United States Patent
`
`[191
`
`[11] Patent Number:
`
`5,549,596
`
`llllllllllllllIllIllll||||lllllllllllllllllllll|||||||||||l||ll|||||||||||l
`USOOSS49596A
`
`
`
` Latina [45] Date of Patent: Aug. 27, 1996
`
`
`
`[54] SELECTIVE LASER TARGETING 0F
`PIGMENTED OCULAR CELLS
`
`5,123,902
`5,312,395
`
`............................... 606/5
`6/1992 Muller et a1.
`5/1994 Tan et a1.
`.................................... 606/9
`
`[75]
`
`Inventor: Mark A. Latina, North Andover, Mass.
`
`[73] Assignee: The General Hospital Corporation,
`Boston, Mass.
`
`FOREIGN PATENT DOCUMENTS
`0172490
`2/1986 European Pat. Off.
`. .................. 606/9
`
`OTHER PUBLICATIONS
`Huis et a1., “Localized Surgical Debridement of RPE by
`O—Switched Neodymiumyag Laser”, Arvo Abs., May 1993,
`. 959.
`P
`Fox et al., “Switched Ruby Laser Irradiation of Melanotic
`Conjunctiva”, ArVO Abs., May 1993, p. 958.,
`
`Primary Examiuer—Angela D' Sykes
`Asszstant Examiner—Sonya Harris-Ogugua
`Attorney, Agent, or Finn—Fish & Richardson RC.
`
`ABSTRACT
`[57]
`A method of damaging pigmented intraocular cells, a patient
`with a disease, which involves selectively damaging pig—
`merited cells in an intraocular area by irradiating the area
`with laser radiation of radiant exposure between about 0.01
`and about 5 Joules/cmz, while sparing nonpigmented cells
`and collagenous structures within the irradiated area. The
`method is useful for the treatment of glaucoma, intraocular
`melanoma, and macular edema.
`
`29 Claims, 6 Drawing Sheets
`
`[21] Appl. NO-3 5459837
`.
`22
`Flrd:
`Ot.2 1
`1 c
`c
`0’ 99
`Related US. Application Data
`
`[
`
`]
`
`5
`
`[63] Continuation of Ser. No. 88,855, Jul. 7, 1993, abandoned.
`[51]
`Int. Cl.6 ..................................................... A61B 17/36
`[52] US. Cl.
`................................................................... 606/4
`[58] Field of Search .................................... 606/2, 3, 4, 5,
`606/6; 607/88, 89
`
`[56]
`
`_
`References Clted
`U.S. PATENT DOCUMENTS
`
`5/1975 Krasnov ...................................... 606/3
`3,884,236
`3/1976 Krasnov ..........
`606/4
`3,943,931
`
`7/1983 Fankhauser et a1.
`606/4
`4,391,275
`....... 606/5
`7/1984 Baron .......................
`4,461,294
`
`..... 606/10
`9/1985 L’Esperance, Jr.
`4,538,608
`
`........................................ 606/6
`4,558,698 12/1985 O’Dell
`
`
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 1
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 1
`
`

`

`US. Patent
`
`Aug. 27, 1996
`
`Sheet 1 of 6
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`5,549,596
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`U.S. Patent
`
`Aug. 27, 1996
`
`Sheet 2 of 6
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`Alcon Research, Ltd.
`Exhibit 1018 - Page 3
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`

`

`US. Patent
`
`Aug. 27, 1996
`
`Sheet 3 of 6
`
`5,549,596
`
`1
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`Initial Beam (E)
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`FIGURE 3'
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 4
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 4
`
`

`

`US. Patent
`
`Aug. 27, 1996
`
`Sheet 4 of 6
`
`5,549,596
`
`
`
`
`
`
`Equation: y=7.53e4 X-1.8895
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`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 5
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 5
`
`

`

`US. Patent
`
`Aug. 27, 1996
`
`Sheet 5 of 6
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`5,549,596
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`FIGURE 5
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 6
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 6
`
`

`

`US. Patent
`
`Aug. 27, 1996
`
`Sheet 6 of 6
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`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 7
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 7
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`1
`SELECTIVE LASER TARGETING OF
`PIGMENTED OCULAR CELLS
`
`5,549,596
`
`2
`SUMMARY OF THE INVENTION
`
`This invention was made with Government support
`under Contract N00014-86—00117 awarded by the Depart-
`ment of the Navy. The Government has certain rights in the
`invention.
`
`STATEMENT AS TO FEDERALLY SPONSORED
`RESEARCH
`
`Studies were supported in part by National Eye Institute
`grant K11 EY00292. The United States government, there-
`fore, has certain rights in this invention.
`This application is a continuation of US. Ser. No. 08/088,
`855, filed Jul. 7, 1993, now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`This invention relates to laser-induced damage to pig-
`mented ocular cells.
`
`Laser treatment of tissues within the eye is commonly
`used to treat diseases such as proliferative diabetic retinopa-
`thy, other forms of neovascularization, open-angle glau-
`coma, cataracts, and intraocular tumors. While laser treat-
`ment has greatly enhanced the performance of many types of
`ocular surgery, certain procedures are adversely affected by
`the nonselective nature of laser-induced tissue damage.
`Current ocular surgery lasers cause tissue coagulation or
`ablation, meaning total removal or vaporization, with no
`ability to discriminate between cell or tissue types.
`For example, two types of ablative laser surgery are now
`commonly used to treat open-angle glaucoma. Glaucoma
`refers to abnormally elevated intraocular pressure, and is
`most commonly caused by increased resistance to outflow of
`the continuously produced aqueous humor. The aqueous
`humor exits the eye through the trabecular meshwork (TM)
`at the periphery of the anterior chamber. The TM consists of
`collagenous beams and plates, covered with phagocytic TM
`cells, and enclosing passages through which the aqueous
`humor flows, finally entering the vasculature via Schlemm’s
`canal. Pathologic changes in the trabecular meshwork are
`thought to be involved in the etiology of certain forms of
`glaucoma.
`The resistance by the TM is lowered, and intraocular
`pressure subsequently decreased, by laser surgery to ablate
`portions of the TM. Laser trabeculoplasty surgery utilizes
`long pulses (of approximately 100 msec) of laser radiation
`at high fluency, which is a measure of radiant exposure, or
`energy per area of tissue (mJ/cmz). This procedure causes
`immediate tissue coagulation and focal burns of the TM, as
`well as subsequent scarring in the burned areas. Aqueous
`flow is thought to be improved in the region adjacent to the
`lasered tissue. The trabeculoplasty procedure results in low-
`ered intraocular pressure which lasts for only a limited time
`period. The scar tissue which forms after the procedure
`severely limits the eflicacy of subsequent trabeculoplasties
`on a given patient. In an alternate procedure, trabeculopunc-
`ture, the absorption of laser radiation at high fluence and
`irradiance (Watts/cmz) causes photodisruption of the TM
`tissue, producing a hole through the TM into Schlemm’s
`canal, the major outflow vessel. The laser-created trabecu-
`lopuncture channel into Schlemm’s canal eventually fills in
`with fibrotic tissue; the beneficial effects of the procedure on
`intraocular pressure are not permanent.
`
`10
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`15
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`20
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`25
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`30
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`35
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`4o
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`45
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`50
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`
`65
`
`The invention features a method of selectively damaging
`pigmented intraocular cells in a patient with a disease, which
`involves selecting an intraocular area containing a pig-
`mented cell and a nonpigmented cell, irradiating the area
`with laser radiation of radiant exposure between about 0.01
`and about 5 Joules/cmz, and damaging the pigmented cell
`without killing the nonpigmented cell.
`In a preferred embodiment, the radiation is delivered in
`pulses with pulse duration of between about
`1 nsec and
`about 2 usec. The radiation may be delivered in a single
`pulse. In a further preferred embodiment, the laser radiation
`impinges upon the intraocular area in a target spot of
`between about 0.05 and about 1.5 mm in diameter. Prefer—
`ably, the laser radiation has a wavelength which is more
`highly absorbed in pigmented than in nonpigmented cells.
`The method involves selectively damaging pigmented
`intraocular cells, such as trabecular meshwork cells, retinal
`pigmented epithelial cells, uveal pigmented cells, melanoma
`cells, or conjunctival pigmented cells. The pigmented cells
`may contain endogenously synthesized pigment, or may be
`phagocytic cells into which exogenous pigment is intro-
`duced by contacting the phagocytic cells with exogenous
`pigment before irradiating the area containing the cells.
`Endogenous pigment refers to pigment synthesized and
`retained within a cell, and exogenous pigment refers to
`pigment within a cell which was not synthesized within the
`same cell. In a preferred embodiment, the phagocytic cell is
`a trabecular meshwork cell and exogenous pigment is intro-
`duced into the aqueous humor by laser irradiation of the iris.
`In yet another embodiment, the intraocular area is trabe-
`cular meshwork, and the laser radiation is delivered via a slit
`lamp delivery system and is directed into the trabecular
`meshwork by a goniolens.
`In another aspect,
`the invention features a method of
`damaging trabecular meshwork cells, involving irradiating a
`region of trabecular meshwork containing cells and collagen
`beams with laser radiation characterized by radiant exposure
`of between about 0.01 and about 5 Joules/cmz, and selec-
`tively damaging the trabecular meshwork cells without
`damaging the collagen beams.
`In a further aspect, the invention features a method of
`damaging conjunctival pigmented epithelial cells in a patient
`with a disease, involving selecting an area of conjunctiva
`containing a pigmented cell and a nonpigmented cell, irra-
`diating the area with laser radiation, wherein the radiation
`has radiant exposure of between about 0.01 and about 5
`Joules/cm2 and damaging the pigmented cell without killing
`the nonpigmented cell.
`In a preferred embodiment,
`the
`radiation is delivered in pulses with pulse duration of
`between about
`1 nsec and about 2 usec. The disease is
`intended to include conjunctival melanoma (benign or
`malignant), pigmented nevus, or other conjunctival diseases
`of pigmented tissues.
`The invention provides a method for selectively damaging
`pigmented intraocular cells, while nonpigmented cells and
`structures are not damaged. The method is useful in the
`treatment of diverse diseases of pigmented tissues within the
`eye. In a preferred embodiment, the method is useful in
`treating glaucoma.
`Other features and advantages of the invention will be
`apparent
`from the following description and from the
`claims.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a diagram of a laser apparatus for use in
`practicing the method of the invention.
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 8
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 8
`
`

`

`5,549,596
`
`3
`FIG. 2 is a diagram of the laser apparatus used in the in
`vitro experiments described below.
`FIG. 3 is a graph depicting Gaussian profiles of three
`beam energies are depicted as E, E2, and E3, Irradiation of
`melanized trabecular meshwork cultures at these energies
`will result
`in a radius of cell killing of r1,
`r2, and r3,
`respectively.
`FIG. 4 is a graph of the threshold energy calculation for
`melanized trabecular meshwork cell (16 hours of 3x107
`melanin/ml challenge) killing using a single pulse Nd:YAG
`laser. Estimated equations of the linear best fit and the
`significance of the coefficients are shown in the box.
`FIG. 5 is a graph of laser parameter ranges for in vitro
`selective cell killing.
`FIG. 6 is a graph of the wavelength dependence of laser
`parameter range for selective cell killing in vitro.
`
`DETAILED DESCRIPTION OF EXEMPLARY
`EMBODIMENTS
`
`The method of the present invention involves selectively
`damaging or killing ocular cells containing pigment, while
`sparing nonpigmented cells and tissue structures within the
`region of irradiation.
`Briefly, an area of intraocular tissue containing pigmented
`cells is irradiated with a laser, such as a q-switched Nd:YAG
`laser. The pigment in the cells makes the pigmented target
`cells optically denser than the nonpigmented surrounding
`cells, and thus more susceptible to laser~induced damage at
`selected laser wavelengths and fluences.
`In a preferred
`mode, laser energy at low fluences, impinging on the target
`tissue areas for short time durations, selectively kills the
`pigmented target cells with minimal damage to surrounding
`cells. The selectivity of tissue damage is of great clinical
`benefit in treating pathological conditions restricted to pig-
`mented cells, including conditions of phagocytic cells which
`can be induced to take up exogenous pigment. For these
`conditions,
`the invention allows killing of affected cells
`while preserving the integrity of unaffected, nonpigmented
`cells and support tissues, thereby minimizing adverse efiects
`of the surgery on the residual function of the ocular tissues.
`The method of the invention utilizes laser irradiation of
`low fluence, or
`radiant exposure. Fluences of about
`0.001—5.0 Joules/cmz, and preferably of about 0.01—1.0
`Joules/cmz, are found to be effective at killing pigmented
`TM cells without causing damage to adjacent, non-target
`cells, when used at short pulse durations, such as a nano-
`second. The desired radiant exposure may be achieved by
`modifying the target spot size, the beam symmetry, or the
`delivered Joules/pulse. In general, the target spot size is
`large compared to those utilized in many previous applica-
`tions of laser therapy to the eye; in preferred embodiments
`of the invention, the target spot size is from about 0.1 to
`about 1 mm in diameter. The large target spot size is possible
`because the method of the invention provides selective cell
`damage based on cell pigmentation, in contrast to prior art
`laser ablation methods, in which tissues are damaged non-
`selectively. For example, in prior techniques which ablate
`tissue by photodisruption, the high fluence which results in
`nonselective tissue ablation follows from the small target
`spot size, typically about 25 pm. It is a major advantage of
`the technique of the invention that focusing is not necessary
`to achieve selective killing; all cells within the irradiation
`field with chromophores which absorb the radiation will be
`affected. A large target area is advantageous: surgical time is
`minimized when the laser apparatus needs to be redirected
`
`5
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`35
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`40
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`45
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`50
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`55
`
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`
`65
`
`4
`fewer times. In a particularly preferred embodiment for
`treatment of the TM,
`the target area corresponds to or
`slightly exceeds the bounds of the TM, allowing irradiation
`of all of the cells within the TM.
`
`Pulse durations of between about 1 nsec and about 2 nsec
`may be utilized. The desired pulse duration is related to the
`type and size of pigment particle within the target cells to be
`damaged. Since the thermal relaxation of a particle is related
`to the particle size of the pigment material, smaller intrac-
`ellular particles require a shorter pulse duration to ensure
`confinement of energy to the target cells. Excessive pulse
`duration, such as more than five nsec or continuous waves,
`may cause nonselective killing of both pigmented and
`nonpigmented cells, as well as disruption of collagenous
`structures; this occurs as the longer laser exposure durations
`allow heat diffusion and resultant disruption of surrounding
`nontarget tissues. In contrast, prior art techniques employing
`ablation of tissue by coagulation utilize distinctly longer
`radiation durations.
`
`More specifically, heat is confined within a spherical
`target for a thermal relaxation time, Tr, which is related to
`the target diameter, d, and the thermal diffusivity constant,
`D, by Tr=d2/4D. During a lengthy laser exposure, greater
`than T,, heat within the target diffuses to surrounding cells
`or structures resulting in coagulation. On the other hand, if
`heat is generated within the target more rapidly than heat can
`diffuse away, target temperatures become much higher than
`their surrounding tissues and thermal diifusion to surround—
`ing structures is minimized. Thus, by choosing a pulse
`duration shorter than the thermal relaxation time of the target
`(i.e., melanin), selective target damage can be achieved.
`Assuming spherical targets of 0.5 to 5 microns, estimates of
`thermal relaxation times for biological targets range from
`10—8 to 10—6 seconds. This is much shorter than the exposure
`time used in the prior art method of argon laser trabeculo-
`plasty (ALT), where non~specific heating and coagulation of
`trabecular tissues results from the long exposure time (0.1
`sec).
`The emission wavelength of the laser may be within either
`the visible or infrared spectra, excluding the absorption lines
`of water. Additional selectivity for target cells may be
`provided by use of an appropriate laser wavelength. For
`example, when applying the method of the invention to
`retinal tissue, incidental absorption by hemoglobin in the
`retinal vessels may be avoided by selecting a wavelength of
`1064 nm for ablating melanin-containing target cells; this
`wavelength is absorbed by melanin but not by hemoglobin.
`For avascular tissue, such as trabecular meshwork, a shorter
`wavelength of 532 nm provides higher absorption by mela—
`nin in pigmented cells and a shallower penetration depth into
`the tissue, as well as reducing the threshold energy for cell
`killing.
`In a preferred mode of practicing the invention, a short-
`pulsed, Nd:YAG q-switched laser
`is used. Generally,
`Nd:YAG lasers emit at 1064 nm, and when frequency
`doubled yield 532 nm output. Both of these wavelengths are
`useful within the eye because they are transmitted by ocular
`media and structures including the cornea, aqueous humor,
`lens, vitreous and sclera.
`There are several types of pigmented cells within the eye,
`which may be advantageously damaged with the method of
`the invention when clinically indicated. These cells acquire
`pigment by either synthesizing melanin endogenously or by
`phagocytosing exogenous pigment. Cell types which syn~
`thesize and retain melanin include the pigmented epithelial
`cells of the retina, ciliary body, and iris, as well as ocular
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 9
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 9
`
`

`

`5,549,596
`
`5
`melanomas. Although TM cells are incapable of synthesiz(cid:173)
`ing melanin, these cells typically acquire pigment by phago(cid:173)
`cytosis from the aqueous humor, which normally contains
`particles of pigmented cellular debris. In addition, the pig(cid:173)
`mentation ofTM cells may be augmented by adding pigment 5
`to the aqueous humor, as is done in one preferred embodi(cid:173)
`ment of the invention. This may be accomplished by inject(cid:173)
`ing a suspension of pigmented particles into the anterior
`chamber of the eye with a fine needle. As the aqueous humor
`with suspended pigment particles flows from the eye 10
`through the TM, the TM cells take up pigment, increasing
`their optical density relative to surrounding nonpigmented
`tissue, and improving the cell selectivity of the laser killing
`of the invention. In a further preferred embodiment, melanin
`is introduced into the aqueous humor by laser iridotomy of 15
`the iris, which releases melanin particles from iris cells into
`the aqueous humor. Other pigments, such as india ink or any
`other nontoxic, insoluble particulate dye, may be introduced
`to phagocytic target cells prior to laser irradiation to ablate
`the pigmented cells.
`In using the selective ablative method of the invention to
`treat glaucoma, the pigmented cells overlying the collag(cid:173)
`enous beams and plates of the TM are killed. While not
`intending to be bound or limited by a theory of why this
`method is beneficial in enhancing outflow of aqueous 25
`humor, the removal of established TM cells while preserving
`an intact collagenous meshwork allows repopulation of the
`meshwork by new, nonpigmented TM cells. While the
`pathophysiologic role of established TM cells in increased
`outflow resistance is unknown, the repopulated TM cells 30
`may better biologically cleanse the outflow pathway, and in
`other ways contribute to the maintainance of normal outflow
`resistance within the meshwork.
`The method is useful for treating any disease where
`killing ocular pigmented cells is beneficial. The pigmented
`target cells may be on the surface of the cornea or conjunc(cid:173)
`tiva, within the cornea or conjunctiva, or may constitute or
`be attached to any of the intraocular regions of the eye, such
`as the inner cornea, iris, ciliary body, lens, vitreous, choroid,
`retina, optic nerve, ocular blood vessels, or sclera, including
`any primary or metastatic tumors within or adjacent to these
`tissues. Specifically, diseases and conditions intended for
`treatment by this method include melanoma in any ocular
`tissue, diabetic retinopathy, and diseases of the retinal pig(cid:173)
`mented epithelium including drusen, macular edema, age(cid:173)
`related macular degeneration, and central serous retinopa(cid:173)
`thy, among others.
`The term "patient" is meant to include any mammalian
`patient to which ocular laser therapy may be administered.
`Patients specifically intended for treatment with the method
`of the invention include humans, as well as nonhuman
`primates, sheep, horses, cattle, goats, pigs, dogs, cats, rab(cid:173)
`bits, guinea pigs, hamsters, gerbils, rats and mice.
`Presently, there are a variety of commercially available
`laser systems for ophthalmic use, which may be adapted to
`perform the method of the invention. The commercially
`available ophthalmic surgical lasers can readily be modified,
`by anyone skilled in laser technology, to have the requisite
`enlarged target spot size, and hence low fluence, required by
`lasers useful in practicing the method of the invention.
`An exemplary laser system used for practicing the present
`invention is shown in FIG. 1. As illustrated, the laser beam
`system consists of a power source 42 and an aiming beam
`source 54. The power source for the preferred mode of the
`invention is an Nd:YAG laser that is q-switched or
`q-switched ruby, with or without a frequency doubler. The
`
`6
`system may include a lens 64 and detector 66 to monitor
`either wavelength or power emitted by the power source 42,
`a component of which is deflected off beam splitter 62. The
`aiming beam source 54 emits a beam which is deflected off
`mirror 56 to another splitter whereat one component is
`deflected through the remainder of the system, while another
`component passes through to a beam stop 60.
`The power source beam 46 and the aiming beam 55
`jointly pass through lens 49, within which they are focused
`to pass through a 100-600 micron optical fiber 50, having
`another mirror 51 therein. The guided beam 40 passes
`through lens 52 then is deflected by mirror 38. The energy
`30 deflects off mirror 38 and into goniolens 34 where it is
`appropriately directed to the target tissue. After further
`deflecting off mirror 36, virtually parallel beams then reach
`the target cells at site 24 in the target eye 12.
`The system may also include a viewing device 32, such as
`a camera or viewpiece, for viewing the aiming light for
`positioning and monitoring the target site 24. An auxiliary
`20 detector may be placed adjacent or proximal to the target site
`24.
`In an embodiment of the invention, laser irradiation is
`delivered through a slit-lamp delivery system such that an
`appropriate radiant exposure is achieved at the focal point of
`the slit-lamp optics.
`Neutral density (ND) filters are used for attenuating the
`primary Nd:YAG laser beams. Helium-Neon laser is used
`for aiming purposes.
`Melanin Phagocytosis by Trabecular Meshwork Cells:
`All experiments were performed using third and fourth
`passage bovine TM cells grown in 6 or 24 well tissue culture
`plates in low glucose D-MEM with 10% fetal bovine serum,
`1% penicillin-streptomycin, and 1% Fungizone (henceforth,
`media; Gibco, Buffalo, N.Y.) at 37 degree Celsius, 5% C02 ,
`35 and 95% humidity. TM cells are non-melanized in their
`normal growth state and serve as control non-melanized TM
`cells.
`To obtain melanized TM cells, confluent TM cell cultures
`were challenged with sepia melanin in media at concentra-
`tions of 1x106 to 3x107 particles/ml and incubated for
`sixteen hours. Prior to incubation, melanin was washed three
`times with D-PBS (Gibco, Ca2+, Mg2+free) and sonicated
`for 10 minutes to obtain a uniform suspension. Melanin
`concentration and size were determined using a Coulter
`45 Multisizer (Coulter Electronics, Hialeah, Fla.). The melanin
`incubation time was standardized to 16 hours. After sixteen
`hours, the cells were washed three times in D-PBS to
`remove the excess melanin and replaced with fresh media
`until irradiation. Just prior to irradiation, the media was
`50 replaced with D-PBS to avoid absorption of laser energy by
`the media. All irradiations occurred within four hours after
`excess melanin was removed.
`As an additional control, TM cells were challenged with
`latex microspheres (3x107 particles/ml, 0.9 J.!m in diameter,
`55 Polyscience, Warrington, Pa.) that contained no chro(cid:173)
`mophore to determine the response of laser irradiation on
`TM cells with particulate material but without a chro(cid:173)
`mophore. Following a 16 hr incubation, the TM cells were
`washed three times in D-PBS and irradiated with the laser
`60 systems described below.
`In vitro monolayer cultures of bovine TM cells demon(cid:173)
`strated avid phagocytosis of sepia melanin. Phase contrast
`photomicrographs of melanin uptake by TM cells at four
`different melanin concentrations (0, 3x106x107 and 3x107
`65 particles/ml) show that the distribution of melanin was
`perinuclear and the amount of melanin phagocytosed
`increased with high incubation concentrations. The incuba-
`
`40
`
`Alcon Research, Ltd.
`Exhibit 1018 - Page 10
`
`

`

`5,549,596
`
`7
`
`tion time of sixteen hours provided a qualitative difference
`in the amount of melanin ingested at these concentrations,
`which was used to differentiate the effects of TM cell
`pigmentation on the threshold energy for cell killing. Exami-
`nation of TM cells challenged with latex microspheres also
`showed avid phagocytosis of latex microspheres with a
`perinuclear distribution of the microspheres.
`Laser Systems:
`Irradiations of TM cells were performed using three laser
`systems. Each laser system had a different pulse duration.
`System-1 was a continuous wave argon-ion laser (Coherent
`Inc., Palo Alto, Calif. model #1100) emitting at 514 nm with
`a 0.1 second exposure time. System 2A was a flashlamp-
`pumped dye laser (Candela Laser Corp., Wayland, Mass.)
`emitting at 590 nm with pulse duration of 8 psec. System 2B
`was a flashlamp—pumped dye laser (Candela Laser Corp.,
`Wayland, Mass., model #88500) emitting at 588 nm with
`pulse duration of 1 nsec. System—3A was a Q-switched
`frequency doubled Nd:YAG laser (Quantel International,
`Santa Clara, Calif. model #YG660A) emitting at 532 nm
`with a 10 nsec pulse duration. System-3B was a Q—switched
`normal mode Nd:YAG laser (Continuum Biomedical Inc.,
`Livermore, Calif.) emitting at 1064 nm with a 10 nsec pulse
`duration. System-3B was used to determine wavelength
`dependency of the threshold response in the nanosecond
`pulse duration domain. The pulse energy was measured
`using an energy power meter (Scientech, Boulder, Colo.,
`model #365) with 110% accuracy. Threshold Response—
`Pulse Duration and Wavelength Dependence Studies
`TM cell cultures were irradiated at various radiant expo-
`sures using the experimental setup shown in FIG. 2. A
`Helium-Neon laser with an output of 3 mW, coupled into the
`optical path using a beam splitter, served as an aiming beam.
`A fiberoptic delivery was utilized for the argon-ion and
`pulsed-dye laser which resulted in a square (flat—top) pulse
`profile. The Nd:YAG lasers had a Gaussian profile. Spot
`diameters ranged from 0.2 to 6 mm. Each irradiation event
`consisted of a single pulse.
`The threshold radiant exposure (mJ/cmz) for cell killing
`was determined for TM cells with varying degree of mela-
`nization (melanin incubation concentration ranging from
`l><106 to 3x107 particles/ml). The threshold radiant exposure
`for TM cell killing was defined as the minimum radiant
`exposure where cell cytotoxicity was observed using a
`fluorescent Live/Dead Viability/Cytoxicity assay (see below
`for detail, Molecular Probes Inc., Eugene, Oreg.). The
`threshold energy for SYSTEM-1 and SYSTEM-2 was mea-
`sured by determining the ratio of dead cells/live cells within
`the irradiation zone. A micrometer equipped inverted fluo-
`rescent microscope (Carl Zeiss IM 35, Goettingen, Ger-
`many) was used to delineate the irradiation zone and count
`the number of dead cells vs. live cells. The Nd:YAG lasers
`(SYSTEM-3A&B) have a Gaussian beam profile which
`required a difi°erent method for determination of threshold
`energy, as described below.
`Method for Quantitation of the Threshold Energy for Cyto-
`toxicity for the Nd: YAG Laser
`In contrast to laser System-1 and System—2, which have a
`uniform spatial energy distribution within the irradiation
`spot, the Nd:YAG laser emits a Gaussian beam with an
`energy distribution profile described by equation 1 and
`depicted in FIG. 3.
`
`E(r)=E,.e‘2"2"””
`
`(Equation 1)
`
`where:
`
`E(r)=Pulse energy at a given radius, r.
`E,:Laser pulse energy.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`r=Radius from center of beam.
`
`w=Constant, defined as Gaussian beam radius at E(r)/E:
`1/e2
`To determine the threshold energy for cell killing, Em, we
`measure the radius of cell killing (by a fluorescent micro-
`scope with a reticule eye piece and the Live/Dead viability
`assay), r,, at diiferent energy levels E,, where i=1, 2, 3, .
`.
`.
`, # of energy levels tested. At threshold energy level, Em, we
`can substitute for E(r) and r in equation 1 to obtain equation
`2.
`
`E,,,=E,-e_2("2”“2)
`
`(Equation 2)
`
`where:
`
`E,,,=Threshold energy for cell killing.
`El. =Laser pulse energy.
`ri =Radius of cell killing at E.
`Algebraic manipulation of equation 2 yields the following
`equation 3, which is in the form of y=mx+b.
`
`2ri2=m21n[E,-]— 2In [E,,,]
`
`(Equation 3)
`
`The linear form of equation 3 easily lends itself to an
`ordinary least square regression method. For example, in
`FIG. 3, Gaussian profiles of three beam energies are depicted
`as E, E2, an