U.S. patent application number 16/416963 was filed with the patent office on 2020-05-07 for cosmetic preservatives as therapeutic corneoscleral tissue cross-linking agents.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. The applicant listed for this patent is David Choohyun Trokel Paik. Invention is credited to David Choohyun Paik, Stephen Lewis Trokel.
Application Number | 20200138787 16/416963 |
Document ID | / |
Family ID | 54072418 |
Filed Date | 2020-05-07 |
View All Diagrams
United States Patent
Application |
20200138787 |
Kind Code |
A1 |
Paik; David Choohyun ; et
al. |
May 7, 2020 |
COSMETIC PRESERVATIVES AS THERAPEUTIC CORNEOSCLERAL TISSUE
CROSS-LINKING AGENTS
Abstract
A composition for opththalmic administration comprising a
formaldehyde releasing agent, sodium bicarbonate, and an
ophthalmically suitable carrier or excipient is provided.
Inventors: |
Paik; David Choohyun;
(Cheltenham, PA) ; Trokel; Stephen Lewis; (New
York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Paik; David Choohyun
Trokel; Stephen Lewis |
Cheltenham
New York |
PA
NY |
US
US |
|
|
Assignee: |
The Trustees of Columbia University
in the City of New York
New York
NY
|
Family ID: |
54072418 |
Appl. No.: |
16/416963 |
Filed: |
May 20, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16133260 |
Sep 17, 2018 |
10292967 |
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16416963 |
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15125558 |
Sep 12, 2016 |
10105350 |
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PCT/US15/20276 |
Mar 12, 2015 |
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16133260 |
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62088383 |
Dec 5, 2014 |
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61952043 |
Mar 12, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 33/00 20130101;
A61K 31/115 20130101; C07K 14/78 20130101; A61K 31/115 20130101;
A61P 27/02 20180101; A61K 31/4166 20130101; A61K 31/4178 20130101;
A61K 33/00 20130101; A61K 31/4178 20130101 |
International
Class: |
A61K 31/4166 20060101
A61K031/4166; A61K 33/00 20060101 A61K033/00; A61K 31/115 20060101
A61K031/115; A61K 31/4178 20060101 A61K031/4178; C07K 14/78
20060101 C07K014/78 |
Goverment Interests
[0003] This invention was made with government support under grant
EY020495 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1-15. (canceled)
16. A method of inhibiting loss of structural integrity of a
collagenous tissue during transplantation-related transport
comprising contacting the collagenous tissue with an amount of a
formaldehyde releasing agent effective to inhibit loss of
structural integrity of the collagenous tissue.
17. The method of claim 16 wherein the collagenous tissue is
contacted with the formaldehyde releasing agent before removal of
the collagenous tissue from a transplant donor.
18. The method of claim 17 wherein the collagenous tissue is
contacted during the transplantation-related transport.
19. The method of claim 16 wherein the formaldehyde releasing agent
is 1-(phenylmethoxy)-methanol,
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea, 1,3-dimethylol-5,5-dimethyl-hydantoin,
N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea,
sodium hydroxymethyl glycinate, 5-bromo-5-nitro-1,3-dioxane,
2-bromo-2-nitropropane-1,3-diol,
3,5,7-triaza-1-azoniatricyclo[3.3.1.13,7]decane,1-(3-chloro-2-propen-1-yl-
)-chloride(1:1), 4,5-dihydroxy-1,3-dimethyl-2-Imidazolidinone,
4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-Imidazolidinone,
tetrahydro-1,3-bis(hydroxymethyl)-2(1H)-pyrimidinone,
tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)-imidazo[4,5-d]imidazole-2,5(1H-
,3H)-dione, polyoxymethylene urea, 4,4-dimethylyoxazolidine,
7a-ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole,
4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-imidazolidinone methylated,
dimethylhydantoin formaldehyde resin,
4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-imidazolidinone,
1,3-bis(hydroxymethyl)-2-imidazolidinone,
N,N'-bis(hydroxymethyl)-urea, 1,3-ethyleneurea,
(Z)-3-(bis(2-hydroxyethyl)amino)-2-(2-hydroxyethyl-(hydroxymethyl)amino)
prop-2-en-1-ol, 1,3,5-trietethyl-1,3,5-tiazinane,
4,5-dihydroxy-2-imidazolidinone,
1-(hydroxymethyl)-5,5-dimethyl-2,4-Imidazolidinedione,
1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane,
4,4'-methylenebis-morpholine, 2-chloro-N-(hydroxymethyl)-acetamide,
N-(hydroxymethyl)-urea, polyoxymethylene melamine,
1,1'-[methylenebis(oxymethylene)]bis-benzene,
1,6-dihydroxy-2-5-dioxahexane
(1,1'-[1,2-ethanediylbis(oxy)]bis-methanol, 2,4-imidazolidinedione,
hydroxymethyl-5-5-dimethyl-2-4-imidazolidinedione,
3-hydroxymethyl-5-5-dimethylimidazolidine-2,4-dione,
dimethoxy-methane, N-methylolethanolamine,
1H,3H,5H-oxazolo[3,4-c]oxazole-7a(7H)-methanol, Bioban N-95
(mixture of 5-methyl.sup.-1- aza-3,7-dioxabicyclo[3.3.0]octane,
5-hydroxymethoxymethyl-1-aZa-3,7-diox abicyclo[3.3.0]octane, and
higher hydroxyalkoxymethyl oligomers),
5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane,
4,4-dimethyl-oxazolidine, 4-ethyl-2-(1-methylethyl)-oxazolidine,
2-(hydroxymethyl)-2-nitro-1,3-propanediol, diethylamine/2-methyl-2
nitro-1,3-propanediol,
dimethylamine-2-methyl-2-nitro-1,3-propanediol,
pyrrolidine/2-methyl 2-nitro-1,3-propanediol, 2-furfural/2-methyl
2-nitro-1,3-propanol, N-hydroxy-2-propanamine,
N-hydroxy-1-propanamine, N-hydroxy-ethanamine,
N-hydroxy-2-methyl-2-propanamine, N-hydroxy-cyclohexanamine,
N-ethyl-N-hydroxy-ethanamine,
1,1'-[methylenebis(oxy)]bis[2-methyl-2-nitro-(9CI)]-propane,
hydroxylamine (HA) nitrone, N-ethylhydroxylamine (EHA) nitrone,
N-propylhydroxylamine (PHA) nitrone, N-t-butyl hydroxylamine
(tBuHA) nitrone, Cyclohexanedicarboxaldehyde
(CHDA)-bis-isopropylhydroxylamine (IPHA) nitrone, N-benzyl
hydroxylamine (N-BzHA) nitrone, or vanillin-isopropylhydroxylamine
(IPHA) nitrone.
20. The method of claim 19 wherein the formaldehyde releasing agent
is 1-(phenylmethoxy)-methanol,
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea, 1,3-dimethylol-5,5-dimethyl-hydantoin,
N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea,
sodium hydroxymethyl glycinate, or
5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane.
21. The method of claim 19 wherein the formaldehyde releasing agent
is
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea.
22-48. (canceled)
49. The method of claims 16, wherein the collagenous tissue is a
heart valve.
50. The method of claims 16, wherein the collagenous tissue is
skin.
51. The method of claims 16, wherein the collagenous tissue is
cornea.
52. The method of claims 16, wherein the collagenous tissue is
sclera.
53. The method of claims 16, wherein the collagenous tissue is
tendon.
54. The method of claims 16, wherein the collagenous tissue is
fascia.
55. The method of claims 16, wherein the collagenous tissue is
bone.
56. The method of claims 16, wherein the collagenous tissue is
cartilage.
57. The method of claim 16, wherein the collagenous tissue is human
tissue.
58. The method of any of claims 16 wherein the contacting is at a
temperature effective to inhibit loss of structural integrity of
the collagenous tissue.
59. The method of claim 58, wherein the temperature is greater than
60.degree. C.
60. The method of claim 58, wherein the temperature is greater than
62.degree. C.
61. A method of altering the refractive power of a cornea
comprising contacting the cornea with a formaldehyde releasing
agent so as to effect cross-linking in the cornea and thereby alter
the refractive power of the cornea.
Description
CROSS-LINKING AGENTS
[0001] This application is a divisional of U.S. application Ser.
No. 16/133,260, filed Sep. 17, 2018, now allowed, which is a
divisional of U.S. application Ser. No. 15/125,558, filed Sep. 12,
2016, now U.S. Pat. No. 10,105,350, issued Oct. 23, 2018, which is
a .sctn.371 national stage of PCT International Application No.
PCT/US2015/020276, filed Mar. 12, 2015, and claims the benefit of
U.S. Provisional Application No. 62/088,383, filed Dec. 5, 2014,
and U.S. Provisional Application No. 61/952,043, filed Mar. 12,
2014, the contents of each of which are hereby incorporated by
reference.
[0002] Throughout this application various publications are
referenced. The disclosures of these documents in their entireties
are hereby incorporated by reference into this application in order
to more fully describe the state of the art to which this invention
pertains.
BACKGROUND OF THE INVENTION
[0004] Collagen is a fundamental protein found in connective tissue
in animals, and it is present in the cornea and sclera of the eye.
Several eye disorders are related to defects in collagen structure
and include keratoconus, keratectasia, progressive myopia, and
possibly glaucoma.
[0005] Keratoconus is a debilitating, progressive eye disorder,
which is believed to occur due to progressive slippage of collagen
lamellae in the cornea, usually bilateral, beginning between ages
10 and 20. The cornea develops a conical shape, causing significant
changes in the refractive power of the eye. While corrective lenses
may help vision, corneal transplant surgery may be necessary if
eyeglasses or contact lenses are inadequate. THE MERCK MANUAL OF
DIAGNOSIS AND THERAPY 722 (Mark H. Beers and Robert Berkow eds.,
17th ed. 1999).
[0006] Keratoconus is estimated to affect 1 person in about 435 to
2000 people in the general population. In its classical form,
keratoconus commences at puberty and progresses into the third to
fourth decade of life Rabinowitz, Y. S., "Keratoconus," Surv.
Opthal. 1998; 43(4):297-319. Thus, its overall impact is magnified
by virtue of the younger population that it afflicts. Clinically,
the disease is marked by progressive thinning of the corneal stroma
with resultant bulging and distortion of the thinned, weakened
areas. This thinning and distortion is documented by optical and
ultrasonic methods. The bulging, distorted cornea creates an
optically imperfect surface to the eye that produces an
increasingly irregular astigmatism and myopia. Contact lenses are
used to correct these optical imperfections when spectacle lenses
are no longer able to compensate for the induced optical
distortion. When contact lens correction fails, only a corneal
transplant will allow restoration of visual function. The need for
corneal transplantation arises when the disease has progressed and
central corneal scar formation occurs, or the distortion is so
great that contact lenses can no longer be worn.
[0007] Although the underlying etiology of keratoconus remains
unclear, there are two main mechanistic theories currently
entertained. The first is related to destabilization of collagen
lamellae through increased degradation via imbalances in endogenous
proteases and/or their inhibitors. In this regard, the scientific
evidence has been somewhat equivocal with some studies showing
increased matrix-metalloproteinase activity and others reporting no
change (reviewed by Collier, S. A., "Is the corneal degradation in
keratoconus caused by matrix-metalloproteinases?" Clin. Exp.
Ophthalmol. 2001; 29:340-344). An alternative theory regards
collagen fibril slippage with no overall tissue loss. Meek, K. M.,
et al. have shown, using synchrotron X-ray scattering, that stromal
lamellar organization is altered with an associated uneven
distribution of collagen fibrillar mass. These changes are
consistent with inter- and/or intra-lamellar slippage within the
stromal layers of the keratoconic cornea, leading to central
thinning. Meek, K. M., et al., "Changes in collagen orientation and
distribution in keratoconus corneas," IOVS 2005; 46(6):1948-1956.
The defect that would allow such slippage could be related to
changes in the collagen to proteoglycan interactions and/or
qualitative changes in the fibrillar collagens. Regarding this
second point, very little is known about the qualitative
biochemical collagen changes that occur in keratoconus. However,
alterations in difunctional collagen cross-linking were reported
decades ago. Cannon, J. and Foster, C. S., "Collagen crosslinking
in keratoconus," IOVS 1978;17(1):63-65; Oxlund, H. and Simonsen, A.
H., "Biochemical studies of normal and keratoconus corneas," 1985;
63:666-669; Critchfield, J. W., et al., "Keratoconus: I.
biochemical studies," Exp. Eye Res. 1988; 46:953-963. Regardless of
the exact mechanism responsible for progressive corneal thinning,
the pathologic changes that take place are accompanied by a loss of
biomechanical strength. In this regard it has been shown that
keratoconic corneas show a decreased stress for a given strain as
compared to controls (i.e., decreased tissue stiffness)
[Andreassen, T. T., et al., "Biomechanical properties of
keratoconus and normal corneas," Exp. Eye Res. 1980; 31:435-441.]
Andreassen, T. T., et al. also found that keratoconus collagen
displayed a decreased resistance to enzymatic digestion with
pepsin, a finding which is consistent with alterations in collagen
cross-linking.
[0008] Current treatments for keratoconus either mask the surface
irregularity with a variety of contact lenses, or attempt to
improve the surface contour with intracorneal ring segments,
lamellar keratoplasty, or excimer laser surgery. Binder, P. S., et
al., "Keratoconus and corneal ectasia after LASIK," J. Refract.
Surg. 2005; 21:749-752. However, the disease is progressive and
none of these options obviates the need for eventual corneal
transplantation.
[0009] Glaucoma is a group of disorders characterized by
progressive damage to the eye at least partly due to increased
intraocular pressure, the aqueous pressure in the eye. Increased
intraocular pressure results from an inadequate aqueous outflow
from the eye due to an obstruction in the trabecular meshwork from
which the eye drains. Collagen is necessary to maintain the
structural integrity of the trabecular meshwork. Rehnberg, M., et
al., "Collagen distribution in the lamina cribosa and the
trabecular meshwork of the human eye." Brit. J. Ophthalmol.
71:886-92 (1987). Open-angle glaucoma can be treated with medical,
laser, or surgical therapy to prevent damage to the optic nerve and
visual field by stabilizing the intraocular pressure. THE MERCK
MANUAL OF DIAGNOSIS AND THERAPY 733-36 (Mark H. Beers and Robert
Berkow eds., 17th ed. 1999).
[0010] In myopia, or nearsightedness, the image of a distant object
is focused in front of the retina because the axis of the eyeball
is too long or the refractory power of the eye is too strong. Rays
of light fall in front of the retina because the cornea is too
steep or the axial length of the eye is too long. Without glasses,
distant images are blurry, but near objects can be seen clearly.
While glasses or contact lenses correct vision, refractive surgery
decreases a patient's dependence on glasses or contact lenses.
Progressive myopia is a condition associated with high refractive
error and subnormal visual acuity after correction. This form of
myopia gets progressively worse over time. THE MERCK MANUAL OF
DIAGNOSIS AND THERAPY 741-43 (Mark H. Beers and Robert Berkow eds.,
17th ed. 1999). The development of severe myopia is associated with
scleral thinning and changes in the diameter of scleral collagen
fibrils in humans. McBrien, N. A., et al., "Structural and
Ultrastructural Changes to the Sclera in a Mammalian Model of High
Myopia." Investigative Ophthalmol. & Visual Sci. 42:2179-87
(2001).
[0011] Refractive surgery alters the curvature of the cornea to
allow light rays to come to focus closer to the retina, thus
improving uncorrected vision. In myopia, the central corneal
curvature is flattened. However, ideal candidates for refractive
surgery are people with healthy eyes who are not satisfied wearing
glasses or contact lenses for their daily or recreational
activities. Candidates for refractive surgery should not have a
history of collagen vascular disease because of potential problems
with wound healing. As keratoconus is a progressive thinning of the
cornea, thinning the cornea further with refractive surgery may
contribute to the advancement of the disease. Huang, X., et al.,
"Research of corneal ectasia following laser in-situ keratomileusis
in rabbits." Yan Ke Xue Bao, 18(2):119-22 (2002). The side effects
of refractive surgery include temporary foreign-body sensation,
glare, and halos. Potential complications include over- and
undercorrection, infection, irregular astigmatism, and, in excimer
laser procedures, haze formation. Permanent changes in the central
cornea caused by infection, irregular astigmatism, or haze
formation could result in a loss of best corrected acuity.
[0012] Keratectasia is the protrusion of a thinned, scarred cornea.
In laser in situ keratomileusis (LASIK), if the laser removes too
much tissue, or the flap is made too deep, the cornea can become
weak and distorted, leading to keratectasia. LASIK is
contraindicated for patients with thin corneas, or those with
keratectasia as a result of a prior LASIK procedure. Rigid gas
permeable contact lenses are the recommended treatment for
correcting vision in these patients. Kim, H., et al., "Keratectasia
after Laser in situ Keratomileusis." Int'l. J. Ophthalmol.
220:58-64 (2006).
[0013] A major breakthrough in the treatment of keratoconus and
related keratectasias has been realized. Recent work by the German
group of Wollensak, Spoerl, and Seiler has shown that cross-linking
corneal collagen through application of riboflavin and ultraviolet
light (UVR) can limit progressive vision loss in keratoconus
patients. This modality represents a method through which
stabilization of the corneal collagen lamellae and has been shown
to prevent the progressive thinning of the cornea and loss of
vision observed in keratoconus patients. This treatment involves
the serial applications of riboflavin (0.1%) onto a
de-epithelialized human cornea followed by exposure of the
riboflavin saturated tissue to ultraviolet radiation in a UVA-370
nanometer wavelength region, at 3 mW/cm.sup.2 radiant energy. The
patient is treated with antibiotic drops to prevent infection and
oral pain medicine after the procedure. Literature accruing over
the past 9 years has described the utility of photochemical
cross-linking using UVA irradiation (.lamda.max=370 nm) with
riboflavin as a photosensitizer (UVR). The work of the German group
of Wollensak, G., Spoerl, E., and Seiler T., has shown that this
method of cross-linking the collagen within the corneal stroma has
proven effective in limiting the progression of corneal thinning,
distortion, and resulting optical degradation of the eye.
Wollensak, G., et al., "Riboflavin/ultraviolet-A-induced collagen
crosslinking for the treatment of keratoconus." Am. J. Ophthalmol.
2003; 135:620-27. Despite these successes, the UVR therapy poses
attendant risks, particularly related to ultraviolet irradiation.
As such, this therapy has yet to gain FDA approval in the US.
[0014] Because riboflavin tissue penetration is limited by the
corneal epithelium, it is necessary to remove the corneal
epithelium by scraping prior to riboflavin application. Removal of
the corneal epithelium exposes the cornea to a risk of infection
and causes significant pain. In addition, keratocyte (Wollensak,
G., et al., "T. keratocyte cytotoxicity of riboflavin/UVA treatment
in vitro." Eye, 18:718-22 (2004); Wollensak, G., et al.,
"Keratocyte apoptosis after corneal collagen cross-linking using
riboflavin/UVA treatment." Cornea, 23(1):43-49 (2004)) and corneal
endothelial cell toxicity (Wollensak, G., et al., "Corneal
endothelial cytotoxicity of riboflavin/UVA treatment in vitro."
Ophthalmic Res., 35:324-28 (2003)) can occur with application of
riboflavin/UVA to the cornea. In a similar manner, application of
this therapy to the posterior sclera has been reported to damage
cells in the photoreceptor, outer nuclear, and retinal pigment
epithelial layers (Wollensak, G., et al., "Cross-linking of scleral
collagen in the rabbit using riboflavin and UVA." Acta
Ophthalmologica Scandinavica, 83:477-82 (2005).
[0015] Clinical trials in Europe (Caporossi, A., et al.,
"Parasurgical therapy for keratoconus by riboflavin-ultraviolet
type A rays induced cross-linking of corneal collagen: Preliminary
refractive results in an Italian study," J. Cataract Refract. Surg.
2006; 32:837-845; Wollensak, G., "Crosslinking treatment of
progressive keratoconus: new hope," Cur. Opin. Ophthal. 2006;
17:356-360) have generated significant interest in initiating
clinical trials in the United States. The early reports from this
therapy were encouraging. After 5 years in the Dresden study,
individuals who underwent this treatment protocol did not yet show
progression of their keratoconus. Based on these encouraging
results, corneal cross-linking therapy has been extended to include
patients with related disorders such as the ectasia that occurs
following LASIK (Laser-Assisted In situ Keratomileusis) and PRK
(Photorefractive Keratectomy) excimer refractive surgery (Binder,
P. S., et al., 2005). These are devastating complications of
keratorefractive surgery in today's clinical practice.
[0016] Anecdotal reports have also emerged reporting the use of
collagen cross-linking as an effective means to control
difficult-to-treat corneal fungal infections and corneal melts.
[0017] Despite these successes, the UVR therapy poses attendant
risks, particularly related to ultraviolet irradiation. As such,
this therapy has encountered difficulty gaining FDA approval and is
currently unavailable in the United States. Because free oxygen
radical formation occurs with riboflavin photolysis (Baier, J., et
al., "Singlet oxygen generation by UVA light exposure of endogenous
photosensitizers," Biophys. J. 2006; 91:1452-1459), this
cross-linking method has a negative impact on cell viability.
Indeed, keratocyte (Wollensak, G., et al., 2004) and corneal
endothelial cell toxicity (Wollensak, G., et al., 2003) does occur
with application of this therapy to the cornea. As a result of such
toxicity, it has been recommended that patients with particularly
thin central corneas (<400.mu.m) not undergo this therapy since
the depth of UVA penetration exposes the endothelial cells (which
are vital to maintaining corneal clarity though water regulation)
to toxic photochemical damage. Furthermore, the long-term risks of
this photochemical exposure are not known. Secondly, deep tissue
penetration by the riboflavin requires removal of the corneal
epithelium, a procedure that increases morbidity and complications.
This requires analgesics and antibiotics following the UVR
cross-linking procedure.
[0018] More recently, a topical self-administered compound has been
suggested for producing a comparable degree of collagen
cross-linking to UVR therapy, as described in U.S. Pat. No.
8,466,203. U.S. Pat. No. 8,466,203 describes a method of
cross-linking collagen in a subject's collagenous tissue by
contacting the collagenous tissue with an amount of a nitrogen
oxide-containing compound, such as a nitroalcohol, to cross-link
the collagen in the collagenous tissue.
[0019] Thus, the growing clinical success of UVA-riboflavin
photochemical corneal cross-linking (CXL) to halt the progression
of keratoconus (KC) and post-LASIK keratectasia suggests that
increasing mechanical tissue strength in vivo can be beneficial.
UVA-riboflavin mediated photochemical cross-linking (CXL) increases
the stiffness of corneal tissue as shown in animal studies using
post-mortem mechanical strip testing. Spoerl et al., Exp Eye Res
66:97-103 (1998). A majority of patients ultimately gain
improvements in topography and gain lines of vision. Raiskup-Wolf
et al., J Cataract Refract Surg 34:796-801 (2008). Application of
CXL has been extended to treat corneal edema, corneal melting, and
corneal infections.
[0020] As clinical trials involving CXL progress in the United
States, suggestions have been made to extend its use to the sclera
as a treatment for progressive myopia (Wollensak et al., J Cataract
Refract Surg 30:689-695 (2004)), since biomechanical weakening
occurs during progressive globe elongation. Scleral cross-linking,
with UVA-riboflavin technology has been reported but may be
difficult to carry out in the posterior region of the sclera
without the use of surgical means. Also, of concern is the
potential of damaging the neural retina during UVA irradiation. The
use of injectable pharmacologic agents that could cross-link the
sclera as an alternative to photochemical activation is being
explored and include glyceraldehyde, glutaraldehyde, genipin, and
nitroalcohols. Hoang et al., IOVS 54:ARVO E-Abstract 5169
(2013).
[0021] The present disclosure serves as an extension of previous
work using nitroalcohols, where the corneal and scleral
cross-linking efficacy of several related though potentially more
potent chemicals from a group known as formaldehyde releasing
agents (FARs) was tested. These compounds are used as preservatives
in a wide array of popular cosmetic and personal care products,
such as skin care products, body wash, fingernail polish and
shampoo, including the former formula for Johnson & Johnson's
"No More Tears" Baby Shampoo. FARs have also been employed in the
textile industry as cross-linking agents to impart anti-wrinkle
properties to clothing. Considering their use in everyday items
that come into direct contact with the human body, examination of
the efficacy and cell toxicity of FARs as tissue cross-linking
agents was a first step in their potential development for clinical
use.
SUMMARY OF THE INVENTION
[0022] This invention provides a method of cross-linking collagen
present in a collagenous tissue comprising contacting the
collagenous tissue with an amount of a formaldehyde releasing agent
effective to cross-link the collagen.
[0023] This invention also provides a method of inhibiting loss of
structural integrity of a collagenous tissue during
transplantation-related transport comprising contacting the
collagenous tissue with an amount of a formaldehyde releasing agent
effective to inhibit loss of structural integrity of the
collagenous tissue.
[0024] This invention also provides a composition for ophthalmic
administration comprising a formaldehyde releasing agent, sodium
bicarbonate, and ophthalmically suitable carriers or
excipients.
[0025] Finally, this invention provides a method of altering the
refractive power of a cornea comprising contacting the cornea with
a formaldehyde releasing agent so as to effect cross-linking in the
cornea and thereby alter the refractive power of the cornea.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1--Net apical displacement over time for a control
cornea and a cornea cross-linked with
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea (diazolidinyl urea) at pH 8.5.
[0027] FIG. 2--Cross-linking efficacy of 5 selected formaldehyde
releasing agents using the ex vivo rabbit corneal cross-linking
simulation setup as compared to a nitroalcohol of
2-nitro-1-propanol (NP).
[0028] FIG. 3--Cross-linking efficacy of 5 selected formaldehyde
releasing agents using the ex vivo rabbit corneal cross-linking
simulation setup as compared to nitroalcohols of
2-bromo-2-nitro-1,3-propanediol (BP) and
2-hydroxymethyl-2-nitro-1,3-propanediol (NT).
[0029] FIG. 4--pH dependent shifts in thermal transition
temperatures for 5 formaldehyde releasing agents (FARs) and
[0030] UVA-riboflavin mediated photochemical cross-linking (CXL)
using an ex vivo corneal cross-linking simulation setup
[0031] FIG. 5--A comparison of pH and concentration dependent
shifts in thermal transition temperatures for 5 formaldehyde
releasing agents (FARs) using cut porcine scleral pieces
[0032] FIG. 6--A direct concentration comparison at 25 mM between 5
formaldehyde releasing agents (FARs) and 2 higher order
nitroalcohols (HONAs) at two different pH values
[0033] FIG. 7--An overview of a disclosed experimental method
DETAILED DESCRIPTION OF THE INVENTION
[0034] This invention provides a method of cross-linking collagen
present in a collagenous tissue comprising contacting the
collagenous tissue with an amount of a formaldehyde releasing agent
effective to cross-link the collagen. In one embodiment, the
collagenous tissue is cornea, sclera, skin, tendon, fascia, bone,
or cartilage. In one embodiment, the collagenous tissue is cornea,
and the cornea is human cornea.
[0035] In one embodiment of the invention, the collagenous tissue
is present in a subject. In a preferred embodiment, the collagenous
tissue is cornea and the subject is afflicted with keratoconus or
keratectasia.
[0036] In one embodiment, the formaldehyde releasing agent is
present in a solution. In another embodiment, the formaldehyde
releasing agent is in an aqueous solution having a pH effective for
cross-linking. In a specific embodiment, the aqueous solution has a
pH value of 7.4. In another specific embodiment, the aqueous
solution has a pH value of 8.5. In one embodiment, the formaldehyde
releasing agent is present in an aqueous solution comprising sodium
bicarbonate.
[0037] In various embodiments, the contacting of the formaldehyde
releasing agent to the collagenous tissue is performed by
intermittent administration of the formaldehyde releasing agent to
the collagenous tissue for a duration of time effective to
cross-link collagen. In various embodiments, the solution is
administered at intervals of one to ten times per day over a period
of one day to one hundred and eighty days. In a specific
embodiment, the solution is administered one to four times per day
over a period of forty-two days. By administered one to ten times
per day, it is meant that all integer unit amounts within the range
are specifically disclosed as part of the invention. Thus, 2, 3, .
. . 8, 9 administrations are included as embodiments of this
invention. Similarly, the administration may be over a period of 2
days, 3 days . . . 178 days, or 179 days, and each integer value of
days is included as an embodiment of this invention.
[0038] In one embodiment of this invention, the solution is
administered as a composition selected from the group consisting of
ophthalmic drops, ophthalmic salve, ophthalmic ointment, ophthalmic
spray, subconjunctival injection, or intravitreal injection,
contact lens, conjunctival insert, ocular time release insert, and
sustained release implant. In a preferred embodiment, the solution
is administered as an ophthalmic drop.
[0039] This invention also provides a method of inhibiting loss of
structural integrity of a collagenous tissue during
transplantation-related transport comprising contacting the
collagenous tissue with an amount of a formaldehyde releasing agent
effective to inhibit loss of structural integrity of the
collagenous tissue. In one embodiment, the collagenous tissue is
contacted with the formaldehyde releasing agent before removal of
the collagenous tissue from the donor subject. In another
embodiment, the collagenous tissue is incubated during transport
from the transplant donor. In a specific embodiment, the transplant
donor is a human. In one embodiment, the collagenous tissue is a
heart valve. In another embodiment, the collagenous tissue is skin.
In another embodiment, the collagenous tissue is cornea. In another
embodiment, the collagenous tissue is sclera. In another
embodiment, the collagenous tissue is tendon. In another
embodiment, the collagenous tissue is fascia. In another
embodiment, the collagenous tissue is bone. In another embodiment,
the collagenous tissue is cartilage. In one embodiment, the
collagenous tissue is human tissue.
[0040] In one embodiment, the contacting is at a temperature
effective to inhibit loss of structural integrity of the
collagenous tissue. In a specific embodiment, the temperature is
greater than 60.degree. C. In another specific embodiment, the
temperature is greater than 62.degree. C.
[0041] This invention also provides a composition for ophthalmic
administration comprising a formaldehyde releasing agent, sodium
bicarbonate, and ophthalmically suitable carriers or
excipients.
[0042] In one embodiment, the formaldehyde releasing agent is in an
aqueous solution having a pH effective for cross-linking. In a
specific embodiment, the aqueous solution has a pH value of 7.4. In
another specific embodiment, the aqueous solution has a pH value of
8.5.
[0043] This invention also provides a method of altering the
refractive power of a cornea comprising contacting the cornea with
a formaldehyde releasing agent so as to effect cross-linking in the
cornea and thereby alter the refractive power of the cornea. In one
embodiment, the refractive power of the cornea is increased. In
another embodiment, the cross-linking effected in the cornea causes
a surface contour of the cornea to change shape. In another
embodiment, the cornea is an isolated cornea. In another
embodiment, the cornea is human cornea.
[0044] In various embodiments, the formaldehyde releasing agent is
1-(phenylmethoxy)-methanol,
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea, 1,3-dimethylol-5,5-dimethyl-hydantoin,
N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea,
sodium hydroxymethyl glycinate, 5-bromo-5-nitro-1,3-dioxane,
2-bromo-2-nitropropane-1,3-diol,
3,5,7-triaza-1-azoniatricyclo[3.3.1.13,7]decane,1-(3-chloro-2-propen-1-yl-
)-chloride(1:1), 4,5-dihydroxy-1,3-dimethyl-2-Imidazolidinone,
4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-Imidazolidinone,
tetrahydro-1,3-bis(hydroxymethyl)-2(1H)-pyrimidinone,
tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)-imidazo[4,5-d]imidazole-2,5(1H-
,3H)-dione, polyoxymethylene urea, 4,4-dimethylyoxazolidine,
7a-ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole,
4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-imidazolidinone methylated,
dimethylhydantoin formaldehyde resin,
4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-imidazolidinone,
1,3.sup.-bis(hydroxymethyl)-2-imidazolidinone,
N,N'-bis(hydroxymethyl)-urea, 1,3-ethyleneurea,
(2)-3-(bis(2-hydroxyethyl)amino)-2-(2-hydroxyethyl-(hydroxymethyl)amino)
prop-2-en-1-ol, 1,3,5-trietethyl-1,3,5-tiazinane,
4,5-dihydroxy-2-imidazolidinone,
1-(hydroxymethyl)-5,5-dimethyl-2,4-Imidazolidinedione,
1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane,
4,4'-methylenebis-morpholine, 2-chloro-N-(hydroxymethyl)-acetamide,
N-(hydroxymethyl)-urea, polyoxymethylene melamine,
1,1'-[methylenebis(oxymethylene)]bis-benzene,
1,6-dihydroxy-2-5-dioxahexane
(1,1'-[1,2-ethanediylbis(oxy)]bis-methanol, 2,4-imidazolidinedione,
hydroxymethyl-5-5-dimethyl-2-4-imidazolidinedione,
3-hydroxymethyl-5-5-dimethylimidazolidine-2, 4-dione,
dimethoxy-methane, N-methylolethanolamine,
1H,3H,5H-oxazolo[3,4-c]oxazole-7a(7H)-methanol, Bioban N-95
(mixture of 5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane,
5-hydroxymethoxymethyl-1-aZa-3,7-diox abicyclo[3.3.0]octane, and
higher hydroxyalkoxymethyl oligomers),
5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane,
4,4-dimethyl-oxazolidine, 4-ethyl-2-(1-methylethyl)-oxazolidine,
2-(hydroxymethyl)-2-nitro-1,3-propanediol,
diethylamine/2-methyl-2nitro-1,3-propanediol,
dimethylamine-2-methyl-2-nitro-1,3-propanediol,
pyrrolidine/2-methyl 2-nitro-1,3-propanediol, 2-furfural/2-methyl
2-nitro-1,3-propanol, N-hydroxy-2-propanamine,
N-hydroxy-1-propanamine, N-hydroxy-ethanamine,
N-hydroxy-2-methyl-2-propanamine, N-hydroxy-cyclohexanamine,
N-ethyl-N-hydroxy-ethanamine,
1,1'-[methylenebis(oxy)]bis[2-methyl-2-nitro-(9CI)]-propane,
hydroxylamine (HA) nitrone, N-ethylhydroxylamine (EHA) nitrone,
N-propylhydroxylamine (PHA) nitrone, N-t-butyl hydroxylamine
(tBuHA) nitrone, Cyclohexanedicarboxaldehyde
(CHDA)-bis-isopropylhydroxylamine (IPHA) nitrone, N-benzyl
hydroxylamine (N-BzHA) nitrone, or vanillin-isopropylhydroxylamine
(IPHA) nitrone. In a preferred embodiment, the formaldehyde
releasing agent is 1-(phenylmethoxy)-methanol,
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea, 1,3-dimethylol -5,5-dimethyl-hydantoin,
N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea,
sodium hydroxymethyl glycinate, or
5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane. In a more preferred
embodiment, the formaldehyde releasing agent is
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea.
[0045] As used herein, "collagenous tissue" refers to any bodily
tissue that contains the protein collagen, such as skin, blood
vessels, heart valve, tendons, fascia, bone, cartilage, tendonous
tissue, and eye tissues such as the cornea, sclera, and retina.
[0046] As used herein, "corneoscleral disorder" is any disease,
condition, or abnormality of the cornea and/or scleral tissue of
the eye involving a loss of stiffness and/or contour changes of the
eye. Thus, the corneoscleral disorder may be keratoconus,
keratectasia, progressive myopia, or glaucoma.
[0047] As used herein, "formaldehyde releasing agent" or
"formaldehyde releaser" (FAR) is a compound, often used as a
preservative in cosmetics, which is able to release formaldehyde,
such as benzyl hemiformal (1-(phenylmethoxy)-methanol),
diazolidinyl urea
(N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'--
hydroxy-methylurea), DMDM hydantoin
(1,3-dimethylol-5,5-dimethyl-hydantoin), imidazolidinyl urea
(N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea-
), sodium hydroxymethyl glycinate, 5-bromo-5-nitro-1,3-dioxane,
bronopol (2-bromo-2-nitropropane-1,3-Diol), quaternium-15
(3,5,7-triaza-1-azoniatricyclo[3.3.1.13,7]decane,1-(3-chloro-2-propen-1-y-
l)-chloride (1:1)), 1,3-dimethyl-4,5-dihydroxyethyleneurea
(4,5-dihydroxy-1,3-dimethyl-2-Imidazolidinone), dimethylol
dihydroxyethyleneurea
(4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-imidazolidinone),
dimethylol propyleneurea
(tetrahydro-1,3-bis(hydroxymethyl)-2(1H)-pyrimidinone),
tetramethylol acetylenediurea
(tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)-imidazo[4,5-d]imidazole-2,5(1-
H,3H)-dione), polyoxymethylene urea (urea polymer with
formaldehyde), 4,4-dimethylyoxazolidine
(3,4,4-trimethyl-oxazolidine with 4,4-dimethyloxazolidine),
7a-ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole,
dihydroxy-dimethylol-ethylene urea methylated
(4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-imidazolidinone
methylated), dimethylhydantoin formaldehyde resin (formaldehyde,
polymer with 5,5-dimethyl-2,4-imidazolidinedione,
dimethylhydroxyethyleneurea
(4,5-dihydroxy-1,3-bis(hydroxymethyl)-2-imidazolidinone),
dimethylolethyleneurea (1,3-bis(hydroxymethyl)-2-imidazolidinone),
dimethylol urea (N,N'-bis(hydroxymethyl)-urea), 2-imidazolidinone
(1,3-ethyleneurea),
(Z)-3-(bis(2-hydroxyethyl)amino)-2-(2-hydroxyethyl-(hydroxymethyl)amino)
prop-2-en-1-ol, 1,3,5-trietethyl-1,3,5-tiazinane, glyoxalurea
(4,5-dihydroxy-2-imidazolidinone), MDM hydantoin
(1-(hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione),
methenamine (1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane),
N,N'-methylenebismorpholine (4,4'-methylenebis-morpholine),
2-chloro-N-(hydroxymethyl)-acetamide, methylol urea
(N-(hydroxymethyl)-urea), polyoxymethylene melamine (urea, polymer
with formaldehyde and 1,3,5-triazine-2,4,6-triamine),
phenylmethoxymehoxymethylbenzene
(1,1'-[methylenebis(oxymethylene)]bis-benzene),
1,6-dihydroxy-2-5-dioxahexane
(1,1'-[1,2-ethanediylbis(oxy)]bis-methanol), hydantoin
(2,4-imidazolidinedione),
hydroxymethyl-5-5-dimethyl-2-4-imidazolidinedione,
3-hydroxymethyl-5-5-dimethylimidazolidine-2, 4-dione, methylal
(dimethoxy-methane), N-methylolethanolamine
(2-(hydroxymethylamino)ethanol),
1H,3H,5H-oxazolo[3,4-c]oxazole-7a(7H)-methanol, Bioban N-95
(mixture of 5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane,
5-hydroxymethoxymethyl-1-aZa-3,7-dioxabicyclo[3.3.0]octane, and
higher hydroxyalkoxymethyl oligomers),
5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane,
4,4-dimethyl-oxazolidine, 4-ethyl-2-(1-methylethyl)-oxazolidine,
2-(hydroxymethyl)-2-nitro-1,3-propanediol,
diethylamine/2-methyl-2nitro-1,3-propanediol,
dimethylamine-2-methyl-2-nitro-1,3-propanediol,
pyrrolidine/2-methyl-2-nitro 1,3-propanediol, 2-furfural/2-methyl
2-nitro-1,3-propanol, N-hydroxy-2-propanamine,
N-hydroxy-1-propanamine, N-hydroxy-ethanamine,
N-hydroxy-2-methyl-2-propanamine, N-hydroxy-cyclohexanamine,
N-ethyl-N-hydroxy-ethanamine,
1,1'-[methylenebis(oxy)]bis[2-methyl-2-nitro-(9CI)]-propane,
hydroxylamine (HA) nitrone, N-ethylhydroxylamine (EHA) nitrone,
N-propylhydroxylamine (PHA) nitrone, N-t-butyl hydroxylamine
(tBuHA) nitrone,
cyclohexanedicarboxaldehyde(CHDA)-bis-isopropylhydroxylamine (IPHA)
nitrone, N-benzyl hydroxylamine (N-BzHA) nitrone, and
vanillin-Isopropylhydroxylamine (IPHA) nitrone.
[0048] The formaldehyde releasing agent can be administered in
admixture with ophthalmically suitable excipients or carriers
suitably selected with respect to the intended form of
administration and as consistent with conventional ophthalmical
practices.
[0049] It is to be understood that the invention is not limited in
its application to the details set forth in the description or as
exemplified. The invention encompasses other embodiments and is
capable of being practiced or carried out in various ways. Also, it
is to be understood that the phraseology and terminology employed
herein is for the purpose of description and should not be regarded
as limiting.
EXAMPLES
First Experimental Details
Cross-Linking Using Formaldehyde Releasing Agents (FARs)
[0050] A listing of formaldehyde releasing agents (FARs) was
gathered from literature review. Sixty-four (64) formaldehyde
releasing agents, regularly found in cosmetics, were identified
from the literature. Each formaldehyde releasing agent was analyzed
with respect to relevant characteristics for cross-linking, such as
molecular weight, carcinogenicity/mutagenicity, toxicity,
hydrophobicity, and commercial availability.
[0051] Based on this analysis, formaldehyde releasing agents were
selected for efficacy screening using an ex vivo rabbit corneal
cross-linking simulation setup, as described below.
[0052] 0.5% proparacaine was applied prior to the cross-linking
solution. A cross-linking solution containing the formaldehyde
releasing agent was then administered via a corneal reservoir for
30 minutes in 0.1M NaHCO.sub.3 at either pH 7.4 or 8.5. The
epithelium was left intact. The control contralateral eye was
treated identically with vehicle.
[0053] Effectiveness of cross-linking was based on shifts in
thermal denaturation temperature (Tm) as measured by differential
scanning calorimetry (DSC) (Perkin-Elmer DSC 6000). Favorable DSC
results were validated using biomechanical inflation tests with
digital image correlation (DIC) as previously described by Myers et
al.
Second Experimental Details
Chemical Registry
[0054] A chemical registry of formaldehyde releasers (FARs)
commonly found in cosmetics and other personal care products (PCPs)
was compiled from a review of the literature. Information used to
assemble this registry included characteristics relevant to tissue
cross-linking such as molecular weight, European Union maximum
allowed concentration (i.e. "max allowed"),
carcinogenicity/mutagenicity, toxicity, hydrophobicity (logP),
efficacy of formaldehyde release, and commercial availability of
the chemicals. From the FARs identified, five compounds with
favorable profiles were selected for cross-linking efficacy and
toxicity evaluation. These compounds include diazolidinyl urea
(DAU), imidazolidinyl urea (IMU), DMDM hydantoin (DMDM), sodium
hydroxymethylglycinate (SMG), and 5-Ethyl-3,7-dioxa-1-azabicyclo
[3.3.0] octane (OCT), which were specifically chosen because of the
vastness of their use in cosmetics and PCPs as well as on their
ability to donate formaldehyde in solution under equilibrium
conditions. The cross-linking efficacy and toxicity of two
additional FARs, bronopol (BP) and
2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD), which are
.beta.-nitroalcohols (BNAs), was included for comparative
purposes.
Chemicals
[0055] Diazolidinyl urea
(N-Hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'--
hydroxy-methylurea [DAD]), imidazolidinyl urea
(N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea
[IMU]), sodium hydroxymethylglycinate (SMG),
5-Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane
(7a-Ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole [OCT]),
2-bromo-2-nitro-1,3-propanediol or bronopol (BP), hydroxypropyl
methyl cellulose (HPMC, 15 centipoise), dextran (high molecular
weight=425-575,000 Da), sodium bicarbonate and
ethylenediaminetetraacetic acid (EDTA) were obtained from
Sigma-Aldrich Corp. (St. Louis, Mo.). DMDM hydantoin was obtained
from Oakwood Products, Inc. (West Columbia, S.C.).
2-hydroxymethyl-2-nitro-1-3-propanediol (HNPD) was obtained from
TCI Chemicals, Inc. (New York, N.Y.). Riboflavin-5-phosphate was
obtained from MP Biomedicals (Santa Ana, Calif.). Dulbecco's
phosphate buffered saline (DPBS) solution (MgCl.sub.2 &
CaCl.sub.2 free) was obtained from Life Technologies (Carlsbad,
Calif.). All chemical solutions and buffers were prepared fresh
using Millipore water (double distilled, de-ionized water,
.rho.=18.2 M.OMEGA.cm at 25.degree. C.) on the day of
cross-linking.
Chemical and Riboflavin-Mediated Photochemical Cross-Linking (CXL)
of the Cornea
[0056] Intact cadaveric rabbit heads with clear corneas were
obtained fresh (within an hour of sacrifice) in adherence with the
ARVO Statement For the Use of Animals in Ophthalmic and Vision
Research. FAR solutions at concentrations equivalent to half the
maximum allowed value (1/2max) were administered in a manner
designed to simulate therapeutic cross-linking in patients. For all
of the corneal experiments (with the notable exception of CXL), the
corneal epithelium was left intact. An 8 mm Hessburg-Barron corneal
reservoir was affixed to the corneal surface using the supplied
syringe vacuum. A single drop of proparacaine (0.5%) was applied to
the corneal surface prior to reservoir application. A buffer
solution containing 0.1M NaHCO.sub.3 at either pH 8.5 or 7.4 was
used. The pH of the sample and buffer mixture was titrated to the
desired pH just prior to application to the eye using an
appropriately concentrated HCl solution. Treatments were conducted
over a 30-minute period at 25.degree. C. with refreshing of the
solution every five minutes. The control contralateral eye was
treated identically with vehicle. Immediately after treatment, a
central 6 mm corneal button was trephined from the treated region
of each eye, was blotted on both sides using a paper towel to
remove excess solution, and was analyzed using differential
scanning calorimetry (DSC) [see below]. A minimum of two
independent determinations were carried out for each condition
described using a fresh cadaver head each time.
[0057] As a comparison, the same ex vivo system was used to conduct
photochemical cross-linking of rabbit cornea as previously
described by Wollensak et al. (Am J Ophthalmol 135:620-627 (2003))
with some changes. To that end, a central 8 mm portion of the
corneal epithelium was debrided using a blunt-end scalpel.
De-epithelialized corneal tissue was pre-soaked in 0.1%
riboflavin-5-phosphate solution in 1.1% HPMC for 5 mins.
Thereafter, the cornea was exposed to UV light (.lamda.max=370 nm)
at an irradiance of 3 mW/cm.sup.2 with an 8 mm aperture for 30 mins
using the Optos XLink Corneal Collagen Cross-Linking System (Optos,
Dunfermline, UK). Riboflavin solution was refreshed every 3 mins
for the course of the treatment. The control contralateral eye was
treated identically without irradiation.
Scleral Tissue Cross-Linking
[0058] Enucleated porcine globes were purchased from Visiontech,
Inc. (Sunnvale, Tex.) and were stored at -80.degree. C. until time
of experimentation (1-2 months). Equatorial scleral strips
approximately 6 mm.times.40 mm in size were obtained from multiple
eyes. These strips were submerged in DPBS solution containing 1 mM
EDTA to inactivate native collagenases and to prevent tissue
dehydration during sample preparation. Each strip was further cut
into smaller 4 mm.times.3 mm pieces. The scleral pieces were
individually transferred to a 24 well plate and were incubated in 1
ml of cross-linking solution in 0.1M NaHCO.sub.3 buffer at either
pH 8.5 or 7.4 for 30 mins at 25.degree. C. without refreshing the
solution. Four concentrations of FAR solution were tested at each
pH: 1) max allowed concentration, 2) 1/2 max allowed concentration,
3) 1/10 max allowed concentration, and 4) 25 mM. Tissue samples
cross-linked with the BNAs BP and HNPD at concentrations of 5 mM
(max allowed for BP), 10 mM, and 25 mM were used as positive
controls. Negative controls were treated identically with vehicle.
Post-treatment, all solutions were aspirated and samples were
washed twice using DPBS to remove remnant cross-linking solution
before being analyzed by DSC. A minimum of three independent
determinations were carried out for each condition using scleral
pieces originating from different porcine globes.
Differential Scanning calorimetry (DSC) and Cross-Link Analysis
[0059] Thermal denaturation temperature (Tm) of all samples was
measured using a Perkin-Elmer DSC 6000 Autosampler (Waltham,
Mass.). Tissue samples were carefully blotted in a standardized,
repetitive manner to remove excess solution/DPBS and transferred to
pre-weighed 50 ul aluminum pans. The pans were immediately
hermetically sealed using a DSC pan sealing press, which is used to
prevent tissue dehydration due to evaporative losses. DSC scans
were run using Pyris software (version 11.0) from 40.degree. C. to
75.degree. C. at a rate of 1.degree. C./min and denaturation curves
representing differential heat flow over time were recorded. DSC
heat flow endotherm data was analyzed using the Pyris data analysis
peak search function using a calculation limit of .+-.0.3.degree.
C. from the apparent thermal denaturation peak.
Statistical Analysis
[0060] T-tests were used to evaluate the significance of observed
differences in T.sub.m between cross-linked and control groups. Due
to the nature of the ex vivo cadaveric system used for corneal
cross-linking, where each cadaver provided the treated eye and
contralateral control, corneal samples were subjected to paired
t-tests. Conversely, scleral samples were subjected to non-paired
t-tests assuming equal variance of data. Significance of all
statistical tests was based on an alpha value of 0.05
(p.ltoreq.0.05). All .DELTA.T.sub.m values are reported in the form
of mean value followed by standard error.
FAR Cytotoxicity Threshold
[0061] Healthy human skin fibroblasts (HSFs) from ATCC (Manassas,
Va.) were cultured in dermal cell basal media (ATCC) using a
serum-free fibroblast growth kit provided by the company (ascorbic
acid, EGF/TGF-.beta.1, glutamine, hydrocortisone, insulin and FB
growth supplements). Cells were grown in 5% CO.sub.2 and 95%
ambient air at 37.degree. C. until confluent. Once confluent, the
cells were detached and seeded into 24 well plates at a density of
5.times.10.sup.4 cells/well and were once again allowed to reach
confluence. Next, cells were treated with FAR solutions over a
range of concentrations (0.001 mM-5 mM) for 24 hrs. Following
cross-linking exposure, all cell media, including FAR solution, was
aspirated and each well was rinsed once with DPBS. Fresh media was
then reintroduced and the cells were allowed to recover for 48 hrs.
Subsequent to cell recovery, cell toxicity was assessed using a
modified version of the trypan blue staining protocol. To that end,
all culture media was aspirated and each well was rinsed with DPBS.
Next, 0.4% trypan blue solution (Gibco, Grand Island, N.Y., USA)
was added to each well for 3 minutes at 25.degree. C. The staining
solution was then aspirated and cells were washed twice with DPBS.
Finally, extent of trypan blue staining and morphology of cells was
visualized using an inverted microscope (Fisher Scientific Cat
#12-560-45).
[0062] FIG. 7 illustrates a general overview of the disclosed
experimental method.
Results
First Experimental Details--Example 1
[0063] In a first example of the present invention,
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea (diazolidinyl urea) was selected and employed in
testing. FIG. 1 depicts the net apical displacement response over
time for a control cornea and a cornea cross-linked through the use
of diazolidinyl urea at pH 8.5. The cross-linked cornea produces a
smaller net displacement than the control cornea. The method for
inflation chamber testing analysis is described in Myers K M, et
al.
[0064] Thus,
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea (diazolidinyl urea) has been shown to be
effective for cross-linking at pH 8.5. In comparison to the
control, the Tm using diazolidinyl urea (DAU) was shifted
1.92.degree. C..+-.0.14.degree. C. (n=2).
[0065] Furthermore, mechanical inflation testing confirmed
increased tissue stiffness in pressure ranges mimicking
physiological pressure (1.875-45 mmHg). Finally, tissue creep was
also diminished under the current loading protocol. See FIG. 1.
First Experimental Details--Example 2
[0066] In a second example of the present invention, several
formaldehyde releasing agents were selected and employed in
testing. FIG. 2 shows an ex vivo rabbit corneal cross-linking
simulation setup used to determine the effects of five selected
formaldehyde releasing agents on the thermal stability of
collagenous tissue as determined by differential scanning
calorimetry (DSC) and measured in upward shifts in thermal
denaturation temperature (Tm). The difference in denaturation
temperature between treated and paired control (AT) represents
cross-linking efficacy. The formaldehyde releasing agents tested
included
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea (diazolidinyl urea; labelled DAU),
N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea
(imidazolidinyl urea; labelled IMU),
5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane (labelled OCT),
1,3-dimethylol-5,5-dimethyl-hydantoin (DMDM hydantoin; labelled
DMDM), and sodium hydroxymethyl glycinate (labelled SHMG). The
cross-linking solution was prepared at half of the maximum allowed
concentration (using
[0067] European regulatory standards) and administered to the right
eye for 30 minutes in 0.1 M NaHCO.sub.3 at either a pH of
approximately 7.4 or approximately 8.5. As a point of comparison,
data from an earlier similar experiments in which a nitroalcohol
(NA), specifically 2-nitro-1-propanol (labelled NP), was studied is
also provided in FIG. 2. The control contralateral eye was treated
identically with vehicle. The corneal epithelium was left intact
for all samples, unless otherwise noted (see DMDM and NP). The
final concentrations were as follows: DAU=9 mM, IMU=7.5 mM,
OCT=10.5 mM, SHMG=18.5 mM, DMDM=16 mM, NP=250 mM. In the
nitroalcohol (NP) experiments, the cross-linking time was twice (60
minutes) the amount of time used for the selected formaldehyde
releasing agents, and the concentration used (250 mM) was
significantly higher than the concentration used for these
formaldehyde releasing agents. A 50 mM
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer at pH 8.5 was used for
the NP experiments. Even with the longer cross-linking times and
higher concentrations,
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea (diazolidinyl urea), sodium hydroxymethyl
glycinate (SHMG), and 1,3-dimethylol-5,5-dimethyl-hydantoin (DMDM
hydantoin) are shown to be significantly more effective as
cross-linking agents.
First Experimental Details--Example 3
[0068] In a third example of the present invention, several
formaldehyde releasing agents were selected and employed in
testing. FIG. 3 shows an ex vivo rabbit corneal cross-linking
simulation setup used to determine the effects of five selected
formaldehyde releasing agents on the thermal stability of
collagenous tissue as determined by differential scanning
calorimetry (DSC) and measured in upward shifts in thermal
denaturation temperature (Tm). The difference in denaturation
temperature between treated and paired control (AT) represents
cross-linking efficacy. The formaldehyde releasing agents tested
included
N-hydroxymethyl-N-(1,3-di(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl)-N'-h-
ydroxy-methylurea (diazolidinyl urea; labelled DAU),
N,N'-methylenebis[N-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]]-urea
(imidazolidinyl urea; labelled IMU),
5-methyl-1-aza-3,7-dioxabicyclo[3.3.0]octane (labelled OCT),
1,3-dimethylol-5,5-dimethyl-hydantoin (DMDM hydantoin; labelled
DMDM), and sodium hydroxymethyl glycinate (labelled SHMG). The
cross-linking solution was prepared at half of the maximum allowed
concentration (using European regulatory standards) and
administered to the right eye for 30 minutes in 0.1 M NaHCO.sub.3
at either a pH of approximately 7.4 or approximately 8.5. As a
point of comparison, comparison runs with two higher order
nitroalcohols (NAs) were performed, where the two nitroalcohols
were 2-bromo-2-nitro-1,3-propanediol (bronopol; labelled BP) and
2-hydroxymethyl-2-nitro-1,3-propanediol (nitrotriol; labelled NT),
as also provided in FIG. 3. The control contralateral eye was
treated identically with vehicle. The corneal epithelium was left
intact for all samples, unless otherwise noted (see DMDM and NP).
The final concentrations were as follows: DAU=9 mM, IMU=7.5 mM,
OCT=10.5 mM, SHMG=18.5 mM, DMDM=16 mM, BP=5 mM, NT=5 mM.
Improved Cross-Linking Efficacy Shown for Formaldehyde Releasing
Agents (FARs) Versus Nitroalcohols (NAs) Using a Hydrogel Model
System
[0069] Shown in Table 1 below are the results from a parallel study
using. a hydrogel functionalized amine cross-linking system
(polyallylamine, or "PAA") previously published for reactions using
nitroalcohols (NAs). Li et al., "Mechanistic and catalytic studies
of .beta.-nitroalcohol crosslinking with polyamine," J Appl Polym
Sci. 2013; 128:3696-3701. The Table summarizes the results obtained
in comparison studies using three of the higher order nitroalcohols
previously reported (the nitrotriol HNPD, the nitrodiol MNPD, and
the brominated nitrodiol known as Bronopol). The cross-linking
efficacy of these nitroalcohol compounds were compared to two of
the instantly disclosed formaldehyde releasing agents (diazolidinyl
urea (DAU) and imidazolidinyl urea (IMU)). The time to gel
formation indicates cross-linking efficacy in this system. In other
words, a shorter cross-linking time indicates that the gel formed
faster, thus indicating greater cross-linking efficacy.
Diazolidinyl urea (DAU) and imidazolidinyl urea (IMU) both
exhibited significantly greater cross-linking efficacy over the
higher order nitroalcohols (NAs), as indicated by the shorter times
to gel formation observed.
TABLE-US-00001 TABLE 1 Cross-linking formation with PAA at
37.degree. C. and pH 7.4 phosphate buffered solution Chemical
cross- linking agent used ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## Cross- linking time to gel formation 29
hr 66 hr 42 hr 1.5 hr 4 hr
[0070] Furthermore, shown in Table 2 below are experimental results
from a study aimed at determining the time and pH dependent release
of formaldehyde from these compounds. Shown are the relative
amounts of formaldehyde produced by diazolidinyl urea (DAU) versus
nitroalcohol (NA) compounds, including
2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD) (a nitro-triol),
2-methyl-2-nitro-1,3-propanediol (MNPD) (a nitro-diol), and
2-nitro-1-propanol (NP) (a nitro-monol).
[0071] In the study corresponding with Table 2, NMR samples were
prepared in NMR tubes as follows: X mg of formaldehyde-releaser,
500 ul of phosphate buffer (pH 7.4), and 500 ul of a solution of
acetonitrile in D20 as internal standard (0.2M). The final
concentration of formaldehyde releaser was 1.5 M, and the final
concentration of acetonitrile was 0.1 M. The NMR tubes were sealed
well and incubated in a water bath at 37.degree. C., and analyzed
at the indicated times of 30 and 60 min. A one-dimensional
.sup.13C-NMR spectrum of each solution was recorded on a Bruker NMR
instrument at 300 MHz. For each spectrum, the area of the
formaldehyde signal at 82.5 ppm was compared with that of the
acetonitrile signal at 1.3 ppm (internal standard).
TABLE-US-00002 TABLE 2 Quantitative determination of formaldehyde
release by 13C-NMR Concentration Concentration Chemicals of
formaldehyde (M).sup.a of formaldehyde (M).sup.b ##STR00006## 2.044
0.319 ##STR00007## 0.041 0.078 ##STR00008## 0.011 0.044
##STR00009## 0.018 0.014 Concentration of formaldehyde-releasing
agent (FAR), including nitroalcohols is 1.5 M. .sup.aConcentration
of free formaldehyde (FA): after the sample was incubated in a
water bath at 37.degree. C. for 30 min. .sup.bConcentration of free
formaldehyde (FA): after the sample was incubated in a water bath
at 37.degree. C. for 1 hour.
[0072] Table 2 indicates that under the conditions studied,
diazolidinyl urea (DAU) released approximately fifty (50) times
more free formaldehyde (2.044M) than the most potent nitroalcohol
(HNPD) (0.041 M) at 30 minutes of incubation time and over four (4)
times as much at 60 minutes of incubation time.
[0073] Thus, Table 2 shows, for each formaldehyde releaser, the
concentration of released formaldehyde after 30 minutes and 1 hour
reaction time in phosphate buffered solution (pH 7.4). In this
experiment, a prototype compound of a group of formaldehyde
releasing agents (FARs), diazolidinyl urea (DAU). The concentration
of formaldehyde released from diazolidinyl urea (DAU) within 30
minutes of standing time in alkali buffered solution was determined
to be approximately fifty (50) times higher than that of the
nitro-triol. In addition, the amount of formaldehyde released from
DAU after 1 hour standing time reduced significantly. This could be
caused by the reaction of released formaldehyde with breakdown
products of the diazolidinyl urea (DAU) starting material.
Diazolidinyl urea (DAU) decomposition products have been reported
(Lehmann et al., "Characterization and chemistry of imidazolidinyl
urea and diazolidinyl urea," Contact Dermatitis 2006; 54:50-8).
Similarly, it is to be noted that particularly in the case of
2-nitro-1-propanol (NP), the released formaldehyde can react with
the starting material to form the nitrodiol or with 2-nitroethane
produced to form the starting material, 2-nitro-1-propanol. This
also holds true for the nitrodiol (MNPD) and the nitrotriol (HNPD),
both of which can form decomposition products during formaldehyde
liberation that could function as a substrate for reaction with the
liberated formaldehyde. Finally, it should be pointed out that, in
general, the levels of H.sub.2CO released from the three different
orders of nitroalcohols (NAs) correspond to the number of potential
H.sub.2CO units released from each molecule. That is, the triol can
theoretically release three (3) moles H2CO per parent molecule, the
diol can release two (2), and the monol can release one (1).
Second Experimental Details--Example 1
Identification of FARs
[0074] From a broad review of the literature, a total of 62
formaldehyde-releasing agents that can potentially be used for
corneal and scleral tissue cross-linking were identified. These
include FARs commonly found in cosmetics and personal care products
as well as those that are used in the textile industry. Table 1
depicts the structures, chemical formulae, toxicity, and other
pertinent information of the seven FARs that were chosen for
evaluation. None of the chemicals that were tested are known
carcinogens. They range in size up to <400 Da with IMU being the
largest at 388 Da and SMG the smallest at 104 Da [MW=127-23
(Na)=104 Da]. In most but not all cases, mutagenicity data is
available, and these chemicals have been found to be non-mutagenic
using Ames, micronucleus and other standard assays. Furthermore,
they exhibit low organismal toxicity as indicated by high
(>1,000 mg/kg,) rat oral LD50 values. The exception is BP which
has a relatively low LD.sub.50 Oral, rat =180 mg/kg.
TABLE-US-00003 TABLE 1 Characteristics of select FARs pertaining to
tissue cross-linking (TXL) in vivo % Max Octanol Allowed Toxicity
Partition Concentration (method, Coefficient (mM species, dose,
Chemical Structure (Log P) conversion) Mutagenicity exposure time)
Diazolidinyl Urea [DAU; CAS No: 78491-02-8; MW: 278.22 g/mol;
Formula: C8H14N4O7] ##STR00010## -5.398 .+-. 0.866 0.5 (17.97 mM)
Non- mutagenic* LD.sub.50 Oral - rat - 2,600 mg/kg; LD.sub.50
Dermal - rabbit - >2,000 mg/kg Imidazolidinyl Urea [IMU; CAS No:
39236-46-9- MW: 388.29 g/mol; Formula: C11H16N8O8] ##STR00011##
-4.930 .+-. 0.959 0.6 (15.45 mM) -- LD.sub.50 Oral - rat - 11,300
mg/kg Sodium Hydroxymethy Iglycinate [SMG; CAS No: 70161- 44-3; MW:
127.07 g/mol; Formula: C3H6NO3.Na] ##STR00012## -1.197 0.5 (39.06
mM) Non- mutagenic.dagger. LD.sub.50 Oral - rat - 2,100 mg/kg,
LD.sub.50 Dermal - rabbit - >2,000 mg/kg DMDM Hydantoin [DMDM;
CAS No: 6440-58-0; MW: 188.18 g/mol; Formula: C7H12N2O4]
##STR00013## -1.078 .+-. 0.654 0.6 (31.88 mM) Non-
mutagenic.dagger-dbl. LD.sub.50 Oral - rat - 3,720 mg/kg; LD.sub.50
Oral - rat - >2,000 mg/kg 5-Ethyl-1-aza- 3,7 dioxabi-
cyclo[3.3.0]octane [OCT; CAS No: 7747-35-5; MW: 143.18 g/mol;
Formula: C7H13NO2] ##STR00014## 0.274 .+-. 0.496 0.3 (20.95 mM) --
LD.sub.50 Oral - rat - >3,600 mg/kg; LD.sub.50 Dermal - rabbit -
1,948 mg/kg Bronopol [BP; CAS No: 52- 51-7; MW: 199.99 g/mol;
Formula: C3H6BrNO4] ##STR00015## 1.150 .+-. 0.631 0.1 (5 mM) Non-
mutagenic.sctn. LD.sub.50 Oral - rat - 180 mg/kg 2-hydroxymetliyl-
2-nitro-l,3- propanediol [HNPD; CAS No: 126-11-4; MW: 151.12 g/mol;
Formula: C4H9NO5] ##STR00016## -0.115 .+-. 0.77 -- Non-
mutagenic.parallel. LD.sub.50 Oral - rat- 1,917 mg/kg; LD.sub.50
Oral - mouse - 10,550 mg/kg *non-mutagenic: Ames, Micronucleus
Assay .dagger.non-mutagenic: Ames - 100% Sodium
hydroxymethylglycinate, Mouse Micronucleus; Rat Hepatocyte/DNA
Repair Assay; In vivo - In vitro Rat Hepatocyte UDS Assay
.dagger-dbl.non-mutagenic: Ames - Salmonella - 55% DMDM - 0.001-5
ul/plate; Salmonella / Mammalian-Microsome Preincubation
Mutagenicity Assay - Salmonella-2.0 ul/plate; mutagenic: L5178
TK+/- Mouse Lymphoma Assay; 0.01-1.0 ug/ml; L5178 TK+/- Mouse
Lymphoma Assay; 0.006-0.2 ul/ml; Chromosome Aberrations Assay;
Chinese Hamster Ovary Cells. 0.3 ul/ml .sctn.non-mutagenic: Ames -
Salmonella - with and without metabolic activation - dose not
specified .parallel.non-mutagenic: Ames - Salmonella with and
without metabolic activation, 1000 ug/plate; Chromosomal
Aberration
Efficacy of Corneal Cross-Linking
[0075] The ability of five FARs (DAU, IMU, SMG, DMDM, OCT) to
cross-link intact cadaveric rabbit cornea, a substrate for
collagenous tissue, was assessed using an ex vivo tissue
cross-linking (TXL) simulation set up. Cross-linking effects were
measured using differential scanning calorimetry (DSC), an assay
method based on changes in thermal denaturation temperature
(T.sub.m). Results indicate that two out of the seven FARs studied,
DAU and SMG, are effective collagen cross-linking agents for the
cornea with the epithelium left intact (in the ex vivo simulation
setup) at half maximum allowed concentrations. DAU was effective at
pH 8.5 and SMG was effective at both pH 8.5 and 7.4. This was
evidenced by shifts in the thermal denaturation temperature of
corneal tissue, as illustrated in FIG. 4.
[0076] With respect to FIG. 4, cadaveric rabbit corneas with intact
epithelia were cross-linked using the FARs DAU, IMU, SMG, DMDM, and
OCT at the indicated concentrations in 0.1M NaHCO.sub.3 buffer for
30 mins. Control samples were treated identically with vehicle. A
0.1% riboflavin-5-phosphate solution in 1.1% hydroxypropyl methyl
cellulose (HPMC, 15 centipoise) was used for CXL with the corneal
epithelium removed. .DELTA.T.sub.m indicates average shifts in the
denaturation temperature of corneal tissue after TXL compared to
the controls as measured by DSC. In this case, each experimental
determination was paired with the contralateral cornea from the
same cadaver head. Dark blue bars depict shifts at pH 8.5 whereas
light blue bars depict shifts at pH 7.4. Error bars represent
standard error. Asterisks indicate significant changes in T.sub.m
following TXL based on paired t-tests on data from at least two
independent trials (p.ltoreq.0.05).
[0077] SMG at pH 8.5 showed the greatest upwards shift in T.sub.m
(.DELTA.T.sub.m=3.573.+-.0.578.degree. C., p<0.05), followed by
DAU at pH 8.5 (.DELTA.T.sub.m=3.398.+-.0.699.degree. C.,
p<0.05). SMG also showed effective cross-linking at pH 7.4
(.DELTA.T.sub.m=2.281.+-.0.697.degree. C., p<0.05). Some
inconsistencies in the shifts in T.sub.m induced by SMG at pH 7.4,
however, were noted and a sample size of n=8 was required to reach
statistical significance. A negative shift in T.sub.m on the order
of .about.0.5.degree. C. was observed for DAU at pH 7.4 and for IMU
at both pH 8.5 and 7.4, but the shift was only significant for IMU
at pH 8.5 (.DELTA.T.sub.m=-0.69.+-.0.697.degree. C., p<0.05).
The lack of effect under these conditions may reflect issues
related to epithelial permeability since both DAU and IMU are
significantly larger than SMG. Furthermore, an increase in T.sub.m
was observed for DMDM at both pH 8.5 and 7.4
(.DELTA.T.sub.m=2.04.+-.0.225.degree. C. and 2.13.+-.0.273.degree.
C., respectively) and for OCT at pH 8.5
(.DELTA.T.sub.m=1.10.+-.0.246.degree. C.). However, these observed
increases in T.sub.m were not statistically significant using
paired controls, which included the contralateral eye for each
sample. Rabbit cornea cross-linked using UVA-riboflavin (CXL)
showed an increase in T.sub.m comparable to values previously
reported. In these results, the .DELTA.T.sub.m following
CXL=1.73.+-.0.487.degree. C. The CXL effect is relatively mild from
a "thermal transition shifting" standpoint if one considers the
potential magnitude of shifts in T.sub.m that may be induced using
chemical agents. Lastly, it is worth noting that corneal tissue
remained clear to visual inspection using either chemical or
photochemical cross-linking treatment.
Efficacy of Scleral Cross-Linking
[0078] The results for scleral tissue cross-linking are generally
comparable to the results for corneal samples, although different
methods of application were used (i.e. refreshing solution every
five mins for corneal experiments and not refreshing for scleral
experiments). In this case, two additional FARs, BP and HNPD, were
also tested. SMG, DAU, and DMDM were found to induce statistically
significant cross-linking effects at pH 8.5 and 7.4 (with the
exception of SMG at pH 7.4) at concentrations as low as half max
allowed. In addition, both a concentration and pH dependent effect
was observed for the FARs, as illustrated in FIG. 5.
[0079] With respect to FIG. 5, porcine scleral tissue was
cross-linked using three different concentrations of FAR solution
in 0.1M NaHCO.sub.3 buffer for 30 mins. Control samples were
treated identically with vehicle. .DELTA.T.sub.m indicates average
shifts in the denaturation temperature of scleral tissue after TXL
compared to the control as measured by DSC. Dark blue bars depict
shifts at pH 8.5 and light blue bars depict shifts at pH 7.4. Error
bars represent standard error. Asterisks indicate significant
changes in T.sub.m following TXL based on non-paired t-tests on
data from three independent trials (p.ltoreq.0.05).
[0080] A notable exception to the concentration dependent effect is
seen in the thermal denaturation data for SMG at pH 7.4 and 39.06
mM, where little change in T.sub.m is observed
(.DELTA.T.sub.m=0.007.+-.0.222.degree. C., p=0.493), although a
dramatic upward shift is seen for the same concentration using a pH
of 8.5 (.DELTA.T.sub.m=9.073.+-.0.450.degree. C., p<0.05). The
reason for this difference is unclear since, in general, upwards
shifts in T.sub.m occur for most FARs, albeit consistently greater
for pH 8.5 over 7.4. It should be noted that SMG is highly basic in
un-buffered solution, requiring the addition of significant amounts
of acid in order to achieve the targeted pH of 7.4. Thus, we
speculate that the procedure for titrating the buffered SMG
solution to pH 7.4 may have impacted the efficacy of TXL in this
case. This phenomenon might also explain the inconsistencies in TXL
efficacy experienced when intact cornea was cross-linked using SMG
at pH 7.4.
[0081] FARs at a concentration of 25 mM were also tested in order
to directly compare the cross-linking "potency" of each FAR in
comparison to the others, as illustrated in FIG. 6.
[0082] With respect to FIG. 6, porcine scleral tissue was
cross-linked using FAR solution at 25 mM in 0.1M NaHCO.sub.3 buffer
for 30 mins. Control samples were treated identically with vehicle.
.DELTA.T.sub.m indicates average shifts in the denaturation
temperature of scleral tissue after TXL compared to the control as
measured by DSC. Dark blue bars depict shifts at pH 8.5 and light
blue bars depict shifts at pH 7.4. Error bars represent standard
error. Asterisks indicate significant changes in T.sub.m following
TXL based on non-paired t-tests from data on three independent
trials (p.ltoreq.0.05).
[0083] A relatively high concentration was chosen for this
comparison for the goal of eliciting a noticeable cross-linking
effect using the BNAs HNPD and BP within 30 mins. DAU showed the
greatest shifts in thermal denaturation temperature at 25 mM for
both pH 8.5 and 7.4 (.DELTA.T.sub.m=7.713.+-.0.226.degree. C. and
4.347.+-.0.538.degree. C., respectively, p<0.05), followed by
SMG (.DELTA.T.sub.m=5.463.+-.0.419.degree. C. and
1.697.+-.0.311.degree. C., respectively, p<0.05), DMDM
(.DELTA.T.sub.m=2.550.+-.0.142.degree. C. and
1.693.+-.0.033.degree. C., p<0.05), and IMU
(.DELTA.T.sub.m=2.543.+-.0.280.degree. C. and
1.280.+-.0.392.degree. C., respectively, p<0.05). OCT, BP, and
HNPD exhibited shifts on the order of .about.0.5.degree. C. for
both pHs (with the exception of HNPD at pH 7.4 which had a negative
.DELTA.T.sub.m), but these shifts were not statistically
significant, when compared to FIG. 5.
Evaluation of FAR Cytotoxicity
[0084] Planar cell culture experiments using Human Skin Fibroblasts
(HSFs) were conducted to determine the toxicity thresholds of the
FARs. The toxicity threshold was taken to be the highest
concentration in mM at which all cells were alive following a 24
hour exposure to the FAR and a 48 hour recovery period. Table 2
shows that, with the exception of BP, the toxicity threshold was
found to lie between 0.1 mM and 1 mM for all FARs. BP was the most
toxic to HSFs, with a toxicity threshold between 0.01 mM and 0.001
mM. These values for BP and HNPD were in agreement with those
recently reported using the same toxicity testing apparatus (Invest
Ophthalmol Vis Sci 55:3247-3257 (2014)).
TABLE-US-00004 TABLE 2 FAR toxicity thresholds for human skin
fibroblasts Concentration (mM) DAU IMU SMG DMDM OCT HNPD BP 5 Dead
Dead Dead Dead Dead Dead Dead 1 Dead Dead Dead Dead Dead Dead Dead
0.1 Alive Alive Alive Alive Alive Alive Dead 0.01 Alive Alive Alive
.Alive Alive Alive Dead 0.001 Alive Alive Alive Alive Alive Alive
Alive Control Alive Alive Alive Alive Alive Alive Alive *Human skin
fibroblasts (Passage 2) were exposed to FARs for 24 hrs followed by
a 48 hr recovery in fresh cell media.
Discussion
[0085] Thus, use of formaldehyde releasing agents may find clinical
utility as a corneal cross-linking/stiffening agent and could have
a significant impact not only on the treatment of keratoconus
(which affects younger individuals) but also on post-PRK and
post-LASIK keratectasias, which are devastating complications of
keratorefractive surgery. These latter mentioned keratectasias are
now emerging as a significant long-term complication (5-10 years)
of LASIK and PRK surgery of unknown epidemiologic proportions
(Binder, et al., 2005). They are also the basis of many of today's
PRK- and LASIK-related medical malpractice litigations in
ophthalmology and optometry.
[0086] The earliest work from Wollensak, Spoerl, and Seiler was
reported in 1998. The initial studies were aimed at identifying
methods useful for corneal collagen cross-linking and included
riboflavin with light exposure, glutaraldehyde, formaldehyde, and
other aldehyde sugars. Spoerl, E., et al., "Induction of
cross-links in corneal tissue," Exp. Eye Res. 1998; 66:97-103;
Spoerl, E. and Seiler, T., "Techniques for stiffening the cornea,"
J. Refract. Surg. 1999; 15:711-713. These studies were followed by
reports which determined the cytotoxic dose of the treatment on
corneal endothelial cells and keratocytes using in vitro cell
culture (Wollensak, G., et al., "Corneal endothelial cytotoxicity
of riboflavin/UVA treatment in vitro," Ophthalmic. Res. 2003;
35:324-328; Wollensak, G., et al., "Keratocyte cytotoxicity of
riboflavin/UVA-treatment in vitro," Eye 2004; 18:718-722) and the
rabbit as a test animal (Wollensak, G., et al., "Endothelial cell
damage after riboflavin-ultraviolet-A treatment in the rabbit," J.
Cataract Refract. Surg. 2003; 29:1786-1790; Wollensak, G., et al.,
"Collagen fiber diameter in the rabbit cornea after collagen
crosslinking by riboflavin/UVA," Cornea 2004; 23:503-507).
Simultaneously, studies were performed which examined biochemical
properties of cross-linked corneal tissue. Basic studies examining
thermal denaturation temperature (Spoerl, E., et al.,
"Thermomechanical behavior of collagen-cross-linked porcine
cornea," Ophthalmologica 2004; 218:136-140) and resistance to
enzymatic digestion (Spoerl, E., et al., "Increased resistance of
crosslinked cornea against enzymatic digestion," Cur. Eye Res.
2004; 29(1):35-40) indicated that the combination of UVA with
riboflavin as a photosensitizer was effective in cross-linking
corneal collagen lamellae. These studies were performed in
conjunction with biomechanical testing which confirmed increases in
Young's modulus (Wollensak, G. and Spoerl, E., "Collagen
crosslinking of human and porcine sclera," J. Cataract Refract.
Surg. 2004; 30:689-95; Kohlhaas, M., et al., "Biomechanical
evidence of the distribution of cross-links in corneas treated with
riboflavin and ultraviolet A light," J. Cataract Refract. Surg.
2006; 32:279-283). Such basic biochemical, biomechanical, and
animal studies were then followed by in vivo experiments aimed at
determining the potential usefulness of this treatment in the
living human eye.
[0087] Several chemical cross-linking agents were tested previously
by the UVR group in comparison studies with the UVR method and
included glucose, ribose, glyceraldehyde, and glutaraldehyde. Of
these, only glyceraldehyde and glutaraldehyde, (i.e. aldehydes)
were found to produce a significant biomechanical effect
(Wollensak, G. and Spoerl, E., 2004). Glutaraldehyde is a well
known cross-linking agent used for tissue cross-linking of
bioprosthetic heart valves and for tissue fixation prior to viewing
by electron microscopy. Its utility as an in vivo cross-linking
agent, however, is limited by its significant cytotoxic effects.
This is true for several other effective yet toxic aldehyde
cross-linking agents, such as formaldehyde and glycoaldehyde.
Glyceraldehyde is a physiologic metabolic product, is generally
considered non-toxic, and could also be potentially used for
topical corneal cross-linking. Another class of cross-linking
compounds that could have utility for in vivo cross-linking is the
iridoid compounds, of which genipin is an example. Nimni, M. E.,
"Glutaraldehyde fixation revisited," Journal of Long-Term Effects
of Medical Implants 2001; 11(3&4):151-161; Jayakrishnan, A. and
Jameela, S. R., "Review: Glutaraldehyde as a fixative in
bioprostheses and drug delivery matrices," Biomaterials 1996;
17:471-484.
[0088] This invention uses a formaldehyde releasing agent to
cross-link collagen in collagenous tissue.
[0089] This invention is an alternative method of tissue
cross-linking in the eye, that is, a reaction of collagen with a
formaldehyde releasing agent.
[0090] This concept has been spurred by recent developments in the
treatment of keratoconus. In this case, collagen cross-linking
using riboflavin/UVA has been used to stabilize corneal collagen
lamellae, preventing the untoward effects of progressive corneal
thinning. Thus, this invention involves the application of
formaldehyde releasing agent-induced cross-linking to the
stiffening of collagen containing tissues for the purpose of
stabilization with therapeutic intent. In some cases, collagen
cross-linking is desirable as a treatment of certain conditions or
to preserve tissue during transplantation as described herein.
[0091] Thus, formaldehyde releasing agents, such as diazolidinyl
urea (DAU), have been shown to be beneficial corneal cross-linking
agents, as indicated by thermal denaturation and biomechanical
inflation testing previously discussed.
[0092] Moreover, with respect to the second experimental details,
both intact cornea and cut scleral tissue pieces were used to test
the cross-linking efficacy of compounds known as formaldehyde
releasing agents (FARs), comparing the effects against two higher
order nitroalcohols (HONAs), BP and HNPD. Three of the FARs were
found to be significantly more effective as tissue cross-linking
agents when compared to the HONAs, showing both pH and
concentration dependent effects. The FARs are a group of compounds
commonly used as preservatives in cosmetics and personal care
products and as fabric cross-linkers in the textile industry (i.e.
for making wrinkle-free clothing), and include bronopol (BP), which
is a well-known compound. They are known to release formaldehyde in
a pH and concentration dependent manner as determined by .sup.13C
NMR equilibrium studies, where formaldehyde release amongst FARs
popularly used in cosmetics, including DAU, IMU, DMDM, and SMG, was
compared.
[0093] FARs in commercial use include O- and N-formal compounds. An
O-formal group is a formaldehyde entity linked to the rest of the
compound via an oxygen atom. An N-formal group is a formaldehyde
entity linked to the rest of the compound via a nitrogen atom and
can be of two types: amide-based (the nitrogen is a part of an
amide) and amine-based (the nitrogen is a part of an amine). The
type of group attached to the N-formal group confers different
release properties. Slower release occurs with the amide-based
N-formals (such as DAU, IMU, and DMDM), which can act as
formaldehyde reservoirs, whereas amine based N-formals like SMG
have been reported to decompose completely under alkaline
conditions and max allowed concentration.
[0094] Based on chemical structure alone, DAU would be predicted as
the most effective cross-linking agent with the ability to release
4 mols of formaldehyde (contains 4 N-formal groups), followed by
HNPD (3 mols), with SMG being the least effective (1 mol). The
amount of formaldehyde actually released in solution by each FAR,
however, is not as easily predictable as evidenced by the pH and
concentration dependent effects noted earlier. The release of
formaldehyde is reported to be facilitated at acid pH for SMG, in
contrast to the other FARs and nitroalcohols which are facilitated
by alkaline pH. Once released from FARs, formaldehyde can react in
a number of ways, including reactions with starting material or
polymerizing, which can occur under equilibrium conditions. In
addition, the availability of reactive substrates under
non-equilibrium conditions (such as in the presence of tissue
amines from cornea and sclera, for example) can drive the reaction
toward formaldehyde release. When used at max allowed concentration
(0.5%) as employed herein, formaldehyde release from SMG has been
reported to be rapid at pH 8.5, which is consistent with its
structure as an amine based N-formal compound.
[0095] Chemical tissue cross-linking (TXL) using FARs were compared
with riboflavin-mediated photochemical collagen cross-linking
(CXL), which is regarded as the "gold standard" of therapeutic
corneal cross-linking. Our value for the increase in thermal
denaturation temperature following CXL is slightly lower than the
shift in the onset of thermal shrinkage (ATI) reported by Spoerl et
al. (Ophthalmologica Journal International d'ophtalmologie
International Journal of Ophthalmology Zeitschrift fur
Augenheilkunde 218:136-140 (2004)) following CXL of the anterior
portion of porcine cornea: .DELTA.T.sub.m=1.733.+-.0.487.degree. C.
vs. .DELTA.T.sub.i=2.5.degree. C. (originally reported as
.DELTA.T.sub.i=5.degree. C. but confirmed to be 2.5.degree. C.
(Invest Ophthalmol Vis Sci 50:1098-1105 (2009))). A 1.9.degree. C.
shift in T.sub.i for porcine cornea cross-linked using the
UVA-riboflavin method was previous reported. Therefore, the
aforementioned values for .DELTA.T.sub.m induced by CXL are similar
to the shifts in T.sub.i induced by CXL as reported previously even
considering the differences in species used (i.e. rabbit vs.
porcine cornea).
[0096] Corneal epithelial permeability is another consideration
that should be borne in mind. These results are favorable since the
ex vivo setup simulates conditions that would be encountered in a
living system. Of particular interest is the fact that
cross-linking effects were induced with the corneal epithelium
intact, suggesting that some of these compounds may be able to pass
through the epithelial barrier (i.e. SMG MW=127 Da). The ability to
induce a cross-linking effect without the need for epithelial
removal, if possible, would be a significant advantage over
riboflavin-mediated collagen cross-linking (CXL). Differences in
transepithelial permeability for IMU, for example, may explain the
lack of cross-linking effect seen in the intact cornea, as
illustrated in FIG. 4. IMU is the largest of the compounds tested
at 388 Da and its size may have hindered passage into the corneal
stroma, accounting for the lack of effect in cornea, while positive
cross-linking effects were observed for the same compound with cut
scleral pieces where permeability was not hindered by an intact
corneal epithelium, as illustrated in FIGS. 5 and 6. Molecular size
is well-known to affect transcorneal permeability, especially for
hydrophilic compounds such as the ones under consideration
here.
[0097] Regarding thermal denaturation as an assay for tissue
cross-linking, several methods have been used previously to
evaluate cross-linking changes intentionally induced in collagenous
tissues by either chemical or photochemical means and include
mechanical testing (either uniaxial strip or inflation testing),
enzymatic digestion, gel electrophoresis, and thermal denaturation.
Thermal denaturation (as thermal shrinkage temperature) was
previously used as an assay measure of chemically and
UVA-riboflavin induced cross-linking of collagenous tissue. Tissue
cross-linking efficacy was evaluated using an automated instrument
that measures change in heat flow over time during the thermal
denaturation of a given substance, which is known as differential
scanning calorimetry (or DSC). Thermal transition temperature is a
concept familiar to the biomaterials industry where it has been
used as a means to evaluate the efficacy of tissue cross-linking
for decades. DSC produces a denaturation curve, which depicts a
major endotherm with the T. value at its peak. In the case of
collagenous tissue, the major endotherm reflects collagen
denaturation, which involves triple helical uncoiling and tissue
shortening. In addition to collagen cross-linking, it is possible
for proteoglycans to be modified in the tissue cross-linking
procedure since the core protein contains potential reactive sites.
However, this is not expected to alter or contribute to the thermal
denaturation of collagen since removal of proteoglycans has been
shown not to alter the T.sub.m of collagenous soft tissue.
[0098] DSC has been used successfully in many tissue types.
including tendon, bone, cartilage, and skin, but there are few
reports regarding cornea. An additional advantage of DSC is that
tissue samples are hermetically sealed, preventing tissue
dehydration, which can introduce experimental error into these
measurements. Changes that can occur in the water content of tissue
are particularly relevant in the case of cornea, which has a large
capacity to swell and/or shrink. Finally, the ease of analyzing DSC
data using the Pyris software adds to the effectiveness of using
DSC for cross-link analysis.
[0099] In order to directly assess the toxicity of these chemicals,
an in vitro cell toxicity experiment was conducted using human skin
fibroblasts (HSFs) and these FARs. The toxicity threshold of all
FARs tested was determined to be below 1 mM with the exception of
bronopol, which was the most toxic (toxicity threshold below 0.01
mM). Past cell toxicity studies using HSFs indicated that genipin
and glutaraldehyde both have toxic thresholds on par with bronopol.
Glyceraldehyde was previously shown to be the least toxic
cross-linking agent for HSFs, with a toxic threshold of 1 mM.
Therefore, the toxicity of FARs lies between the toxicity of
glutaraldehyde and glyceraldehyde, with glyceraldehyde being the
least toxic. The cell toxicity thresholds determined are not
designed to provide direct clinical information regarding
potentially applicable concentrations, but rather, as a means to
compare toxicity between compounds.
[0100] Finally, with regards to safety, owing to their widespread
use in cosmetics and by the textile industry, where workplace
hazards are closely scrutinized, the FARs have been extensively
tested in European safety studies by the Scientific Committee on
Cosmetics and Non-food Products following the commission of
Cosmetic Products Directive 76/768/EC by the Council of the
European Communities in 1976. The result of the Cosmetic Directive
was a delineation of which FARs can appear in cosmetics and
personal care products and at what concentrations. The maximum
allowed concentrations of FARs as defined in the Cosmetic Directive
were adapted on the belief that working within the maximum allowed
value would be a good starting point in evaluating effects that
could be induced in patients.
[0101] In conclusion, the aforementioned disclosure has
demonstrated a novel therapeutic application for formaldehyde
releasing agents commonly employed in consumer personal care
products. Two of these agents, DAU and SMG, have shown effective
cross-linking abilities in intact cornea and cut scleral pieces as
indicated by shifts in thermal denaturation temperature (Tm). In
light of the current growing therapeutic cross-linking application
in both the cornea and sclera, FARs may have therapeutic potential
in the treatment of diseases such as keratoconus and myopia.
Continued screening of FARs from the compiled registry could lead
to the identification of additional potent cross-linking
agents.
[0102] Additional references relating to this invention include the
following: Abraham, V. C., et al., "High content screening applied
to large-scale cell biology," Trends in Biotechnology 2004;
22(1):15-22; Amano, S., et al., "Comparison of central corneal
thickness measurements by rotating scheimpflug camera, ultrasonic
pachymetry, and scanning-slit corneal topography," Ophthalmology
2006; 113:937-941; Bailey, A. J., "Molecular mechanisms of ageing
in connective tissues," Mech. Aging Dev. 2001; 122:735-55; Banse,
X., et al., "Cross-link profile of bone collagen correlates with
structural organization of trabeculae," Bone 2002; 31(1):70-76;
Bednarz, J., et al., "Effect of three different media on serum free
culture of donor corneas and isolated human corneal endothelial
cells," Br. J. Ophthalmol. 2001; 85:1416-1420; Brady, J. D. and
Robins, S. P., "Structural characterization of pyrrolic cross-links
in collagen using a biotinylated Ehrlich's reagent," J. Biol. Chem.
2001; 276(22):18812-18818; Chiou, A. G. Y., et al., "Clinical
corneal confocal microscopy," Survey of Ophthalmology 2006;
51(5):482-500; Eyre, D. R., et al., "Cross-linking in collagen and
elastin," Ann. Rev. Biochem. 1984; 53:717-748; Lackner, B., et al.,
"Repeatability and reproducibility of central corneal thickness
measurement with pentacam, orbscan, and ultrasound," Optometry and
Vision Science 2005; 82:892-899; Lee, M. Y. and Dordick, J. S.,
"High-throughput human metabolism and toxicity analysis," Current
Opinion in Biotechnology 2006; 17:619-627; McLaren, J. W., et al.,
"Corneal thickness measurement of confocal microscopy, ultrasound,
and scanning slit methods," Am. J. Ophthalmol. 2004; 137:1011-1020;
Naor, J., et al., "Corneal endothelial cytotoxicity of diluted
providone-iodine," J. Cataract Refract. Surg. 2001; 27:941-947;
Sady, C., et al., "Advanced Maillard reaction and crosslinking of
corneal collagen in diabetes," Biochem. Biophys. Res. Com.
1995;214(3):793-797; Sell, D. R. and Monnier, V. M., "Structure
elucidation of a senescence cross-link from human extracellular
matrix," J. Biol. Chem. 1989; 264(36):21597-21602; Skinner, S. J.
M., "Rapid method for the purification of the elastin cross-links,
desmosine and isodesmosine," J. Chromatog. 1982; 229:200-204;
Wollensak, G., et al., "Stress-strain measurements of human and
porcine corneas after riboflavin-ultraviolet-A-induced
cross-linking," J. Cataract Refract. Surg. 2003; 29:1780-1785.
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