U.S. patent application number 13/879440 was filed with the patent office on 2014-03-27 for hydrogel formulation for dermal and ocular delivery.
This patent application is currently assigned to RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY. The applicant listed for this patent is SivaNaga S. Anumolu, Manjeet Deshmukh, Marion Gordon, Anupa Menjoge, Patrick J. Sinko. Invention is credited to SivaNaga S. Anumolu, Manjeet Deshmukh, Marion Gordon, Anupa Menjoge, Patrick J. Sinko.
Application Number | 20140086975 13/879440 |
Document ID | / |
Family ID | 45938590 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140086975 |
Kind Code |
A1 |
Sinko; Patrick J. ; et
al. |
March 27, 2014 |
HYDROGEL FORMULATION FOR DERMAL AND OCULAR DELIVERY
Abstract
Formulations of cross-linkable polymers, capable of forming
non-toxic and biocompatible hydrogels in situ, containing at least
one of doxycycline or minocycline. Methods of using the hydrogels
for treating the skin or ocular tissues of mammals exposed to
vesicant compounds such as sulfur mustard (SM), nitrogen mustard
(NM) or half mustard (2-chloroethyl ethyl sulfide (CEES)) are also
disclosed.
Inventors: |
Sinko; Patrick J.;
(Annandale, NJ) ; Deshmukh; Manjeet; (Edison,
NJ) ; Anumolu; SivaNaga S.; (Edison, NJ) ;
Menjoge; Anupa; (Detroit, MI) ; Gordon; Marion;
(Princeton, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sinko; Patrick J.
Deshmukh; Manjeet
Anumolu; SivaNaga S.
Menjoge; Anupa
Gordon; Marion |
Annandale
Edison
Edison
Detroit
Princeton |
NJ
NJ
NJ
MI
NJ |
US
US
US
US
US |
|
|
Assignee: |
RUTGERS, THE STATE UNIVERSITY OF
NEW JERSEY
New Brunswick
NJ
|
Family ID: |
45938590 |
Appl. No.: |
13/879440 |
Filed: |
October 21, 2010 |
PCT Filed: |
October 21, 2010 |
PCT NO: |
PCT/US10/53566 |
371 Date: |
August 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61393653 |
Oct 15, 2010 |
|
|
|
Current U.S.
Class: |
424/429 ;
424/178.1; 514/1.1; 514/152; 514/21.6; 514/8.7; 514/9.6 |
Current CPC
Class: |
A61K 9/06 20130101; A61K
31/65 20130101; A61K 9/0048 20130101; A61K 47/10 20130101; A61K
9/0051 20130101; A61K 47/34 20130101; A61K 33/40 20130101; A61K
9/0014 20130101 |
Class at
Publication: |
424/429 ;
514/152; 424/178.1; 514/9.6; 514/8.7; 514/21.6; 514/1.1 |
International
Class: |
A61K 31/65 20060101
A61K031/65; A61K 9/00 20060101 A61K009/00; A61K 47/48 20060101
A61K047/48 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under Grant
U54AR055073 awarded by the National Institute of Arthritis and
Musculoskeletal and Skin Diseases. Accordingly, the U.S. Government
has certain rights in this invention.
Claims
1. A formulation comprising a cross-linkable polymer capable of
forming a non-toxic and biocompatible hydrogel in situ and at least
one of doxycycline or minocycline.
2. The formulation of claim 1 adapted for ocular or topical
administration to a mammal.
3. The formulation of claim 1, wherein, upon administration to a
mammal, said cross-linkable polymer forms a hydrogel having said at
least one of doxycycline or minocycline entrapped in the
hydrogel.
4. The formulation of claim 1, wherein said cross-linkable polymer
is a linear or branched PEG.
5. The formulation of claim 1, wherein said polymer is derivatized
with --SH, --NH.sub.2, --COOH, or any combination thereof.
6. The formulation of claim 1, further comprising a cross-linker,
wherein the ratio of polymer to the cross-linker is from about
0.05:10 to about 10:0.05.
7. The formulation of claim 6, wherein said ratio is between 1:1
and 1:2.
8. The formulation of claim 4, wherein said PEG is a linear or
multi-arm having from 2 to 8 arms, has a molecular weight of
1000-100,000 Da, and is derivatized with multiple thiol groups.
9. The formulation of claim 8, wherein the cross-linker is selected
from the group consisting of TP, NHS, vinylsulfone, maleimide,
BM[PEO].sub.3 (1,8-bis-maleimido-triethylene-glycol), BM[PEO].sub.4
(1,11-bis-maleimidotriethyleneglycol), BMH (bis-maleimido-hexane)
or BMOE (bis-maleimidoethane) and combinations thereof.
10. The formulation of claim 1, further comprising an oxidizing
agent, and wherein said polymer is self-cross-linkable.
11. The formulation of claim 10, wherein said oxidizing agent is
hydrogen peroxide.
12. The formulation of claim 1, wherein the concentration of said
at least one of doxycycline or minocycline is 0.1-12% (w/v).
13. The formulation of claim 1, wherein the at least one of
doxycycline or minocycline is unmodified.
14. The formulation of claim 1, wherein the at least one of
doxycycline or minocycline is coupled to the polymer through
degradable bonds.
15. The formulation of claim 14, wherein said degradable bonds are
enzyme-sensitive peptide linker bonds, self-immolative linker
bonds, acid and base-sensitive linker bonds, pH sensitive linker
bonds, multifunctional organic linking agent bonds, multifunctional
inorganic crosslinking agent bonds or peptidic backbone bonds.
16. The formulation of claim 15, wherein said peptide backbone
bonds contain a sequence
CH.sub.3CO--(X--Z--Z).sub.x--(Y--Z--Z).sub.y--CONH.sub.2, where
X=Lys, Glu, Asp or diaminobutyric acid; Y=Cys, homocysteine or
1-amino-2-methyl-2-propanethiol; Z=.beta.-Ala, Gly, Ala, or GABA
(gamma-amino butyric acid); x and y are interchangeable; x is
between 1 to 4; y is between 1 to 4; minimum number of Z-spacer on
the peptide backbone=2; maximum number of Z-spacer on the peptide
backbone=4.
17. The formulation of claim 15, wherein said peptide linker bonds
comprise at least one of Leu-Gly, Glu(Leu-Gly).sub.2, Arg-Gly-Asp,
Arg-Gly-Asp-Cys, Gly-Arg-Gly-Asp-Ser, Gly-Arg-Gly-Asp-Ser-Pro, or
cyclic Arg-Gly-Asp-Tyr-Lys.
18. The formulation of claim 1, wherein the at least one of
doxycycline or minocycline is bound to a targeting moiety.
19. The formulation of claim 18, wherein the targeting moiety is a
peptide.
20. The formulation of claim 19, wherein said peptide is selected
from the group consisting of an RGD peptide, EGF peptide, DV3
(LGASWHRPDKC) peptide, a LYP peptide (CGNKRTRGC), membrane-binding
domain of IGFBP3 (QCRPSKGRKRGFCW), fMLF, mannose, transferrin
ligand, and monoclonal antibodies.
21. A method of treating the skin or eyes of a mammal exposed to a
vesicant compound comprising administering to said exposed skin or
eyes of said mammal a formulation according to claim 1.
22. The method of claim 21, wherein said vesicant compound is
selected from the group consisting of sulfur mustard (SM), nitrogen
mustard (NM) and half mustard (2-chloroethyl ethyl sulfide
(CEES)).
23. The method of claim 21, wherein said formulation administered
in a form of an ocular formulation to an eye of said mammal.
24. The method of claim 23, wherein said ocular formulation is
fabricated as a contact lens.
25. The method of claim 21, wherein said formulation is
administered in a form of a topical formulation to a portion of
said mammal skin exposed to said vesicant compound.
26. A contact lens fabricated from a hydrogel formulation according
to claim 1.
27. A hydrogel contact lens comprising at least one of doxycycline
or minocycline in an amount effective for the controlled and
sustained delivery of said doxycycline or minocycline to the
surface of the eye from said hydrogel.
28. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present invention claims priority benefit under 35
U.S.C. .sctn.119(e) of U.S. Provisional Patent Application Ser. No.
61/393,653, filed Oct. 15, 2010, the disclosure of which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to compositions of
polyalkylene oxide hydrogels, as well as methods of making and
using the hydrogel for wound healing applications. More
particularly, embodiments relate to compositions comprising
functionalized polyethylene glycol (PEG) in new and useful
configuration hydrogels.
[0005] 2. Description of the Related Art
[0006] Vesicants such as sulfur mustard, nitrogen mustard and half
mustard are potent cytotoxic and mutagenic agents. Sulfur mustard
(SM) has the chemical formula bis(2-chloroethyl) sulfide and is a
well known chemical warfare agent. It was first used as such during
World War I and has been subsequently used in over 10 additional
conflicts.
[0007] Nitrogen mustard and half mustard are analog derivatives of
SM and, just like SM, also have potential as chemical warfare
agents. Half mustard (CEES) has the chemical formula 2-chloroethyl
ethyl sulfide, and nitrogen mustard (NM) encompasses a class of
chloroalkyl amines, the three most common species including
bis(2-chloroethyl)ethyl amine (HN1); bis(2-chloro-ethyl)methyl
amine (HN2) and tris(2-chloroethyl)amine (HN3). Many countries have
been stockpiling these derivatives for use as chemical warfare
agents, but none have ever been used.
[0008] The toxicity between SM, NM and CEES varies. Nevertheless,
exposure to any one of these agents can cause devastating injuries
to the eyes, skin and respiratory system, with the eyes being the
most sensitive tissue to exposure. SM, for example, exhibits a
threshold in the eyes of 12 mgmin/m.sup.3, as compared to 200
mgmin/m.sup.3 for the skin. Thus, even low doses of SM, NM, or CEES
induce incapacitation, visual impairment and panic. While the
molecular mechanisms for SM, NM, or CEES induced injury are
unclear, these all exhibit DNA, RNA and protein alkylation and
cause inflammation, tissue damage and cell death.
[0009] MMPs are a family of enzymes that enhance the action of many
activating factors during inflammatory response and contribute to
tissue degradation. MMP-9 has been identified as a potential target
of therapy for SM, NM, and/or CEES damage since it was found that
its expression and activation quantitatively increases over time in
response to SM exposure. The cornea is clinically impaired by such
exposure exhibiting chronic inflammation and increased MMP
activity. Decreased MMP-9 activity in humans has been found to
correlate with accelerated wound healing. Hence, intervention
targeting of both the inflammatory response and increased protease
expression could provide a therapeutic approach for the treatment
of vesicant induced corneal wounds.
[0010] Doxycycline is a long acting semi-synthetic tetracycline
analog, which is well recognized for its therapeutic efficacy in
treating MMP mediated ocular surface diseases, such as rosacea,
recurrent epithelial erosions and sterile corneal ulcerations.
Doxycycline has been found to inhibit MMP-9 activity in vivo in the
corneal epithelial cells of experimental dry eye as well as in
vitro in human corneal epithelial cells. Treatment with doxycycline
has been shown to be beneficial in attenuating acute and delayed
ocular injuries caused by SM exposure. The drug is an inexpensive,
FDA approved antibiotic that likely promotes wound healing by
reducing inflammation and protease activity.
[0011] Minocycline is also a long acting semi-synthetic
tetracycline analog that exhibits neuro-protective, anti-apoptotic,
and anti-inflammatory effects. Recently, it has been shown to
inhibit macrophage inflammation and T cell activation, as well as
inhibiting the inflammatory effects of the enzyme 5-lipoxygenase.
Much like doxycycline, minocycline also has been shown to inhibit
MMP-9 activity in vivo, particularly after cerebral ischemia. It
has been previously suggested that this MMP inhibitory action could
be a central link for minocycline's neuroprotective,
anti-apoptotic, and anti-inflammatory effects. For at least this
reason, minocycline is also a good candidate for promoting wound
healing.
[0012] The blood-ocular barriers, which include the blood-aqueous
and blood-retina barriers protect the eye, but prevent drug
distribution to the anterior and posterior chambers, limiting
ocular bioavailability. Drug diffusion into the eyes from the
systemic circulation is slow and inefficient. Most drugs applied to
the eye surface as solutions have ocular bioavailability in the
range of about 10% with most of the drug being cleared by local
systemic absorption. Solutions are in contact with the eye surface
for a very short period of time as the tear film quickly washes
them away.
[0013] The contact time, local drug concentration and thereby
duration of action can be prolonged by designing topical
formulations with higher viscosities. The ideal drug delivery
system for corneal wound repair should be nontoxic, transparent,
easy to administer, possess rheological properties to maintain its
structural integrity, provide a microbial barrier, release the drug
in a controlled and sustained manner and decrease the time of wound
healing. There are very few-controlled drug delivery systems
reported for corneal wound repair applications. Although
doxycycline and minocycline are commercially available in a wide
variety of dosage formulations including tablets, capsules and
suspensions, topical ocular doxycycline eye drop formulations are
to this day compounded by a pharmacist. Since there are currently
no ocular formulations commercially available for doxycycline,
there is a critical need for a controlled release doxycycline
delivery system that can be easily applied to the eye to promote
wound healing.
SUMMARY OF THE INVENTION
[0014] The instant invention addresses these and other needs by
providing, in one aspect, a formulation comprising a cross-linkable
polymer capable of forming non-toxic and biocompatible hydrogels in
situ and at least one of doxycycline or minocycline. In certain
embodiments, the formulation is adapted for topical or ocular
administration. Preferably, though not exclusively, the formulation
is administered in a liquid form and forms a hydrogel in situ.
[0015] In certain embodiments, the cross-linkable polymer of the
instant formulation is a linear or branched PEG having 2-8 arms,
and preferably having a molecular weight of 1000-100,000 Da. The
polymer of the instant invention may be derivatized with reactive
groups such as, for example, --SH, --NH.sub.2, --COOH, or any
combination thereof.
[0016] In certain embodiments of the invention, the formulation
also comprises a cross-linker, wherein the ratio of polymer to the
cross-linker is from about 0.05:10 to about 10:0.05, and, more
preferably, between 1:1 and 1:2. In different embodiments, the
cross-linker may be selected from thiopyridine (TP),
N-Hydroxysuccinimide (NHS), vinylsulfone, maleimide,
BM[PEO].sub.3-(1,8-bis-maleimido-tri-ethylene-glycol),
BM[PEO].sub.4-(1,11-bis-maleimido-triethylene-glycol),
BMH-(bis-maleimido-hexane) or BMOE-(bis-maleimidoethane) and
combinations thereof.
[0017] In other embodiments, the polymer is self-crosslinkable upon
reaction with an oxidizing agent such as, without limitation,
hydrogen peroxide.
[0018] According to any of the embodiments of the instant
invention, the concentration of said at least one of doxycycline or
minocycline is 0.1-12% (w/v). In certain embodiments, the at least
one of doxycycline or minocycline is unmodified. In other
embodiments, the at least one of doxycycline or minocycline is
coupled to the polymer through degradable bonds. In different
embodiments, the degradable bonds are enzyme-sensitive peptide
linker bonds, self-immolative linker bonds, acid and base-sensitive
linker bonds, pH sensitive linker bonds, multifunctional organic
linking agent bonds, multifunctional inorganic crosslinking agent
bonds and peptidic backbone bonds.
[0019] In some embodiments the at least one of doxycycline or
minocycline is bound to a targeting moiety, such as, for example,
an RGD peptide, EGF peptide, DV3 peptide (LGASWHRPDKC), LYP peptide
(CGNKRTRGC), membrane-binding domain of IGFBP3 (QCRPSKGRKRGFCW),
fMLF, mannose, transferrin ligand or monoclonal antibodies. While
not limited thereto, in one embodiment the doxycycline or
minocycline is bounded by at least one of its hydroxyl
residues.
[0020] While the foregoing exemplifies in situ administration, in
alternative embodiments, particularly with respect to ocular
administration, the hydrogel may be provided in a prepared dosage
form adapted for release of the active agent in accordance with the
foregoing. In one aspect, an ocular dosage form includes the
hydrogel fabricated as a contact lens having the active ingredient
(e.g. doxycycline or minocycline). Such a dosage form may also
include additional excipients or ingredients for such purposes, as
defined herein or otherwise known in the art. The prepared dosage
forms of the instant invention are not limited to a contact lens
and may also be adapted for alternative uses, as generally
understood in the art.
[0021] In another aspect, the invention provides a method of
treating the skin or eyes of a mammal exposed to a vesicant
compound by administering to the mammal a formulation according to
any of the embodiments of the instantly claimed formulation. In
certain embodiments, the vesicant compound is sulfur mustard (SM),
nitrogen mustard (NM) or half mustard (2-chloroethyl ethyl sulfide
(CEES)). In certain embodiments, the formulation is administered in
the form of an ocular formulation to an eye of the mammal. In other
embodiments, the formulation is administered in the form of a
topical formulation to a portion of the mammal's skin exposed to
the vesicant compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is an illustration of hydrogel preparation scheme in
which a hydrogel is formed using multi-arm thiol-containing PEG
with multi-arm PEG cross-linker containing S-TP;
[0023] FIG. 2 illustrates optical transmission of 5% (1:1), 7.5%
(1:1), 7.5% (1:2), 10% (1:1), 15% (1:2), 15% (1:1), and 22.5% (1:2)
hydrogels;
[0024] FIG. 3 demonstrates the influence of strain on G' (A) and
G'' (B) of 5% (1:1), 7.5% (1:1), 7.5% (1:2), 15% (1:1), and 15%
(1:2) hydrogels, which depicts the range of linear visco-elasticity
for the hydrogels;
[0025] FIG. 4 demonstrates the influence of frequency on G' (A) and
G'' (B) of 5% (1:1), 7.5% (1:1), 7.5% (1:2), 15% (1:1), and 15%
(1:2) hydrogels;
[0026] FIG. 5 demonstrates the effect of polymer concentration and
crosslinking density on the swelling kinetics of 5% (1:1), 7.5%
(1:1), 7.5% (1:2), 10% (1:1), 15% (1:1), 15% (1:2), and 22.5% (1:2)
hydrogels;
[0027] FIG. 6 is an illustration of the cumulative amount of
doxycycline released as a function of time for hydrogels: 10%
(1:1), 15% (1:2), 15% (1:1), and 22.5% (1:2), with the data fitted
using a two-phase exponential association equation in GraphPad
Prism 4 software with the fit varying from 0.87 to 0.99;
[0028] FIG. 7 is an illustration of the cumulative amount of
doxycycline permeated as a function of time through cornea exposed
to different concentrations of CEES and NM;
[0029] FIG. 8 depicts H & E staining to visualize the histology
of CEES and NM-exposed corneas treated for 24 h with doxycycline in
solution or in a hydrogel, in which the damaged area is where the
epithelium meets the stroma;
[0030] FIG. 9 depicts the immuno-fluorescent staining of corneas
exposed to CEES and NM and subsequently treated with doxycycline
either in solution or hydrogel;
[0031] FIG. 10 is a collection of images of rabbit corneas exposed
to SM and subsequently treated with doxycycline hydrogel, including
pictures of the rabbit cornea after doxycycline hydrogel
application;
[0032] FIG. 11 depicts H & E staining to visualize the
histology of NM-exposed corneas treated for 24 h with minocycline
drops;
[0033] FIG. 12 depicts H & E staining to visualize the
histology of NM-exposed corneas treated for 24 h with minocycline
PEG hydrogel;
[0034] FIG. 13 depicts H & E staining to visualize the
histology of NM-exposed corneas treated for 24 h with NDH4417
drops;
[0035] FIG. 14 depicts H & E staining to visualize the
histology of NM-exposed corneas treated for 24 h with NDH4417 PEG
hydrogel;
[0036] FIG. 15 provides photos of mice treated with minocycline
hydrogel (0 h, 4 h and 20 h) applied to skin;
[0037] FIG. 16 depicts H & E staining to visualize the
histology of dose dependent effects of NM exposure on mouse
skin;
[0038] FIG. 17 depicts H & E staining to visualize the
histology of time dependent effects of NM exposure on mouse
skin;
[0039] FIG. 18 demonstrates marked edema overtime when mouse skin
is exposed to NM;
[0040] FIG. 19 demonstrates increased mRNA expression levels of
both IL-1.beta. and TNF-.alpha. at 24, 72 and 168 h after exposure
to NM on mouse skin;
[0041] FIG. 20 demonstrates the permeation profile of 3[H] mannitol
through NM exposed mouse skin;
[0042] FIG. 21 demonstrates the permeation profile of
FITC-dextran's through NM exposed mouse skin; and
[0043] FIG. 22 demonstrates the permeation profile of rhodamine 123
through NM exposed mouse skin.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0044] Unless characterized otherwise, technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
For purposes of the present invention, the following terms are
described below.
[0045] "PEG" is used herein as an abbreviation for polyethylene
glycol. PEGs are included within the broader class of polyalkylene
oxides, which include PEG as well as polypropylene glycols and
polyglycol copolymers. PEG can have a range of molecular weights.
The PEG molecular weight range contemplated for use in the present
invention is from about 1000 to about 100,000 Da. PEG can be
linear, branched, multi-arm, or a combination of branched and
multi-arm. Various PEGs can be derivatized with various groups,
such as activated ester (N-hydroxy succinimide ester),
p-nitrophenyl, aldehyde, amine, thiol, activated thiol
(thio-pyridine activated thiol, for example), vinyl sulfone,
maleimide, aminooxy, hydrazine, tosyl, and iodoacetamide.
[0046] "Surface modification" includes chemical treatment of
nanogel particles or aggregated nanogel particles (ANPs) to modify,
for example, the surface charge/charge density,
hydrophobicity/hydrophilicity, or both. The actual chemical
treatment can be performed on the final material, such as the
nanogel particle, or ANP, or it be performed on a precursor
material, such as the scaffold or nanocarrier.
[0047] "Agent" includes without limitation any therapeutic,
palliative, cosmetic and/or prophylactic compositions, including
without limitation small molecules, drugs, biologicals, recombinant
peptides, proteins and nucleic acids and immunochemicals, as well
as diagnostic and imaging compositions, as may be further indicated
by the context. In some uses, the term can relate to other types of
compositions, as indicated by the context.
Solubility Terms
[0048] Unless indicated otherwise, either expressly or by
implication, the following solubility terms are used as described
in Table 1 below (reproduced from Stegemann et al., "When Poor
Solubility Becomes an Issue: From Early Stage to Proof of Concept,"
Eur. J. Pharm. Sci., 31, 250 (2007)).
TABLE-US-00001 TABLE 1 Solubility definition in the USP Parts of
solvent Solubility Solubility Description forms required for one
part range assigned (solubility definition) of solute (mg/ml)
(mg/ml) Very soluble (VS) <1 >1000 1000 Freely soluble (FS)
From 1 to 10 100-1000 100 Soluble From 10 to 30 33-100 33 Sparingly
soluble From 30 to 100 10-33 10 (SPS) Slightly soluble (SS) From
100 to 1000 1-10 1 Very slightly soluble From 1000 to 10,000 0.1-1
0.1 (VSS) Practically insoluble >10,000 <0.1 0.01 (PI)
[0049] The instant invention addresses the needs of the prior art
by providing in one aspect a composition suitable for ocular or
topical administration and comprising a cross-linkable,
polymer-based hydrogel and an active agent entrapped therein.
[0050] The hydrogels evaluated in the current study are formed in
situ, in other words, they are liquids upon instillation and
undergo a phase transition at physiological pH to form the
hydrogel. This occurs by covalent intermolecular crosslinking of
polymer chains through reversible thioester bonds, resulting in
biodegradable viscoelastic hydrogels. In the current invention,
doxycycline/minocycline loaded fast forming PEG hydrogels were
designed for the treatment of simulated mustard injuries using
CEES, NM and SM vesicants and evaluated in exposed rabbit corneas
or on the skin ion the back of the mice or rats.
[0051] There are many variations, all of which are considered
embodiments of the invention, of the general scheme for hydrogel
preparation. One such embodiment as shown in the FIG. 1 uses
multi-arm thiol-containing PEG with multi-arm PEG cross-linkers
containing S-TP. Upon mixing the multi-arm PEG with the
multifunctional cross-linker under proper conditions of pH, reagent
concentrations and temperature, covalent bonds are formed due to
the reaction of the thiol group with the S-TP. Other bi-functional
or multifunctional cross-linkers such as NHS, vinylsulfone or
maleimide groups can be used to form the gel upon mixing with a
multi-arm PEG thiol. The transition from a liquid to a hydrogel
will occur when the network of inter-molecularly crosslinked PEG
molecules reaches a particular molecular weight, which depends on
many factors. In this embodiment, the cross-linker contains both
the chemo-selective group needed for hydrogel formation and an
oxidizing agent that oxidizes thiol groups present on the polymers
or copolymers. The present invention is directed, in part, to
materials and methods for the preparation and use of hydrogels
incorporating chemistries allowing for timed degradation and/or
release of active agents, which may be embedded therein by covalent
or non-covalent means.
[0052] Hydrogels (polymer/copolymer, cross-linker, and/or active
agents) can be applied to wounded or CEES, NM and SM exposed skin
or eyes as a solution, where it is converted into the hydrogel in
situ due to the intermolecular crosslinking of
polymer/copolymer/cross-linker chains. The hydrogel stays in the
applied space and provide controlled-release of active agents
(e.g., doxycycline minocycline, etc.)
[0053] Preferably, the polymer comprises at least two thiol groups,
and may be a homopolymer or a copolymer.
[0054] The hydrogels of the present invention include at least one
cross-linkable polymer, which is cross-linked to entrap the
therapeutic agent, which is may be encapsulated, for example,
within a liposome. Any cross-linkable polymer bearing two or more
functional or reactive groups capable of participating in a
cross-linking reaction to form a matrix of the invention may be
used. Such functional groups include but are not limited to amino,
carboxyl, thiol and hydroxyl groups, or combinations thereof; and
reactive groups include vinylsulfone, maleimide, pyridyldithio and
other moieties capable of reacting with the aforementioned
functional groups, among others.
[0055] A preferred polymer is one on which at least two thiol
groups are present and is cross-linked with a thiol-reactive
bi-functional cross-linking reagent in the presence of the
encapsulated therapeutic agent, thus forming a cross-linked polymer
with the liposome encapsulated therapeutic agent physically
entrapped therein. Selection of the appropriate polymer, the
concentration in the matrix, the extent of functional groups
capable of participating in cross-linking, the type of
cross-linking agent, and the extent of cross-linking, and other
factors may be governed by the amount of liposome encapsulated
therapeutic agent present in the composition in order to achieve
the desired controlled release properties of the composition, or
retention of the liposomes within the composition.
[0056] Examples of suitable polymers for the preparation of the
polymer on which at least two thiol groups are present include
either homopolymers or copolymers. By way of non-limiting example,
suitable polymers, which may be chemically modified to comprise
thiol groups, include polyalkylene oxides such as poly(ethylene
glycol) [also known as poly-ethylene glycol or PEG, polyethylene
oxide or PEO], carboxymethylcellulose, dextran, poly-vinyl alcohol,
N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone,
poly-1,3-dioxo-lane, poly-1,3,6-trioxane, polypropylene oxide, a
copolymer of ethylene/maleic anhydride, a polylactide/polyglycolide
copolymer, a polyaminoacid, a copolymer of poly(ethylene glycol)
and an amino acid, or a polypropylene oxide/ethylene oxide
copolymer.
[0057] Such polymers are then derivatized or further polymerized to
introduce thiol groups, and chemical modification of the polymer
may be necessary as a step prior to the further derivatization to
incorporate thiol groups. In certain embodiments, for example, a
polymer of the present invention may be derived from a
poly(ethylene glycol) (PEG) derivative, for example,
.alpha.,.omega.-dihydroxy-PEG or .alpha.,.omega.-diamino-PEG, but
other derivatives are embraced herein. In certain embodiments the
polymer comprising thiol groups may be, for example, a polymer of
.alpha.,.omega.-diamino-poly(ethylene glycol) and thiomalic acid; a
polymer of .alpha.,.omega.-dihydroxy poly(ethylene glycol) and
thiomalic acid; or a polymer of .alpha.,.omega.-dicarboxy-PEG
subunits and lysine, wherein the free carboxy groups on the lysine
residues are derivatized to form thiol groups.
[0058] These polymers are only examples of possible choices, as the
skilled artisan will be aware of numerous alternatives. The
selection of the polymer, or combinations thereof, may be guided by
the desired properties of the final product such as, for example,
the duration of release of the therapeutic agent and the release
kinetics. In certain embodiments, a product of the invention may
comprise more than one polymer component in order to provide two or
more different release characteristics. In certain embodiments,
more than one therapeutic agent may be included.
[0059] In certain preferred embodiments, a polymer of the present
invention is derived from a poly(ethylene glycol) (PEG) derivative,
for example, .alpha.,.omega.-dihydroxy-PEG or
.alpha.,.omega.-diamino-PEG, but other derivatives are embraced
herein. Examples of such polymers with particular molecular weights
include .alpha.,.omega.-dihydroxy-PEG.sub.3,400;
.alpha.,.omega.-dihydroxy-PEG.sub.1,400;
.alpha.,.omega.-diamino-PEG.sub.3,400 and
.alpha.,.omega.-diamino-PEG.sub.1,000. PEG is known to be a
particularly nontoxic polymer. These derivatized PEG subunit
polymers may be used as amino- and hydroxy-containing polymers for
cross-linking, or may be further derivatized, for example, to
prepare the polymer on which at least two thiol groups are present
by derivatization with thiomalic acid. Thio-malic acid (also known
as mercaptosuccinic acid) may be replaced by dimercaptosuccinic
acid, thereby doubling the number of sites available for
cross-linking. Increasing the extent of cross-linking the matrix
results in a gel with smaller pores and thus regulates the rate of
release of the active agent.
[0060] In certain embodiments, the cross-linking can be performed
before, during, or after the matrix is administered to a mammal.
For example, the cross-linking reaction can be initiated in vitro,
and the mixture, while undergoing cross-linking, may administered
to a mammal, wherein the administered composition continues to
cross-link and harden in situ. In certain embodiments, a
cross-linked matrix after formation can be administered.
[0061] In certain embodiments, the polymer moieties may be
cross-linked by reagents capable of forming covalent bonds between
the functional groups, such as but not limited to homobifunctional
and heterobifunctional cross-linking agents. As described above,
one preferred moiety is a thiol group. In certain embodiments, a
preferred cross-linking agent is one that forms thioether bonds,
such as a vinylsulfone or maleimide, but the invention is not so
limiting. Other cross-linking reagents, such as a
pyridyldithio-containing reagent, or oxidation, may be used to
generate reducible cross-links. Combinations of cross-linking
reagents may be used, to provide a ratio of cross-link types, which
generate the desired release characteristics of the composition. In
certain embodiments, the preferred thiol-containing polymer has
from 2 to about 20 thiol groups, preferably from about 3 to about
20 thiol groups, and most preferably from about 3 to about 8 thiol
groups. In certain embodiments, the thiol groups on the polymer are
sterically hindered.
[0062] Various conditions and/or reagents may be used to effect the
cross-linking of the polymer, depending on the particular
functional groups on the polymer. By way of non-limiting example,
the conditions that cause cross-linking of the thiol groups on a
thiol-containing polymer may be reaction in the presence of an
oxidizing agent or reaction with a cross-linking agent. The
oxidizing agent may be by way of non-limiting example, molecular
oxygen, hydrogen peroxide, dimethylsulfoxide, and molecular iodine.
The cross-linking agent may be a bifunctional disulfide-forming
cross-linking agent or a bifunctional thioether-forming
cross-linking agent. In a preferred embodiment, the cross-linking
agent is a long-chain cross-linking agent, with a molecular weight
of about 300 to about 5,000 Da. Non-limiting examples of suitable
cross-linking agent include
1,4-di-[3',2'-pyridyldithio(propion-amido)butane];
.alpha.,.omega.-di-O-pyridyldi-sulfidyl-poly(ethylene glycol); a
vinyl sulfone such as .alpha.,.omega.-divinylsulfone-poly(ethylene
glycol); 1,11-bis-maleimidotetraethylene glycol; and
.alpha.,.omega.-diiodoacetamide-poly(ethylene glycol).
[0063] For other functional groups or a combination of a thiol
group and another group, any appropriate bi-functional
cross-linking agent may be selected which will achieve the desired
cross-linking of the functional groups and formation of the
cross-linked polymer.
[0064] In certain embodiments, the release rate of the therapeutic
or other agent in the composition of the invention may be regulated
by the biodegradability of the cross-linked polymer matrix, the
liposome or combination thereof. In certain embodiments, the
degradation rate may be adjusted by varying the ratio or types of
cross-links of the matrix, and the stability or lability thereof,
in the composition. For example, the ratio of reducing
agent-sensitive disulfide bonds, esterase-sensitive ester bonds,
and stable thioether bonds may be selected to provide the desired
release kinetics of the active agent. In certain embodiments, the
release rate is adjusted by adjusting the pore size of the pores in
the hydrogel matrix.
[0065] In certain embodiments, the polymer is PEG-based (i.e.,
comprises more than 50% w/w of PEG which may be straight or
branched). The hydrogel is based on intermolecular cross-linking of
soluble PEG polymers, which form an insoluble, high molecular
weight PEG hydrogel matrix. Active agents may be loaded into this
hydrogel prior to the cross-linking reaction, so that the hydrogel
will serve as a depot for the sustained release of that agent. The
chemical reaction used for forming the hydrogel by cross-linking
polymer and cross-linker. Non-limiting examples of methods to
accomplish this includes: (i) using chemo-selective pairs of
reactive groups, for example, the cross-linker may comprise a
thiol-reactive group such as vinylsulfone or maleimide, S-TP or NHS
that will react with thiol groups on PEG; and (ii) using oxidizing
agents like H.sub.2O.sub.2 for the cross-linking reaction.
General Procedure for Hydrogel Formation
[0066] Hydrogels are formed in situ by reaction between a
multivalent copolymer or PEG polymer and cross-linker in aqueous
medium. Several combinations are possible: (i) the PEG polymer or
copolymer contain thiol groups whereas the cross-linker has
thiol-reactive S-TP, NHS, vinylsulfone, maleimide etc. groups; or
(ii) the polymer or copolymer containing thiol groups cross-linked
with oxidizing agents like H.sub.2O.sub.2 etc. The hydrogels
disclosed herein can be obtained over a broad concentration range
of the polymers or copolymers, and cross-linkers. The concentration
ranges of the polymer or copolymer is 1%-20% (w/v) and that of the
cross-linker is 1%-15% (w/v). The ratios of the polymer or
copolymer to the cross-linker in the hydrogel vary from 0.05:10 to
10:0.05 and preferably between 1:1 and 1:2. Either single type of
polymer/copolymer and cross-linker is used or a combination of
different types of unmodified and modified copolymer or polymer and
cross-linkers is used.
[0067] The formulation of the instant invention may be prepared by
reacting a PEG-SH or PEG-COOH having from 2 to 8 arms and a
molecular weight from about 1 to 20 kDa with cross-linkers
comprising S-TP and/or NHS in DMF to obtain PEG-AA-S-TP or
PEG-AA-NHS, wherein AA is derived from the cross-linker and is
selected from GABA (gamma-amino butyric acid); AHA (6-aminohexanoic
acid), AOA (8-aminooctanoic acid), GABA-GABA, AHA-AHA, AOA-AOA,
AHA-GABA, AOA-GABA, AHA-GAA and combinations thereof.
Polymers for Hydrogel Formation.
[0068] Linear or multi-arm (2 to 8) PEGs contain multiple thiol
groups within a molecular weight range of 1000-100,000 Da. Polymers
can be unmodified or modified with active agents (timed-release
mechanism, other degradation mechanism, or degradation preventing
mechanisms) prior to hydrogel formation.
Copolymer Containing Thiol Groups.
[0069] The invention can be extended to copolymers containing
repeating units of thiol groups. For example, copolymer like
poly[poly(ethylene glycol)-alt-poly (mercaptosuccinic acid)] in the
molecular weight range of 10,000 to 100,000 Da. Copolymers can also
be unmodified or modified with active agents (timed-release
mechanism, other degradation mechanism, or degradation-preventing
mechanism) prior to hydrogel formation.
Polymer Containing Peptide Thiol Groups.
[0070] The invention can be extended to polymers containing
repeating units of peptide thiol groups such as polycysteine in the
molecular weight range of 1,000 to 100,000 Da. These polymers can
also be unmodified or modified with active agents prior to hydrogel
formation.
Cross-Linkers for Hydrogel Formation.
[0071] Cross-linkers containing functional groups like S-TP, NHS,
vinyl sulfone and maleimide groups or thiol groups are used for
hydrogel formation through thioether or disulfide bonds.
Cross-linkers can be linear or branched and contain 2-8 functional
groups, with molecular weights in the range of 1-20 kDa.
Cross-Linkers Containing Vinylsulfone Groups.
[0072] The cross-linkers containing terminal vinylsulfone (VS)
functional groups like 1,6-Hexane-bis-vinylsulfone (HBVS) can also
be used.
Cross-Linkers Containing Maleimide Groups (MA).
[0073] Cross-linkers containing terminal maleimide groups like
BM[PEO].sub.3-(1,8-bis-maleimidotriethyleneglycol) or
BM[PEO].sub.4-(1,11-bis-maleimidotriethyleneglycol) or
BMH-(bis-maleimidohexane) or BMOE (bis-baleimidoethane) can also be
used.
Cross-Linkers Containing Thiol Groups.
[0074] Thiol-containing cross-linkers such as dithiothreitol,
polycysteines, PEG-thiol's or 4-arm thiol, 8-arm thiol can be
used.
Active Agents.
[0075] In certain embodiments, the active agent includes
doxycycline and/or minocycline. The instant invention is not
limited to the forgoing may include one or a combination of any of
the following active agents: anti-inflammatory drugs, doxycycline,
minocycline, NSAID analogs, NSAID-ache (NSAID-acetylcholin-esterase
complexes, steroidal anti-inflammatory drugs, anti-cancer drugs,
HIV protease inhibitors, monoclonal antibodies, imaging agents, and
combinations thereof. In certain other embodiments, the agent is
selected from one or more of the following: indomethacin,
sancycline, a sancycline analog, olvanil, an olvanil analog,
retro-olvanil, a retro-olvanil analog, olvanil carbamate,
budesonide, a budesonide analog, methylprednisolone, a
methylprednisolone analog, dexamethasone, a dexamethasone analog,
camptothecin, carboplatin, doxorubicin, paclitaxel, saquinavir
mesylate, amprenavir, ritonavir, indinavir, nelfinavir mesylate,
tipranavir, darunavir, atazanavir sulfate, a coloring dye, an FD
and C dye, a visible/near infrared fluorescence dye, fluorescein,
methylene blue, rhodamine, dansyl, Alexa, a cyanine dye, Hilvte,
indocyanine green, and combinations thereof. More preferably, the
agent is doxorubicin.
[0076] For passive entrapment, the agent may be unmodified or
coupled to the PEG through degradable bonds (prodrugs) like
enzyme-sensitive peptide linkers, self-immolative linkers, acid and
base-sensitive linkers, pH sensitive linkers, multifunctional
organic linking agents, multifunctional inorganic crosslinking
agents and/or peptidic backbones represented as:
CH.sub.3CO--(X--Z--Z).sub.x--(Y--Z--Z).sub.y--CONH.sub.2,
[0077] where X=Lys, Glu, Asp or diaminobutyric acid; Y=Cys,
homocysteine or 1-amino-2-methyl-2-propanethiol; Z=.beta.-Ala, Gly,
Ala, or GABA (gamma-amino butyric acid); x and y are
interchangeable; x is between 1 to 4; y is between 1 to 4; minimum
number of Z-spacer on the peptide backbone=2; maximum number of
Z-spacer on the peptide backbone=4.
[0078] In variations, the active agent may further comprise a
targeting moiety. The targeting moiety may be a peptide, and
preferably such a peptide is an RGD peptide. In certain other
embodiments, the targeting group is selected from an RGD peptide,
EGF peptide, DV3 (LGASWHRPDKC) peptide, a LYP peptide (CGNKRTRGC),
a membrane-binding domain of IGFBP3 (QCRPSKGRKRGFCW), fMLF,
mannose, transferrin ligand and monoclonal anti-bodies. When the
drug is doxycycline, the linker used may also be any of following:
Leu-Gly, Glu(Leu-Gly).sub.2, Arg-Gly-Asp-Cys, Gly-Arg-Gly-Asp-Ser,
Gly-Arg-Gly-Asp-Ser-Pro, cyclic Arg-Gly-Asp-Tyr-Lys or any peptide
with Arg-Gly-Asp. While not limiting to the invention, in such
embodiments, doxycycline and/or minocycline may be linked to
targeting moieties or linkers by any of its hydroxyl groups.
[0079] In variations, the active agent may contain a targeting unit
selected from the targeting groups listed above.
[0080] Either for passive entrapment or timed release, a single
active agent can be used or combinations of active agents can be
used, and the active agent content in the hydrogel formulation may
vary from 0.1-12% (w/v). Thus, in different embodiments, the active
agent content is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%.
[0081] In certain embodiments, the timed release of the active
agent is from about 1 minute to about 1440 h. In certain
embodiments, the timed release of the active agent is from about 1
min to about 720 h. In certain embodiments, the timed release of
the active agent is from about 1 min to about 490 h. In certain
embodiments, the timed release of the active agent is from about 1
min to about 360 h. In certain embodiments, the timed release of
the active agent is from about 1 min to about 119 h. In certain
embodiments, the timed release of the active agent is from about 1
min to about 96 h. In certain embodiments, the timed release of the
active agent is from about 1 min to about 72 h. In certain
embodiments, the timed release of the active agent is from about 1
min to about 48 h. In certain embodiments, the timed release of the
active agent is from about 1 min to about 24 h. In certain
embodiments, the timed release of the active agent is from about 1
min to about 12 h. In certain embodiments, the timed release of the
active agent is from about 1 min to about 6 h.
[0082] The general procedure for release of active agents from
hydrogels preferably involves the following processes:
[0083] (i) Passive entrapment and release. In one embodiment, the
active agent(s) are physically entrapped into the hydrogel by
mixing it in the formulation (polymer/copolymer and cross-linker)
prior to hydrogel formation. In alternative embodiments, the
agent(s) may be incorporated into a second phase (e.g.
nanoparticle, microparticle, or the like) and passively dispersed
in the hydrogel. The active agent content in the hydrogel
formulation may vary from 0.1-12% (w/v) and the formulation may
contain one active agent or a combination of active agents.
[0084] (ii) Active entrapment and release. In other embodiments,
the active agent(s) may be covalently attached into the hydrogel by
and releasable as provided herein.
[0085] While the foregoing exemplifies in situ administration, in
alternative embodiments, particularly with respect to ocular
administration, the hydrogel may be provided in a prepared dosage
form adapted for release of the active agent in accordance with the
foregoing. In one aspect, the ocular dosage form includes a contact
lens fabricated, at least in part, from the active ingredient (e.g.
doxycycline or minocycline) containing hydrogel of the instant
invention. Such a dosage form may also include additional
excipients or ingredients for such a purposes. To this end, the
contact lens may be comprised entirely of the active
agent-containing hydrogel in accordance with the foregoing.
Alternatively, it may be comprised of a combination of the
foregoing hydrogel with one or more additional hydrogels, polymers,
or other components known in the art for use in preparing a hard or
soft contact lens. In either embodiment, the contact lens is
adapted to be fully or partially biodegradable such that the lens
releases the active agent over a period of time. The prepared
dosage forms of the instant invention are not limited to a contact
lens and may also be adapted for alternative uses in accordance
with the teaching herein and as generally understood in the
art.
[0086] The invention is described more fully by way of the
following non-limiting examples. All references cited above and
hereafter in this document are hereby incorporated by reference in
their entirety herein.
EXAMPLES
Example 1
Preparation of Doxycycline Hydrogel Using 8 Arm PEG-SH and 8 Arm
PEG NHS
[0087] A. Preparation of Sodium Phosphate Buffer (0.1 M, pH
8.00.+-.0.05)
[0088] A solution of sodium phosphate dibasic (1 M, Catalog
#S-9763, Sigma Aldrich, St. Louis, Mo.) was prepared in a
volumetric flask by dissolving 14.2 grams of salt in 100 mL of
deionized (DI) water. Similarly, the solution of sodium phosphate
monobasic (1M, Catalog # S-0751, Sigma Aldrich, St. Louis, Mo.) was
prepared in another flask by dissolving 12.0 grams of salt in 100
mL DI water. 9.32 mL of sodium phosphate dibasic and 0.68 ml of
sodium phosphate monobasic solutions were transferred to a beaker.
DI water (80.0 mL) was added to the beaker and the pH of the
solution was measured as described in example 3A. The pH was
adjusted to 8.00 using 0.1 N sodium hydroxide solution (Catalog #
SS276-4, Fisher Scientific, Suwanee, Ga.). The buffer was
transferred to a volumetric flask and DI water was added to adjust
the final buffer volume to 100 mL. Unless otherwise indicated, all
reference to DI refers to deionized water. Likewise, unless
otherwise indicated, all reference to PB in examples refers to 0.1
M phosphate buffer, pH, 8.00.
[0089] B. Preparation of Cross-Linker Solution
[0090] 8-arm PEG.sub.20kDa-[NHS].sub.8 (10 mg, Catalog # SUNBRIGHT
PTE-200GS, NOF America Corporation, White Plains, N.Y.) was weighed
in a centrifuge tube and dissolved in PB (100 .mu.L). The
Doxycycline (0.6 mg) was added to this solution and vortexed (<1
minutes) to make a clear solution.
[0091] C. Preparation of Polymer Solution Containing the
Nanocarrier
[0092] 8-arm PEG.sub.20kDa-[SH].sub.8 (Hexa-glycerine,
octa-(thioethylene)poly(ethylene glycol) ether)) (5 mg, Catalog #
SUNBRIGHT PTE-200SH, NOF America Corporation, White Plains, N.Y.)
was weighed in a centrifuge tube and dissolved in PB (100 .mu.L) by
vortexing for <1 minutes.
[0093] D. Preparation of Hydrogel (0.2 mL)
[0094] The cross-linker solution (100 .mu.L) containing the
nanocarrier was transferred to a glass vial (12.times.32 mm, SepCap
clear vial, Catalog #C4011-80, National Scientific Company,
Rockwood, Tenn.) followed by the polymer solution (100 .mu.L). The
solution mixture was allowed to stand at room temperature
(24.degree. C.). The hydrogel solution started becoming more and
more viscous after 30 Sec and ceased to flow from the inverted tube
in 48 Sec indicating the formation of hydrogel.
[0095] Hydrogel examples with passively entrapped drugs are
summarized in Table 2.
TABLE-US-00002 TABLE 2 Polymer/cross- Time for linker hydrogel
Cross-linker Polymer Nanocarrier composition formation PEG.sub.20
kDa- PEG.sub.20 kDa- Doxycycline 1:2 (10 and 60 minutes [NHS].sub.8
[SH].sub.8 20% w/v) PEG.sub.40 kDa- PEG.sub.20 kDa- Doxycycline 1:1
(5% w/v) 90 minutes [NHS].sub.8* [SH].sub.8 PEG.sub.20 kDa-
PEG.sub.20 kDa- Doxycycline 1:1 (7.5% w/v) 85 minutes [NHS].sub.8*
[SH].sub.8 PEG.sub.40 kDa- PEG.sub.20 kDa- Doxycycline 1:2 (5 and
10% 75 minutes [NHS].sub.8* [SH].sub.8 w/v) PEG.sub.20 kDa-
PEG.sub.20 kDa- Doxycycline 1:1 (10% w/v) 65 minutes [NHS].sub.8
[SH].sub.8 PEG.sub.40 kDa- PEG.sub.20 kDa- Doxycycline 1:1 (15%
w/v) 55 minutes [NHS].sub.8* [SH].sub.8 PEG.sub.40 kDa- PEG.sub.20
kDa- Doxycycline 1:2 (15 and 30 minutes [NHS].sub.8* [SH].sub.8 30%
w/v) *Hydrogels prepared using the procedure described in example
1.
Example 2
Preparation of Doxycycline Hydrogels Using 8 Arm
PEG-SH(H.sub.2O.sub.2)
[0096] Phosphate buffer is prepared as in example 1.
[0097] A. Preparation of Polymer Solution
[0098] 8-arm PEG.sub.20kDa-[SH].sub.8 (4 mg, Catalog # SUNBRIGHT
PTE-200SH, NOF America Corporation, White Plains, N.Y.) was weighed
in a centrifuge tube and dissolved in PB (100 .mu.L) by vortexing
for <1 minutes.
[0099] B. Preparation of Hydrogel (0.2 mL)
[0100] The polymer solution (100 .mu.L) containing the nanocarrier
was transferred to a glass vial (12.times.32 mm, SepCap clear vial,
Catalog #C4011-80, National Scientific Company, Rockwood, Tenn.)
followed by H.sub.2O.sub.2 solution (1.9 .mu.L) was added into the
polymer solution. The solution mixture was allowed to stand at room
temperature (24.degree. C.). The hydrogel solution started becoming
more and more viscous after 40 Sec and ceased to flow from the
inverted tube in 78 Sec indicating the formation of hydrogel.
[0101] Hydrogel examples with passively entrapped drugs are
summarized in Table 3.
TABLE-US-00003 TABLE 3 Total wt of Time for Polymer H.sub.2O.sub.2
polymers hydrogel (% w/v) Nanocarrier volume (.mu.l) (% w/v)
formation PEG.sub.20 kDa- Doxycycline 1.9 4 75 minutes [SH].sub.8
(4%) PEG.sub.40 kDa- Doxycycline 5.4 8 55 minutes [SH].sub.8* (8%)
*Hydrogels prepared using the procedure described in example 2.
Example 3
Preparation of Minocycline Hydrogel Using 8 Arm PEG-SH and 8 Arm
PEG NHS
[0102] Phosphate buffer is prepared as in example 1.
[0103] A. Preparation of Polymer Solution
[0104] 8-arm PEG.sub.20kDa-[SH].sub.8 (10 mg, Catalog # SUNBRIGHT
PTE-200SH, NOF America Corporation, White Plains, N.Y.) was weighed
in a centrifuge tube and dissolved in PB (100 .mu.L) by vortexing
for <1 minutes.
[0105] B. Preparation of Cross-Linker Solution Containing the
Nanocarrier
[0106] 8-arm PEG.sub.20kDa-[NHS].sub.8 (10 mg, Catalog # SUNBRIGHT
PTE-200GS, NOF America Corporation, White Plains, N.Y.) was weighed
in a centrifuge tube and dissolved in PB (100 .mu.L). The
Minocycline (0.6 mg,) was added to this solution and vortexed
(<1 minutes) to make a clear solution.
[0107] C. Preparation of Hydrogel (0.2 mL)
[0108] The cross-linker solution (100 .mu.L) containing the
nanocarrier was transferred to a glass vial (12.times.32 mm, SepCap
clear vial, Catalog # C4011-80, National Scientific Company,
Rockwood, Tenn.) followed by the polymer solution (100 .mu.L). The
solution mixture was allowed to stand at room temperature
(24.degree. C.). The hydrogel solution started becoming more and
more viscous after 30 Sec and ceased to flow from the inverted tube
in 48 Sec indicating the formation of hydrogel.
[0109] Hydrogel examples with passively entrapped drugs are
summarized in Table 4.
TABLE-US-00004 TABLE 4 Polymer to cross- Time for linker hydrogel
Cross-linker Polymer Nanocarrier ratio formation PEG.sub.20 kDa-
PEG.sub.20 kDa-[SH].sub.8 Minocycline 1:2 48 Sec. [NHS].sub.8
Example 4
Preparation of Minocycline Hydrogels Using 8 Arm PEG-SH and 8 Arm
PEG TP
[0110] Phosphate buffer and polymer solutions, were prepared as in
example 1.
[0111] A. Preparation of Cross-Linker Solution Containing the
Nanocarrier
[0112] 8-arm PEG.sub.20kDa-[TP].sub.8 (10 mg) was weighed in a
centrifuge tube and dissolved in PB (100 .mu.L)). The Doxycycline
(0.6 mg,) was added to this solution and vortexed (<1 minutes)
to make a clear solution.
[0113] B. Preparation of Hydrogel (0.2 mL)
[0114] The cross-linker solution (100 .mu.L) containing the
nanocarrier was transferred to a glass vial (12.times.32 mm. SepCap
clear vial, Catalog # C4011-80, National Scientific Company,
Rockwood, Tenn.) followed by the polymer solution (100 mL). The
solution mixture was allowed to stand at room temperature
(24.degree. C.). The hydrogel solution started becoming more and
more viscous after 30 Sec and ceased to flow from the inverted tube
in 48 Sec indicating the formation of hydrogel.
[0115] Hydrogel examples with passively entrapped drugs are
summarized in Table 5.
TABLE-US-00005 TABLE 5 Polymer to cross- Time for Cross-linker
Polymer linker hydrogel (%, w/v) (%, w/v) Nanocarrier ratio
formation 8 arm-PEG.sub.20 kDa- 8 arm-PEG.sub.20 kDa- Doxycycline
1:1 <10 sec. [NHS].sub.8 (5%) [SH].sub.8 (5%) 8 arm-PEG.sub.20
kDa- 8 arm-PEG.sub.20 kDa- Doxycycline 1:1 <10 sec. [NHS].sub.8*
(8%) [SH].sub.8 (8%) *Hydrogels prepared using the procedure
described in example 4.
Example 5
Optical Transmission Properties of Hydrogels
[0116] Phosphate buffer, cross-linker solution, polymers and
hydrogels were prepared as it in Example 1.
[0117] The hydrogels were screened for their potential application
as drug delivery systems for corneal wound repair. Different
hydrogel compositions [5% (1:1), 7.5% (1:1); 7.5% (1:2); 10% (1:1);
15% (1:1); 15% (1:2); and 22.5% (1:2)] were analyzed for their
Optical transmission (OT) properties. The hydrogels (200 .mu.L)
were placed in a quartz cuvette containing distilled water and
transmission of light was measured at 480 nm using a UV-Vis
spectrophotometer. A cuvette containing only distilled water was
used as reference. All OT studies were done in triplicate and the
mean SEM reported. One-way analysis of variance (ANOVA) was used to
determine the effect of hydrogel composition on its OT. Hydrogels
with OT 90% were classified as transparent; those in the 10-90%
range were classified as translucent, and those 10% as opaque.
[0118] The % OT of various hydrogels is shown in FIG. 2 and as can
be seen from the figure, all hydrogels used in this study are
transparent. It was also observed that a change in hydrogel
composition produces a statistically significant effect (p<0.05)
on their OT properties. An increase in the concentration of
8-arm-PEG-SH and/or 8-arm-PEG-NHS resulted into a slight decrease
in the transparency of the hydrogels. The transparent
characteristic of these hydrogels could be beneficial for their use
as ocular drug delivery systems.
Example 6
Rheology of Hydrogels
[0119] Phosphate buffer, and cross-linker solution, polymers and
hydrogels were prepared as in Example 1.
[0120] The rheological measurements of hydrogels (prepared in
example 1) [5% (1:1), 7.5% (1:1), 7.5% (1:2), 15% (1:1) and 15%
(1:2)] were performed using a rheometer with cone plate geometry at
37.degree. C. (plate diameter: 25 mm, gap: 3 mm, 2.degree. angle).
The hydrogel samples were equilibrated on the plate for 5 min to
reach the running temperature before each measurement. The
viscoelastic properties of the hydrogels were evaluated by strain
sweep test (FIG. 3) and frequency sweep test (FIG. 4). Both tests
are used to obtain the rheological parameters G' (storage/elastic
modulus), G'' (loss/viscous modulus) and loss tangent/phase angle
(tan .delta.=G''/G'). G' represents the elastic storage of energy
and is a measure of how well-structured a hydrogel is. G''
represents the viscous energy dissipation and changes depending on
the viscosity of the hydrogel.
[0121] The strain sweep test results suggest that G' dominates in
both the formulations and this is supported by the results obtained
from the frequency sweep test. Since G' was one order higher than
G'', the hydrogels are more elastic than viscous in the
investigated frequency range. FIGS. 2 and 3 also show that G' is
independent of frequency and strain whereas G'' is weakly dependent
on both. The hydrogels crosslinked in a 1:2 ratio have slightly
higher G' and G'' than hydrogels crosslinked in a 1:1 ratio. This
can be attributed to the formation of denser and stronger
crosslinking networks in 1:2 hydrogels.
[0122] A change in hydrogel composition resulted in a statistically
significant effect (p<0.001) on the mechanical strength of the
hydrogels. The hydrogels containing higher concentrations of
polymers [15% (1:1) and (1:2)] showed a higher and constant G'
under increasing frequency, suggesting that the hydrogels have the
ability to resist structural changes under strain. The small tan
.delta. values indicate that G' is the dominant feature in all the
hydrogels and that variations in hydrogel composition do not result
in extreme variations in rheological parameters. The rheological
data show that the hydrogels have good viscoelastic properties,
which might help prolong their ocular residence time and prevent
structural break-age. An increased contact time in turn may lead to
an increased duration of pharmacological response.
Example 7
Hydrogels Swelling Studies
[0123] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0124] The degree of swelling for different NHS hydrogels (prepared
in example 1) [5% (1:1); 7.5% (1:1); 7.5% (1:2); 10% (1:1); 15%
(1:1); 15% (1:2); and 22.5% (1:2)] was measured. Hydrogels were
placed in a vial and weighed (initial weight) prior to being
immersed in PBS (pH 7.4) and placed in an incubator at 37.degree.
C. The degree of swelling of the hydrogels was calculated by
weighing the vials after removing the PBS at predetermined time
intervals. The buffer was replaced after every measurement and the
hydrogels were allowed to swell until equilibrium is reached. FIG.
5 shows the degree of swelling expressed as percent swelling
plotted against time for 5% (1:1), 7.5% (1:1), 7.5% (1:2), 10%
(1:1), 15% (1:1), 15% (1:2) and 22.5% (1:2) hydrogels. The
hydrogels in this study showed a relatively lower degree of
swelling (<7%).
[0125] The hydrogels crosslinked in a 1:1 ratio initially swelled
rapidly, and then gradually reached equilibrium. Furthermore, the
hydrogels crosslinked in a 1:2 ratio showed a much lower degree of
swelling (<3%) than hydrogels crosslinked in a 1:1 ratio
(<7%). A change in hydrogel composition resulted in a
statistically significant effect (p<0.001) on the degree of
swelling. Hence, a smaller pore size of the hydrogels obtained from
increasing the polymer concentration or crosslinking ratio results
in a lower degree of hydrogel swelling.
Example 8
Drug Loading Efficiency of Hydrogels
[0126] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0127] The 10% (1:1), 15% (1:1), 15% (1:2), and 22.5% (1:2) NHS
hydrogels (prepared in example 1) loaded with 0.25% w/v of
doxycycline were used for drug loading and release studies. The
hydrogels were dissected into small pieces and suspended in 5 mL
PBS (pH 7.4). The suspension was sonicated for 30 min to completely
extract doxycycline from the hydrogel. The amount of doxycycline
extracted was quantified by RP HPLC analysis at a wavelength of 350
nm. 0.01M oxalic acid, acetonitrile and methanol (70:18:12) were
used as mobile phase at a flow rate of 1 mL/min.
[0128] After extraction, suspension containing the hydrogel was
stored for several days at 4.degree. C. and then reanalyzed to
ensure the complete extraction of doxycycline from the hydrogel.
Doxycycline was stable under the storage conditions, as determined
by HPLC analysis. Doxycycline loading efficiency results show that
22.5% (1:2), 15% (1:2), 15% (1:1) and 10% (1:1) hydrogels resulted
in doxycycline loading efficiencies of 44.7, 47.5, 51.4 and 48.2%,
respectively. Higher drug loading efficiency was observed when
equivalent ratios of the polymers were used.
Example 9
In Vitro Drug Release from Hydrogels
[0129] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0130] In vitro release of doxycycline from the NHS hydrogels were
studied on a Franz diffusion cell apparatus with a diameter of 5 mm
and a diffusion area of 0.636 cm.sup.2. A polycarbonate membrane
(0.4.mu.) was sandwiched between the lower cell reservoir and the
glass cell-top containing the sample for doxycycline release
studies. The receiving compartment (volume 5.1 mL) was filled with
PBS (pH 7.4). The system was maintained at 37.degree. C. using a
circulating water bath and a jacket surrounding the cell. The
receiving medium was continuously stirred (600 rpm) with a magnetic
bar to avoid stagnant aqueous diffusion layer effects. 200 .mu.L
sample of each hydrogel formulation containing 0.25% w/v
doxycycline was prepared and placed in the donor compartment, which
was then sealed with parafilm and aluminum foil to prevent
evaporation.
[0131] Aliquots (200 .mu.L) were collected from the receiver
compartment at predetermined intervals and replaced with equal
volume of PBS to maintain sink conditions throughout the study. The
concentration of doxycycline in the release medium was determined
using RP HPLC. The cumulative amount of doxycycline released from
the hydrogel was determined using a calibration curve. A plot of
cumulative amount of doxycycline released (.mu.g/cm.sup.2) as a
function of time (h) (FIG. 6) demonstrates that doxycycline
entrapped in the hydrogel shows sustained drug release for about 7
days (168 h) with 80 to 100% of doxycycline being released from
different formulations. From FIG. 5, it appears that as the total
concentration of the polymers increased in the hydrogels, the
release of doxycycline was sustained. Also, as the crosslinking
density increases from 1:1 to 1:2 in the hydrogels, a slower
sustained doxycycline release was observed.
Example 10
Treatment of CEES (Half Mustard, 2-Chloroethyl Ethyl Sulfide) and
NM (Nitrogen Mustard, Bis-Chloroethyl-Methyl-Amine) Using Rabbit
Corneas
[0132] A rabbit cornea organ culture model system adapted from
Foreman, et al., Exp. Eye. Res., 62(5), 555-564 (1996) was used to
evaluate healing after exposure to model vesicants CEES and NM,
followed by subsequent treatment with doxycycline drops or
doxycycline hydrogels. Rabbit eyes were stored in DMEM (with
penicillin, streptomycin, amphotericin B and gentamicin) and
transported to the laboratory on ice. Corneas with a surrounding 2
mm scleral rim were dissected from the eye and placed with the
epithelial side facing down into spot plates containing a small
amount of DMEM to prevent drying of the epithelium. The corneal
endothelial concavity was then filled with DMEM containing 0.75%
agar at 50.degree. C. and this mixture was allowed to set (usually
within 1 min).
[0133] Corneas were then inverted and transferred to 60 mm sterile
tissue culture dishes and cultured at 37.degree. C. in a humidified
5% CO.sub.2 incubator in the presence of medium (500 mL high
glucose DMEM, 5 mg ciprofloxacin, 5 mL of 100.times.MEM-NEAA, 5 mL
RPMI 1640 vitamin solution and 50 mg ascorbic acid). To moisten the
epithelium, 500 L of medium was added drop wise to the surface of
the corneal epithelium every 8 h. The level of medium in dishes was
allowed to rise only to the corneal-scleral rim. All agents were
added drop wise to the central cornea. Either 200 nmoles CEES
(dissolved first in absolute ethanol and then DMEM) or 100 nmoles
NM (dissolved first in saline and then DMEM) were applied onto the
cornea and allowed to remain there unwashed for 2 h. The 2 h time
period approximately simulates the time that would pass before an
exposure is recognized (based on the delayed times for tearing and
pain) and medical help is secured.
Example 11
Permeation of Doxycycline Through Vesicant Exposed Rabbit's
Cornea
[0134] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0135] Corneas untreated with either of the above vesicants (CEES
or NM; example 10) were used as controls. After 2 h, the corneas
were placed horizontally on the receptor compartment with the
endothelial surface facing the receiver compartment of the Franz
diffusion cell set up. The donor half cell was carefully placed on
top of the receptor half cell and clamped. 200 L sample of 15%
(1:2) hydrogel encapsulating 0.25% w/v doxycycline was placed in
the donor compartment. Aliquots (200 .mu.L) were collected from the
receiver compartment at predetermined intervals and replaced with
equal volume of PBS to maintain sink conditions through out the
study. The concentration of doxycycline in the release medium was
determined using a RP HPLC as described above. The cumulative
amount of doxycycline permeated through the corneas was determined
using a calibration curve. All permeation experiments were done in
triplicate and the results reported as mean SEM. Two way ANOVA was
used to determine the statistical significance of permeation
between different treatment groups.
[0136] The permeation profiles of doxycycline through CEES and NM
exposed corneas were evaluated for 24 h using a Franz diffusion
cell apparatus. FIG. 7 shows a plot of the cumulative amount of
doxycycline permeated (.mu.g/cm.sup.2) as a function of time (h).
The permeability of doxycycline through CEES and NM exposed corneas
was significantly higher than untreated corneas (p<0.0001) by
2.5 to 3.3 fold. The cumulative amount of doxycycline permeated
through vesicant-exposed corneas is almost equal to the cumulative
amount released in 24 h, which verifies that corneal epithelium no
longer acts as a barrier for permeation of drugs after
exposure.
Example 12
Wound Healing Efficacy of Doxycycline PEG Hydrogels on Corneas
Exposed to CEES and NM
[0137] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0138] The CEES or NM-exposed corneas (Example 10) were incubated
for 2 h at 37.degree. C. Medium was replaced with fresh medium
after 2 h and then each cornea was treated with doxycycline.
Doxycycline solution (2M in 50 .mu.L) was added drop wise to the
central cornea 3 times over the subsequent 24 h, whereas 15% (1:2)
doxycycline hydrogel (6M in 50 .mu.L) was applied once. After 24 h,
the corneas were put in cryomolds containing Tissue-Tek O.C.T.
compound with the epithelial side facing down and placed on ice for
15 min before snap freezing them in liquid nitrogen. Corneas were
stored at -80.degree. C. until sectioned for histology and
immunofluorescence (IF) analysis. The 10 m corneal sections were
stained using a modified Hematoxylin & Eosin (H & E)
staining method. The corneal sections were fixed in a Pen-Fix
solution for 60 s, stained with H & E, dehydrated through
graded alcohols, immersed in xylene and covered with a cover slip.
Digital images were captured with a light microscope at 40.times.
magnification.
[0139] The hydrogel formed a thin transparent film, and likely
because of its high water content, the hydrogel was retained in
place for the entire duration of the study (24 h). The histology of
the control cornea showed an epithelium with normal thickness and
an intact stroma with corneal keratocytes separated by
extracellular matrix. The controls treated with hydrogel (not
shown) and doxycycline hydrogel were very similar to the controls
demonstrating that the hydrogel did not cause damage to the cornea.
The CEES-exposed corneas exhibited a loss of distinctness of the
epithelial-stromal border with frequent dipping into the stroma
(also known as pitting). This visible damage, seen in foci
throughout the cornea at the epithelial-stromal junction was
expected, since this is the known target area of vesicants. In
addition, the cells of the anterior stroma were swollen.
[0140] CEES-exposed corneas treated with doxycycline in solution
have an epithelial-stromal border that appears more normal when
compared to those without treatment. The epithelial cell layer
demonstrated less pitting, looking more like control tissues.
CEES-exposed corneas treated with doxycycline hydrogel were similar
to those treated with doxycycline solution, and thus were also much
more normal in appearance than CEES-exposed corneas. The flattening
of the epithelial-stromal border suggests that these corneas are
perhaps more like controls than the CEES-exposed corneas treated
with doxycycline solution. CEES causes mild damage, and therefore
the difference in corneal wound healing efficacy between
doxycycline solution and the doxycycline hydrogel would be expected
to be minimal.
[0141] The most significant differences that were seen indicated
that the hydrogel ameliorated pitting. The micrographs confirm the
data from FIG. 8 showing that vesicant exposure damages the cornea
and doxycycline treatment acts to maintain the basement membrane
zone integrity. Severe damage to the epithelium with NM causes
epithelial cell sloughing, epithelial cell dissociation and
pitting. The epithelium is detached from the stroma and the
epithelial cells are separated, apparently having lost their cell
to cell junctions. Where the epithelium is still attached, the
basal cell nuclei appear to be more distant from the stroma than in
controls.
[0142] When treated with doxycycline in solution for 24 h after NM
exposure, the epithelial-stromal border is somewhat improved.
However there are still many areas where the epithelium is detached
from the stroma and in areas where the epithelium and stroma are
still attached, the basal cell nuclei were more distant from the
stroma than in controls. For NM-exposed samples treated with
doxycycline hydrogel, the epithelium remains attached to the
basement membrane in most areas and shows a significant improvement
in the appearance of the epithelial-stromal border. Doxycycline in
solution probably did not show superior efficacy because drop wise
application on a curved surface would favor a low retention time
and only a small percentage of the doxycycline would be expected to
remain in the wound area. The PEG doxycycline hydrogel on the other
hand showed a great improvement over the NM exposed corneas.
Example 13
Detection of MMP-9 in Vesicant Exposed and Treated Corneas by
Immunofluorescence
[0143] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0144] The sectioned corneas (10 m) were fixed in 100% methanol for
10 min at -20.degree. C. After rinsing with PBS nonspecific binding
was blocked with 5% normal goat serum for 1 h. The blocking agent
was removed and the sections were incubated with primary mouse anti
human MMP-9 monoclonal antibodies (1:400) overnight at 4.degree. C.
Sections were blotted and washed four times with PBS/Tween and
incubated for 1 h at RT in the dark with Alexa-Fluor 488-conjugated
goat anti-mouse IgG secondary antibodies (1:1000). The sections
were washed with PBS/Tween, counterstained with DAPI for 5 min,
mounted with Prolong gold and cover slipped. Negative controls
replaced primary antibodies with PBS. Digital epifluorescent images
were captured from a light microscope at 494 nm excitation and 517
nm emission and acquired at 10.times. magnification.
[0145] The IF staining of corneas exposed to CEES and NM, and
subsequently treated with doxycycline either in solution or
hydrogel for 24 h are shown in FIG. 9. In the controls, a very
small amount of MMP-9 staining (green) is seen under the basal
epithelial cells in the basement membrane zone. The staining at the
apical epithelial cells is typical of the corneal epithelium's
auto-fluorescence. Nuclei were stained blue. For CEES exposed
corneas, there is a moderate increase in MMP-9 staining observed in
the basement membrane zone, apical cells and a small amount
throughout the epithelium. For CEES-exposed corneas subsequently
treated with doxycycline in solution, there is a slight, if any,
decrease in staining. However, applying a doxycycline hydrogel
after CEES exposure reduces immuno-reactivity in the basement
membrane zone and returning the sub epithelial expression to its
original low MMP-9 levels.
[0146] For NM-exposed cornea, a drastic increase in MMP-9 staining
was observed at the basement membrane zone, reflecting the greater
wounding by of NM. The corneas exposed to NM, then treated with
doxycycline in solution showed a less intense level of
fluorescence. However, in this treatment group, there remained many
areas where the epithelial cells were totally detached from the
stroma. In this case there were few epithelial cells to secrete
MMP-9 and thus there was a lower intensity of fluorescence in those
areas. For NM-exposed samples treated with doxycycline hydrogel,
the fluorescence was significantly less intense than NM exposure
without treatment. Most areas show the epithelium to be in contact
with the stroma. Hence intervention of MMP-9 activity with
doxycycline hydrogels should be pursued as a potential treatment
option for healing of mustard injuries in the eye.
Example 14
Wound Healing Efficacy of Doxycycline PEG Hydrogels on Corneas
Exposed to Sulfur Mustered (SM)
[0147] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0148] Doxycycline hydrogel (25 .mu.L) solution was applied to the
SM exposed rabbit eyes (in the cul de sac). After different time
points (1 day, 3 day . . . 28 days) (FIG. 10) the corneas were put
in cryomolds containing Tissue-Tek O.C.T. compound with the
epithelial side facing down and placed on ice for 15 min before
snap freezing them in liquid nitrogen. Corneas were stored at
-80.degree. C. until sectioned for histology and immunofluorescence
(IF) analysis. The 10 m corneal sections were stained using a
modified Hematoxylin & Eosin (H & E) staining method. The
corneal sections were fixed in a Pen-Fix solution for 60 s, stained
with H & E, dehydrated through graded alcohols, immersed in
xylene and covered with a cover slip. Digital images were captured
with a light microscope at 40.times. magnification (FIG. 11). H
& E data showed that doxycycline hydrogels improved wound
healing efficacy compared to the controls, untreated, doxycycline
eye drops and placebo hydrogel treated groups after SM exposure,
evidenced by increased survival rates and signs of wound
healing.
Example 15
Wound Healing Efficacy of Minocycline Drops on Corneas Exposed to
NM Preparation of Organ Cultures
[0149] Eyes arrived from the vendor (Pelfreez, Ark.) in DMEM+
Penicillin streptomycin, Gentamicin and Amphotericin B. The Corneas
were dissected from eyes with about 2-3 mm of scleral rim. Some
corneas were directly prepared for frozen sections to serve as
controls. Other corneas were prepared for organ culture. These were
laid epithelial side down into rounded wells of (Coors) spot plates
wetted with a small amount of DMEM medium, then filled with 0.75%
agar (Sigma) in 1.times.DMEM (Gibco). After the agar hardened, the
cornea was flipped into a 60 mm glass petri dish (Pyrex), putting
the epithelial side up. Medium containing high glucose DMEM,
MEM-NEAA, RPMI 1640, Ciprofloxacin and ascorbic acid was applied to
the dish up to the corneal-scleral rim. Corneas were allowed to
stabilize in the 37.degree. C. culture incubator overnight. During
this time, medium was replaced twice. All additions (whether
medium, NM and counteragents) were added via pipette onto the
central cornea. The following day corneas were for the experimental
set up, receiving exposure to NM as a test cornea, or medium as a
control.
[0150] A. Preparation of Minocycline Drops
[0151] Minocycline were prepared as eyedrops at 200 mM
concentrations to 1 mg of Minocycline was dissolved in 10 ml
teargen ophthalmic solution and vortex for 1 min.
[0152] B. Application of Minocycline Drops
[0153] Minocycline solution (2M in 50 .mu.L) was added drop wise to
the NM exposed central cornea 3 times over the subsequent 24 h.
After 24 h, the corneas were put in cryomolds containing Tissue-Tek
O.C.T. compound with the epithelial side facing down and placed on
ice for 15 min before snap freezing them in liquid nitrogen.
Corneas were stored at -80.degree. C. until sectioned for histology
and immunofluorescence (IF) analysis. The 10 m corneal sections
were stained using a modified Hematoxylin & Eosin (H & E)
staining method. The corneal sections were fixed in a Pen-Fix
solution for 60 s, stained with H & E, dehydrated through
graded alcohols, immersed in xylene and covered with a cover slip.
Digital images were captured with a light microscope at 40.times.
magnification (FIG. 11).
Example 16
Wound Healing of Minocycline PEG Hydrogels on Corneas Exposed to
NM
[0154] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1.
[0155] A. Wound Healing Efficacy of Minocycline PEG Hydrogels
[0156] The procedure for the preparation of phosphate buffer, and
NM exposure was used as it is, as mentioned in example 3.
[0157] B. Application of Minocycline Hydrogels on NM Exposed
Central Cornea
[0158] Minocycline hydrogel (200 .mu.L) solution was applied drop
wise to the NM exposed central cornea. After 24 h the corneas were
put in cryomolds containing Tissue-Tek O.C.T. compound with the
epithelial side facing down and placed on ice for 15 min before
snap freezing them in liquid nitrogen. Corneas were stored at
-80.degree. C. until sectioned for histology and immunofluorescence
(IF) analysis. The 10 m corneal sections were stained using a
modified Hematoxylin & Eosin (H & E) staining method. The
corneal sections were fixed in a Pen-Fix solution for 60 s, stained
with H & E, dehydrated through graded alcohols, immersed in
xylene and covered with a cover slip. Digital images were captured
with a light microscope at 40.times. magnification (FIG. 12).
Example 17
Wound Healing Efficacy of NDH4417 Drug Drops on Corneas Exposed to
NM
[0159] Phosphate buffer preparation, NM exposure and preparation of
organ cultures was performed as in example 13.
[0160] A. Preparation of NDH-4417 Drops
[0161] NDH4417 were prepared as eye drops at 200 .mu.L
concentration. 0.62 mg of NDH 4417 was dissolved in 50 ul DMSO
(final concentration of 0.5%, maximum amount okay for cells). The
dissolved compound in DMSO was vortexed and coated around the vial.
10 ml Teargen ophthalmic solution was added rapidly via pipette
while vortexing.
[0162] B. Application of NDH4417 Drops
[0163] Drug solution (2M in 50 .mu.L) was added drop wise to the NM
exposed central cornea three times over the subsequent 24 h. After
24 h, the corneas were put in cryomolds containing Tissue-Tek
O.C.T. compound with the epithelial side facing down and placed on
ice for 15 min before snap freezing them in liquid nitrogen.
Corneas were stored at -80.degree. C. until sectioned for histology
and immunofluorescence (IF) analysis. The 10 m corneal sections
were stained using a modified Hematoxylin & Eosin (H & E)
staining method. The corneal sections were fixed in a Pen-Fix
solution for 60 s, stained with H & E, dehydrated through
graded alcohols, immersed in xylene and covered with a cover slip.
Digital images were captured with a light microscope at 40.times.
magnification (FIG. 13).
Example 18
Wound Healing of Olvanil RetroOH-8 (NDH-4417) Drugs PEG Hydrogels
on Corneas Exposed to NM
[0164] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 1 and as in example 13.
[0165] A. Preparation of Drug Solution
[0166] NDH4417 (8.5 mg) was dissolved in 250 .mu.L DMSO (final
concentration of 0.5%, maximum amount okay for cells).
[0167] B. Preparation of Hydrogel (0.2 mL)
[0168] The polymer solution (100 .mu.L) containing the nanocarrier
was transferred to a glass vial (12.times.32 mm, SepCap clear vial,
Catalog # C4011-80, National Scientific Company, Rockwood, Tenn.)
followed by drug solution (1 .mu.L). The cross-linker solution (99
.mu.L) was transferred to a polymer and drug solution. The solution
mixture was allowed to stand at room temperature (24.degree. C.).
The hydrogel solution started becoming more and more viscous after
25 Sec and ceased to flow from the inverted tube in 45 Sec
indicating the formation of hydrogel.
[0169] C. Application of NDH4417 Hydrogels on NM Exposed Central
Cornea
[0170] NDH4417 hydrogel (200 .mu.L) was applied to the NM exposed
central cornea. After 24 h the corneas were put in cryomolds
containing Tissue-Tek O.C.T. compound with the epithelial side
facing down and placed on ice for 15 min before snap freezing them
in liquid nitrogen. Corneas were stored at -80.degree. C. until
sectioned for histology and immunofluorescence (IF) analysis. The
10 m corneal sections were stained using a modified Hematoxylin
& Eosin (H & E) staining method. The corneal sections were
fixed in a Pen-Fix solution for 60 s, stained with H & E,
dehydrated through graded alcohols, immersed in xylene and covered
with a cover slip. Digital images were captured with a light
microscope at 40.times. magnification (FIG. 14).
Example 19
Minocycline PEG Additive Hydrogels for Mice Back Model
[0171] Phosphate buffer, cross-linker solution, polymers, and
hydrogels were prepared as in Example 3
[0172] A. Preparation of Hydrogel (0.2 mL)
[0173] The cross-linker solution (100 .mu.L) containing nanocarrier
(0.25% minocycline) was transferred to a glass vial (12.times.32
mm, SepCap clear vial, Catalog # C4011-80, National Scientific
Company, Rockwood, Tenn.), followed by polymer solution (100 .mu.L)
containing additives 5% v/v glycerin, 4% w/v PVP and 5% v/v PEG
600. The solution mixture was allowed to stand at room temperature
(24.degree. C.). The hydrogel solution started becoming more and
more viscous after 30 Sec and ceased to flow from the inverted tube
in 48 Sec indicating the formation of hydrogel.
[0174] Hydrogel solution was applied on the back of the SKH-1 mice
after 30 sec (once solution is viscous). It was observed that after
4 h hydrogel stays on the skin (FIG. 15). After 20 h it was
observed hydrogel disappears.
Example 20
Dose and Time Dependent Effects of NM on Mouse Skin
[0175] A SKH-1 hairless mouse model was used to assess NM dermal
wound progression in vivo. NM dissolved in acetone was applied
topically to the dorsal skin of mice and left in the hood overnight
to degas. A standard circular template (15 mm) was used to ensure
uniform exposure area of NM on all mice. For the dose response
study, topical wounds were created by application of 5, 25, 50, 75
and 100 .mu.mmoles NM. The mice were euthanized by CO.sub.2 gas and
punch biopsies were collected at 24 h after exposure to evaluate
the dose dependent effects of NM on dermal wound formation. The
dose of NM at which a lesion forms and mice survive for at least
168 h was chosen for time response, inflammatory biomarkers and
permeation studies. For the time response study, topical wounds
were created by application of 5 .mu.moles of NM (which was based
on the results of the dose response study) and punch biopsies were
collected from the wounded skin at 0, 24, 72 and 168 h. The skin
samples were fixed in 10% formalin and histology evaluated by
H&E staining. Both the dose and time response treatments
included five mice in each group.
[0176] Histology of NM exposed mouse skin indicated a dose and time
dependent wound progression (FIGS. 16 and 17). A dose dependent
investigation was conducted at 24 h after NM administration (FIG.
16). All mice exposed to NM at doses higher than 5 .mu.moles
survived for less than a week demonstrating that NM is extremely
toxic when applied topically. NM induced wound progression was
monitored for upto 168 h after exposure (FIG. 17). Histology of
skin samples obtained 24 h after NM exposure, mainly showed edema,
pyknotic nuclei in the epidermis, inflammatory infiltration and
discontinuity of the skeletal muscle. Indeed, in FIG. 16, which
provides histological slides illustrating the dose dependent
effects of NM exposure on mouse skin visualized by H & E
staining (10.times.), the arrows in B denote pyknotic nuclei in
epidermis and discontinuous skeletal muscle in the dermis. Arrows
in C, D, E and F denote pyknotic nuclei in epidermis, separation of
epidermis from dermis and discontinuous skeletal muscle in dermis.
The separation of epidermis from the dermis progresses in a dose
dependent manner from 5-100 .mu.moles. In FIG. 17, the arrows in B
denote pyknotic nuclei in epidermis and discontinuous skeletal
muscle in the dermis. Arrows in C denote absence of nuclear
staining in the epidermis, separation of epidermis from dermis and
discontinuous skeletal muscle in dermis. Arrows in D denote dermal
necrosis with, reepithelization from the wound edges characterized
by epidermal hyperplasia and hyperkeratosis.
[0177] In the skin samples obtained 72 h after NM exposure, edema,
separation of the epidermis from the dermis, death of the epidermal
cells (no nuclear staining) and discontinuity of the skeletal
muscle was observed. At 168 h after NM exposure, reepithelization
in the epidermis (a sign of wound healing) from the wound edges was
observed along with necrosis in the dermis. The results of the time
response study indicate that the NM (5 .mu.moles) induced wound
progresses from 0-72 h and wound healing initiates between 72-168 h
in SKH-1 mice.
Example 21
Determine the Significance of NM Exposure Time on Edema
[0178] Punch biopsies from the dorsal wounded skin of mice were
collected at 0, 24, 72 and 168 h. The weight of the tissue was
measured and the results were reported as mean.+-.SEM of five mice
in each treatment group. One-way analysis of variance (ANOVA) was
used to determine the significance of NM exposure time on edema.
The tissue weight of biopsy from NM exposed skin was measured to
determine the extent of edema. FIG. 18 indicates that dermal edema
is dependent on the duration of NM exposure. A significant increase
(p<0.05) in tissue weight compared to the control was observed
at 24, 72 and 168 h after exposure to NM, demonstrating marked
edema in a time dependent manner, with edema being highest at 24
h.
Example 22
Determine the Significance of NM Exposure Time on mRNA Levels of
the Inflammatory Biomarkers IL-1.beta. and TNF-.alpha.
[0179] The punch biopsy samples from the wounded skin of mice
collected at 0, 24, 72 and 168 h were snap frozen in liquid
nitrogen and stored at -80.degree. C. until further analysis of
RNA. The results were reported as mean.+-.SEM of five mice in each
treatment group. One way ANOVA was used to determine the
significance of NM exposure time on mRNA levels of the inflammatory
biomarkers IL-1.beta. and TNF-.alpha..
[0180] The RNA isolation and reverse transcription were performed
using methods known in the art. Briefly, total RNA was isolated
from the tissue samples using TRIzol reagent, according to the
manufacturer's instructions. Eppendorf phase lock gel, a product
that eliminates interface-protein contamination during phenol
extraction and was added during centrifugation. The RNA pellet was
dissolved in RNA storage solution and RNA quantitated
spectrophometrically at 260 nm. Total RNA (1 .mu.g) was
reverse-transcribed into cDNA using a High Capacity cDNA Reverse
Transcription Kit for reverse transcriptase-polymerase chain
reaction (RT-PCR). A minus reverse transcriptase reaction was used
as a control.
[0181] The mRNA levels of IL-1.beta. and TNF-.alpha. were measured
from cDNA samples by TaqMan gene expression assay. To compensate
for variations in input RNA amounts, and efficiency of reverse
transcription, GAPDH an endogenous control gene was also
quantified, and results were normalized to these values.
[0182] FIG. 19 shows the mRNA levels of IL-1.beta. and TNF-.alpha.
in NM exposed skin. The mRNA levels of both the inflammatory
markers increased at 24, 72 and 168 h after NM exposure relative to
control skin, with peak activity at 168 h. Only the mRNA levels of
IL-1.beta. at 168 h after NM exposure were found to be
significantly higher than the control (p<0.05).
Example 23
Measurement of Permeability of Molecular Markers Through NM Exposed
Skin
[0183] The damage caused by vesicant exposure on the integrity and
barrier properties of skin was evaluated by measuring skin
permeability, at various time points following NM exposure. [3H]
mannitol (molecular weight=182.17) was used as a hydrophilic
marker; FD-4, FD-10, FD-20 and FD-40 were used as molecular weight
markers; Rhodamine 123 (molecular weight=380.82) was used as a
lipophilic marker. The permeability profiles were evaluated on mice
skin collected at 0 (controls), 24, 72 and 168 h after exposure to
NM.
[0184] Permeability studies were carried out using a Franz
diffusion cell apparatus (diameter of 5 mm and diffusional area of
0.636 cm.sup.2) as previously described by Anumolu et al. (Anumolu
et al., 2009). Mouse skin was excised and placed in PBS (Phosphate
buffered saline, pH 7.4) for 1 h prior to being sandwiched between
the lower cell reservoir and the glass cell-top. The receiving
compartment (volume 5.1 mL) was filled with PBS and the medium was
continuously stirred (600 rpm) with a magnetic bar to avoid
stagnant aqueous diffusion layer effects. The system was maintained
at 37.degree. C. using a circulating water bath and a jacket
surrounding the cell. 200 .mu.L of each marker in PBS was placed in
the donor compartment, which was then sealed with parafilm and
aluminum foil to prevent evaporation. Aliquots (200 .mu.L) were
withdrawn from the receiver compartment at predetermined intervals
and replaced with equal volume of PBS to maintain sink conditions
through out the study. The specific activity of [3H] mannitol
permeated was measured with a LSC. The amounts of FD-4, FD-10,
FD-20, FD-40 and rhodamine 123 markers that permeated through the
skin samples were measured using a fluorescent plate reader (Ex=485
nm; Em=520 nm). All permeability studies were conducted in
triplicate and the results were reported as the mean.+-.SEM.
Student's t-test was used to determine the effect of NM exposure
time on the permeation of molecular markers. FIGS. 20, 21 and 22
show the cumulative amount of molecular marker permeated
(cpm/cm.sup.2 or ng/cm.sup.2) as a function of time (h).
[0185] The permeation of the hydrophilic marker 3[H] mannitol
increased significantly through mice skin exposed to NM for various
time periods compared to the control (FIG. 20 which provides the
permeation profile of 3[H] mannitol (hydrophilic marker) through NM
exposed mouse skin.). The order of permeation of mannitol is 72
h>168 h>24 h>control (table 6). The effect of molecular
weight on permeability through intact and NM damaged skin was
studied using different molecular weight markers of FITC-dextran's
(FIG. 21). Irrespective of their molecular weight, the
FITC-dextran's, at all time points post NM exposure exhibited a
significant increase in permeation compared to control skin (Table
6). The order of permeation of FD-4, FD-10, FD-20 and FD-40 is 72
h>168 h>24 h>control. The permeability coefficients of
FITC dextran's through control and NM exposed skin declined with
increasing molecular weight (FD-4>FD-10>FD-20>FD-40)
demonstrating that permeability through skin is molecular weight
dependent. The permeability of rhodamine 123 through control skin
is much higher than that of the other molecular markers evaluated
in the current study. The order of permeation of the lipophilic
marker rhodamine 123 through NM exposed skin is 24 h>168 h>72
h>control (FIG. 22). All the molecular markers showed an
increased permeation through NM exposed skin compared to the
control, which demonstrates that the barrier property is
compromised in vesicant exposed skin.
TABLE-US-00006 TABLE 6 Permeability coefficients of different
molecular markers through skin exposed to NM for 0, 24, 72 and 168
h Permeability coefficient (cm h.sup.-1) .times. 10.sup.-6
Molecular Control (No 24 h after NM 72 h after NM 168 h after NM
marker NM exposure exposure exposure exposure 3[H] mannitol 27.45
.+-. 2.47 96.36 .+-. 13.68 1091 .+-. 216 157.7 .+-. 18.6 Rhodamine
123 161.5 .+-. 75.58 889.7 .+-. 124.4 477.1 .+-. 14.14 692.2 .+-.
18.32 FD-4 15.23 .+-. 1.76 28.55 .+-. 9.38 1270 .+-. 43.21 1054
.+-. 42.03 FD-10 5.06 .+-. 0.54 9.52 .+-. 1.40 278.7 .+-. 10.18
157.2 .+-. 9.85 FD-20 1.84 .+-. 0.15 3.15 .+-. 0.34 147 .+-. 8.53
43.82 .+-. 2.34 FD-40 1.67 .+-. 0.56 2.60 .+-. 0.38 75 .+-. 28.72
16.61 .+-. 0.66
* * * * *