# Patent

## Title
Treatment solution and method for preventing posterior capsular opacification by selectively inducing detachment and/or death of lens epithelial cells

## Abstract

A treatment solution used to prevent posterior capsular opacification is applied or introduced into the lens capsular bag before, during, or after cataract surgery. The treatment solution comprises an ion transport mechanism interference agent, which either alone or in combination with other treatment agents such as an osmotic stress agent and an agent to establish a suitable pH, selectively induces detachment and/or death of lens epithelial cells such that posterior capsular opacification is prevented. While the ion transport mechanism interference agent is capable of interfering with the cellular mechanisms and cell ion distribution of a broad range of cells, a concentration of agent is selected such that the treatment solution interferes selectively with the cellular mechanisms of lens epithelial cells while leaving other ocular cells substantially unharmed. The treatment solution selectively induces cellular death and/or detachment of lens epithelial cells while other ocular cells and tissue remain substantially unharmed and without lengthy preoperative pre-treatment.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 10/244,878, filed on Sep. 17, 2002, which is herein incorporated by reference in its entirety.


TECHNICAL FIELD
The present invention relates to novel treatment solutions and methods comprising an ion transport mechanism interference agent, alone or in combination with other agents, used to prevent posterior capsular opacification by selectively inducing detachment and/or cell death of lens epithelial cells without damaging other ocular cells and tissue and without lengthy preoperative treatment.
BACKGROUND OF THE INVENTION
The predominant role of the lens of the human eye is to focus light rays that have passed through the cornea and aqueous humour onto the retina. The structure and metabolism of the lens contributes directly toward maintaining its integrity and transparency. The lens is composed entirely of epithelial cells in different stages of maturation and is relatively unusual in that tissue is never discarded during the maturation process. As new lens cells are formed, older cells are displaced toward the interior of the lens. The lens soon becomes isolated from a direct blood supply and depends on the aqueous and vitreous humors for both nutrition and elimination pathways. The optical characteristics of the lens are much dependent on lens cells maintaining a constant cell volume and dense packing of the fibers to reduce the volume of intercellular space. Maintaining its delicate structure therefore becomes an essential characteristic of the lens. The lens has evolved its unique capabilities to maintain constant cell volume by regulating its ion, sugar, amino acid, and water balances.
A cataract of the human eye is the interruption of the transmission of light by loss of lens transparency. Cataracts, which cause blurring and clouding of vision, are by far the most common cause of low visual acuity. The clouded lens can be removed by surgical procedure, i.e. extra-capsular cataract extraction (ECCE). ECCE comprises the removal of the clinical nucleus with cortical cleanup using either manual or automated vacuuming techniques. The posterior and equatorial capsule is left intact as an envelope or bag into which a posterior chamber intraocular lens can be inserted. If the posterior capsule and zonules are intact, this lens will ordinarily remain in place throughout the patient's life without any complications. During the operation, the anterior portion of the lens capsule is carefully opened and the cataract is removed. The intraocular lens is inserted into the remaining (posterior) portion of the capsule. This results in a loss of natural lens accommodation.
Standard cataract surgery employs a procedure known as phaco-emulsification. This process agitates the lens content causing break-up of the lens material, which is then sluiced out of the lens capsule by the phaco-emulsification probe that simultaneously injects and extracts a washing solution. Both dead and live lens epithelial cells detached by the treatment will be washed out by this sluicing action.
Progress is being made in the development of new treatments that involve retention of the anterior lens capsule and the replacement of the lens content with injectable material.
After this surgery, vision is restored but one of the most frequent complications of prevailing cataract surgery is the proliferation of lens epithelial cells (LECs) after cataract surgery. Posterior Capsular Opacification (PCO), also known as secondary cataract, is the most frequent complication following extra-capsular surgery, occurring in about twenty to forty percent of patients. In the past decade, results from a number of experimental and clinical studies have led to a better understanding of the pathogenesis of PCO.
The main cause of PCO is the proliferation and migration of residual LECs to the posterior lens capsule. Despite the care taken by surgeons to remove most of the residual lens epithelial cells, they are difficult to remove. Many LECs are, therefore, left behind in the lens capsular bag at the end of the surgical procedure. The proliferation of the LECs causes the membrane or envelope into which the intraocular lens is placed to become cloudy over time. Proliferation of LECs is also a significant problem in the new cataract treatments utilizing, for example, injectable lenses. The cloudy membrane is called an “after cataract” or PCO. The symptoms of PCO are identical to those of cataract, causing vision to gradually fade and eventually leading to blindness if not treated.
Much effort has been made to prevent or minimize formation of PCO. These efforts can be broadly categorized into three areas: surgical improvement, lens design improvement and chemical prevention.
A number of surgical strategies have been developed to attempt prevention of PCO. These have involved the use of various surgical instruments and the application of laser, ultrasound, and freezing techniques. Once PCO has occurred, YAG laser capsulotomy is a simple, quick procedure in which a laser beam is used to create an opening in the center of the cloudy capsule. There are, however severe risks associated with YAG laser capsulotomy. These mainly include the development of retinal detachment, glaucoma, cystoid macula oedema.
Much interest has centered on the type of material from which the intraocular lens is made and the profile of the intraocular lens' edge. Biconvex and planer convex polymethylacrylate as well as silicone plate hepatic intraocular lenses and lenses with sharp optic edges are reported to have a beneficial effect on PCO. Significant advances have been made in this area particularly by Alcon's Acrysof lens. These lenses are however relatively expensive and introduce complications or constraints of their own.
There have been other attempts to destroy or prevent proliferation of LECs making use of chemical agents. In British Patent Application 0122807.1, filed on Sep. 21, 2001, the hypothesis was that by decreasing intracellular sorbitol concentration in LECs by modulating sorbitol pathways via an aldose reductase inhibitor, PCO could be prevented by inducing death of LECs by osmotic shock. However, in order to modulate the sorbitol pathways, the proposed treatment involved pretreating the lens capsular bag for a period of up to 48 hours prior to surgery and is, therefore, not easily incorporated into current cataract surgery.
Other chemical therapies have been attempted. For example, the use of immunotoxin-conjugated antibody specific for LECs can reduce but not completely prevent the incidence of PCO. See Clark et al, J. Cataract Refract. Surg. December 1998; 24(12): 1614-20 and Meacock et al, J. Cataract Refract. Surg. May 2000; 26(5):716-21. The use of ethylenediamine tetraacetic acid, Trypsin and DISPASE® (Neutral Protease) has also been used to separate the LECs but can damage the zonules and surrounding tissues. See Nishi, J. Cataract Refract. Surg. January 1999; 25(1):106-17 and Nishi et al, Opthalmic Surg. August 1991; 22(8):444-50. Other agents that have been tested for the prevention of PCO include RGD peptides to inhibit the migration and proliferation of lens epithelial cells; anti-mitotic drugs, such as Mitomycin C and 5-fluorouracil. See Chung H S, Lim S J, Kim H B. J Cataract Refract Surg. October 2000; 26(10):1537-42; Shin D H, Kim Y. Y., Ren J. et al. Ophthalmology. 1998; 105:1222-1226.
Unfortunately most chemical agents described above, when used at their effective doses, demonstrate unacceptable levels of toxicity to surrounding ocular tissues including ocular cells such as corneal endothelial cells (CEDCs) and retinal pigmented epithelial cells (RPECs).
BRIEF SUMMARY OF THE INVENTION
In accordance with the present invention, the disadvantages of prior means of preventing PCO as discussed above, have been overcome. The present invention prevents PCO by rapidly and selectively inducing detachment and/or cell death of lens epithelial cells (LECs) without significantly damaging other ocular cells and tissues, is easily incorporated into existing standard cataract surgery and into future potential new treatments, and allows for easy removal of residual lens epithelial cells during or after cataract surgery. PCO prevention is accomplished via application of treatment solution or solutions. The treatment solution is applied or introduced into the lens capsular bag before, during, or after cataract surgery. The treatment solution comprises an ion transport mechanism interference agent, which either alone or in combination with other treatment agents such as an osmotic stress agent and an agent to establish a suitable pH, selectively induces detachment and/or death of lens epithelial cells such that posterior capsular opacification is prevented. While the ion transport mechanism interference agent is capable of interfering with the cellular mechanisms and cell ion distribution of a broad range of cells, a concentration of agent is selected such that the treatment solution interferes selectively with the cellular mechanisms of lens epithelial cells while leaving other ocular cells substantially unharmed. The treatment solution selectively induces cellular death and/or detachment of lens epithelial cells while other ocular cells and tissue remain substantially unharmed and without lengthy preoperative pre-treatment.
The treatment solution or solutions, which may be applied or introduced before, during, or after cataract surgery, may selectively and rapidly remove the LECs by: (i) inducing selective detachment of the LECs from their substrates or membranes through interference with normal cellular functions, causing eventual cellular death or at least allowing for easy removal of LECs; (ii) inducing selective death of LECs through interference with normal cellular functions; and/or (iii) inducing susceptibility of the LECs to osmotic stress through interference with normal cellular functions, ultimately leading to cellular detachment and/or death. The present invention allows for chemical agents that interfere with the ion transport mechanisms of a broad range of cells to specifically and uniquely target the LECs.
The ocular treatment solutions and methods prevent PCO by utilizing an ion transport mechanism interference agent to induce substantially greater incidence of detachment and/or death of LECs than other ocular cells. The ion transport mechanism interference agent of the present invention is capable of interfering, either directly or indirectly, with intracellular, extracellular and/or intercellular mechanisms that regulate the flow of ions and water across the cellular membrane of a LEC. This interference may cause cells to shrink and/or swell, resulting in cell detachment and/or death. A concentration of agent is selected such that, while the treatment solution comprising the ion transport mechanism interference agent causes significant detachment and/or death of lens epithelial cells, it does not cause the death and/or detachment of a significant percentage of other ocular cells. More particularly, the ion transport mechanism interference agent may include a co-transport interference agent, a sodium pump interference agent, an exchange interference agent, or a channel interference agent.
The ion transport mechanism interference agents may themselves induce cell detachment and/or death of LECs or may sensitize LECs such that the LECs are rendered incapable of sufficiently responding to an additional agent that causes osmotic stress, selectively detaching and/or killing the LEC and preventing PCO. This additional agent is an osmotic stress agent such as an osmolyte that induces osmotic shock in the LECs causing cell detachment and/or death.
The treatment solutions of the present invention have a suitable pH selected according to a selective concentration of a particular osmotic stress. The pH of the solutions can be adjusted by any acids, bases or other agent that increases or decreases the concentration of hydrogen ions in the solution.
These and other advantages and novel features of the present invention, as well as details herein, will be more fully understood from the following description.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
FIG. 1 is a diagram illustrating the cell viabilities of different ocular cells (LECs, RPECs and CEDCs) after solutions comprising varying concentrations of NaCl are introduced;
FIG. 2 is a diagram illustrating cell viabilities of LECs after application of increasing concentrations of NaCl in the absence (diamonds) and the presence (circles) of 30 μM frusemide, a co-transport interference agent;
FIG. 3 is a diagram illustrating cell viabilities of total LECs after application of the solutions comprising increasing concentrations of NaCl at varying pH levels;
FIG. 4 is a diagram illustrating cell viabilities of RPECs after application of solutions comprising increasing concentrations of NaCl at varying pH levels;
FIG. 5 is a diagram illustrating cell viabilities of LECs and RPECs after application of treatment solutions comprising ≦200 mM NaCl, increasing concentrations of a co-transport interference agent, frusemide, and 3 μl of 5N NaOH;
FIG. 6 is a diagram illustrating cell viabilities of LECs, RPECs, and CEDCs after application of treatment solutions comprising ≦200 mM NaCl, increasing concentrations of a co-transport interference agent, bumetanide, and 3 μl of 5N NaOH;
FIG. 7 is three photographs illustrating the effects of treatment on primary cultured LECs with the treatment solutions of the present invention at (A) zero, (B) five and (C) ten minutes after washing;
FIG. 8 is four photographs illustrating the effects of treatment on primary LECs in a human organ culture PCO model with the treatment solutions of the present invention (A) before treatment, (B) five minutes after treatment, (C) ten minutes after treatment, and (D) after washing.
FIG. 9 is four photographs illustrating the effects of treatment on RPECs with a treatment solution comprising ≦200 mM NaCl, 90 μM frusemide, and 3 μl of 5N NaOH at (A) zero, (B) five, and (C) ten minutes post-treatment and (D) after washing;
FIG. 10 is a diagram illustrating cell viabilities of LECs, RPECs and CEDCs after application of treatment solutions comprising ≦200 mM NaCl, 90 μM frusemide and 3 μl of 5N NaOH;



DETAILED DESCRIPTION OF THE INVENTION
While the invention will be described in connection with one or more preferred embodiments, it will be understood that the invention is not limited to those embodiments. On the contrary, the invention includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the claims concluding this specification.
The present invention prevents PCO by rapidly and selectively inducing detachment from the lens capsule and/or death of lens epithelial cells via an ion transport mechanism interference agent, either alone or in combination with other agents, without substantially damaging other ocular cells and tissue and without lengthy pre-operative treatment. This selective detachment and/or death is made possible by the specific nature of LECs, which differentiates them from other cells in the eye. The result is that, at the doses and concentrations of agents used, LECs are detached, killed, and/or removed from the lens, whilst other ocular cells and tissue are left unharmed.
Water is effectively in thermodynamic equilibrium across the plasma membrane of human cells. Because cell membranes of virtually all human cells are highly permeable to water, cell volume is determined by the cellular content of osmotically active solutes (electrolytes and osmolytes) and by the osmolarity and/or tonicity of the extracellular fluid. Generally, intracellular and extracellular tonicity are the same; under physiological conditions, most mammalian cells are not exposed to large ranges of tonicity. The osmolarity of body fluids is normally about 285 mOsm/kg H2O and is regulated within extremely narrow limits (±3%) by body fluid homeostasis. However, under pathophysiological conditions, disturbances of body fluid homeostasis can be encountered. Plasma fluid osmolarities ranging between about 220 mOsm/kg H2O and about 350 mOsm/kg H2O have been observed. Under these extreme conditions the cells of the body would, in the absence of volume-regulating mechanisms, swell and shrink by twenty to thirty percent, respectively.
Lens epithelial cells, like other cells, use cell volume regulatory processes to defend against changes in extracellular osmolarity and to maintain constant cell volume. Volume regulatory mechanisms for lens epithelial, fibre and ciliary epithelial cells have been described earlier. See for example, C. W. McLaughlin et al, Amer. J. Phsiol. Cell Physiol. September 2001; 281 (3): C865-75; J. J. Zhang et al, Exp. Physiol. March 1997: 82(2): 245-59; J. J. Zhang et al, Exp. Physiol. September 1994: 79(5): 741-53; J. J. Zhang et al, J. Physiol. Mar. 1, 1997; 499(Pt. 2): 379-89. After osmotic perturbation, there ensues a volume regulatory phase in which cells tend to return to the volume they had in an isotonic medium. Changes in cell volume activate specific metabolic and membrane transport pathways that result in the net accumulation or loss of osmotically active solutes. Regulatory volume decrease (RVD) is the process used to decrease or maintain cell volume in response to low extra-cellular osmotic pressure or tonicity. Regulatory volume increase (RVI) is the process used to increase or maintain cell volume in response to high extra-cellular osmotic pressure or tonicity. When cell solute content (i.e., intracellular tonicity) or extracellular tonicity is altered, rapid transmembrane water flow occurs to restore equilibrium. Because the plasma membrane is highly compliant, water flow causes cell swelling or shrinkage.
One way that cells alter their intracellular ion concentrations to establish equilibrium is through the use of ion transport mechanisms located on the surface of the cells. These mechanisms move ions into or out of cells, altering intracellular ion strength and, as a result, osmotic pressure. For example, the ion transport mechanisms activated during RVD in various cell types involve conductive K+ and Cl− channels (separate, conductive K+ and Cl− transport pathways), K−—Cl− co-transport and functionally coupled K+—H− and Cl−—HCO3− exchange. The ion transport mechanisms activated during RVI in various cell types involve Na+—K+-2Cl− co-transport, and functionally coupled Na+—H+ and Cl−—HCO3− exchange.
There is a surprising diversity between different cell types in the nature and reaction of ion transport mechanisms. See Hoffmann and Simonsen,
Physiol Rev. 1989 April; 69 (2):315-82. For example, cells have certain volume set-points at which the cells capable of undergoing RVD and RVI are doing neither. Different cells have different volume set-points at which given transport mechanisms turn on or off See Parker, J. C Am J Physiol. 1993, 265:C1191-1200; Parker J C et al. J Gen Physiol. June 1995; 105(6):677-99. The set point or threshold at which lens cells turn off their RVI and RVD processes is different from other ocular cells. Cell shape and interaction of integrins with the extra-cellular matrix play a critical role in determining cell detachment, survival or inducing programmed cell death in adherent cells. This differentiates the LECs from other cells in the eye and the body.
Interfering with the ion transport mechanisms of lens epithelial cells prevents these cells from adequately responding to a hypoosmotic or hyperosmotic extra-cellular environment or other cellular stresses. The failure of the cells to respond to these stresses triggers immediate or eventual cell detachment and/or cell death.
Lens epithelial cells that are unable or rendered incapable of responding to stresses may have several responses. One response is cell death. Another possible response is detachment of the cell from its underlying substrate or membrane. For example, the cell may shrivel such that the cell can no longer maintain its attachment to the substrate, which may be associated with rearrangement of the cell cytoskeleton. Cell shrinkage may also trigger adherent cells to rearrange their focal adhesion contacts and cytoskeletal attachments to those contacts. The detachment ultimately causes cellular death and/or allows for easy removal of the cell. Cell death may lead to cell detachment from surrounding cells and the extra-cellular matrix (ECM) and cell detachment may occur without immediate cell death. For example, disruption of extracellular matrix contacts has been shown to induce anoikis, a form of apoptosis induced by cell detachment from its substation cells. Another possible response by lens epithelial cells is a weakening or sensitizing of the cell, such that the cell becomes vulnerable to osmotic stress or shock. For example, once a cell is sensitized, it is rendered incapable of appropriately responding to osmotic shock or stress such that introduction of shock or stressors to the sensitized cell will cause detachment and/or cellular death. The treatment solutions and methods of the present invention prevent PCO by selectively and rapidly eliciting at least one of these responses from LECs while leaving other ocular cells substantially unharmed.
In one aspect, the ocular treatment solutions and methods of the present invention prevent PCO by inducing substantially greater incidence of detachment of the LECs from their substrates or membranes and/or cell death than other ocular cells such as corneal endothelial cells (CEDCs) and retinal pigmented epithelial cells (RPECs) via an ion transport mechanism interference agent. Therefore, a significant advantage of the present invention is that treatment with the agents of the present invention prevents PCO by allowing for removal from the lens capsular bag of a substantial percentage of LECs, while other ocular cells and tissue are not significantly harmed.
The ion transport mechanism interference agent is an agent capable of interfering, either directly or indirectly, with intracellular, extracellular and/or intercellular mechanisms that regulate the flow of ions and water across or within the cellular membrane of a lens epithelial cell. The ion transport mechanism interference agent interferes with normal cellular ion distribution mechanisms and consequently the functions of cells. The ion transport mechanism interference agent of the present invention, either alone or in combination with other agents, disturbs the normal distribution of LEC cell ions and thereby selectively induces, either directly or indirectly, LEC detachment and/or death to prevent PCO. A concentration of the agent is selected such that the treatment solution interferes with ion transport mechanisms of LECs causing detachment and/or death of LECs, but does not substantially interfere with ion transport mechanisms of other ocular cells. While LECs may be easily removed from the lens capsular bag due to cell detachment and/or death, other ocular cells remain substantially unharmed.
The ion transport mechanism interference agent is capable of interfering with one or more of the following cellular mechanisms in an LEC: (1) co-transport mechanisms (for example, K+—Cl− co-transport, Na+—Cl− co-transport, Na+—K+-2Cl− co-transport and amino acid transport); (2) the sodium pump; (3) ion exchanges (for example, functionally coupled Na+—H+ and Cl−—HCO3− exchanges; functionally coupled K+—H+ and Cl−—HCO3− exchange, Cl−—Cl− exchange, Ca2−—Na+ exchange); and (4) ion channels (for example, potassium channels, chloride channels, volume-sensitive organic osmolyte and anion channel (VSOAC) and other anion channels). The ion transport mechanism interference agent may activate or inhibit either directly or indirectly these cellular mechanisms.
In hypotonic media, vertebrate cells initially swell by osmotic water equilibration but subsequently regulate their volume by a net loss of KCl and a concomitant loss of cell water to restore normal cell volume. Cell swelling activates transport pathways that result in the net efflux of potassium, chloride and organic osmolytes. The ion transport mechanisms activated during RVD in various cell types involve conductive K+ and Cl− channels (separate, conductive K+ and Cl− transport pathways), K+—Cl− co-transport, and functionally coupled K+—H+ and Cl−—HCO3− exchange. For example, potassium and chloride are lost from the cell primarily through activation of the K+—Cl− co-transport or through separate potassium and anion channels.
In contrast, in hypertonic media, vertebrate cells initially shrink by osmotic water equilibration but subsequently regulate their volume by a net gain of KCl and a concomitant uptake of cell water to restore normal cell volume. Cell shrinkage activates transport pathways that result in the net influx of chloride, sodium, and potassium. The ion transport mechanisms activated during RVI in various cell types involve Na+—Cl− or Na+—K+-2Cl− co-transport, and functionally coupled Na+—H+ and Cl−—HCO3 exchange.
Examples of ion transport mechanism interference agents include, but are not limited to:
n-ethylmaleimide
valinomycin
gramicidin
catecholamines
calcium
Ionophore A23187 (including, for example, Ionophore A23187 plus calcium)
calmodulin
pimozide
loop diuretics (e.g., frusemide, bumetanide, ethacrynic acid, piretanide, torasemide)
amiloride
di-isothiocyano-disulfonyl stilbene (DIDS)
staurosporine (SITS)
niflumic acid
stilbene derivatives (e.g. 4,4′-dinitrostilbene-2′2-disulfonic acid)
quinine
Cytochalasin B
In general there are four classes of ion transport mechanism interference agents: (1) co-transport interference agents; (2) sodium pump interference agents; (3) exchange interference agents; and (4) channel interference agents. As reflected below, it is understood that an ion transport mechanism interference agent may fall into one or more of the four preceding classes.
A co-transport interference agent is an agent capable of interfering, either directly or indirectly, with the co-transport mechanisms of a cell. More particularly, the co-transport interference agent activates or inhibits the co-transport mechanisms of an LEC. A concentration of co-transport interference agent is selected such that the treatment solution induces substantially greater incidence of detachment and/or death of LECs than other ocular cells. Examples of co-transport mechanisms include the K+—Cl− or Na+—K+-2Cl− cotransports. Examples of co-transport interference agents include:
n-ethylmaleimide (NEM) (including, for example, n-ethylmaleimide plus rubidium)
iodoacetamide
frusemide (also known as furosemide),
bumetanide
mercury (Hg2+)
and any other agent capable of capable of activating or inhibiting a co-transport mechanism of a lens epithelial cell.
A sodium pump interference agent is an agent capable of interfering, either directly or indirectly, with the Na/K/ATPase pump, known as the sodium pump, mechanism of a cell. More particularly, the sodium pump interference agent activates or inhibits the Na/K/ATPase pump of an LEC. For example, LECs contain impermeant, anionic macromolecules and the colloid-osmotic swelling, resulting from entry of diffusible ions and water (Gibbis-Donnan equilibrium), is constantly counteracted by the operation of ion-extruding pumps that extrude ions at a rate equal to that of the dissipative leak entry. A concentration of a sodium pump interference agent is selected such that the treatment solution induces substantially greater incidence of detachment and/or death of LECs than other ocular cells. An example of a sodium pump interference agent includes ouabain and any other agent capable of activating or inhibiting a sodium pump of a lens epithelial cell.
Yet another type of ion exchange mechanism interference agent is an exchange interference agent. An exchange interference agent is an agent capable of interfering, either directly or indirectly, with the ion exchanges of a cell. More particularly, the exchange interference agent activates or inhibits the ion exchange mechanisms of a LEC, which not only results in intracellular and extracellular ion re-distribution but may also alter intracellular pH. Examples of ion exchange mechanisms include K+—H+ and Cl−—HCO3 exchanges and Na+—H+ exchange functionally coupled to Cl−—HCO3 exchange. For example, any stimuli that induce potassium and chloride loss by activation of the K+—H+ and Cl−—HCO3− exchanges of an LEC is an exchange interference agent. Examples of exchange interference agents include:
staurosporine (SITS)
amiloride
di-isothiocyano-disulfonyl stilbene (DIDs)
calmodulin (CaM)
copper (Cu2+)
hydrogen (H+)
and any other agent capable of activating or inhibiting an exchange channel of a lens epithelial cell.
A channel interference agent is an agent capable of interfering, either directly or indirectly, with the ion channels of a cell. More particularly, the channel interference agent activates or inhibits the ion channels of an LEC. Examples of ion channel include the potassium, chloride and VSOAC transport pathways. For example, any stimulus that induces potassium chloride and organic osmolyte loss by activation of potassium and chloride channels is a channel interference agent. A concentration of a channel interference agent is selected such that the treatment solution induces substantially greater incidence of detachment and/or death of LECs than other ocular cells. Examples of channel interference agents include:
ketoconazole (KETO)
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)
1,9,-dideoxyforskolin (DDF)
tamoxifen
verapamit
quinine
barium (Ba2+)
diphenylamine-2-carboxylate (DPC)
anthracene-9-carboxylic acid
indocrinone (MK-196)
pimozide
and any other agent capable of activating or inhibiting an ion channel of a lens epithelial cell.
Using the agents of the present invention, a concentration of agent or agents is selected such that application of the treatment solution results in LEC cell viability of preferably less than about ten percent, more preferably less than about five percent, and more preferably approaching zero percent. The desired cell viability for other ocular cells such as RPECs and CEDCs is preferably greater than about seventy percent, more preferably about ninety percent, and more preferably approaching one hundred percent.
Under these circumstances, the treatment solution comprising the agent, perhaps in combination with other agents, selectively induces LECs detachment and/or death and induces substantially greater incidence of detachment and/or death of LECs than other ocular cells. As shown in the examples, cellular detachment and/or death may be evidenced by viewing the cellular area before or after the area has been rinsed, washed, or in any way agitated such that detached and/or dead LECs may be removed.
The agents of the present invention described above (referred to hereafter as the primary agent) selectively induce detachment and/or death of an LEC, directly or indirectly, alone or in combination with additional agents described below or other agents and/or chemicals. For example, the ocular treatment solutions and methods of the present invention may comprise an osmotic stress agent, in addition to the primary agent, wherein the solution induces substantially greater incidence of cell detachment and/or death of lens epithelial cells than corneal endothelial cells and retinal pigmented epithelial cells to prevent posterior capsular opacification.
An osmotic stress agent is an agent capable of inducing osmotic shock in an LEC. Osmotic shock in an LEC may be caused by any cellular condition that alters normal intracellular or extracellular ion equilibrium. For example, changes in the extracellular concentrations of osmotically active solutes and various stimuli that affect the activity of the specific ion transport systems involved in cell volume control (e.g., hormones, growth factors and other external stimuli, the cell membrane potential, and cytoplasmic second messengers) may induce osmotic shock. Large transcellular ion fluxes may shock cellular volume homeostasis. Hypoosmotic stress can be caused by exposure of cells to a hypotonic solution or a solution with a hypoosmotic osmolarity. Conversely, hyperosmotic stresses can be caused by exposure of cells to a hypertonic solution or a solution with a hyperosmotic osmolarity. An increase or decrease in the cellular concentration of osmotically active ions and organic solutes may also shock cellular volume regulation and activate membrane transport processes involved in the RVD or RVI response.
The application or introduction of an osmotic stress agent is appropriate where the primary agent sensitizes the LEC to osmotic shock (for example, by inhibiting an ion transport mechanism) and where the osmotic stress agent itself induces cell detachment and/or death. For example, if the ion transport mechanism interference agent inhibits an essential ion transport mechanism such as the sodium-potassium pump, the LEC is incapable of defending itself against external osmotic stresses or shocks, thereby enhancing its sensitivity to extracellular osmotic pressure caused by an additional osmotic stress agent. The failure of LECs to respond to osmotic stresses or shocks may trigger cell death through activation of death-signaling processes.
However, it is understood that the primary agents may also be osmotic stress agents such that a primary agent may both selectively interfere with the ion transport mechanisms of LECs and induce osmotic shock.
More particularly, the osmotic stress agent has a hyperosmotic or hypoosmotic osmolarity. A hypoosmotic solution is one that has a lower solute concentration than that normally found in physiological conditions; a hyperosmotic solution has a higher solute concentration than that normally found in physiological conditions. In other words, the osmotic stress agent creates extracellular hypertonicity or hypotonicity.
Any physiologically tolerable electrolytes and osmolytes, any solute that contributes to osmotic strength, are suitable for inducing osmotic stresses or shocks. Examples of osmolytes include:
salts (such as NaCl);
amino acids (such as taurine, glycine, and alanine);
sugars (such as mannitol, myo-inositol, betaine, glycerophosphorylcholine, betains, urea, sucrose and glucose);
and any other organic or inorganic osmolytes.
It is noted that while the osmolyte may be sodium chloride, as described above, the chloride may be replaced by iodide, bromide, nitrate, thiocyanate, fluoride, sulfate, isethionate, gluconate, and any other acceptable substitute; similarly, sodium may be replaced by potassium, cesium, rubidium, lithium, francium, and any other acceptable substitute.
The osmotic stress agent and the primary agent may be applied separately, simultaneously or sequentially in the treatment of PCO.
The treatment solution may preferably have a pH from about 5 to 11, depending on the agents and concentrations used. In a preferred embodiment, a treatment solution comprising approximately ±200 mM NaCl has a pH preferably in the range of about 8 to 10. The pH of the treatment solution may be adjusted by varying the concentrations of the primary agents or the osmotic stress agent or by the introduction of an additional chemical agent that alters the pH. The pH can be adjusted by any acids or bases or any agent that increases or reduces the hydrogen ion concentration.
The treatment will work with a range of concentrations of primary agents, osmotic stress agents and agents which adjust pH. In accordance with the present invention, suitable concentrations of agents are selected such that an agent that may be capable of interfering with the cellular mechanisms of a broad range of cells selectively interferes with the ion exchange mechanisms of LECs, causing LEC detachment and/or death. Application of the treatment solution results in a significant percentage of LECs that are detached and/or killed whilst a low percentage of other ocular cells are harmed. At one end of the range of concentrations, the efficiency of the treatment solution is reduced, resulting in longer times to kill the LEC cells. At the other end of the range the medicament is prone to damaging other cells in the eye. Suitable concentrations of both the primary agent, the osmotic stress agent and the agents which adjust pH should be selected such that PCO may be prevented by selective cell death and/or detachment of LECs without significant damage to other ocular cells and ocular tissue. Preferably, concentrations of the agent or agents should be selected such that, upon application, cell viability for LECs is less than about ten percent, more preferably less than about five percent, and more preferably approaching zero percent and the cell viability for other ocular cells such as RPECs and CEDCs is greater than about seventy percent, more preferably greater than about ninety percent, and more preferably approaching one hundred percent.
The agents described above and the concentrations in which they are introduced are interrelated. For example, a high pH treatment solution will kill LECs with lower levels of osmolarity. Examples of suitable concentrations of certain agents are provided in the examples below. Moreover, the concentrations can be adjusted to suit the particular needs of prevailing cataract surgery techniques and the needs of the patient.
The treatment solution of the present invention may be administered before, during, or after cataract surgery to prevent PCO. The anterior portion of the lens capsule is opened and the cataract removed. The intraocular lens may then be inserted into the posterior portion of the capsule. Alternatively, the lens nucleus may be replaced with an injectable intraocular lens. The residual LECs may or may not be removed using vacuuming or other surgical techniques. Phaco-emulsification, laser or other techniques may be employed to remove the lens contents. The treatment solution or treatment solutions may be applied or introduced topically at any time before, during or after this process. The ocular region may then be washed to remove residual cells.
As described above, the treatment solution may comprise the primary agent, the osmotic stress agent, and any chemical agent used to adjust the pH of the solution. Alternatively, separate solutions comprising the primary agent, the osmotic stress agent, and/or the chemical agent to adjust pH may be applied or introduced either sequentially or simultaneously to prevent PCO. As described above, the primary agent may also be an osmotic stress agent. The solution or solutions may be applied via syringe, dropper, probe used for phaco-emulsification or any other suitable or convenient means of application.
An additional advantage of the present invention is that the treatment solutions induce relatively rapid detachment and/or death of lens epithelial cells such that the treatment of the present invention is easily incorporated into cataract surgery. Detachment of lens epithelial cells may occur within about thirty minutes of application of the solution or agents of the present invention, with cell death occurring within several hours. Preferably, detachment occurs within about fifteen minutes of application but is dependent on the concentrations and agents selected.
Whether the treatment solutions and methods of the present invention have induced substantially greater incidence of cell detachment and/or death of LECs than other ocular cells or whether such treatment is selective may be determined by a comparative assessment of cell viability after treatment or any other acceptable method of determining effectiveness. More particularly, the treatment solutions and methods should prevent PCO while causing only minimal physiological damage to other ocular cells as described. Preferably, concentrations of the agent or agents should be selected such that, upon application, cell viability for LECs is less than about ten percent and the cell viability for other ocular cells such as RPECs and CEDCs is greater than about seventy percent. It is understood that the treatment solution or solutions may comprise additional agents, chemicals, proteins, and/or substances and may comprise more than one primary agent or osmotic stress agent. Additional chemical or biological components may be added to improve the properties of the treatment solution or solutions, for example, physiological tolerance, viscosity or storage capability.
The treatment solution may be packaged in kits or other packaging or stored either as a unitary solution or, as described above, in separate solutions for later application. For example, a kit or package may include a solution containing the ion transport mechanism interference agent packaged separately from a solution containing the osmotic stress agent. Furthermore, the treatment solution may be packaged in kits or packaging containing fluids used during phaco-emulsification or in kits used for removal of lens content prior to injection of an injectable intraocular lens.
EXAMPLES
Comparative Example 1
As described below, the confluent monolayers of LECs, CEDCs, and RPECs grown in 96-well flat-bottom plates are exposed to varying concentrations of NaCl (an osmolyte) solution. Two hundred μl of NaCl solution (prepared as described below) are added to each well at concentrations of 137 mM, 170 mM, 340 mM, 680 mM, 1360 mM and 2000 mM NaCl, respectively. The solutions have a pH of 8.0±0.4. The concentrations of NaCl correspond to final osmolarities of about 285 to 4000 mOsm/L, respectively. Within about five to ten minutes after addition of hypertonic NaCl, some of the LECs began to shrink, round up and lose adhesion from the monolayer in a dose-dependent manner. This effect is not seen as significantly in the CEDCs and RPECs.
Cell viabilities of LECs, CEDCs and RPECs in response to increasing hyperosmotic NaCl are shown in FIG. 1 (LECs shown in light gray diamonds, RPECS in dark gray squares, CEDCs in black diamonds). Each point represents the mean of two experiments. At a hypertonic stress of about 1380 mM NaCl, about forty percent of the LECs survive while more than eighty percent of CEDCs and RPECs survive, evidencing the different osmotic tolerance between these cells.
The primary and first passages of human lens epithelial cells, human corneal endothelial cells, and human retinal-pigmented epithelial cells are cultured. The LECs, the CEDCs, and the RPECs are isolated from post-mortem eyes by using standard techniques of cell isolation and enzyme digestion. See Uebersax E D, Grindstaff R D and Defoe D M Exp Eye Res. March 2000; 70(3): 381-90; Wagner L M, Saleh S M, Boyle D J, and Takemoto D J. Mol Vis. Mar. 14, 2002; 8:59-66; Cammarata P R, Schafer G. Chen S W, Guo Z, and Reeves R E. Invest Ophthalmol Vis Sci. February 2002; 43(2):425-33; Rakic J M, Galand A, and Vrensen G F. Exp Eye Res. November 2000; 71(5):489-94. LECs and CEDCs are cultured in Eagle's minimum essential medium (MEM). RPECs are cultured in Ham's F10 medium, supplemented with 2 mM glutamine, 25 mmol/L Hepes, pH 7.4, 10 U/mL penicillin, and 10 μg/mL streptomycin and 15% heat-inactivated foetal cow serum (FCS). The primary cultured LECs, CEDCs, and RPECs are sub-cultured at 104 cells/wells in 96-well flat-bottom plates until they reached 90 to 100% confluence. These cells are grown to confluence in an incubator with a humidified atmosphere of 5% CO2 at 37° C. The cells are kept in a confluent state for overnight before assays.
Hyperosmotic solutions of NaCl are prepared by first making a 2 M NaCl stock solution by adding 58.44 grams of NaCl to 500 ml dH2O (NaCl supplied by Sigma Chemical co, Dorset, England). A desired concentration of osmotic NaCl solution is then prepared by diluting the 2M stock solution with distilled water (dH2O). For example, to make a hyperosmotic solution of 170 mM NaCl, the 2M NaCl stock solution is diluted with dH2O at a ratio of 1 to 10.1; to make a hyperosmotic solution of 340 mM NaCl, the 2M NaCl stock solution is diluted with dH2O at a ratio of 1 to 4.56. Final osmolarities (mOsm/kg/H2O) are determined by a freezing point depression method using a Cryoscopic Osmometry.
After treatment, the treated cells are thoroughly washed, and cultured with fresh media for twelve hours before cell viability is assessed. The capability of these cells to survive at the indicated hypertonic stress levels is assessed by an MTT assay 12-24 hours post-exposure.
MTT assay is an assay to assess mitochondria function, performed as described by Mosmann (Mosmann, T. J Immunol Methods. Dec. 16, 1983; 65(1-2):55-63. The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; supplied by Sigma Chemical Co.) is dissolved in Phosphate's Balanced Salt (PBS) Solutions at 5 mg/ml and sterilized by passage through a 0.22 μm Millipore filter. MTT stock solution is added to growth media at a ratio of 1 to 10. Cells are incubated with this growth media for three to four hours. In cells that are alive at the time of adding MTT, mitochondrial dehydrogenase cleaves the tetrazolium ring into a dark blue formazan reaction product, which builds up in the cell. MTT-reacted cultures are observed under brightfield microscopy without further processing. Cells in each well are scored as MTT-positive if they are completely filled with reaction product (in the case of unattached cells) or contain many formazan crystals emanating from multiple foci (in the case of attached or spread cells). MTT-reacted cultures are then lysised by dimethyl sulfoxide (DMSO) and read in a Microplate Reader to obtain a measure of cell viability. The number of live cells (MTT-positive) per well is determined at the specified concentrations and expressed as a percentage of total cells (demonstrated in FIG. 1).
Example 2
Confluent monolayers of LECs are given three washes with Hank's Balanced Salt Solutions (HBSS) to remove FCS before the experiment. LECs are prepared in 96-well culture plates as in Example 1. These cells are exposed for about five to ten minutes to 200 μl/well of treatment solutions comprising increasing concentrations ( ̃135 mM,  ̃170 mM,  ̃340 mM,  ̃680 mM,  ̃1360 mM and  ̃2000 mM) of NaCl (an osmolyte) in the presence and the absence of 30 μM frusemide (a co-transport interference agent, an ion transport mechanism interference agent). The treatment solutions have a pH of 8.0±0.4. After treatment, the treated cells are thoroughly washed, and cultured with fresh media. The viability of the treated LECs was determined by MTT assay 12-24 hours post-treatment as described in Example 1.
After this treatment, the same phenomenon of cell shrinkage and detachment as observed in Comparative Example 1 also occurs in the presence of frusemide, but the changes are much more dramatic. FIG. 2 shows that in the absence of frusemide (depicted in diamonds) about 40% of LECs survive after a rapid hypertonic shock with 1380 mM NaCl. However, after inhibition of co-transport mechanisms using frusemide (depicted in circles), the osmotic tolerance of LECs is dramatically reduced, and almost no cells survive after rapid hypertonic shock with 340 mM NaCl. This result shows survival capability of the LECs shifts from hypertonic shock with 200 mM NaCl in the absent of frusemide to 170 mM NaCl in the presence of the frusemide.
Each point represents the mean±standard deviation (S.D) of three experiments. A stock solution of 30.3 mM frusemide is prepared by first dissolving 20 mg frusemide in 100 μl DMSO to make sure it dissolved and then adding it to 1.9 ml of the desired concentration of hyperosmotic NaCl. To get 30 μM frusemide, five μL of frusemide stock solution is then added to five ml of the desired concentration of NaCl solution (which is prepared as described in Comparative Example 1).
Comparative Example 3
This example demonstrates effects of pH on LECs osmotic survival. Confluent monolayers of the LECs are prepared as described in Comparative Example 1. A control group of cells are exposed to a solution comprising physiological 135 mM NaCl at a pH of 7.4±0.3. A different group of cells are exposed for ten minutes to 200 μl/well of the solutions comprising increasing concentrations of NaCl ( ̃170 mM, and  ̃340 mM) at varying pH (8.0±0.4, 8.4±0.5, 8.9±0.8, and 10.6±0.5).
The pH of the solutions is adjusted by directly adding 0, 1, 3, or 5 μl of 5N NaOH stock solution to 10 ml of the selected hyperosmotic NaCl solutions (which are prepared as described in Example 1). The corresponding final pH was determined by using a professional pH meter. Final pH in 10 ml of the NaCl solution without addition of NaOH gave a reading of pH about 8.0±0.4. Final pH in 10 ml of the NaCl solution with 1 μl of 5N NaOH gave a pH reading of about 8.4±0.5. Final pH in 10 ml of the NaCl solution with 3 μl of 5N NaOH gave a pH reading of about 8.9±0.8. Final pH in 10 ml of the NaCl solution with 5 μl of 5N NaOH gave a reading of about 10.6±0.5.
Cell viability of the treated cells is determined by MTT assay 12-24 hours post-treatment as described in Example 1. Cell viability of LECs after application of solutions is depicted in FIG. 3. As shown, the gray bar represents the control group in which LECs were exposed to physiological 135 mM NaCl. The black and white bars represent the LECs exposed to 170 mM NaCl and 340 mM NaCl, respectively, with increasing pH. As shown, LECs sensitivity to hypertonic stress increases with increasing pH and increasing NaCl concentrations. At pH of about 10.6±0.5, about greater than seventy five percent of LECs burst and die within five minutes. At pH of about 8.9±0.8, the LECs detach effectively from their substrates but do not burst significantly.
Comparative Example 4
This example demonstrates effects of extracellular pH on the RPECs osmotic survival. Confluent monolayers of RPECs are prepared as described in Example 1. These cells are exposed for ten minutes to 200 μl/well of the solutions comprising increasing concentrations of NaCl (137 mM, 170 mM, 340 mM, 680 mM, 1360 mM and 2000 mM) at varying pH (8.4±0.5 and 10.6±0.5). The solutions of NaCl are prepared as described in Example 1. The pH of the solutions is adjusted with additions of 1 μl and 5 μl of 5N NaOH as described Example 3.
The viability of the treated cells was determined by MTT assay 12 to 24 hours post-treatment as described in Example 1. Cell viability of RPECs after application of the solutions is depicted in FIG. 4 (pH 8.4±0.5 depicted in diamonds; pH 10.6±0.5 depicted in circles). Each point represents the mean±standard deviation of three experiments. As shown, there is a dramatic decrease in the survival level of RPECs exposed to hypertonic NaCl when the pH level is raised.
Example 5
Confluent monolayers of LECs and RPECs are prepared as described in Example 1. These cells are exposed for about ten minutes to 200 μl/well of treatment solutions comprising: (i) ≦200 mM hyperosmotic NaCl; (ii) increasing concentrations (10 μM, 30 μM, 60 μM, 90 μM, and 120 μM) of co-transport interference agent, frusemide, and (iii) 3 μl of 5N NaOH.
The treatment solutions containing frusemide are prepared as described in Example 2, by diluting stock solution of frusemide with ≦200 mM hyperosmotic NaCl solution (prepared as described in Example 1). For example, to get a hyperosmotic NaCl solution with 60 μM frusemide, 10 μL of frusemide stock solution is added to 5 mL hyperosmotic NaCl solution. The pH of the solutions is adjusted with 3 μl of 5N NaOH.
The viability of the treated cells is determined by MTT assay 12 to 24 hours post-treatment as described in Example 1. Cell viability of LECs (circles) and RPECs (diamonds) are depicted in FIG. 5. Each point represents the mean±standard error (s.e.m) of six experiments for the HLECs and four experiments for the HRPECs. As shown, at 10 μM frusemide, more than 50% of the LECs shrink, are lost or die. Between 30 μM and 120 μM frusemide, about >90% of LECs are removed or killed. On the other hand, between 30 μM and 90 μM frusemide, few RPECs are removed or killed. Therefore, for example, in about 200 mM hyperosmotic NaCl solution at this pH, about 15 μM to about 100 μM are effective concentrations at which no significant harm was done to RPECs.
Example 6
Confluent monolayers of LECs, CEDCs and RPECs are prepared as described in Example 1. These cells are exposed for about ten minutes to 200 μl/well of treatment solutions comprising: (i) ≦200 mM hyperosmotic NaCl, (ii) increasing concentrations (14 μM, 28 μM, 56 μM, 112 μM, and 224 μM) of bumetanide, a co-transport interference agent, and (iii) 3 μl of 5N NaOH.
The treatment solutions containing Bumetanide are prepared as follows. A stock solution of 1.4 M bumetanide is prepared. A second stock solution of 14 mM bumetanide is then made by diluting 1 μL of the 1.4 M bumetanide solution with 1 mL hyperosmotic NaCl solution (prepared as described in Example 1). The final desired concentrations of bumetanide are obtained by diluting the 14 mM bumetanide stock solution with NaCl solution to get concentrations of approximately 0 μM (control) 14 μM, 28 μM, 56 μM, 112 μM, and 224 μM, respectively. The pH of the solutions is adjusted with 3 μL of 5 N NaOH.
The viability of the treated cells was determined by MTT assay 12 to 24 hours post-treatment as described in Example 1. FIG. 6 depicts cell viability of LECs (circles), RPECs (diamonds) and CEDCs (squares). Each point represents the mean±s.e.m and standard deviations of six experiments for LECs and three experiments for RPECs and CEDCs, respectively. As shown, at 28 μM, 112 μM, and 224 μM bumetanide, nearly all of the LECs shrink, detach and/or die under phase-contrast microscopic observation, and there are almost no cells left after washing. Nearly 100% cells show MTT negative response. On the other hand, at these same concentrations, less than about 20% RPECs and 30% CEDCs are killed or removed. Accordingly, 28 μM, 112 μM, and 224 μM bumetanide are suitable therapeutic concentrations for these particular treatment solutions.
Example 7
Confluent monolayers of LECs are prepared as described in Example 1 and grown on a glass cover-slip instead of 96-well flat bottom culture plates. The LECs are then thoroughly washed to remove serum.
Ten milliliters (10 mL) of the treatment solution containing ≦200 mM NaCl, 90 μM frusemide and 3 μl of 5N NaOH is prepared in a deep 35 mm petri-dish.
The cover-slips are then transferred to the petri-dish containing the treatment solution and are kept in the treatment solution for five to ten minutes. The treatment solution is then poured away, and the cover-slips along with detached LECs are thoroughly rinsed with a fresh HBSS solution with slight pressure using an aspiration/irrigation probe. After immediate examination and photography using inverted phase contrast microscope, the LECs are kept in culture for seven days with fresh medium to examine whether any residual cells would survive.
FIG. 7 depicts images of LECs taken using an inverted phase-contrast microscope and their osmotic responses during treatment. Photograph 7A depicts, at zero minutes, confluent LECs covering the entire area. Photograph 7B depicts, at five minutes post-treatment, the LECs rounded up and separating from their substrate. Photograph 7C depicts, at ten minutes post-treatment and after washing, a substantially clear substrate after the cells have been washed away.
Example 8
In order to assess the effects of the therapy in an environment close to that in vivo a lens organ culture PCO model is used (model was originally developed by Liu et al (Liu C S, Wormstone I M, Duncan G, Marcantonio J M, Webb S F, Davies, P D. Invest Ophthalmol Vis Sci. April 1996; 37 (5):906-14). Human lenses are isolated from donor eyes (ages between 19 to 89 years), and then glued onto a tailor-made stainless steel stand. Under an operating microscope, the anterior portion of the lens capsule 100, which is about 6 mm in diameter, is carefully opened using the technique of continuous curvilinear capsulorhexis. The lens nucleus is then hydroexpressed with HBSS and the remaining lens fibres carefully removed. The posterior capsule 200 and equatorial capsule 300 are left intact as envelopes (bags), stored in plastic culture dishes (deep 35 mm petri-dish) and covered with Eagle's Minimum Essential Medium growth medium (MEM) supplemented with 10% to 15% FCS. The human LECs in the organ culture model are then allowed four to five days to recover from the surgical trauma.
Two groups of organ cultured PCO models were established. The first group of ten lenses was established as a control and kept in organ culture without treatment. All of these lenses developed confluent mono- or multiple-layered proliferated LECs on the anterior capsule 100 and posterior capsule 200 after about four to seven days.
The second group of twenty lenses, after four to five days of culture, also had a monolayer of the proliferated LECs. This group was thoroughly washed to remove serum prior to the introduction of the treatment solutions. Ten milliliters (10 mL) of treatment solutions comprising: (i) ≦200 mM NaCl, (ii) about 90 μM frusemide, or about 28 μM bumetanide, or about 112 μM bumetanide, and (iii) 3 μl of 5N NaOH are prepared in a deep 35 mm petri-dish. The solutions are prepared as described in other examples. The organ cultures are then transferred to the petri-dish containing the treatment solutions and are kept in the treatment solution for five to ten minutes. The organ cultures are then transferred using forceps into another petri-dish. The capsules along with detached LECs in the PCO organ culture are then thoroughly rinsed with a fresh HBSS solution with slight pressure using an aspiration/irrigation probe. After immediate examination, the organ cultures are kept in culture for seven days with fresh medium to examine whether any residual cells would survive.
The effect of this treatment on LECs in PCO organ culture model is examined on inverted microscope using phase contrast optics. They are photographed with T-Max 400 film (Kodak) at zero, five, and ten minutes and at twelve hours post-treatment. As shown in FIG. 8, within five to ten minutes of treatment, all of the LECs round up and lose adhesion from the monolayer and the lens capsule. Phase contrast micrograph 8A depicts at low magnification (field of view is 1 mm×0.75 mm) the proliferated LECs after a sham ECCE operation on the anterior 100 and posterior capsule 200 six to seven days after dissection and before treatment. Phase contrast micrograph 8B depicts at high magnification (field of view is 3.4 mm×1.5 mm) the LECs starting to round up and detach from the lens capsule five minutes after treatment. Phase contrast micrograph 8C depicts at high magnification the majority of treated cells detached from the monolayer ten minutes after treatment. Phase contrast micrograph 8D depicts at low magnification that, after washing, the lens capsule was devoid of LECs ten minutes after treatment.
Example 9
In this example, the effects of the treatment on primary RPECs are examined. To assess whether there are any side effects on other ocular cells, the primary cultured RPECs were cultured in 6-well flat-bottom culture plate until they reached 90 to 100% confluence in Ham's F10 medium, supplemented with 2 mM glutamine, 25 mmol/L Hepes, pH 7.4, 10 U/mL penicillin, and 10 μg/mL streptomycin and 15% heat-inactivated foetal cow serum (FCS). The cultures were kept in an incubator with a humidified atmosphere of 5% CO2 at 37° C. and were kept in a confluent state for overnight before assays.
The RPEC cultures were thoroughly washed to remove serum prior to the introduction of the treatment solutions. Twenty milliliters (20 mL) of the treatment solution comprising (i) ≦200 mM NaCl, (ii) about 90 μM frusemide, or about 28 μM bumetanide, or about 112 μM bumetanide, and (iii) 6 μl of 5N NaOH is prepared in a deep 35 mm petri-dish as described in previous examples. Five milliliters (5 ml) of the treatment solution is then added to the confluenced RPECs in each well of 3 wells in a 6-well culture plate. After about 10 minutes, the solution is removed completely and the treated RPECs are thoroughly rinsed with a fresh HBSS solution with slight pressure using an aspiration/irrigation probe. After immediate examination, the primary RPEC cultures are kept in culture for seven days with fresh medium to examine whether any residual cells would survive.
The effect of treatment on these primary RPECs are examined on inverted microscope using phase contrast optics. They are photographed with T-Max 400 film (Kodak) at zero, five, and ten minutes and at twelve hours post-treatment. The RPECs are not significantly affected by ten minutes exposure to the treatment solutions. FIG. 9 depicts a representative example of the effects of the treatment on these cells. Photograph 9A depicts RPECs at zero minutes. Photograph 9B depicts RPECs at five minutes post-treatment. Photograph 9C depicts RPECs ten minutes post-treatment. Photograph 8D depicts RPECs after the cells were washed following treatment.
Example 10
To assess whether there is any side effect of this treatment on other ocular cells, confluent monolayers of LECs, CEDCs and RPECs are prepared as described in Example 1. These cells were exposed for about ten minutes to 200 μl/well of treatment solutions comprising ≧200 mM NaCl, 90 μM frusemide, and 3 μl of 5N NaOH.
The viability of the treated cells was determined by MTT assay 12 to 24 hours post-treatment as described in Example 1. Cell survival response of LECs, RPECs and CEDCs to the treatment are depicted in FIG. 10. Each point represents the mean±s.e.m. of four experiments. As shown, ten minutes after treatment, the treatment solution induced substantially greater incidence of cell detachment and/or death, measured by cell viability, of LECs than CEDCs and RPECs. Specifically, more than seventy percent of CEDCs and eighty percent of RPECs survived after the treatment. This can be compared to a less than ten percent survival rate for LECs.
Example 11
The effects of treatment on organ cultured corneal buttons is examined to determine whether the treatment would cause damage to surrounding ocular tissues in an environment close to in vivo. Cornea is chosen because of its sensitivity and the danger of direct exposure to treatment. Corneal organ cultures are washed with HBSS and placed endothelial-side up on a tailor-made stainless steel stand. They are then stored in a sterile culture dish containing MEM growth medium covering only the bottom of the culture dish, providing a humidified chamber to prevent drying of the epithelium. The endothelial corneal concavity is then filled with MEM growth medium supplemented with 10% to 15% FCS, and cultured at 37° C. in a 5% CO2 incubator.
The organ cultures are divided into control and treatment groups. The cultures are first washed three times with HBSS and then their endothelial surfaces are exposed for ten minutes to either physiological NaCl (137 mM, 293±9 mOsm/L) or the treatment solution.
For the treatment group, ten milliliters (10 mL) of treatment solutions comprising (i) ≦200 mM NaCl, (ii) about 90 μM frusemide or about 112 μM bumetanide, and (iii) 3 μl of 5N NaOH are prepared in a deep 35 mm petri-dish. The organ cultures are transferred to the petri-dish containing the treatment solution and are kept in the treatment solution for more than ten minutes. The organ cultures are then transferred using forceps to another petri-dish containing fresh HBSS. The corneal organ cultures are then thoroughly rinsed with a fresh HBSS solution with slight pressure using an aspiration/irrigation probe. After the treatment, the corneal organ culture is incubated in fresh medium for six hours.
For the control group, instead of the treatment solution described above, a fresh HBSS was applied to the corneal organ cultures for ten minutes.
To assess toxicity of the treatment on corneal endothelial cells, the corneal organ culture buttons are examined by both inverted microscope using phase contrast optics and fluorescent-confocal microscopy. Under confocal microscope, the morphology of actin filaments of corneal endothelial cells is examined to establish evidence for the intact architecture of the cells. The endothelium of these corneal organ cultures is fixed before and after treatment, and stained with FITC-conjugated phalloidin. Confocal laser-scanning images of corneal endothelium demonstrate no significant differences between untreated (control) and treated groups. The endothelial sheet in the treated group maintains the same integrity as those in the untreated group. The corneal endothelial cells remain as an evenly distributed monolayer with the cells keeping their hexagonal appearance in the control as well as in the treated organ cultures. There is no apparent damage to corneal endothelium cells.
Many modifications and variations of the present invention are possible in light of the above teachings. For example, other ion transport mechanism interference agents may be used than those discussed above in detail such as the following which may be appropriate: valinomycin, bromine, theophylline and other alkylxanthines, icosanoids, phorbol ester (e.g. TPA), potassium-sparing diuretics (e.g., triamterene, spironolactone, and potassium canrenoate), calyculin A, okadaic Acid (OA), propranolol and its analogs, Angiotensin II, Ketoconazole (CDC), arachidonic acid, ianthanum, trifluoperazine, idoxifene, 2-aminisobutyric acid, 17 beta-oestradiol, Bradykinin, phosphatidylinositol 4,5-biphosphate (PIP2), inositol 1,4,5, triphosphate (IP3), prostaglandins (PGE2), adenosine 3′5′-cyclic monophosphate (cAMP), guanosine 3′5′-cyclic monophosphate (cGMP), serine/threonine protein phosphatases (S/T-Ppases), protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinase (MAPK), SRC family tyrosine kinases (SFKs), polyunsaturated fatty acids, phospholipase C (PLC), phosphatases (PP), G-proteins, Rubidium, Copper (Cu2+), Nitrate (NO3−), Barium (Ba2+), Chloride (Cl−), Potassium (K+), agents that change intracellular magnesium and hydrogen concentration, and agents that decrease extracellular sodium, chloride or potassium concentrations. It should be appreciated that there are other ion transport mechanism interference agents as well. Thus, it is to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as described hereinabove.

## Claims

1. An ocular treatment solution comprising: an ion transport mechanism interference agent and an osmotic stress agent wherein the solution induces substantially greater incidence of detachment of lens epithelial cells than corneal endothelial cells and retinal pigmented epithelial cells to prevent posterior capsular opacification and wherein the solution has a hyperosmotic osmolarity.

2. An ocular treatment solution comprising: an ion transport mechanism interference agent and an osmolyte, wherein the solution induces substantially greater incidence of detachment of lens epithelial cells than corneal endothelial cells and retinal pigmented epithelial cells to prevent posterior capsular opacification; wherein the osmolyte is an amino acid, a sugar, chloride, urea, sodium, organic or inorganic salt or a combination thereof and wherein the solution has a hyperosmotic osmolarity.

3. An ocular treatment solution as recited in claim 2 wherein the amino acid is selected from the group consisting of taurine, glycine, and alanine.

4. An ocular treatment solution as recited in claim 2 wherein the osmolyte comprises a sugar.

5. An ocular treatment solution as recited in claim 1 wherein the sugar is mannitol, myo-inositol, betaine, glycerophosphorylcholine, betains, urea, sucrose or glucose.

6. An ocular treatment solution as recited in claim 2 wherein the osmolyte comprises chloride.

7. An ocular treatment solution as recited in claim 2 wherein the chloride is sodium chloride.

8. An ocular treatment solution as recited in claim 2 wherein the chloride is selected from the group consisting of potassium chloride, cesium chloride, rubidium chloride, lithium chloride, and francium chloride.

9. An ocular treatment solution as recited in claim 2 wherein the osmolyte comprises urea.

10. An ocular treatment solution as recited in claim 2 wherein the osmolyte comprises sodium.

11. An ocular treatment solution as recited in claim 2 wherein the osmolyte comprising sodium is selected from the group consisting of sodium chloride, sodium iodide, sodium bromide, sodium nitrate, sodium thiocyanate, sodium fluoride, sodium nitrate, sodium sulfate, sodium isethionate, and sodium gluconate.

12. An ocular treatment solution as recited in claim 2 wherein the osmolyte comprises a salt.

13. An ocular treatment solution as recited in claim 2 wherein the solution has a pH from about 5 to 11.

14. An ocular treatment solution as recited in claim 2 wherein the solution has a pH from about 8 to 10.

15. An ocular treatment solution as recited in claim 13 wherein the ion transport mechanism interference agent is a co-transport interference agent.

16. An ocular treatment solution as recited in claim 15 wherein the co-transport interference agent is selected from the group consisting of n-ethylmaleimide, iodoacetamide, frusemide, bumetanide, and mercury.

17. An ocular treatment solution as recited in claim 15 wherein the co-transport interference agent is selected from the group consisting of frusemide and bumetanide.

18. An ocular treatment solution as recited in claim 2 wherein the ion transport mechanism interference agent comprises a loop diuretic.

19. The solution of claim 18 wherein the loop diuretic is selected from the group consisting of ethacrynic acid, piretanide, and torasemide.

20. An ocular treatment solution as recited in claim 2 wherein the ion transport mechanism interference agent is selected from the group consisting of n-ethylmaleimide, valindomycin, gramicidin, catecholamines, calcium, ionophore A23187, calmodulin, pimozide, amiloride, di-isothiocyano-disulfonyl stilbene, staurosporine, niflumic acid, quinine, and cytochalasin B.

21. An ocular treatment solution as recited in claim 2 wherein the ion transport mechanism interference agent comprises a stilbene derivative.

22. The solution of claim 21 wherein the stilbene derivative is 4,4′-dinitrostilbene-2,2′-disulfonic acid.

23. An ocular treatment solution as recited in claim 13 wherein the ion transport mechanism interference agent is a sodium pump interference agent.

24. An ocular treatment solution as recited in claim 23 wherein the sodium pump interference agent is ouabain.

25. An ocular treatment solution as recited in claim 13 wherein the ion transport mechanism interference agent is an exchange interference agent.

26. An ocular treatment solution as recited in claim 25 wherein the exchange interference agent is selected from the group consisting of amiloride, staurosporine, di-isothiocyano-disulfonyl stilbene, and calmodulin.

27. An ocular treatment solution as recited in claim 25 wherein the exchange interference agent further comprises copper.

28. An ocular treatment solution as recited in claim 25 wherein the exchange interference agent comprises hydrogen.

29. An ocular treatment solution as recited in claim 13 wherein the ion transport mechanism interference agent is a channel interference agent.

30. An ocular treatment solution as recited in claim 24 wherein the channel interference agent is selected from the group consisting of ketoconazole, 5-nitro-2-(3-phenylpropylamino) benzoic acid, 1,9,-dideoxyforskolin, tamoxifen, verapamit, quinine, calcium, diphenylamine-2-carboxylate, anthracene-9-carboxylic acid, indocrinone, and pimozide.

31. An ocular treatment solution as recited in claim 24 wherein the channel interference agent comprises barium.

32. An ocular treatment solution as recited in claim 2 further comprising an agent capable of adjusting the pH of said solution.

33. An ocular treatment solution as recited in claim 2 wherein the agent capable of adjusting the pH is a hydroxide and wherein the treatment solution has a pH from about 8 to 10.

34. An ocular treatment solution as recited in claim 33 wherein the hydroxide solution comprises sodium hydroxide.

35. An ocular treatment solution as recited in claim 2 wherein the ion transport mechanism interference agent is frusemide and wherein the treatment solution has a pH from about 8 to 10.

36. An ocular treatment solution as recited in claim 35 wherein frusemide is present in the treatment solution in a concentration ranging from about 15 μM to about 100 μM.

37. An ocular treatment solution as recited in claim 2 wherein the ion transport mechanism interference agent is bumetanide and wherein the treatment solution has a pH from about 8 to 10.

38. An ocular treatment solution comprising: an ion transport mechanism interference agent and an osmotic stress agent to prevent posterior capsular opacification, the solution having a pH from about 5 to about 11 and the solution rapidly inducing detachment of lens epithelial cells.

39. An ocular treatment solution comprising: an ion transport mechanism interference agent and an osmotic stress agent wherein the solution induces substantially greater incidence of detachment of lens epithelial cells than corneal endothelial cells and retinal pigmented epithelial cells to prevent posterior capsular opacification and wherein the solution has a hypoosmotic osmolarity.