>
`Inventor/Applicant (for US only): HEREKAR, Satish
`V. [US/US]; 820 La Para Avenue, Palo Alto, California Published:
`.
`.
`.
`94306 (US).
`.
`— without international search report and to be republished
`upon receiptof that report (Rule 48.2(g))
`
`oN. a MR.NE.SNTD.TG)" CI, CM, GA, GN, GQ,
`
`>
`
`eee? >
`
`.
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`
`(10) International Publication Number
`WO 2011/019940 A2
`
`17 February 2011 (17.02.2011)
`
`(51) International Patent Classification:
`A61K 47/38 (2006.01)
`A6I1K 31/2605 (2006.01)
`A61K 47/42 (2006.01)
`AGLK 9/08 (2006.01)
`A61K 31/14 (2006.01)
`AG6IP 27/02 (2006.01)
`Int
`ti
`| Application Number:
`International
`Application Number:
`PCT/US2010/045356
`
`21)
`(21)
`
`(22) International Filing Date:
`
`12 August 2010 (12.08.2010)
`
`(25) Filing Language:
`(26) Publication Language:
`(30) Priority Data:
`61/233,315
`
`12 August 2009 (12.08.2009)
`
`English
`English
`
`US
`
`(71) Applicant (for ail designated States except US): SEROS
`MEDICAL, LLC [US/US]; 226 W. Edith Avenue, Unit
`#23, Los Altos, California 94022 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national 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, FIL 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.
`(§4) 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, FL, FR, GB, GR, HR, HU,IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SL SK,
`
`(72)
`
`(75)
`
`Inventor; and
`
`(74) Agents: LUPKOWSKI, Market al.; Squire, Sanders &
`Dempsey L.L.P., 275 Battery Street,Suite 2600, San Fran-
`cisco, California 94111 (US).
`
`
`
`
`
`wo2011/019940A2|IMITINMINIIMTNIAAAAAAT
`
`(54) Title: DEUTERATED WATER AND RIBOFLAVIN SOLUTION FOR EXTENDING SINGLET OXYGEN LIFETIMES
`IN TREATMENT OF OCULAR TISSUE AND METHOD FOR USE
`
`FIG. 1C
`
`(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 oculartis-
`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.
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`DEUTERATED WATER AND RIBOFLAVIN SOLUTION FOR
`
`EXTENDING SINGLET OXYGEN LIFETIMES IN TREATMENT OF
`
`OCULAR TISSUE AND METHOD FOR USE
`
`This application claims benefit of U.S. Patent Application No. 61/233,315
`
`which wasfiled on August 12, 2009.
`
`BACKGROUND OF THE INVENTION
`
`[0001]
`
`This invention relates to compositions, methods and delivery systems for promoting
`
`10
`
`cross-linking of proteins in tissue using ultraviolet irradiation of a solution of water
`
`containing riboflavin or its analogues, particularly in ocular tissue (such as tissue in the
`
`sclera, cornea, prepapillary region, etc.).
`
`[0002]
`
`Therapies are knownor are underlaboratory investigation for promoting structural
`
`enhancementof stromal and scleral tissues by application of ultraviolet A radiation to
`
`15
`
`riboflavin in a water solution on oculartissue in the presence of oxygen-containing
`
`atmosphere. The present inventor has determined that singlet oxygen lifetimes have an
`
`evident impact on degree of cross-linking densities of protein such as collagen, a main
`
`structural componentof stromal and scleral tissues.
`
`[0003] Literature reports that deuterated water can increase singlet oxygen lifetimes in
`
`20
`
`various methods for generating singlet oxygen. This invention takes advantage ofthis
`
`discovery in a new context. A search of the literature has found no reports or suggestions of
`
`the present methodology and compositions. Reference is made to the collection of references
`
`supplied by the inventor to the Patent and Trademark Office for consideration.
`
`[0004] Collagen cross-linking (CXL) in ophthalmology,as it currently exists in Europe
`
`25
`
`(whereit is approved), provides a biomechanical basis of increased corneal strength (i.e.,
`
`stability & stiffness) as a result of the formation of covalent bonding betweencollagen
`
`strands. This occurs when a photo-sensitizer, riboflavin (Vitamin B-2) is applied to the de-
`
`epithelialized surface of the cornea. This epithelial protective tissue over the cornea is
`
`surgically debrided(1.e., surgically removed) so the riboflavin can pass(i.e., be absorbed)
`
`30
`
`into the stroma (collagen layers) of the cornea. After the riboflavin saturates the stroma,it is
`
`exposed to UVA light (approximatcly 365 nm). This excitation of the riboflavin by the UVA
`
`results in the creation of free radicals that interact with amino acids and carbonyl groups in
`
`1
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`neighboring collagen molecules to form the strong covalent chemical bonds. Debride refers
`
`to removal of dead, contaminated or adherenttissue or foreign material.
`
`[0005]
`
`The primary emphasis in the application of CXL for ophthalmology has been in the
`
`treatment of keratoconus, which is prevalent in about one in 2,000 people in the US and
`
`Europe, with a slightly higher percentage in Asian countries. This condition is manifested by
`
`a weak cornea which becomestoo elastic and stretches, causing it to bulge forward. This
`
`changes the curvature of the cornea which almost always leads to poorvisual acuity (not
`
`correctable with glasses and/or soft contact lenses) that requires the use of rigid gas
`
`permeable lens. Thus, when the cornea begins losing its shape (i.e., becomes cone shaped
`
`10
`
`instead of spherical) the person typically becomes nearsighted and will develop irregular
`
`astigmatism, which causes the blurring of vision. As this condition progresses, this person
`
`may develop scarring and a very irregular corneal curvature. If the person cannot be helped
`
`with the rigid contact lens, then he/she will require a corneal transplantation.
`
`[0006]
`
`There are other conditions/corneal diseases where the cornea can becomestretched
`
`15
`
`and distorted. One of these, where CXLis currently being utilized, is in corneal ectasia. This
`
`condition involvesstretching of the cornea (collagen tissue) that occurs after refractive
`
`surgeries, such as laser in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK).
`
`Other corneal diseases in which CXL has been tried successfully include corneal ulceration
`
`(possible sequelae to bacterial, viral or fungal infections) and bullous keratopathy (excess
`
`20
`
`fluid accumulation causing corneal edema).
`
`[0007]
`
`The existing procedure of CXL has beenclinically proven (in Europe) to be safe.
`
`However, in its current form, the procedure is very rudimentary with a numberof significant
`
`limitations including but not limited to:
`
`the procedure takes too long (approximately one
`
`hourin total); removal of the corneal epithelium (i.e., debridement) is required, making the
`
`25
`
`procedure invasive and uncomfortable for the patient intra-operatively and for 3-4 days
`
`following surgery; and, it is not fully measurable for accuracy. These limitations clearly
`
`preclude the use of CXL for many corneal treatments that would require a fast and highly
`
`accurate process for stiffening and stabilizing the cornea.
`
`30
`
`SUMMARY OF THE INVENTION
`
`[0008] According to the invention, a solution of deuterated water containing a riboflavin-
`
`based photosensitizer (Rf aka Vitamin B2) is provided in order to extendlifetimes of
`
`UVA/Rf photo-generated intra-stromal singlet oxygen, in combination with UVA delivery
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`profiles of pulsing, fractionation, and optionally auxiliary stromal/Rf hyper-oxygenation in
`
`order to accelerate protein cross-linking density rates in oculartissue.
`
`[0009]
`
`This invention is based upon the discovery that there is a correlation between the
`
`concentration of dissolved [singlet] oxygen in irradiated ocular tissue and the efficiency of
`
`cross-linking with the photo-sensitizer riboflavin. Our studies have demonstrated that a
`
`100% deuterated water (D20)solution with 0.1% riboflavin in solution increases singlet
`
`oxygen lifetimes by about an order of magnitude (10X or more). Further studies have shown
`
`that the such application of deuterated water does not induce endothelial cell apoptosis.
`
`[0010] Our studies have also shownthat by delivering optimized combinations of
`
`10
`
`deuterated water, riboflavin, and UVA dosage on an intact (undebrided) epithelium,
`
`equivalent cross-link densities are rapidly achieved with reduced adverse effects as compared
`
`to current treatments. Lower concentrations of deuterated water with regular water, for
`
`example, yields shorter singlet oxygen lifetimes. These lifetimes have approximately a linear
`
`relationship to the concentration of deuterated water. These lower concentrations are
`
`15
`
`considered acceptable in therapies known or being developed in the art of corneal cross-
`
`linking.
`
`[0011] Our experiments have shown various correlations such as between the following:
`
`the concentration of D2O and reactive oxygen species (ROS) lifetimes; the UVA fluence and
`
`ROS concentration; and, the dissolved oxygen consumption and UVAfluence.
`
`20
`
`[0012] Deuterated waterrefers to water containing a higher-than-normal proportion of the
`
`hydrogen isotope deutcrium, cithcr as deuterium oxide, D2O or 7H20, or as deuterium
`
`protium oxide, HDO or 'H?HO. Conventional water is water that has a normal proportion of
`
`deuterium isotope, such as in tap water to distilled water.
`
`[0013]
`
`The invention will be better understood by reference to the following detailed
`
`25
`
`description in connection with the accompanying drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0014]
`
`Figures 1A-1C areillustrations of a first method according to the invention.
`
`[0015]
`
`Figures 2A-2Dare illustrations of a second method according to the invention.
`
`[0016]
`
`Figure 3 is a schematic diagram of a delivery system accordingto the invention.
`
`30
`
`[0017]
`
`Figure 4 is a graph showingthe relationship between D2O and ROS lifetimes.
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`[0018]
`
`Figure 5 is a graph showing the relationship between ROS concentration and UVA
`
`irradiation (fluence).
`
`[0019]
`
`Figure 6 is a graph showing the relationship between rate of oxygen consumption
`
`and UVAirradiation.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0020]
`
`The invention is embodied in methods, compositions and delivery systems,
`
`particularly in relation to therapies for strengthening and re-shaping ocular tissue. The
`
`formulation invention includes a composition or substance comprising a solution of
`
`deuterated water (between 100 wt% D2O and 10 wt% D2O in water) containing a riboflavin
`
`10
`
`based photo-sensitizer, carboximethylcellulose (CMC), and benzalkonium chloride (BAC).
`
`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
`
`refers to ~0.1 gm in 100mL). The molecular weight of Rf is: ~378 gm/L. In some
`
`embodiments, the concentration of the riboflavin based photo-sensitizer, such as Rf aka
`
`15
`
`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
`
`some embodiments, the concentration of BAC,is 0.2% or about 0.2%. In some
`
`embodiments, the concentration of BAC is between X and Y, or more narrowly, V and W. In
`
`20
`
`certain embodiments, the formulation invention includes deuterated water between 100%
`
`D20 and 10% D2O in water containing, the riboflavin based photo-sensitizer Rf aka Vitamin
`
`B2 ofabout 0.1% Molar concentration, carboximethylcellulose (CMC) of about 0.2%, and
`
`benzalkonium chloride (BAC) of about 0.02%. Embodiments of the invention include any
`
`value or range of D2O between 10% and 100% in the formulation.
`
`25
`
`[0021] Referring to Figures 1A-1C, a method according to the invention is illustrated.
`
`Figure 1A depicts an intact cornea 11 comprising an epithelium 12 with underlying stromal
`
`tissue 14. As shownin Figure 1B, the formulation 16 according to the invention is applied as
`
`a spray or droplets to the epithelium 12 in the presence of ambient oxygen (in the air) to an
`
`undebrided corneal surface. The period of exposure of formulation is several minutes.
`
`In
`
`30
`
`some embodiments, the period of exposure can be between | to 2, 2 to 3, 3 to 5, 5 to 7, 7 to
`
`10, or greater than 10 minutes. Bursts of spray or droplets are applied over the affected area
`
`for the duration of the soaking cycle.
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`[0022]
`
`Then the solution-soaked stromal region 14 is irradiated with ultraviolet A 18, as
`
`shown in Figure 1C. The UVAirradiation treatment may be continuous(i.e., irradiation
`
`without interruption) for a period ranging from | to 15 minutes or fractionated (turned on and
`
`off for a few seconds to a minute) or pulsed (brief bursts of high irradiance with ON times in
`
`the 1 microsecond to millisecond range, and frequencies in the 1 killohertz to 500 killohertz
`
`range. The irradiation creates reactive oxygen species (ROS) that cause the desired
`
`crosslinking of proteins 20. In one preferred embodiment, which maximizesbenefits
`
`efficiently, the irradiation is pulsed and fractionated, to promote the production ofsinglet
`
`oxygen, or reactive oxygen species (ROS) in the intrastromal region to thereby promote the
`
`10
`
`desired cross-linking of proteins 20 during the lifetimes of the reactive oxygen.
`
`[0023]
`
`In embodiments of the invention, there may be various compounds which can
`
`function as both preservative and penetration enhancers. These compoundsinclude,, but are
`
`not limited to benzalkonium chloride (BAC) and sodium ethylenediaminetetraacetate
`
`(EDTA), as,well as viscosity agents such as carboxymethylcellulose (CMC)ordextran. BAC
`
`15
`
`(~0.02%) and EDTA (~0.1%) enhance penetration ofthe riboflavin and D2O water
`
`formulation. CMC (~0.2%) or dextran (~20%) enhancethe lubricity and the formation ofa
`
`persistent, broader, and more uniform corneal tear film before and during the procedure. This
`
`allows greater absorption of the active ingredients of the formulation into the cornea.
`
`[0024]
`
`Significantly, the riboflavin formulation can also be manufactured with a high
`
`20
`
`concentration of dissolved oxygen. This oxygen enrichment enables the production of greater
`
`ROS concentration in a shorter period of time, and, in turn, this makes higher UVA irradiance
`
`practical. However, it should be noted that there may be other meansto diffuse oxygen gas
`
`into the stroma, which might include, among others, the use of a device that would deliver
`
`such oxygen gas to the corneal surface. This oxygen gas then diffuses (albeit slowly) into the
`
`stroma, thereby increasing dissolved oxygen.
`
`In summary, the ability to increase dissolved
`
`oxygen in the stroma enables the use of a higher UVA irradiance exposureto the collagen
`
`tissue. This concept of embedding dissolved oxygen in the stroma means that optimum
`
`cross-linking (i.e., adequate stiffness of the cornea with minimal side effects) can be achieved
`
`in a shorter period of time. A means to manufacture the enriched oxygen deuterated Rf
`
`30
`
`formulation, that will provide up to and over 1 year of extended shelf life, is contemplated by
`
`this invention. The components of the formulation invention, which are set forth hereinabove,
`
`may be optimized for penetration rate, pH, hypotonicity, and lubricity by the proportions that
`
`each component is used within the formulation.
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`[0025] Referring to Figures 2A-2D,a further method accordingto the invention is
`
`illustrated. The intact cornea 11 (Figure 1A) comprises an epithelium 12 with underlying
`
`stromal tissue 14. The corneal surface is debrided to remove the surface layer and expose the
`
`underlying tissue (Figure 2B) in a debrided region 13. A solution 16 according to the
`
`invention is applied as a spray or droplets to the debrided region 13 in the presence of
`
`ambient oxygen (in the air) to the (Figure 1B). The period of exposure is several minutes.
`
`The same ranges of exposure disclosed in the embodiments of Figures 1 A-C apply to Figures
`
`2A-D. Bursts of spray or droplets are applied over the affected area for the duration ofthe
`
`soaking cycle. Due to the debriding, the stromal tissue 14 is soaked to a greater depth than
`
`10
`
`the embodiments of Figures 1A-C. Then the solution-soaked stromal region 14 is irradiated
`
`with ultraviolet A 18 (Figure 2D). As described above, the UVAirradiation treatment may be
`
`continuous or fractionated (turned on and off for extended periods) or pulsed (bricf bursts of
`
`high illumination for an extended period), or most preferably pulsed and fractionated, to
`
`promote the production of singlet oxygen, or reactive oxygen species (ROS) in the deep
`
`15
`
`intrastromal region to thereby promote the desired cross-linking of proteins 20 during the
`
`lifetimes of the reactive oxygen.
`
`[0026]
`
`The process of soaking the formulation, on either a debrided or undebrided surface,
`
`results in diffusing oxygen into the stroma. For a undebrided surface, the penetration is to a
`
`depth of up to about 0.5 mm. The penctration into debrided surfaces is greater than 0.5 mm.
`
`20
`
`The UVAirradiation in the presence of oxygen promotes singlet oxygen species generation.
`
`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 hypcer-oxygenation accclerates protein
`
`cross-linking density rates in the ocular tissue. Our studies have shown that the use of a 100%
`
`25
`
`deuterated water solution with 0.1% riboflavin in solution increases singlet oxygen lifetimes
`
`byat least an order of magnitude (10X or more) without inducing endothelial cell apoptosis.
`
`Jt is well knownin the arts that the current cross-linking procedure may inducc the following
`
`side effects: (1) stromal haze due to keratocyte apoptosis; (2) endothelial cell density loss.
`
`[0027]
`
`In another embodiment, the formulation includes a combination of lower
`
`30
`
`percentages of dcutcratcd watcr, lower concentrations of riboflavin or lower dosages of UVA
`
`on intact (un-debrided) epithelium may be employed for equivalent cross-link densities as
`
`comparedto current acceptable corneal cross-linking standards for CXL procedures. In
`
`various embodiments, the ranges of components and delivery parameters of the formulation
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`are as follows: 100% D20 to 1%; 0.1% Rf to 0.01%; 0.02% BAC to .01%; 0.2% CMCto
`
`0.1%; 5.4 J/em2 UVAto 2.5J/cm2; 30 minutes or less UVA exposure.
`
`[0028]
`
`Figure 4 showsthe singlet oxygen lifetime in deuterated water as a function of
`
`concentration of D2O. Figure 4 demonstrates the correlation of ROS lifetimes to varying
`
`D20 solvent (0% to 100%) in the 0.1% Rf solution under normoxic (i.e., ambient oxygen
`
`dissolved into the test sample at room temperature by natural diffusion conditions in collagen
`
`and 0.1% Rf matrices. As shown in Figure 4, lower concentrations of deuterated water with
`
`regular water, for example, yield shorter singlet oxygen lifetimes. The relationship between
`
`singlet oxygen lifetime and D2O concentration in regular water is approximately linear, as
`
`10
`
`shownin Figure 5. This data was generated by a custom built photon counter and dissolved
`
`oxygen probe, which was excited by a frequency tripled Nd:Yag laser for time-resolved
`
`measurements.
`
`[0029] The inventor has measured reactive oxygen species (ROS) in vitro in aerated collagen
`
`and riboflavin under UVAillumination and has foundan increase in the ROS duration of
`
`15
`
`about 4.5 wSecs for HzO with no D20 to a duration of over 45 uSecs for a 100% deuterated
`
`solvent D,O (See Figure 5). Figure 5 showsa stronglinear correlation of ROS concentration
`in a normoxic collagen and Rf matrix as UVAirradianceis varied from about 3 mW/cm? to
`about 50 mW/cm’.
`
`[0030] Figure 6 showsthe inverse correlation of dissolved oxygen concentration (due to
`
`20
`
`consumption from varying ROS generation) with varying UVAirradiance in a normoxic
`
`collagent+0.1% Rf matrix. A 500% factor is shown in the example below.
`
`[0031] A system using dual UVA/Blue sourcesis able to provide pulsed irradiances up to
`150 mW/cm”, with pulsing frequencies at up to 200 kHz andis, for example, set to deliver
`
`25
`
`pulses at a 20 kHz (50 uSecs) pulse repetition frequency, and a duty cycle of about 20%
`
`(intermittency). This is a 40 usec UVA OFFperiod and a 10 wSec high intensity UVA ON
`
`period applied cyclically. It is believed that the 10 pSec UVA ON pulse rapidly generates a
`
`maximal new population of ROS molecules in the targeted stroma just as the previously
`
`stimulated ROS population is about to be depleted or otherwise be consumed through
`
`30
`
`quenching mechanisms in the local microenvironment. It is believed that the 40 uSec UVA
`
`OFFperiod provides sufficient time for chemical interactions in the microenvironment to
`
`effect cross-linking of proteins, specifically collagen, in the target region. The singlet oxygen
`
`population in the presence of the aerated deuterated solvent survives for an extended duration
`
`of about 40 Secs, while the UVA is OFF.
`
`

`

`WO 2011/019940
`
`PCT/US2010/045356
`
`[0032]
`
`Ina specific embodiment, during the extended reactive lifetime of singlet oxygen,
`
`rapid cross-linking reactions are induced in the carbonyl (aldehyde) groups of collagen while
`
`dissolved oxygen Oz, riboflavin, and singlet oxygen species (ROS) are consumed (as long as
`
`present in sufficient concentrations) by Type I (energy transfer) mechanisms. (From photo-
`
`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).
`
`Analoguesof riboflavin may also be employed, such as 3-methyl-riboflavin tetraacetate.
`
`[0033] As riboflavin molecules degrade and transform through such singlet oxygen
`
`regenerative timing cycles, they generate reduced fluorescence intensity in the 530nm-570nm
`
`10
`
`band in response to UVA which may thereby signal a riboflavin “reinstillation” cycle.
`
`[0034]
`
`In addition to increased endothelial safety due to reduced riboflavin concentration
`
`requirements, the use of viscous carboxy-methyl-cellulose (CMC) in the present formulation
`
`forms a corneal film of thickness ~ 501M-200uM, which provides added UVAprotection to
`
`the endothelium. Pulsed UVA applied as herein described (instead of CW UVA)provides
`
`15
`
`for a reduced apoptotic effect on both keratocytes and endothelial cells.
`
`[0035]
`
`The formulation (D20 + CMC + BAC) provides for faster penetration and
`
`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
`
`20
`
`thickness, epithelialization state (whether or not debrided), sensitizer pre-oxygenation,
`
`viscosity and ambicnt oxygen cnvironment of the stroma. Some amountof dissolved oxygen
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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 fractionationis able to
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`25
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`overcomethe dissolved oxygen limitations inherent in ambient diffusion. Depending on the
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`depth of cross-linking desired, a pause in the UVAirradiation (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]
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`Total cross-linking treatment times and singlet oxygen/riboflavin molecular
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`efficiencies are significantly enhanced dueto this timed UVA/oxygen modulation sequencing
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`with minimized UVA dosage but with minimal or no loss in effectivity and little or no
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`increase in toxicity.
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`[0038] Singlet oxygen concentration as generated according to this method is highly linearly
`
`correlated to UVAirradiance. Figure 6 showsthe rate of dissolved oxygen consumption in
`the collagen at 15 mW/cm/’in twotest samples. Each test sample included collagen and 0.1%
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`Rf solution. Figure 6 showsthe inverse correlation of dissolved oxygen concentration (due to
`
`consumption from varying ROS generation) with varying UVAirradiance in a normoxic
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`collagent+0.1% Rf matrix. A 500% modulation factor is shown in the graph below. Here we
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`show that a 500% increase in UVA irradiance increasesthe rate of dissolved oxygen
`
`consumption by 500%. Increased UVA (See Figure 6) irradiance also generates
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`correspondingly increased cross-link densities with correspondingly greater dissolved oxygen
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`consumption during exposure.
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`[0039] While the main focus persistent in prior art publications is on collagen cross-linking
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`in the stroma, the inventor has concludedthat 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
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`15
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`such cross-linking as well.
`
`[0040] DO is non-toxic andis 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
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`20
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`amount of a viscosity agent which maintains film thickness and extends UV protection c) an
`
`effective amount of an agent imparting a hypotonic solution (i.e., 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 oxygenlifetimes, 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
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`reaction with UVAirradiation directed (via a lampor fiber) at the ocular tissue in the
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`presence of oxygen (such as ambient air). The lifetimes of singlet oxygen released by the
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`UVAradiation for promoting protein cross-linking in the ocular tissue are extended by the
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`formulation . The viscosity agent imparting viscosity control may be or contain CMCat a
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`30
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`concentration between [1%] and [90%]. The penctrating cnhancing agent may be or contain
`
`0.02% or less BAC, and the photosensitizing agent may be or contain riboflavin or its
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`analogues.
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`[0042] Referring to Figure 3, an appropriate delivery system 100 may be the content ofa
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`substancein 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
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`outlets 110, 112, coupled via tubes 114, 116 to a pair of spray dispensing devices 118, 120
`
`each to be mounted on frame 122, 124 over an eye 126, 128 of a patient to provide sterile
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`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 fora period of a few minutes) via fiber optic cables 136, 138.
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`The same controller 134 may be coupled to and control the pumps 104, 106 to meter the
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`delivery of the solution according to the invention. The delivery system provides for dual
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`10
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`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 sequentially is also an embodiment ofthis invention.
`
`[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/cm’ to >100mW/em’andat up to 100kHz
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`15
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`frequency. A simple nomogram might be: 10 kHz Pulsing Frequency with 50% duty cycle
`
`(50 psecs ON/S50 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
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`20
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`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 CLAIMEDIS:
`
`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 substanceinto ocular tissue;
`
`an effective amount of deuterated water for extending singlet oxygen lifetimes;
`
`and
`
`an effective amount ofa 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
`
`oculartissue in the presence of oxygen, such thatthe 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 | further including conventional water
`
`in mixture with the deuterated water, the deuterated water exceeding one percent ofthe total
`
`solution.
`
`3.
`
`The substance according to claim 1 wherein the concentration of D2O in
`
`the solution is between 10% and 100%.
`
`4.
`
`A substance for ocular treatment comprising:
`
`a) an effective amountof 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 thatthe lifetimes of singlet oxygen released by the
`
`ultraviolet A radiation are extended for promoting protein cross-linking in the ocular tissue.
`
`OoDTNnHBWYN
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`10
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`11
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`12
`
`—OnBBWONOeK
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`10
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`11
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`12
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`1]
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`WO 2011/019940
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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 including conventional water
`
`in mixture with the deuterated water, the deuterated water exceeding one percentofthe total
`
`solution.
`
`8.
`
`A de