(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
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`17 February 2011 (17.02.2011) (10) International Publication Number
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`(43) International Publication Date
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`WO 2011/019940 A2
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`(51)
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`International Patent Classification:
`A61K 47/38 (2006.01)
`A61K 31/205 (2006.01)
`A61K 47/42 (2006.01)
`A61K 9/08 (2006.01)
`A61K 31/14 (2006.01)
`A61P 27/02 (2006.01)
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`(81)
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`(21)
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`International Application Number:
`PCT/US2010/045 3 5 6
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`(22)
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`International Filing Date:
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`12 August 2010 (12.08.2010)
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`(25)
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`(26)
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`(30)
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`(71)
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`(72)
`(75)
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`(74)
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`Filing Language:
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`Publication Language:
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`English
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`English
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`Priority Data:
`61/233,315
`
`12 August 2009 (12.08.2009)
`
`US
`
`Applicant flor all designated States except US): SEROS
`MEDICAL, LLC [US/US]; 226 W. Edith Avenue, Unit
`#23, Los Altos, California 94022 (US).
`
`Inventor; and
`Inventor/Applicant (for US only): HEREKAR, Satish
`V. [US/US]; 820 La Para Avenue, Palo Alto, California
`94306 (US).
`
`Agents: LUPKOWSKI, Mark et a1.; Squire, Sanders &
`Dempsey L.L.P., 275 Battery Street,Suite 2600, San Fran-
`cisco, California 9411 1 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, R0, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`Published:
`
`without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(g))
`
`(54) Title: DEUTERATED WATER AND RIBOFLAVIN SOLUTION FOR EXTENDING SINGLET OXYGEN LIFETIMES
`IN TREATMENT OF OCULAR TISSUE AND METHOD FOR USE
`
`FIG. 1 C
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`(57) Abstract: A solution of deuterated water containing a riboflavin-based photosensitizer is provided in order to extend life-
`times of UVA/Rf photo-generated intra-stromal singlet oxygen, in combination with UVA delivery profiles of pulsing, fractiona-
`tion, and optionally auxiliary stromal/Rf hyper-oxygenation in order to accelerate protein cross-linking density rates in ocular tis-
`sue. A 100% deuterated water solution with 0.1% riboflavin in solution increases singlet oxygen lifetimes by at least an order of
`magnitude without inducing endothelial cell apoptosis, thereby also permitting use of some combination of lower percentages of
`deuterated water, lower concentrations of riboflavin or lower dosages of UVA on intact (un—debrided) epithelium for equivalent
`cross-link densities compared to current acceptable corneal cross-linking procedures. Lower concentrations of deuterated water
`with regular water, for example, yields shorter singlet oxygen lifetimes in approximately linear proportion to concentration, which
`are considered acceptable in therapies known or being developed in the art of corneal cross-linking.
`
`
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`W0 201 1/019940
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`PCT/US2010/045356
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`DEUTERATED WATER AND RIBOFLAVIN SOLUTION FOR
`
`EXTENDING SINGLET OXYGEN LIFETIMES IN TREATMENT OF
`
`OCULAR TISSUE AND METHOD FOR USE
`
`This application claims benefit of US. Patent Application No. 61/233,315
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`which was filed on August 12, 2009.
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`BACKGROUND OF THE INVENTION
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`[0001]
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`This invention relates to compositions, methods and delivery systems for promoting
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`cross—linking of proteins in tissue using ultraviolet irradiation of a solution of water
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`containing riboflavin or its analogues, particularly in ocular tissue (such as tissue in the
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`sclera, cornea, prepapillary region, etc.).
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`[0002]
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`Therapies are known or are under laboratory investigation for promoting structural
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`enhancement of stromal and scleral tissues by application of ultraviolet A radiation to
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`riboflavin in a water solution on ocular tissue in the presence of oxygen—containing
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`atmosphere. The present inventor has determined that singlet oxygen lifetimes have an
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`evident impact on degree of cross—linking densities of protein such as collagen, a main
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`structural component of stromal and scleral tissues.
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`[0003] Literature reports that deuterated water can increase singlet oxygen lifetimes in
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`various methods for generating singlet oxygen. This invention takes advantage of this
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`discovery in a new context. A search of the literature has found no reports or suggestions of
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`the present methodology and compositions. Reference is made to the collection of references
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`supplied by the inventor t0 the Patent and Trademark Office for consideration.
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`[0004] Collagen cross—linking (CXL) in ophthalmology, as it currently exists in Europe
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`(where it is approved), provides a biomechanical basis of increased corneal strength (i.e.,
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`stability & stiffness) as a result of the formation of covalent bonding between collagen
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`strands. This occurs when a photo—sensitizer, riboflavin (Vitamin B—2) is applied to the de—
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`epithelialized surface of the cornea. This epithelial protective tissue over the cornea is
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`surgically debrided (i.e., surgically removed) so the riboflavin can pass (i.e., be absorbed)
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`into the stroma (collagen layers) of the cornea. After the riboflavin saturates the stroma, it is
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`exposed to UVA light (approximately 365 nm). This excitation of thc riboflavin by the UVA
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`results in the creation of free radicals that interact with amino acids and carbonyl groups in
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`neighboring collagen molecules to form the strong covalent chemical bonds. Debride refers
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`to removal of dead, contaminated or adherent tissue or foreign material.
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`[0005]
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`The primary emphasis in the application of CXL for ophthalmology has been in the
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`treatment of keratoconus, which is prevalent in about one in 2,000 people in the US and
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`Europe, with a slightly higher percentage in Asian countries. This condition is manifested by
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`a weak cornea which becomes too elastic and stretches, causing it to bulge forward. This
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`changes the curvature of the cornea which almost always leads to poor visual acuity (not
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`correctable with glasses and/or soft contact lenses) that requires the use of rigid gas
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`permeable lens. Thus, when the cornea begins losing its shape (i.e., becomes cone shaped
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`instead of spherical) the person typically becomes nearsighted and will develop irregular
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`astigmatism, which causes the blurring of vision. As this condition progresses, this person
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`may develop scarring and a very irregular corneal curvature. If the person cannot be helped
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`with the rigid contact lens, then he/she will require a corneal transplantation.
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`[0006]
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`There are other conditions/corneal diseases where the cornea can become stretched
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`and distorted. One of these, where CXL is currently being utilized, is in corneal ectasia. This
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`condition involves stretching of the cornea (collagen tissue) that occurs after refractive
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`surgeries, such as laser in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK).
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`Other corneal diseases in which CXL has been tried successfully include corneal ulceration
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`(possible sequelae to bacterial, viral or fungal infections) and bullous keratopathy (excess
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`fluid accumulation causing corneal edema).
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`[0007]
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`The existing procedure of CXL has been clinically proven (in Europe) to be safe.
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`However, in its current form, the procedure is very rudimentary with a number of significant
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`limitations including but not limited to:
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`the procedure takes too long (approximately one
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`hour in total); removal of the corneal epithelium (i.e., debridement) is required, making the
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`procedure invasive and uncomfortable for the patient intra—operatively and for 3—4 days
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`following surgery; and, it is not fully measurable for accuracy. These limitations clearly
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`preclude the use of CXL for many corneal treatments that would require a fast and highly
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`accurate process for stiffening and stabilizing the cornea.
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`SUMMARY OF THE INVENTION
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`[0008] According to the invention, a solution of deuterated water containing a riboflavin—
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`based photosensitizer (Rf aka Vitamin B2) is provided in order to extend lifetimes of
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`UVA/Rf photo—generated intra—stromal singlet oxygen, in combination with UVA delivery
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`profiles of pulsing, fractionation, and optionally auxiliary stromal/Rf hyper—oxygenation in
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`order to accelerate protein cross—linking density rates in ocular tissue.
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`[0009]
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`This invention is based upon the discovery that there is a correlation between the
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`concentration of dissolved [singlet] oxygen in irradiated ocular tissue and the efficiency of
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`cross—linking with the photo—sensitizer riboflavin. Our studies have demonstrated that a
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`100% deuterated water (D20) solution with 0.1% riboflavin in solution increases singlet
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`oxygen lifetimes by about an order of magnitude (10X or more). Further studies have shown
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`that the such application of deuterated water does not induce endothelial cell apoptosis.
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`[0010] Our studies have also shown that by delivering optimized combinations of
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`deuterated water, riboflavin, and UVA dosage on an intact (undebrided) epithelium,
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`equivalent cross—link densities are rapidly achieved with reduced adverse effects as compared
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`to current treatments. Lower concentrations of deuterated water with regular water, for
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`example, yields shorter singlet oxygen lifetimes. These lifetimes have approximately a linear
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`relationship to the concentration of deuterated water. These lower concentrations are
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`considered acceptable in therapies known or being developed in the art of corneal cross—
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`linking.
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`[0011] Our experiments have shown various correlations such as between the following:
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`the concentration of D20 and reactive oxygen species (ROS) lifetimes; the UVA fluence and
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`ROS concentration; and, the dissolved oxygen consumption and UVA fluence.
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`[0012] Deuterated water refers to water containing a hi gher—than—normal proportion of the
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`hydrogen isotopc dcutcrium, cithcr as dcutcrium oxidc, D20 or 2H2O, or as dcutcrium
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`protium oxide, HDO or 1HZHO. Conventional water is water that has a normal proportion of
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`deuterium isotope, such as in tap water to distilled water.
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`[0013]
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`Thc invcntion will bc bcttcr undcrstood by rcfcrcncc to thc following dctailcd
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`description in connection with the accompanying drawings.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0014]
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`Figures lA—lC are illustrations of a first method according to the invention.
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`[0015]
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`Figures 2A—2D are illustrations of a second method according to the invention.
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`[0016]
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`Figure 3 L a schematic diagram of a delivery system according to the invention.
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`[0017]
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`Figure 4 is a graph showing the relationship between D20 and ROS lifetimes.
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`[0018]
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`Figure 5 is a graph showing the relationship between ROS concentration and UVA
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`irradiation (fluence).
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`[0019]
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`Figure 6 is a graph showing the relationship between rate of oxygen consumption
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`and UVA irradiation.
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`DETAILED DESCRIPTION OF THE INVENTION
`
`[0020]
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`The invention is embodied in methods, compositions and delivery systems,
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`particularly in relation to therapies for strengthening and re—shaping ocular tissue. The
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`formulation invention includes a composition or substance comprising a solution of
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`deuterated water (between 100 wt% D20 and 10 wt% D20 in water) containing a riboflavin
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`based photo—sensitizer, carboximethylcellulose (CMC), and benzalkonium chloride (BAC).
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`In one embodiment, the riboflavin based photo—sensitizer is Rf aka Vitamin B2. It should be
`
`noted that all concentrations are, unless otherwise specified, wt/vol (for example, 0.1% Rf
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`refers to ~01 gm in lOOmL). The molecular weight of Rf is: ~378 gm/L. In some
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`embodiments, the concentration of the riboflavin based photo—sensitizer, such as Rf aka
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`Vitamin B2, is between X and Y, or more narrowly, V and W.
`
`In some embodiments, the
`
`concentration of carboximethylcellulose, is 0.2% or about 0.2%. In some embodiments, the
`
`concentration of carboximethylcellulose is between X and Y, or more narrowly, V and W. In
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`some embodiments, the concentration of BAC, is 0.2% or about 0.2%. In some
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`embodiments, the concentration of BAC is between X and Y, or more narrowly, V and W. In
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`certain embodiments, the formulation invention includes deuterated water between 100%
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`D20 and 10% D20 in water containing, the riboflavin based photo—sensitizer Rf aka Vitamin
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`B2 of about 0.1% Molar concentration, carboximethylcellulose (CMC) of about 0.2%, and
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`benzalkonium chloride (BAC) of about 0.02%. Embodiments of the invention include any
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`value or range of D20 between 10% and 100% in the formulation.
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`[0021] Referring to Figures lA—lC, a method according to the invention is illustrated.
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`Figure 1A depicts an intact cornea l 1 comprising an epithelium 12 with underlying stromal
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`tissue 14. As shown in Figure 1B, the formulation 16 according to the invention is applied as
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`a spray or droplets to the epithelium 12 in the presence of ambient oxygen (in the air) to an
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`undebrided corneal surface. The period of exposure of formulation is several minutes.
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`In
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`some embodiments, the period of exposure can be between 1 to 2, 2 to 3, 3 to 5, 5 to 7, 7 to
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`10, or greater than 10 minutes. Bursts of spray or droplets are applied over the affected area
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`for the duration of the soaking cycle.
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`[0022]
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`Then the solution—soaked stromal region 14 is irradiated with ultraviolet A 18, as
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`shown in Figure 1C. The UVA irradiation treatment may be continuous (i.e., irradiation
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`without interruption) for a period ranging from 1 to 15 minutes or fractionated (turned on and
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`off for a few seconds to a minute) or pulsed (brief bursts of high irradiance with ON times in
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`the 1 microsecond to millisecond range, and frequencies in the 1 killohertz to 500 killohertz
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`range. The irradiation creates reactive oxygen species (ROS) that cause the desired
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`crosslinking of proteins 20. In one preferred embodiment, which maximizes benefits
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`efficiently, the irradiation is pulsed and fractionated, to promote the production of singlet
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`oxygen, or reactive oxygen species (ROS) in the intrastromal region to thereby promote the
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`desired cross—linking of proteins 20 during the lifetimes of the reactive oxygen.
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`[0023]
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`In embodiments of the invention, there may be various compounds which can
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`function as both preservative and penetration enhancers. These compounds include,, but are
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`not limited to benzalkonium chloride (BAC) and sodium ethylenediaminetetraacetate
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`(EDTA), as,well as viscosity agents such as carboxymethylcellulose (CMC) or dextran. BAC
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`(~0.02%) and EDTA (~0. 1%) enhance penetration of the riboflavin and D20 water
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`formulation. CMC (~0.2%) or dextran (~20%) enhance the lubricity and the formation of a
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`persistent, broader, and more uniform corneal tear film before and during the procedure. This
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`allows greater absorption of the active ingredients of the formulation into the cornea.
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`[0024]
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`Significantly, the riboflavin formulation can also be manufactured with a high
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`concentration of dissolved oxygen. This oxygen enrichment enables the production of greater
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`ROS concentration in a shorter period of time, and, in turn, this makes higher UVA irradiance
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`practical. However, it should be noted that there may be other means to diffuse oxygen gas
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`into the stroma, which might include, among others, the use of a device that would deliver
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`such oxygen gas to the corneal surface. This oxygen gas then diffuses (albeit slowly) into the
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`stroma, thereby increasing dissolved oxygen.
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`In summary, the ability to increase dissolved
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`oxygen in the stroma enables the use of a higher UVA irradiance exposure to the collagen
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`tissue. This concept of embedding dissolved oxygen in the stroma means that optimum
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`cross—linking (i.e., adequate stiffness of the cornea with minimal side effects) can be achieved
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`in a shorter period of time. A means to manufacture the enriched oxygen deuterated Rf
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`formulation, that will provide up to and over 1 year of cxtcndcd shelf life, is contemplated by
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`this invention. The components of the formulation invention, which are set forth hereinabove,
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`may be optimized for penetration rate, pH, hypotonicity, and lubricity by the proportions that
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`each component is used within the formulation.
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`[0025] Referring to Figures 2A—2D, a further method according to the invention is
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`illustrated. The intact cornea 11 (Figure 1A) comprises an epithelium 12 with underlying
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`stromal tissue 14. The corneal surface is debrided to remove the surface layer and expose the
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`underlying tissue (Figure 2B) in a debrided region 13. A solution 16 according to the
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`invention is applied as a spray or droplets to the debrided region 13 in the presence of
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`ambient oxygen (in the air) to the (Figure 1B). The period of exposure is several minutes.
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`The same ranges of exposure disclosed in the embodiments of Figures lA—C apply to Figures
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`2A—D. Bursts of spray or droplets are applied over the affected area for the duration of the
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`soaking cycle. Due to the debriding, the stromal tissue 14 is soaked to a greater depth than
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`the embodiments of Figures lA—C. Then the solution—soaked stromal region 14 is irradiated
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`with ultraviolet A 18 (Figure 2D). As described above, the UVA irradiation treatment may be
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`continuous or fractionatcd (turncd on and off for cxtcndcd pcriods) or pulscd (bricf bursts of
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`high illumination for an extended period), or most preferably pulsed and fractionated, to
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`promote the production of singlet oxygen, or reactive oxygen species (ROS) in the deep
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`intrastromal region to thereby promote the desired cross—linking of proteins 20 during the
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`lifetimes of the reactive oxygen.
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`[0026]
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`The process of soaking the formulation, on either a debrided or undebrided surface,
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`results in diffusing oxygen into the stroma. For a undebrided surface, the penetration is to a
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`dcpth of up to about 0.5 mm. Thc pcnctration into dcbridcd surfaccs is grcatcr than 0.5 mm.
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`The UVA irradiation in the presence of oxygen promotes singlet oxygen species generation.
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`The deuterated water with riboflavin extends lifetimes of UVA/Rf photo—generated intra—
`
`stromal singlet oxygen This in combination with UVA delivery profiles of pulsing,
`
`fractionation, and optionally auxiliary stromal/Rf hyper—oxygenation accclcratcs protcin
`
`cross—linking density rates in the ocular tissue. Our studies have shown that the use of a 100%
`
`deuterated water solution with 0.1% riboflavin in solution increases singlet oxygen lifetimes
`
`by at least an order of magnitude (10X or more) without inducing endothelial cell apoptosis.
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`lt is wcll known in the arts that the current cross—linking proccdurc may inducc thc following
`
`side effects: (1) stromal haze due to keratocyte apoptosis; (2) endothelial cell density loss.
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`[0027]
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`In another embodiment, the formulation includes a combination of lower
`
`pcrccntagcs of dcutcratcd watcr, lowcr conccntrations of riboflavin or lowcr dosagcs of UVA
`
`on intact (un—debrided) epithelium may be employed for equivalent cross—link densities as
`
`compared to current acceptable corneal cross—linking standards for CXL procedures. In
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`various embodiments, the ranges of components and delivery parameters of the formulation
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`are as follows: 100% D20 to 1%; 0.1% tho 0.01%; 0.02% BAC to .01%; 0.2% CMC to
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`0.1%; 5.4 J/cm2 UVA to 2.5J/cm2; 30 minutes or less UVA exposure.
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`[0028]
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`Figure 4 shows the singlet oxygen lifetime in deuterated water as a function of
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`concentration of D20. Figure 4 demonstrates the correlation of ROS lifetimes to varying
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`D20 solvent (0% to 100%) in the 0.1% Rf solution under normoxic (i.e., ambient oxygen
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`dissolved into the test sample at room temperature by natural diffusion conditions in collagen
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`and 0.1% Rf matrices. As shown in Figure 4, lower concentrations of deuterated water with
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`regular water, for example, yield shorter singlet oxygen lifetimes. The relationship between
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`singlet oxygen lifetime and D20 concentration in regular water is approximately linear, as
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`shown in Figure 5. This data was generated by a custom built photon counter and dissolved
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`oxygen probe, which was excited by a frequency tripled Nd:Yag laser for time—resolved
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`measurements.
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`[0029] The inventor has measured reactive oxygen species (ROS) in vitro in aerated collagen
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`and riboflavin under UVA illumination and has found an increase in the ROS duration of
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`about 4.5 uSecs for H20 with no D2O to a duration of over 45 uSecs for a 100% deuterated
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`solvent D20 (See Figure 5). Figure 5 shows a strong linear correlation of ROS concentration
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`in a normoxic collagen and Rf matrix as UVA irradiance is varied from about 3 mW/cm2 to
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`about 50 mW/cmz.
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`[0030] Figure 6 shows the inverse correlation of dissolved oxygen concentration (due to
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`consumption from varying ROS generation) with varying UVA irradiance in a normoxic
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`collagen+0. 1% Rf matrix. A 500% factor is shown in the example below.
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`[0031] A system using dual UVA/Blue sources is able to provide pulsed irradiances up to
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`150 mW/cmz, with pulsing frequencies at up to 200 kHz and is, for example, set to deliver
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`pulses at a 20 kHz (50 uSecs) pulse repetition frequency, and a duty cycle of about 20%
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`(intermittency). This is a 40 uSec UVA OFF period and a 10 uSec high intensity UVA ON
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`period applied cyclically. It is believed that the 10 uSec UVA ON pulse rapidly generates a
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`maximal new population of ROS molecules in the targeted stroma just as the previously
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`stimulated ROS population is about to be depleted or otherwise be consumed through
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`quenching mechanisms in the local microenvironment. It is believed that the 40 uSec UVA
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`OFF period provides sufficient time for chemical interactions in the microenvironment to
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`effect cross—linking of proteins, specifically collagen, in the target region. The singlet oxygen
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`population in the presence of the aerated deuterated solvent survives for an extended duration
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`of about 40 uSecs, while the UVA is OFF.
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`[0032]
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`In a specific embodiment, during the extended reactive lifetime of singlet oxygen,
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`rapid cross—linking reactions are induced in the carbonyl (aldehyde) groups of collagen while
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`dissolved oxygen 02, riboflavin, and singlet oxygen species (ROS) are consumed (as long as
`
`present in sufficient concentrations) by Type 11 (energy transfer) mechanisms. (From photo—
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`chemistry competitive mechanisms of radicals formation are known: electron transfer or
`
`Type I; and, energy transfer, Type II.) This on—off cycle repeats every 50 uSecs (at 20 kHz).
`
`Analogues of riboflavin may also be employed, such as 3—methyl—riboflavin tetraacetate.
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`[0033] As riboflavin molecules degrade and transform through such singlet oxygen
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`regenerative timing cycles, they generate reduced fluorescence intensity in the 530nm-570nm
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`band in response to UVA which may thereby signal a riboflavin “reinstillation” cycle.
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`[0034]
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`In addition to increased endothelial safety due to reduced riboflavin concentration
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`requirements, the use of viscous carboxy—methyl—cellulose (CMC) in the present formulation
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`forms a corneal film of thickness ~ SOHM—ZOOHM, which provides added UVA protection to
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`the endothelium. Pulsed UVA applied as herein described (instead of CW UVA) provides
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`for a reduced apoptotic effect on both keratocytes and endothelial cells.
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`[0035]
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`The formulation (D20 + CMC + BAC) provides for faster penetration and
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`clearance, reducing pre—treat soak times and end—product clearance periods. The use of BAC
`
`as a penetration enhancer has been previously reported in the literature.
`
`[0036]
`
`The rate of diffusion of dissolved oxygen through the stroma depend on corneal
`
`thickness, epithelialization state (whether or not debrided), sensitizer pre—oxygenation,
`
`viscosity and ambicnt oxygcn cnvironmcnt of the stroma. Somc amount of dissolved oxygcn
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`will continue to migrate into the stroma and sclera. However, during UVA irradiation a
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`much larger consumption of local dissolved oxygen occurs than can be supplied through
`
`ambient diffusion The formulation, and the use of UVA pulsation and fractionation is able to
`
`overcome the dissolved oxygen limitations inherent in ambient diffusion. Depending on the
`
`depth of cross—linking desired, a pause in the UVA irradiation (of the order of seconds to
`
`minutes) cycle may permit dissolved oxygen to permeate deeper in the stroma before
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`localized consumption due to ROS generation.
`
`[0037]
`
`Total cross—linking treatment times and singlet oxygen/riboflavin molecular
`
`efficiencies are significantly enhanced due to this timed UVA/oxygen modulation sequencing
`
`with minimized UVA dosage but with minimal or no loss in effectivity and little or no
`
`incrcasc in toxicity.
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`[0038] Singlet oxygen concentration as generated according to this method is highly linearly
`
`correlated to UVA irradiance. Figure 6 shows the rate of dissolved oxygen consumption in
`
`the collagen at 15 mW/cm2 in two test samples. Each test sample included collagen and 0.1%
`
`Rf solution. Figure 6 shows the inverse correlation of dissolved oxygen concentration (due to
`
`consumption from varying ROS generation) with varying UVA irradiance in a normoxic
`
`collagen+0.l% Rf matrix. A 500% modulation factor is shown in the graph below. Here we
`
`show that a 500% increase in UVA irradiance increases the rate of dissolved oxygen
`
`consumption by 500%. Increased UVA (See Figure 6) irradiance also generates
`
`correspondingly increased cross—link densities with correspondingly greater dissolved oxygen
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`10
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`consumption during exposure.
`
`[0039] While the main focus persistent in prior art publications is on collagen cross—linking
`
`in the stroma, the inventor has concluded that cxtra—ccllular matrix (ECM)/protcoglycans may
`
`play a role in the stromal cross—linking process and may form inter—molecular and intra—
`
`molecular collagen/proteoglycan cross—links. The object of this proposed method includes
`
`15
`
`such cross—linking as well.
`
`[0040] D20 is non—toxic and is readily available. One supplier is Sigma Aldrich, from
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`which a 10 gram vial costs about $40.
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`[0041] A generalized formulation for cross—linking according to the invention may be
`
`characterized as: a) an effective amount of a penetration enhancing agent; b) an effective
`
`20
`
`amount of a viscosity agent which maintains film thickness and extends UV protection c) an
`
`effective amount of an agent imparting a hypotonic solution (Le, a solution which has an
`
`osmolarity less than ~ 295mOsol, and is adjusted by the salt NaCl); d) an effective amount of
`
`an agent for extending singlet oxygen lifetimes, e) an effective amount of a photosensitizing
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`agent, and, f) an effective amount of deuterated water forming a solution. The formulation is
`
`configured upon delivery to ocular tissue (through its delivery mechanism and the like) for
`
`reaction with UVA irradiation directed (via a lamp or fiber) at the ocular tissue in the
`
`presence of oxygen (such as ambient air). The lifetimes of singlet oxygen released by the
`
`UVA radiation for promoting protein cross—linking in the ocular tissue are extended by the
`
`formulation . The viscosity agent imparting viscosity control may be or contain CMC at a
`
`30
`
`conccntration between [1%] and [90%]. The pcnctrating cnhancing agent may be or contain
`
`0.02% or less BAC, and the photosensitizing agent may be or contain riboflavin or its
`
`analogues.
`
`[0042] Referring to Figure 3, an appropriate delivery system 100 may be the content of a
`
`substance in a single use dose container 102 and an appropriate applicator subsystem
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`comprising one or more medical grade peristaltic pumps 104, 106 in a housing 108 having
`
`outlets 110, 112, coupled via tubes 114, 116 to a pair of spray dispensing devices 1 18, 120
`
`each to be mounted on frame 122, 124 over an eye 126, 128 of a patient to provide sterile
`
`delivery of the substance to the affected area of each eye, a region about 8 mm in diameter.
`
`Irradiation ports 130, 132 mounted to the frame 122, 124 provide directed radiation, which is
`
`controlled by a UVA source and controller 134 that delivers the prescribed irradiation dosage
`
`(e.g., fractionated pulsed UVA for a period of a few minutes) via fiber optic cables 136, 138.
`
`The same controller 134 may be coupled to and control the pumps 104, 106 to meter the
`
`delivery of the solution according to the invention. The delivery system provides for dual
`
`delivery of the formulation, i.e., delivery simultaneously to both eyes. The system further
`
`provides for dual irradiation of UVA to each eye simultaneously. Although delivery and
`
`irradiation to one cyc or scqucntially is also an cmbodimcnt of this invcntion.
`
`[0043]
`
`Features of the invention are advantageous when exciting the sensitizer, since one
`
`can select the duty cycle with an OFF time of about 50 usecs (ROS lifetime). The peak
`
`amplitudes can be dynamically set from 3mW/cm2 to >100mW/cm2 and at up to 100kHz
`
`frequency. A simple nomogram might be: 10 kHz Pulsing Frequency with 50% duty cycle
`
`(50 usecs ON/SO usecs OFF).
`
`[0044]
`
`This invention has been explained with respect to specific embodiments. Other
`
`embodiments will be evident to those of ordinary skill in the art. For example, cross—linking
`
`may be employed for treatment of maladies or used in procedures including keratoconus,
`
`myopia, presbyopia, LASIK, cataract, and corneal transplantation. Therefore, it is not
`
`intended that the invention be limited, except as indicated by the amended claims.
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`WHAT IS CLAIMED IS:
`
`OOQOMAWNH
`
`11
`
`12
`
`meNH
`
`1.
`
`A substance for ocular treatment comprising:
`
`carboxy-methyl-cellulose for providing viscosity control and protection against
`
`incident ultraviolet radiation;
`
`an effective amount of benzalkonium chloride (BAC) as a penetration enhancer of
`
`the substance into ocular tissue;
`
`an effective amount of deuterated water for extending singlet oxygen lifetimes;
`
`and
`
`an effective amount of a riboflavin-based photosensitizer in solution with the
`
`deuterated water and carboxy—methyl-cellulose,
`
`said solution being configured for reaction with ultraviolet A radiation directed at
`
`ocular tissue in the presence of oxygen, such that the lifetimes of singlet oxygen released by the
`
`ultraviolet A radiation are extended for promoting protein cross-linking in the ocular tissue.
`
`2.
`
`The substance according to claim 1 fiarther including conventional water
`
`in mixture with the deuterated water, the deuterated water exceeding one percent of the total
`
`solution.
`
`3.
`
`The substance according to claim 1 wherein the concentration of D20 in
`
`the solution is between 10% and 100%.
`
`4.
`
`A substance for ocular treatment comprising:
`
`a) an effective amount of a Viscosity agent for film thickness control and UV
`
`protection;
`
`b) an effective amount of an agent imparting a hypotonic solution;
`
`[what does
`
`hypotonic solution mean]
`
`c) an effective amount of an agent for extending singlet oxygen lifetimes;
`
`d) an effective amount of a photosensitizing agent that aborbs UV radiation;
`
`e) an effective amount of deuterated water forming a solution;
`
`said solution being configured for reaction with ultraviolet A radiation directed at
`
`ocular tissue in the presence of oxygen, such that the lifetimes of singlet oxygen released by the
`
`ultraviolet A radiation are extended for promoting protein cross-linking in the ocular tissue.
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`5.
`
`The substance according to claim 4 wherein the viscosity agent imparting
`
`viscosity control comprise CMC at a concentration between [1%] and [90%].
`
`6.
`
`The substance according to claim 4 wherein the photosensitizing agent
`
`comprises riboflavin.
`
`7.
`
`The substance according to claim 4 further