four
times a day, and lyophilized to give the product (300 mg) in 68%
yield and characterized by 1H NMR, estimated sulfation
SD=1.0-1.5.
8. Preparation of P-OSFHA-2 (DS-I) (GM-331101)
The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (6 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.4 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated FHA-2 was dissolved in water, dialyzed, and lyophilized
as in 1. to give the product (320 mg) in 70% yield and
characterized by 1H NMR, estimated sulfation SD=1.0-1.5.
9. Preparation of P-OSMeHA (DS-I) (GM-131101)
The TBA salt of MeHA (DS-I) (from MW 950 kDa HA) was prepared from
0.5 mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1.
The resulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to
which the required excess (6 mol/equiv of available hydroxy groups
in MeHA) of pyridine-sulfur trioxide complex (0.8 g) was added.
After stirring for 3 h at 40.degree. C., the reaction was quenched
by adding 100 mL of water, and crude material was precipitated by
adding 150 mL of cold ethanol saturated with anhydrous sodium
acetate, and then collected by filtration. The resulting crude
partially O-sulfated MeHA was dissolved in water, dialyzed, and
lyophilized as in 1 to give the product (510 mg) in 60% yield,
which was shown by 1H NMR to have an estimated sulfation
SD=1.0-1.5.
10. Preparation of P-OSFHA-I (DS-2) (GM-231201)
TBA salt of FHA-I (FHA-I from MW 950 kDa HA) was prepared by adding
0.5 mL of tributylamine to the FHA-I (0.25 g) in 50 mL of distilled
water and processing as above in 1. The resulting salt (FHA-I-TBA)
was dissolved in 25 mL of DMF to which the required excess (6
mol/equiv of available hydroxy group in HA) of pyridine-sulfur
trioxide complex (0.4 g) was added. After stirring for 3 h at
40.degree. C., the reaction was quenched by addition of 50 mL of
water, and the crude material was precipitated by adding 75 mL of
cold ethanol saturated with anhydrous sodium acetate, and then
collected by filtration. The resulting crude partially O-sulfated
HA was dissolved in distilled water and dialyzed against 100 mM of
NaCl solution for two days, changing the solution four times a day,
and lyophilized to give the product (280 mg) in 68% yield and
characterized by 1H NMR, estimated sulfation SD=1.0-1.5.
11. Preparation of P-OSFHA-2 (DS-2) (GM-331201)
The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (6 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.4 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated FHA-2 was dissolved in water, dialyzed, and lyophilized
as in 1 to give the product (300 mg) in 69% yield and characterized
by 1H NMR, estimated sulfation SD=1.0-1.5.
12. Preparation of P-OSMeHA (DS-2) (GM-131201)
The TBA salt of MeHA (DS-I) (from MW 950 kDa HA) was prepared from
0.5 mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1.
The resulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to
which the required excess (6 mol/equiv of available hydroxy groups
in MeHA) of pyridine-sulfur trioxide complex (0.8 g) was added.
After stirring for 3 h at 40.degree. C., the reaction was quenched
by adding 100 mL of water, and crude material was precipitated by
adding 150 mL of cold ethanol saturated with anhydrous sodium
acetate, and then collected by filtration. The resulting crude
partially O-sulfated MeHA was dissolved in water, dialyzed, and
lyophilized as in 1 to give the product (560 mg) in 64% yield,
which was shown by 1H NMR to have estimated sulfation
SD=1.0-1.5.
13. Preparation of LMW-F-OSFHA-I (DS-I) (GM-212101)
The TBA salt of LMW FHA-I (FHA-I from MW 53 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-I (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-I-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated HA was dissolved in distilled water and dialyzed against
100 mM of NaCl solution for two days, changing the solution four
times a day, and lyophilized to give the product (300 mg) in 71%
yield and characterized by 1H NMR, estimated sulfation
SD=1.5-2.0.
14. Preparation of LMW-F-OSFHA-2 (DS-I) (GM-312101)
The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(LMW FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated LMW FHA-2 was dissolved in water, dialyzed, and
lyophilized as in 1. to give the product (260 mg) in 65% yield and
characterized by 1H NMR, estimated sulfation SD=1.5-2.0.
15. Preparation of LMW-F-OSMeHA (DS-I) (GM-112101)
The TBA salt of LMW MeHA (DS-I) (from MW 53 kDa HA) was prepared
from 0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water
as in 1. The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL
of DMF to which the required excess (12 mol/equiv of available
hydroxy groups in MeHA) of pyridine-sulfur trioxide complex (1.8 g)
was added. After stirring for 3 h at 40.degree. C., the reaction
was quenched by adding 100 mL of water, and crude material was
precipitated by adding 150 mL of cold ethanol saturated with
anhydrous sodium acetate, and then collected by filtration. The
resulting crude partially O-sulfated MeHA was dissolved in water,
dialyzed, and lyophilized as in 1 to give the product (480 mg) in
60% yield, which was shown by 1H NMR to have a estimated sulfation
SD=1.5-2.0.
16. Preparation of LMW-F-OSFHA-I (DS-2) (GM-212201)
The TBA salt of LMW FHA-I (FHA-I from MW 53 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-I (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-I-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated HA was dissolved in distilled water and dialyzed against
100 mM of NaCl solution for two days, changing the solution four
times a day, and lyophilized to give the product (300 mg) in 70%
yield and characterized by 1H NMR, estimated sulfation
SD=1.5-2.0.
17. Preparation of LMW-F-OSFHA-2 (DS-2) (GM-312201)
The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(LMW FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated LMW FHA-2 was dissolved in water, dialyzed, and
lyophilized as in 1 to give the product (300 mg) in 68% yield and
characterized by 1H NMR, estimated sulfation SD=1.5-2.0.
18. Preparation of LMW-F-OSMeHA (DS-2) (GM-112201)
The TBA salt of LMW MeHA (DS-I) (from MW 53 kDa HA) was prepared
from 0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water
as in 1. The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL
of DMF to which the required excess (12 mol/equiv of available
hydroxy groups in MeHA) of pyridine-sulfur trioxide complex (1.6 g)
was added. After stirring for 3 h at 40.degree. C., the reaction
was quenched by adding 100 mL of water, and crude material was
precipitated by adding 150 mL of cold ethanol saturated with
anhydrous sodium acetate, and then collected by filtration. The
resulting crude partially O-sulfated MeHA was dissolved in water,
dialyzed, and lyophilized as in 1 to give the product (550 mg) in
63% yield, which was shown by 1H NMR to have an estimated sulfation
SD=1.5.
19. Preparation of F-OSFHA-I (DS I) (GM-232101)
The TBA salt of FHA-I (FHA-I from MW 950 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-I (0.25 g) in 50 mL of
distilled water and processing as above in I. The resulting salt
(FHA-I-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 400 C, the reaction was quenched by addition of 50 mL of
water, and the crude material was precipitated by adding 75 mL of
cold ethanol saturated with anhydrous sodium acetate, and then
collected by filtration. The resulting crude partially O-sulfated
HA was dissolved in distilled water and dialyzed against 100 mM of
NaCl solution for two days, changing the solution four times a day,
and lyophilized to give the product (300 mg) in 68% yield and
characterized by 1H NMR, estimated sulfation SD=1.5.
20. Preparation of F-OSFHA-2 (DS-I) (GM-332101)
The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated FHA-2 was dissolved in water, dialyzed, and lyophilized
as in 1. to give the product (310 mg) in 70% yield and
characterized by 1H NMR, estimated sulfation SD=1.5-2.0.
21. Preparation of F-OSMeHA (DS-I) (GM-132101)
The TBA salt of MeHA (DS-I) (from MW 950 kDa HA) was prepared from
0.5 mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1.
The resulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to
which the required excess (12 mol/equiv of available hydroxy groups
in MeHA) of pyridine-sulfur trioxide complex (1.6 g) was added.
After stirring for 3 h at 40.degree. C., the reaction was quenched
by adding 100 mL of water, and crude material was precipitated by
adding 150 mL of cold ethanol saturated with anhydrous sodium
acetate, and then collected by filtration. The resulting crude
partially O-sulfated MeHA was dissolved in water, dialyzed, and
lyophilized as in 1 to give the product (500 mg) in 60% yield,
which was shown by 1H NMR to have an estimated sulfation
SD=1.5-2.0.
22. Preparation of F-OSFHA-I (DS-2) (GM-232201)
The TBA salt of FHA-I (FHA-I from MW 950 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-I (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-I-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 40.degree. C., the reaction was quenched by addition of
50 mL of water, and the crude material was precipitated by adding
75 mL of cold ethanol saturated with anhydrous sodium acetate, and
then collected by filtration. The resulting crude partially
O-sulfated HA was dissolved in distilled water and dialyzed against
100 mM of NaCl solution for two days, changing the solution four
times a day, and lyophilized to give the product (300 mg) in 69%
yield and characterized by 1H NMR, estimated sulfation
SD=1.5-2.0.
23. Preparation of F-OSFHA-2 (DS-2) (GM-332201)
The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by
adding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of
distilled water and processing as above in 1. The resulting salt
(FHA-2-TBA) was dissolved in 25 mL of DMF to which the required
excess (12 mol/equiv of available hydroxy group in HA) of
pyridine-sulfur trioxide complex (0.8 g) was added. After stirring
for 3 h at 400 C, the reaction was quenched by addition of 50 mL of
water, and the crude material was precipitated by adding 75 mL of
cold ethanol saturated with anhydrous sodium acetate, and then
collected by filtration. The resulting crude partially O-sulfated
FHA-2 was dissolved in water, dialyzed, and lyophilized as in 1. to
give the product (300 mg) in 69% yield and characterized by 1H NMR,
sulfation SD=1.5-2.0.
24. Preparation of F-OSMeHA (DS-2) (GM-132201)
The TBA salt of MeHA (DS-I) (from MW 950 kDa HA) was prepared from
0.5 mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1.
The resulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to
which the required excess (12 mol/equiv of available hydroxy groups
in MeHA) of pyridine-sulfur trioxide complex (1.6 g) was added.
After stirring for 3 h at 40.degree. C., the reaction was quenched
by adding 100 mL of water, and crude material was precipitated by
adding 150 mL of cold ethanol saturated with anhydrous sodium
acetate, and then collected by filtration. The resulting crude
partially O-sulfated MeHA was dissolved in water, dialyzed, and
lyophilized as in 1 to give the product (550 mg) in 63% yield,
which was shown by 1H NMR to have estimated sulfation
SD=1.5-2.0.
Example III
Preparation of Fluorescent SAGEs and Palmitoylated SAGE
a. Preparation of LMW-F-OSFHA-I(DS-2) Fluorescent Conjugate
LMW-F-OSFHA-1 (50 mg) and NHS (40 mg) were dissolved in 10 mL
water, and then Alexa Fluor488 (1 mg) in 4 mL DMF was added. And
then pH was adjusted to 4.75. After that, 100 mg EDCl was added in
solid form. The pH was kept at 4.75 by adding NaOH solution. The
solution was stirred for overnight at room temperature with
aluminum foil coverage. Then the product was purified by dialyzing
against distilled water (cut-off MW 3500) and following
methanol/water solution (50/50, v/v), further purified by gel
filtration column (Sephadex G-25). Further purified by PD-10 column
and lyophilized to dry to obtain 40 mg.
b. Preparation of LMW-P-OSMeHA(DS-I) Fluorescent Conjugate
LMW-P-OSMeHA (50 mg) and NHS (40 mg) were dissolved in 10 mL water,
and then Alexa Fluor488 (1 mg) in 4 mL DMF was added. And then pH
was adjusted to 4.75. after that, 100 mg EDCl was added in solid
form. The pH was kept at 4.75 by adding NaOH solution. The solution
was stirred for overnight at room temperature with aluminum foil
coverage. Then the product was purified by dialyzing against
distilled water (cut-off MW 3500) and following methanol/water
solution (50/50, v/v), further purified by gel filtration column
(Sephadex G-25), and further purified by PD-10 column and
lyophilized to dry to obtain 43 mg.
c. Preparation of Palmitoylated SAGE
LMW-P-OSMeHA (50 mg) was dissolved in 50 mL of DMF, triethylamine
(0.3 mL) was added to the DMF solution while stirred. After 5 min,
the palmitoyl chloride (0.5 mL) was added. The resulting mixture
was kept stirring for overnight. The solution was evaporated, and
the residue was dissolved in distilled water, dialyzed for one day
(change water four times), and lyophilized to dry to obtained 35
mg.
Example IV
SAGEs are Potent Inhibitors of P-Selectin, Human Leukocyte Elastase
and the Interaction of RAGE with all of its Ligands
Materials. Polyclonal goat anti-human RAGE, recombinant human high
mobility box protein-1 (HMGB-I), recombinant human P-selectin/Fc
chimera, recombinant human RAGE/Fc chimera, human azurocidin and
polyclonal goat anti-human azurocidin were purchased from R&D
Systems (Minneapolis, Minn.). Human S1006 calgranulin was from
Calbiochem (San Diego, Calif.). The advanced glycation end-product
carboxymethyl lysine-bovine serum albumin (CML-BSA) was obtained
from MBL International (Woburn, Mass.). U937 human monocyte cells
were obtained from American Type Culture Collection (Manassas,
Va.). Protein A, horse radish peroxidase-conjugated rabbit
anti-goat IgG, carbonate-bicarbonate buffer and bovine serum
albumin blocker (10.times.) were obtained from Piercenet (Rockford,
Ill.). Calcein AM, Dulbecco's modified Eagle's medium (DMEM),
ethylenediamine tetraacetic acid (EDTA), fetal bovine serum (FBS),
HEPES, non-essential amino acids,
penicillin/streptomycin/L-glutamine solution, RPMI-1640 without
L-glutamine and sodium bicarbonate were obtained from Invitrogen
(Carlsbad, Calif.). High-bind 96-well microplates were obtained
from Corning Life Sciences (Corning, N.Y.). All other chemicals not
specified were purchased from Sigma-Aldrich (St. Louis, Mo.).
Cell culture. U937 monocytes were grown in suspension culture at
37.degree. C. in humidified 5% CO2-95% air in RPMI-1640
supplemented with 10% heat inactivated FBS, 2 mM L-glutamine, 1 mM
sodium pyruvate, 0.1 mM MEM non-essential amino acids, 100 units/ml
penicillin and 100 mg/ml streptomycin. Experiments were performed
on cells from passages 1-5.
Cell Binding Assays. The effect of SAGEs on binding of U937
monocytes to P-selectin or RAGE was studied in high-bind micro
plates coated with 8 .mu.g/ml protein A (50 .mu.g/well) in 0.2 M
carbonate-bicarbonate buffer (pH 9.4). Plates were washed with
phosphate buffered saline containing 1% BSA (PBS-BSA), and
P-selectin-Fc or RAGE-Fc chimera (50 .mu.L containing 1 .mu.g) was
added to each well and incubated for 2 h at room temperature or
overnight at 4.degree. C., respectively. Following incubation,
wells were washed twice with PBS-BSA. Fifty (50) .mu.L of SAGEs (0
to 1,000 .mu.g/ml) serially diluted in 20 mM HEPES buffer
(containing 125 mM NaCl, 2 mM calcium and 2 mM magnesium) were
added to each well and incubated at room temperature for 15 min. As
a negative control, 50 .mu.L of 10 mM EDTA was added to select
wells to prevent cell binding through sequestration of calcium. At
the end of the incubation period, 50 .mu.L of U937 cells (105
cells/well, calcein-labeled, according to the manufacturers
instructions) were added to each well and plates were incubated an
additional 30 min at room temperature. The wells were then washed
thrice with PBS, and bound cells were lysed by addition of 100
.mu.L of Tris-TritonX-100 containing buffer. Fluorescence was
measured on a microplate reader using excitation of 494 nm and
emission of 517 nm.
Solid phase binding assays. Solid phase binding assays were used to
study the ability of SAGEs to inhibit RAGE binding to its ligands.
For studies of the effect of SAGEs on RAGE binding to its ligands,
polyvinyl 96-well plates were coated with 5 .mu.g/well of specific
ligand (CML-BSA, HMGB-I or S100b calgranulin). Plates were
incubated overnight at 4.degree. C. and washed thrice with
PBS-0.05% Tween-20 (PBST). Separately, RAGE-Fc chimera (100 .mu.L
containing 0.5 .mu.g/ml in PBST-0.1% BSA) was incubated with an
equal volume of serially diluted SAGEs (0.001 to 1,000 .mu.g/ml in
PBST-BSA) overnight at 4.degree. C. The following day, 50 .mu.L of
RAGE-SAGE mix was transferred to each respective ligand-coated well
and incubated at 37.degree. C. for 2 h. Wells were then washed four
times with PBST. To detect bound RAGE, 50 .mu.L of anti-RAGE
antibody (0.5 .mu.g/ml) was added to each well, the mixture was
incubated for 1 h at room temperature, and wells were washed again
four times with PBST. Horse-radish peroxidase conjugated secondary
antibody (1:10,000 antibody dilution in PBST; 50 .mu.L per well)
was added, wells were incubated for 1 h at room temperature, and
then washed once with PBST. A colorimetric reaction was initiated
by addition of 50 .mu.L of tetramethyl benzidine chromogen (TMB
single solution chromogen) and terminated after 15 min by addition
of 50 .mu.L of 1 N HCl. Absorbance at 450 nm was read using an
automated microplate reader.
Enzymatic assays. To characterize SAGE inhibition of the cationic
PMN protease HLE an activity assay (Fryer A, Huang Y-C, Rao G,
Jacoby D, Mancilla E, Whorton R, Piantadosi C A, Kennedy T, Hoidal
J. Selective O-desulfation produces nonanticoagulant heparin that
retains pharmacologic activity in the lung. J Pharmacol Exp Ther
282:208-219, 1997) was employed, which measured the ability of
purified HLE to cleave a chromogenic substrate. HLE (100 nM) was
incubated with SAGE (1-100 nM) in 0.5 M HEPES buffer for 15 min.
Following incubation, the elastase substrate
Suc-Ala-Ala-Val-p-nitroanaline (p-NA) was added to the reaction
mixture to a final concentration of 0.3 mM. The hydrolysis of p-NA
released was followed for 15 min by measuring the absorbance at 405
nm. In order to characterize the ability of SAGEs to activate
Factor XII or complement, activity assays were employed that are
similar to those recently used to screen for toxicity of
adulterated commercial heparin (Kishimoto T K, Viswanathan K,
Ganguly T, Elankumaran S, Smith S, Pelzer K, Lansing J C,
Sriranganathan N, Zhao G, Galcheva-Gargova Z, Al-Hakim A, Bailey G
S, Fraser B, Roy S, Rogers-Cotrone T, Buhse L, Whary M, Fox J, Nasr
M, Dal Pan G J, Shriver Z, Langer R S, Venkataranam G, Austen K F,
Woodcock J, Sasisekharan R. Contaminated heparin associated with
adverse clinical events and activation of the contact system. N
Engl J Med 358:2457-2467, 2008; Guerrini M, Beccati D, Shriver Z,
Naggi A, Viswanathan K, Bisio A, Capila I, Lansing J C, Guglieri S,
Fraser B, Al-Hakim A, Gunay N S, Zhang Z, Robinson L, Buhse L, Nasr
M, Woodcock J, Langer R, Venkataraman G, Linhardt R J, Casu B, Torn
G, Sasisekharan R. Oversulfated chondroitin sulfate is a
contaminant in heparin associated with adverse clinical events. Nat
Biotech 26:669-675, 2008). Pooled human plasma (5 .mu.l) was
incubated with 100 .mu.l of SAHA (0.1 to 1000 .mu.g/ml) in 0.05 M
HEPES containing Triton X-100 for 5 min at 5.degree. C. Amidolytic
activity specific for Hageman factor was determined by adding 0.5
mM D-cyclohydrotyrosyl-Glyc-L-Arg-p-NA and following the change in
absorbance at 405 nm (Silverberg M, Dunn J T, Garen L, Kaplan A P.
Autoactivation of human Hageman factor. Demonstration using a
synthetic substrate. J Biol Chem 255:7281-7286, 1980).
Amidolytic activity specific for active kallikrein was determined
by adding D-Pro-Phe-Arg-/7-NA and following change in absorbance at
450 nm.
Results: The results of the assays are shown in Table 2. SAGEs are
potent inhibitors of P-selectin. The competitor-mediated
displacement of U937 human monocytes, which firmly adhere to
P-selectin through P-selectin glycoprotein ligand-1 (PSGL-I), was
studied using fluorescently-labeled cells. FIG. 3 shows that a SAGE
inhibits U937 binding to P-selectin with a 50% inhibitory
concentration (IC50) of 0.5 .mu.g/ml.
TABLE-US-00002 TABLE 2 SAGE In Vitro Data P-Selectin/ RAGE/
Leukocyte Hageman SAGE PSGL RAGE/Mac-1 CML-BSA RAGE/S100B
RAGE/HMGB1 Elastase Factor GM-111101 0.14 0.042 2.27 1.56 0.455
0.22 4 GM-111201 0.017 0.033 0.082 0.077 1.033 0.54 0.4 GM-112101
2.164 0.113 0.002 0.042 0.537 0.187 0.4 GM-112201 0.496 0.152 0.005
0.017 0.634 0.117 0.4 GM-131101 5.61 0.51 6.09 31.069 2.68 0.285 NR
GM-131201 0.5 0.3 0.044 0.06 1.66 0.42 0.4 GM-132101 0.0794 0.0297
0.015 0.004 0.501 0.127 0.4 GM-132201 0.22 0.004 0.1 0.04 0.228
0.24 0.4 GM-211101 6.53 2.11 NR NR NR 0.133 NR GM-211201 NR NR NR
NR 0.54 400 GM-212101 5.13 6.51 10.5 15.671 NR 0.357 NR GM-212201
0.019 0.015 0.217 0.4 GM-231101 12.048 NR NR 14.6 NR 0.185 400
GM-231201 0.126 28.4 2.579 8.82 NR 0.487 400 GM-232101 0.883 4.78
0.041 0.02 0.408 0.19 4 GM-232201 0.579 0.142 0.019 0.021 0.51
0.202 0.4 GM-311101 NR 4.7 2.452 7.358 NR 0.439 NR GM-311201 NR NR
NR NR 0.52 400 GM-312101 0.897 0.34 0.299 0.56 0.454 0.261 NR
GM-312201 0.036 0.009 0.059 0.075 0.47 0.4 GM-331101 8.958 NR NR
4.557 NR 0.375 400 GM-331201 1.682 2.28 NR 0.353 40 GM-332101 0.108
0.015 0.021 0.371 0.184 40 GM-332201 38.3 0.0879 0.096 0.22 1.074
0.392 4 IC50 values (ug/ml) Heparin 0.3 0.11 0.39 1.29 0.04 0.21
0.4 ODSH 1.1 0.09 8.6 4.2 0.23 0.22 NR
TABLE-US-00003 TABLE 3 IC.sub.50 values (.mu.g/mL) for GM-1111
batches % Sulfur Leukocyte Hagemann (elemental SAGE RAGE/CML-BSA
RAGE/S100B RAGE/HMGB1 Elastase Factor analysis GM-111101 2.27 1.51
0.438 0.22 4 12% GM-111102 0.0815 0.0293 0.734 0.201 NA 14%
GM-111103 0.0108 tbd 0.409 0.211 0.4 12% GM-111104 6.2 4.33 62.8
0.321 NA 4% GM-111105 20.7 2.45 89.4 0.447 400 3% GM-111106 0.00499
0.0343 0.299 0.337 4-400 7%
Second, as highly sulfated polyanions, alkylated and sulfated SAGEs
are potent inhibitors of polymorphonuclear leukocyte proteases.
FIG. 4 shows that alkylated and/or fluoroalkyated and sulfated
SAGEs inhibit human leukocyte elastase (HLE) with impressively
potent IC50 values. Specifically, the non-alkylated, fully
O-sulfated HA (F-OSHA) shows an 0.66 nM IC50 for HLE. For the
modified and sulfated HA derivatives, IC50 values were 1.89 nM for
partially O-sulfated .box-solid. carboxymethylated HA (P-OSCHMHA);
1.97 nM for partially O-sulfated HA (P-OSHA); and 3.46 nM for
partially O-sulfated methylated HA (P-OSMEHA; GM-131101).
Third, SAGEs are extremely potent inhibitors of RAGE. SAGEs
inhibited the interaction of RAGE and amphoterin (HMGB-I) with an
IC50 of 1.1 .mu.g/ml (FIG. 5), the interaction of RAGE and S100
calgranulins with an ICs0 of 60 ng/ml (FIG. 6), and the binding of
RAGE to the AGE product carboxymethyl-lysine BSA with an IC50 of 44
ng/ml (FIG. 7). These values are 5 to 10-fold more potent than
corresponding levels of RAGE-ligand inhibition we have measured
with heparin and 2-0,3-0 desulfated heparin.
Fourth, SAGEs are potent inhibitors of the proliferation of human
keratinocytes. In these assays human neonatal epidermal
keratinocytes were cultured in the presence and absence of SAGEs or
other inhibitors, and proliferation was measured by adding a dye
that is reduced in direct proportion to the number of viable cells
present. In the figures displayed, absorbance values were
normalized to controls, which were assigned a relative absorbance
of 1.0. FIG. 8 shows that the high molecular weight partially
O-sulfated HA (P-OSHA) and fully O-sulfated HA (F-OSHA) were more
effective than the heparin derivative 2-0, 3-0 desulfated heparin
(ODSH) at preventing keratinocyte proliferation when added in
concentrations of 100 .mu.g/ml. Higher sulfation appears
advantageous, since partially sulfated carboxymethylated HA
(P-OSCMHA) and methylated HA (P-OSMEHA; GM-131101) were less active
at inhibiting keratinocyte proliferation. FIG. 9 shows that overall
lower molecular weight (LMW) derivatives were more effective than
higher molecular weight derivatives at reducing keratinocyte
proliferation in this assay. In particular, the LMW partially
O-sulfated MeHA (LMW-P-OMEHA; GM-111201), partially O-sulfated CMHA
(LMW-P-OSCMHA) and partially O-sulfated 53 kDa HA, itself
(LMW-P-OSHA), all reduced proliferation relative to controls at
concentrations as low as 1 .mu.g/ml.
GM-1111 can be prepared from different sources of hyaluronic acid
with different polydispersities and initial average molecular
weights. The in vitro biochemical results, in vivo biological
activities, and alkylation/sulfation levels vary based on the size
and solubility of the starting HA. To our surprise, starting with
poorly soluble HA of size 60-70 kDa resulted in low levels of
methylation, sulfation, and dramatically reduced biological
activities. In contrast, starting with readily soluble HA of sizes
40-60 kDa (whether obtained commercially at this size or prepared
by partial depolymerization of a higher molecular weight HA
starting material) resulted in reproducible levels of methylation,
sulfation, and high biological activity.
Example V
In Vivo Studies for the Treatment of Rosacea and Inflammation
Materials and Methods
Chemicals. GM-111101, GM-131101, GM-312101 and GM-212101 were
evaluated. Medium was bought from American Type Culture Collection
(ATCC). EpiLife medium was purchased from Invitrogen (Madison,
Wis.).
Cells. The Human Dermal Fibroblasts (nHDF) were purchased from
ATCC. Human neonatal epidermal keratinocytes (HEKn) were obtained
from Invitrogen (Madison, Wis.).
Cytotoxicity. 4,000 nHDF cells were seeded 100 .mu.l medium in each
well of 96-well flat-bottomed microplates, and incubated at
37.degree. C. in 5% CO2 for 12 hours. All the medium was changed
with complete medium containing a variety of sulfated hyaluronan
(HA) derivatives at final concentrations of 10, 100, 1000, 10000,
100000, 1000000 ng/ml to each column. At 48 hours, 20 .mu.l MTS
(Promega, Madison, Wis.) was pipetted into each well, and cells
were further incubated for 2 hours. The absorbance of the samples
was measured at 490 nm using a 96-well plate reader.
Skin irritation test in mice. GM-111101 and GM-212101 were tested
in vivo to determine the dermal irritation potential to the skin of
the mice. The two test agents were prepared with two different
concentrations individually, 0.1 mg/ml and 1 mg/ml. 10% formic acid
and PBS were used as positive and negative control respectively
(n=6). Balb/c mice, which had not been used in previous experiments
and were observed to be free from any skin irritation, trauma, or
adverse clinical signs prior to initiation of the studies, were
randomized and grouped for designed test conditions. The back of
the animals was clipped free of fur with an electric clipper at
least 4 hours before application of the sample. Just prior to test
substance application, each mouse received four parallel epidermal
abrasions with a sterile needle at the bottom area of the test site
while the upper area of the test site remained intact. Under
anesthesia, two 0.5-ml samples of the test solution were applied to
the entire test site by introduction under a double gauze layer to
an area of skin approximately 2.5 cm square. The patches were
backed with plastic, covered with a non-reactive tape and the
entire test site wrapped with a bandage. Animals were returned to
their cages. After a 24 hr exposure to the agent, the bandage and
soaked test gauze were removed. The test sites were wiped with tap
water to remove any remaining test compound. At 24 and 72 hours
after compound application, the test sites were examined for dermal
reactions in accordance with the FHSA-recommended Draize scoring
criteria. The Primary Irritation Index (P.I.I.) of the test article
will be calculated following test completion. A material producing
a P.I.I, score of greater than or equal to 5.00 would be considered
positive; the material would be considered a primary irritant to
the skin. Animals. Balb/c mice were purchased commercially from a
vendor approved by the Univ. of Utah veterinary medicine department
and vivarium. After they are quarantined for the prescribed period
following receipt, they are ready for use.
LL37 peptide and SAGE injection rosacea models. Chronic disease of
the skin leaves an indelible mark on the patient's life,
especially, as in the case of rosacea, when it presents on the
face. As a species, we react positively or adversely to one
another's appearance, instinctively recoiling from those who appear
abnormal and unlike ourselves. While skin diseases are not often
life-threatening, they are life-altering in ways that normal
individuals do not fathom. Rosacea is one of those life-altering
illnesses. Rosacea is a common disfiguring facial skin disease
affecting 3% of the U.S. population, or about 14 million Americans,
with onset usually between the ages of 30 and 50 years. It strikes
primarily in Caucasians of Celtic descent, and appears to be more
problematic and common in women than men. Over a third of patients
have a family history, strongly suggesting an inherited illness.
The condition is particularly stigmatizing because of the common
misconception that the facial redness and the knobby nose of
rosacea are the consequence of excessive alcohol consumption.
Rosacea presents as several clinical phenotypes. The most common
presentation is characterized by transient or persistent central
flushing and erythema of the face, with dilated capillaries on the
cheeks and nose. This phenotype ranges from a "ruddy complexion" to
persistent, readily visible dilated vessels. This subtype is
without an effective topical treatment. In a second common
presentation, papules and pustules occur on the central convexities
of the face, frequently superimposed upon a background of erythema.
On biopsy, the skin is richly infiltrated with PMNs. Papulopustular
rosacea is treated empirically with topical or systemic
antibiotics, but is not clearly related to documented cutaneous
infection. Macrolides, either topically or systemically, been most
often used as therapy, and may produce improvement not because of
their anti-bacterial activity so much as their ability to retard
PMN chemotaxis into areas of inflammation. In a third and
fortunately, more rare phenotype, rosacea produces rhinophyma from
hyperplasia of both the sebaceous glands and connective tissue of
the nose, creating the classical "W. C. Fields" nose, or "whiskey
nose".
Treatment of this condition requires surgical excision of the
tissue, or laser therapy, to remove hypertrophied tissue. In a
fourth phenotype, rosacea can produce dry, itchy eyes with
irritation of the lids (blepharitis), photosensitivity, blurred
vision and conjunctivitis. Prolonged disease can result in
keratitis and even corneal scarring. This phenotype, which arises
from inflammation of the lids and the meibomian glands on the lower
lid surface, is common and overlaps clinically with the "dry-eye"
syndrome seen frequently by ophthalmologists. The occurrence of
rosacea exclusively on sun-exposed facial areas in those with fair
skin and light eyes points to a pathogenic role for solar radiation
in causing the condition.
The pathogenesis of rosacea has recently been elucidated (Yamasaki
K, Di Nardo A, Bardan A, Murakami M, Ohtake T, Coda A, Dorschner R
A, Bonnart C, Descargues P, Hovnanian A, Morhenn V B, Gallop R L.
Increased serine protease activity and cathelicidin promotes skin
inflammation in rosacea. Nat Med 13:975-980, 2007; Bevins C L, Liu
F-T. Rosacea: skin innate immunity gone awry? Nat Med 13:904-906,
2007). In elegant work Yamasaki et al. demonstrated that the skin
involved with rosacea contains high levels of cathelicidins and
their processing protease SCTE. Cathelicidins, a major family of
antimicrobial peptides in mammals, are expressed in leukocytes and
epithelial cells of many organs, where they mediate innate immune
responses to bacteria (Nizet V, Ohtake T, Lauth X, Trowbridge J k,
Rudisill J, Dorschner R A, Prestonjamasp V, Piraino J, Huttner K,
Gallop R L. Innate antimicrobial peptide protects the skin from
invasive bacterial infection. Nature 414:454-457, 2001).
Cathelicidins also signal vascular growth, PMN migration and wound
healing (Bevins, ibid). Human cathelicidin is secreted by
keritinocytes as an 18-kDa hCAP18 pro-peptide and cleaved by SCTE
to the C-terminal active anti-microbial peptide LL-37, designated
after its 37 amino acid length and two N-terminal leucines.
Skin involved with rosacea demonstrates inappropriately high levels
of both cathelicidins and SCTE without clearly inciting microbial
invasion as a stimulus. When placed in the culture media covering
primary human keritinocytes LL-37 greatly augments production of
chemotactic cytokines such as IL-8. When injected intradermally
into mice in levels similar to those found in rosacea-involved
skin, LL-37 produces erythema and stimulates prominent dermal
infiltration by PMNs. Intradermal injection of SCTE also produces
skin erythema and PMN infiltration of the dermis in wild type, but
not in cathelicidin`7" mice. Thus, there is a growing body of
evidence which supports the concept that rosacea is mediated by V
local over-expression of pro-inflammatory, cationic skin peptides
which produce the inflammation, excessive angiogenesis and
sebaceous hyperplasia characteristic of the disease.
Because nearly every patient with rosacea can tell which of his
parents has a pattern of reactive facial flushing and blushing, it
is apparent to clinicians that rosacea is genetically driven. To
produce a model of rosacea, LL-37 was injected intradermally every
12 h for 48 h. This model produced erythema of the skin and
prominent intradermal infiltration of polymorphonuclear leukocytes
(PMNs), as reported by Yamasaki et al. Balb/c mice were shaved
prior to study to expose an area of skin on the back. Twenty-four
hours later, we injected 40 .mu.L of vehicle (phosphate buffered
saline, PBS), cationic peptide (at 320 .mu.M concentration in PBS),
SAGE (320 to 1,280 .mu.M in PBS), or cationic peptide+SAGE mix
(peptide+1.times. to 4.times. molar concentrations of SAGE)
intradermally into the shaved skin using a 31 gauge needle in a
manner designed to raise an intact epidermal bleb, thereby
identifying that administration was at the level of the lower
epidermis or dermis. SAGE selected for injection were chosen from
over twelve newly synthesized SAGEs, which were tested extremely
active in biochemical assays as inhibitors of human leukocyte
elastase (as another cationic protein) and as antagonists for
activation of the receptor for advanced glycation end-products
(RAGE) by four common ligands.
SAGEs and peptides were mixed together in PBS prior to injection
and allowed to incubate 15 min at room temperature before being
injected. Injections were repeated every 12 h thereafter.
Forty-eight hours after the initial injection (four injections in
total), animals were lightly anesthetized with 25 mg/kg
pentobarbital When the mouse was asleep, the area of injected skin
was photographed to visually record the severity of erythema and
edema. The intensity of erythema was assessed as a redness score
(from 1 to 5), and the area of erythema was measured with calipers.
The area of injected skin was then excisionally biopsied using a 6
mm hole punch for hematoxylin-eosin staining to examine the
histopathologic changes and to assess PMN infiltration through
measurement of myeloperoxidase (MPO) activity. One representative
image of skin surface and histology from each skin was viewed under
high power viewing under a microscope.
SAGE topical treatment rosacea model. Balb/c mice were shaved in
time from LL37 exposure on area of skin on the back. We then began
topical application of a hyaluronan-cased emollient containing 5%
SAGEs (active emollient) or hyaluronic acid based emollient alone
to this area of skin every 12 hours. Twenty-four hours later, we
injected 40 .mu.L of vehicle (PBS) or cationic peptide (at 320
.mu.M concentration) subcutaneously into the shaved skin in the
manner described previously. Injections and topical emollient
applications were repeated every 12 h thereafter. Forty-eight hours
after the initial injection (four injections in total), animals
were lightly anesthetized as described previously. The area of
injected skin was photographed to visually record the severity of
erythema and edema. The intensity of erythema was assessed as a
redness score (from 1 to 5), and the area of erythema was measured
with calipers. The area of injected skin was then excisionally
biopsied using a 6 mm hole punch for H&E staining to examine
the histopathologic changes and to assess PMN infiltration through
measurement of myeloperoxidase (MPO) activity.
SAGE dermis penetration. Balb/c mice were shaved prior to study to
expose an area of skin on the back. Topical application of SAGE was
carried out to the skin every 12 h. Forty-eight hours later animals
were euthanized and the skin was biopsied. Sections of skin were
then studied by fluorescence microscopy to determine the depth to
which SAGEs penetrate into the skin.
Croton oil inflammation model. As another model of PMN-mediated
skin inflammation, croton oil was employed. Croton oil contains
phorbol esters, which activate protein kinase C in skin cells. As a
result, skin cell produce abundant chemokines and chemotaxins which
signal the influx of PMNs from the circulation. Activated PMNs
produce erythema and edema of skin tissues. Croton oil induced
inflammation is a commonly employed model of PMN-mediated skin
inflammation in the screening of anti-inflammatory compounds for
dermatologic use.
To produce this model, croton oil (Sigma-Aldrich, St. Louis, Mo.)
was mixed as a 0.8% solution in acetone. Using a pipette, 10 .mu.l
were painted onto each side of one ear of the mouse, with the other
ear remaining as a control. At 4, 8 and 24 hours later, ear
thickness was measured near the top of the ear distal to the
cartilaginous ridges. Change in ear thickness from control was then
taken as an index of edema. The intensity of erythema was assessed
as a redness score (from 1 to 5), and the area of erythema was
measured with calipers. Following the 24 hour measurements, mice
were euthanized and ear punch biopsies (6 mm hole punch) were taken
immediately, weighted, frozen and stored at -800 C for H&E
staining to examine the histopathologic changes and to assess PMN
infiltration through measurement of myeloperoxidase (MPO) activity.
A single investigator performed all ear measurements and biopsies
in order to standardize the procedure and reduce error. The
remainder of ears were removed, embedded and frozen for
immunohistochemistry.
Myeloperoxidase (MPO) assay. For each mouse, tissue biopsies (6 mm
diameter hole punch) were taken immediately, weighted, frozen and
stored at -800 C. Tissue MPO activity was measured using a method
by Suzuki et. al. (Suzuki K, Ota H, Sasagawa S, Sakatani T,
Fujikura T. Assay method for myeloperoxidase in human
polymorphonuclear leukocytes. Anal Biochem 132:345-352, 1983) as
modified by Young et. al. (Young J M, Spires D A, Bedord C J,
Wagner B, Ballaron S J, Deoung L M. The mouse ear inflammatory
response to topical arachidonic acid. J Invest Dermatol 82:367-371,
1984). Each mouse tissue biopsy was placed in 0.75 mL of 80 mM
phosphate-buffered saline (PBS) pH 5.4 containing 0.5%
hexadecyltrimethyl-ammonium bromide (HTAB). Each sample was
homogenized for 45 s at 4.degree. C. with a small laboratory Tissue
Tearor Homogenizer Model 985-370 (Biospec Products, Bartlesville,
Okla.). The homogenate was transferred quantitatively to a
microcentrifuge tube with an additional 0.75 mL HTAB in PBS. The
1.5 mL sample was centrifuged at 12,000.times.g for 15 min,
maintained at 4.degree. C. Triplicate 30 uL samples of the
resulting supernatant were added to 96-well microtier plate wells.
For the MPO assay, 200 uL of a mixture containing 100 uL of 80 mM
PBS (pH 5.4), 85 uL of 0.22 M PBS (pH 5.4), and 15 uL of 0.017%
hydrogen peroxide were added to each well. 20 uL of 18.4 mM
tetramethylbenzidine HCl in 8% aqueous dimethylformamide was added
to start the reaction. Microtiter plates were incubated at
37.degree. C. for 3 min, and then placed on ice. The reaction was
stopped with the addition of 30 uL of 1.46 M sodium acetate. MPO
enzyme activity was assessed at an absorbance wavelength of 630 nm.
MPO activity was expressed as optical density (OD)/biopsy.
Statistical Analyses. All experiments were performed in triplicate
for in vitro tests. Significance differences between samples were
calculated by comparison of means using the Aspin-Welch test.
Significance was declared at <0.05.
Results
LL37 peptide and SAGE injection rosacea models. To determine if
direct neutralization of cationic cathelicidins prevented their
inflammatory activity in the skin, LL37 only, SAGE (GM-111101)
only, vehicle (PBS) only, or mixture of LL37 and SAGE were
subcutaneously injected into the shaved back area of mice every 12
h thereafter. After 48 hours, mice were sacrificed and gross
pictures in different treatment groups were taken (FIGS. 10a and
10b). Histological studies using hematoxylin and eosin staining
showed increased number of leukocytic infiltration and marked
dermal edema, whereas SAGE administration immediately after
challenge resulted in the inhibition of skin swelling response
(FIGS. 10c and 10d). Individual results of dermal scoring were
expressed by erythema area (FIG. 10f) and erythema redness score
(FIG. 10g). After 48 hours, the SAGE treated group demonstrated a
dramatically decreased area of erythema and a significant reduction
of redness score. Myeloperoxidase activity was measured in the
tissue punch biopsies taken 48 hr after injection as an index of
PMN infiltration. SAGE co-administration with LL-37 peptide
significantly reduced MPO activity by 50% (FIG. 10e). Therefore,
co-injection of SAGE with LL-37 peptide substantially induced the
inflammatory activity of the LL-37 cathelicidin peptide. This
indicates that SAGEs inhibit LL-37 mediated inflammation and would
be useful treatments for rosacea.
SAGE Topical Rosacea Treatment Model. Topical treatment of SAGE
(GM-111101) is used to test if treatment remote in time from LL-37
exposure can also prevent peptide-induced skin inflammation.
Therefore, after the LL-37 injection into the mouse back skin area,
SAGE was applied right after. The gross pictures showed strong
edema and erythema at 48 hours after the LL-37 application (FIG.
11), while topical treatment with SAGE significantly decreased the
redness and its affected area both for immediately treatment and 12
h delayed treatment (FIG. 11). The H&E staining indicated much
more leukocytic infiltration and dermal edema than the two SAGE
treatment groups, which was in agreement with the results of SAGE
as inhibitor of skin swelling response and MPO activities (FIG.
11). These results indicate that SAGEs can be applied topically in
a conventional and pharmaceutically acceptable emollient to treat
the cathelicidin-mediated inflammation of rosacea.
SAGE dermis penetration. To determine the level of which SAGE
penetrates into the dermis. SAGE compound fluorescent-GM-212101 and
fluorescent-GM-111101 were used as test article, and 0.1 mg/ml, 1
mg/ml and 10 mg/ml fluorescent compound were applied on the abraded
and untouched skin area of Balb/c mice. After 24 hours, mice were
sacrificed and the whole tested skin area was excised and
photographed under both natural light and long wavelength UV light
condition. (FIG. 12) Layers of fluorescence were observed under
both natural and UV light condition for both inner and outer
treatment area skin. Significant penetration of SAGEs was
distributed even on a micrometer-length scale.
Cytotoxicity and Skin Irritation in Vivo Tests. The cytotoxicity of
SAGE derivatives (SAGEs) GM-131101, GM-312101 and GM-212101 was
evaluated in nHDF cells and the results are demonstrated in FIG.
13a. All compounds were also found to be non-toxic to the nHEK
cells up to 10 mg/ml concentration (FIG. 13b). For the in vivo skin
irritation tests, gross pictures of mice in different treatment
groups were represented in FIG. 13c-13j. The Primary Irritation
Index of the test substances was calculated to be 0.00 for both
GM-111101 and GM-212101; No irritation were observed on the skin of
the mice (FIG. 14). Both the GM-111101 and GM-212101 have not been
found cytotoxic. The concentration threshold of all SAGEs could be
determined from this test. Under the conditions of this test, the
test agents would not be considered a primary skin irritant; as
defined in the guidelines of the FHSA Regulations, 16 CFR 1500, a
substance with an empirical score of less than 5.00 is not a
primary irritant to the skin. These results indicate that SAGEs are
non-irritating themselves for skin and can be employed as safe
treatments for inflammatory skin disorders.
Croton oil inflammation model. The application of croton oil to
mouse skin was used as a convenient and highly reproducible model
of PMN-mediated skin inflammation. This model was used to test the
anti-inflammatory activity of SAGEs. Gross pictures of mice in
different treatment groups were represented in FIGS. 15a and 15b,
as well as the ear thickness measured for both the treated and
untreated ears, and compared in all the five groups. Individual
results of dermal scoring were expressed by erythema for abraded
area and intact area. The results showed significant reduction of
redness and thickness in the SAGE (GM-111101) treatment groups
compared with non-treatment groups (FIGS. 15g and 15h).
Histopathology exams revealed that in croton oil painted ears,
there was an increased number of leukocytic infiltrations and
marked dermal edema, whereas SAGE administration immediately after
challenge resulted in the inhibition of ear swelling response,
which was comparable to that of vehicle-treated mice. These
histological findings further confirmed those of the measurement
data. MPO activity was also measured in the ear punch biopsies
taken after croton oil application. SAGE treatment every 4 hr
starting immediately after croton oil application significantly
reduced MPO activity (FIG. 15f). These results indicate that SAGEs
can be employed as topical treatments for inflammatory skin
disorders other than rosacea.
Hyaluronic Acid (HA) Topical Treatment in Rosacea Model. The
previous topical treatment of LL-37 rosacea model was used to
compare SAGE (GM-111101) vs. HA. The results clearly showed that HA
in the topical administration did not alleviate inflammation (FIG.
16b, 16e, and IT) and MPO activities (FIG. 16d). Conversely, SAGE
possessed highly enhanced anti-inflammatory properties (FIG. 16c-f)
and could be considered as an inhibitor of inflammatory and
rosacea. These data indicate that the pharmacologic activity of
SAGEs is not inherent in hyaluronic acid, but require the novel
chemical modifications of hyaluronan. In Vivo Acute Intravenous
Toxicity Study in Rats. The objective of the study was to evaluate
the acute intravenous toxicity of SAGEs GM-111101 and GM-212101
when administered as a single dose to rats and also to evaluate the
toxicity of GM-111101 when administered once daily for a period of
seven days at a single dose level.
One dose group consisting of three male and three female
Sprague-Dawley rats was exposed to a single dose of 3 mg/kg
GM-111101, then a single dose of 10 mg/kg GM-111101 one week
following and finally once daily doses of 10 mg/kg GM-111101 for a
total of seven days initiating one week following the last single
dose. Two groups of three male and three female Sprague-Dawley rats
were exposed to GM-111101 at doses of 30 or 100 mg/kg. In addition,
two groups of three male and three female Sprague-Dawley rats were
exposed to GM-212101 at doses of 30 or 100 mg/kg. Two groups of
three male and three female Sprague-Dawley rats were exposed to
0.9% Sodium Chloride for Injection and were used as negative
control groups. All doses were administered at a dose volume of 1
ml/kg by intravenous injection via the caudal tail vein. Dose
calculations were determined based upon the most recently
documented body weight.
All animals exposed acutely (single dose) were observed immediately
following injection, and again at 2 hours and 4 hours following
dose administration on Day 0 for apparent signs of clinical
toxicity. In addition, all surviving animals were observed once
daily on days 1-14 for apparent signs of clinical toxicity. All
animals exposed once daily for seven days were observed once daily
from days 1-14 for apparent signs of clinical toxicity. Body weight
was recorded on day 0 (Acute dose) or day 1 (Repeat dose) prior to
dose administration, on day 6 or 7, and on day 14, prior to
termination. Gross necropsy evaluations were performed on each of
the surviving animals on day 14 of the study. Animals that died on
study underwent a gross necropsy examination immediately following
observation of mortality.
Clinical signs of moderate abnormal gait, moderate ataxia, and
reddish-orange discolored urine were observed in one female animal
from the 30 mg/kg GM-212101 dose group within the first 2 hours
following dose administration. These observations, with the
exception of mild ataxia, were no longer present as of the 4 hour
observation period and remained that way throughout the remainder
of the study. The observation of ataxia was no longer present on
the day following dose administration. In addition, one male rat
from the 100 mg/kg GM-212101 dose group was found dead within the
first four minutes following dose administration. All animals
gained body weight throughout the study period. There were no
visible lesions observed at necropsy with the exception of dark red
foci measuring -1-2 mm in diameter throughout the thymus in the one
animal from the 100 mg/kg GM-212101 dose group that died on study.
Based upon the results of this study, GM-111101 did not produce
signs of toxicity at any of the dose levels evaluated, including
single acute doses of 3, 10, 30, and 100 mg/kg and a seven day
repeat dose of 10 mg/kg. Therefore, the no observable effect level
(NOEL) for intravenous exposure to GM-111101 in rats is considered
to be at least 100 mg/kg. GM-212101 produced signs of toxicity or
mortality at doses of 30 and 100 mg/kg. Therefore, the NOEL for
intravenous exposure to GM-212101 in rats is considered to be 10
mg/kg. Due to the absence of mortality observed at all doses of
GM-111101 and mortality observed in only 17% of the animals at a
dose of 100 mg/kg GM-212101, the intravenous LD50 in rats for
GM-111101 and GM-212101 is considered to be greater than 100 mg/kg.
These results indicate that SAGEs are safe to employ as systemic or
injected treatments for diseases.
Example VI
Investigation of SAGEs for Treating Age-Related Macular
Degeneration
Activated complement and RAGE induce angiogenic and
pro-inflammatory signaling in cultured RPE cells. Experiments
studying the biology of RAGE in cultured RPE cells using the
ARPE-19 human RPE cell line were constructed. As shown in
immunoblots (FIG. 17), ARPE-19 cells express at least 4 isoforms of
RAGE ranging from 45-50 kDa in cell lysates, and secrete these
isoforms into conditioned media. When cells were grown on plates
coated with the AGE product CML-BSA, expression of all four RAGE
isoforms was markedly upregulated (compare right immunoblot to that
on the left in FIG. 17). Because RAGE ligation activates the
transcription factor NF-KB, enhanced expression of RAGE greatly
promotes pro-inflammatory signaling. The addition of the
nonanticoagulant heparin 2-O, 3-0 desulfated heparin (ODSH) to this
system prevented up-regulation of RAGE expression by blocking
interaction of CML-BSA with RAGE on ARPE-19 cells. This would
prevent "feed-forward" pro-inflammatory increases in RAGE
expression itself by RAGE ligation.
The ability of AGE products to induce RPE cell apoptosis was
investigated. ARPE-19 cells were grown to confluence on round
coverslips, then transferred to new dishes for exposure to 25 .mu.M
AGE-BSA for 40 h. Apoptosis was then assayed with Molecular Probes
Fixable Live/Dead Cell Stain Kit (LI 1101, Eugene, Oreg.). Each
panel shows the entire coverslip and an enlarged view of the
live/dead interface. Nuclei are stained red with DAPI; dead cells
are stained green. A. BSA control. Some dead cells are noted at the
edges of the coverslip, but live and dead cells are interspersed at
the interface. B. AGE-BSA (25 .mu.M). Significant cell death is
seen around the edges, and the boundary between live and dead cells
is stark. C. AGE-BSA (25 .mu.M)+ODSH (200 .mu.M). ODSH appears to
provide some protection but significant apoptosis still occurs. D.
AGE-BSA (25 .mu.M)+P-OSMeHA (200 .mu.M) (GM-111101). As shown in
FIG. 18, AGE treatment of cultured ARPE-19 cells induced prominent
apoptosis (FIG. 18B), measured by green staining with the Live/Dead
Cell Stain kit (Molecular Probes). Apoptosis is reduced by
concomitant incubation of cells with ODSH (FIG. 18C) but is almost
completely prevented by an equivalent concentration of the SAGA
P-OSMeHA (GM-111101) (FIG. 18D). Apoptosis appeared to advance
inward from the edges of cells cultured on round cover slips. RAGE
is prominently expressed in RPE cells where it may be selectively
expressed on the basal membrane as in other human epithelial cells.
Thus, in culture, AGE might initially be able to access and ligate
basally located RAGE only at the edges of monolayers, producing a
wave of cell death that predictably advances inward. In contrast,
AGE in drusen, which accumulates between retinal pigment epithelium
and Bruch's membrane, would have ready access to basally located
RAGE, optimally positioning AGE/RAGE signaling to mediate the
localized RPE apoptosis that constitutes so-called "geographic
atrophy" in age-related macular degeneration. Thus, the SAGE
GM-111101 almost completely prevents AGE-induced RPE apoptosis.
These results indicate that SAGEs might be effective in treatment
of important eye diseases causing blindness, such as age-related
macular degeneration.
SAGEs are non-toxic and non-anticoagulant. When O-sulfated and
methylated HA (P-OSMeHA), fully O-sulfated and
pentafluoropropylated HA (F-OSFHA-I) and fully O-sulfated and
methylated HA (F-OSMeHA) were applied to cultured human skin
epithelial cells or fibroblasts studied with a cell toxicity assay
(CellTiter96.RTM. Aqueous One assay, Promega), the SAGEs do not
inhibit proliferation or produce cell toxicity, even at
concentrations of 1 mg/ml. The SAGEs also are non-anticoagulant.
Low molecular weight sulfated and fluoroalkylated HAs demonstrate
no anti-Xa and <0.2 U/mg anti-IIa anticoagulant activities,
compared to 150 U/mg each for unfractionated heparin.
Unlike heparin, highly-charged polyanionic polymers are potent
inducers of the intrinsic or contact coagulation cascade through
activation of Hageman factor (factor XIIa), secondarily activating
kinins. SAGEs were screened for their ability to stimulate
intrinsic coagulation (activation of Hageman factor). Pooled human
plasma was incubated with heparin or low molecular weight (LMW)
sulfated and fluoroalkylated HAs (LMW-OSFHA-1, LMW-OSFHA-2) and
amidolytic activity was determined using the substrate
D-cyclohydrotyrosyl-Gly-Arg-p-NA. As shown in FIG. 19, low
molecular weight (50 kDa) SAGEs appear even safer than commercial
medical heparin when tested for ability to activate Factor XII,
even at SAGE concentrations 10 to 100-fold higher than those
achieving pharmacologic inhibition of inflammation.
Example VII
In Vitro Anti-Inflammatory Activity of GM-1111
GM-1111 (generally for several lots of GM-1111 in addition to
GM-111101) has potent activity inhibiting P-selectin, in vitro,
blocking proteolytic activity of cationic PMN proteases and
disrupting the interaction of RAGE with its ligands. Selectins are
the initial adhesion molecules used by PMNs, monocytes and
lymphocytes to marginate and roll along the blood vessel wall until
binding such targets as the intercellular adhesion molecule-1
(ICAM-1). The competitor-mediated displacement of U937 human
monocytes, which firmly adhere to P-selectin through P-selectin
glycoprotein ligand-1 (PSGL-1), was studied using
fluorescent-labeled cells.
As a highly sulfated polyanion, GM-1111 is also a potent inhibitor
of PMN proteases such as human leukocyte elastase (HLE). An
experiment was conducted in which HLE was incubated with GM-1111
and standard control in HEPES buffer, following incubation, the
elastase substrate, Suc-Ala-Ala-Val-pNA was added to the reaction
mixture and hydrolysis was assessed. GM-1111 inhibited the PMN
protease HLE activity with IC.sub.50 of 131 nM.
GM-1111 also inhibited ligation of RAGE by high mobility box group
protein-1 (HMGB-1), an "alarmin" secreted by monocytes and
macrophages as an inflammation producing cytokine and also released
by necrotic cells into areas of injury, with an IC.sub.50 of 29 nM.
SAGE's also inhibit the ability of monocytes and lymphocytes to
ligate RAGE on vascular endothelium with the Mac-1 (CD11b/18)
counter-ligand and to use RAGE as an adhesion molecule essential
for exiting the circulation into areas of inflammation.
Because SAGEs block both the Mac-1 (CD11b/CD18) ligand ICAM-1 and
RAGE (which is the alternate adhesion molecule to ICAM-1), SAGEs
can also block migration of leukocytes into areas of inflammation
by blunting attachment to nearby vessel walls. These cumulative
results suggest that GM-1111 may provide considerable
anti-inflammatory activity and might retard influx of CD4+
lymphocytes into the ocular epithelial environment.
A. GM-1111 Binds to Cultured Corneal Epithelial Cells
Corneal epithelial cells were exposed to Alexa Fluor.RTM.
633-labeled GM-1111, stained for actin to identify the sub-membrane
cytoplasm and studied with confocal microscopy. Cultures exposed to
GM-1111 demonstrated bright red fluorescence at 647 nm at the cell
surface when excited at 633 nm, indicating that GM-1111 avidly
binds to cultured corneal epithelial cells (FIG. 20).
Confocal images (FIG. 21) graphically demonstrate these findings
and show concentration dependent fluorescence in these cultured
epithelial cells. Regions of intense fluorescence likely represent
GM-1111 material aggregation. These findings demonstrate that
GM-1111 can bind to epithelial corneal cells when applied
topically, acting as an artificial barrier covering to retain
sodium and water at the epithelial surface. This avid binding to
epithelial cells may be related to the polyanionic charges of the
molecule. The natural lubricating properties of GM-1111 can provide
a mucus-like effect for the eye, restoring some of the function in
dry eye lost by goblet cell atrophy and inadequate production of
endogenous mucins, while localizing SAGE anti-inflammatory effects
at specific target areas for dry eye disease.
B. GM-1111 Increased Tear Production and Tear Film Stability In
Vivo
Treatment of rodents with cholinergic receptor blockade
(scopolamine) and exposure to desiccating environment model results
in ocular surface epithelial changes resembling human
keratoconjunctivitis sicca. A scopolamine-induced dry eye study was
conducted in mice. Dry eye was induced by four-times-daily
subcutaneous injections of 0.5 mg of scopolamine and a desiccating
environment for 16 hours/day over 21 days. Starting on day 1, mice
received either an ophthalmic solution of GM-1111 at 5%,
cyclosporine 0.05% ophthalmic emulsion (Restasis.RTM.), or saline
solution 0.9%. Each group consisted of 10 mice. Tear production was
evaluated using the Phenol Red Thread Test (PRTT) and tear film
stability was evaluated measuring the Tear Break Up Time (TBUT).
Slit lamp examination was conducted during the study and findings
were documented using Draize scoring. Tissue samples from globes,
eyelids, conjunctiva and lacrimal glands will be examined
microscopically.
The study showed that GM-1111 is well tolerated with no related
adverse reactions reported during the in-life observations, and no
GM-1111-related irritation was observed. GM-1111 was significantly
better than vehicle in increasing tear production and performed
comparable to cyclosporine 0.05% (FIG. 22). GM-1111 was also
significantly better than vehicle in increasing the stability of
the tear film and performed comparable to cyclosporine 0.05%, for
this parameter, as well (FIG. 22).
Results from the dry eye mouse study did not indicate any relevant
associated adverse events after 21 days of topical application.
Throughout this application, various publications are referenced.
The disclosures of these publications in their entireties are
hereby incorporated by reference into this application in order to
more fully describe the compounds, compositions and methods
described herein. Various modifications and variations can be made
to the compounds, compositions and methods described herein. Other
aspects of the compounds, compositions and methods described herein
will be apparent from consideration of the specification and
practice of the compounds, compositions and methods disclosed
herein. It is intended that the specification and examples be
considered as exemplary.
* * * * *