WO2000074648A2 - Prevention de la regression dans la keratoplastie de refraction - Google Patents

Prevention de la regression dans la keratoplastie de refraction Download PDF

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WO2000074648A2
WO2000074648A2 PCT/US2000/013600 US0013600W WO0074648A2 WO 2000074648 A2 WO2000074648 A2 WO 2000074648A2 US 0013600 W US0013600 W US 0013600W WO 0074648 A2 WO0074648 A2 WO 0074648A2
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comeal
composition
refractive
comea
collagen
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WO2000074648A3 (fr
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Bruce J. Sand
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SUNRISE TECHNOLOGIES INTERNATIONAL Inc
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SUNRISE TECHNOLOGIES INTERNATIONAL Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A61K9/0051—Ocular inserts or implants

Definitions

  • Refractive keratoplasty All methods used to alter the anterior corneal radius of curvature for the correction of refractive error are collectively known as "refractive keratoplasty.”
  • Refractive keratoplasty is based upon the hypothesis that the cornea is responsible for about eighty per cent (80%) of the light bending power of the eye. Nominal modification of the front surface radius will, therefore, result in marked changes in the focusing power of the cornea.
  • the correction of nearsightedness (myopia) requires that the front radius be lengthened or flattened, while steepening of the central cornea, or shortening of the radius, is performed for the correction of farsightedness (hypermetropia).
  • refractive keratoplasty can be surgical, laser-assisted, and conductive.
  • Laser- assisted procedures include laser in situ keratomileusis (LASIK), photo-ablative refractive keratectomy (PRK), and laser thermal keratoplasty (LTK).
  • PRK and LASIK have in common the use of the excimer ultraviolet laser for photodisruption and the removal of corneal stromal collagen.
  • PRK requires that the epithelium be removed entirely in the area of ablation, while LASIK-induced epithelial damage results from the microkeratome that is used to cut a thin corneal flap beneath which the stroma is ablated.
  • Radiofrequency keratoplasty and laser thermal keratoplasty each cause thermal epithelial trauma and subsequent apoptosis.
  • the stabilizing heat-labile hydrogen bonding of the collagen fibrillar triple helices are also destroyed by the thermal keratoplasty method. All of the techniques lead to stromal remodeling.
  • Tissues can be defined by their (1) optical properties (absorption, scattering and scattering an isotropy), (2) thermal properties (heat capacity and heat diffusivity), (3) mechanical properties (viscoelasticity, tensile strength and rupture points), (4) chemical composition (water and other endogenous and exogenous absorbers), (5) anatomy (physical arrangement of organelles, cells and tissues) and (6) physiology (tissue and organismal metabolic status and function).
  • the photothermal effects of the laser-tissue interactions come under the influence of the energy requirements of the phase changes of the water, tissue desiccation, formation of steam vacuoles within the tissue, and the mechanical effects of the rapidly expanding steam vacuoles trapped within the tissue.
  • Tissue desiccation radically changes the optical characteristics of tissues, especially their absorption characteristics of infrared laser irradiation. In addition to the optical property changes, water loss reduces the thermal conductivity and specific heat of the tissue.
  • thermal history is a dynamic function and must therefore be constantly monitored in order to attain accurate refractive correction.
  • Each method of corneal recurvature requires some mode of traumatic intervention, although some methods are more invasive than others.
  • the trauma is compounded.
  • the efficacy and predictability of each method becomes an important issue.
  • Treatments have been developed that are directed to reducing the fibrosis that accompanies wound healing and induces subsequent corneal scarring.
  • Such treatments include antimetabolites, immunosupressants, corticosteroids, and growth factors such as TGF- ⁇ .
  • TGF- ⁇ growth factors
  • the methods and compositions of the present invention provide therapeutic agents designed to accurately intersect with certain stages of the corneal wound-healing process and inhibit the repair mechanisms that contribute substantially to refractive regression.
  • a method of thermal refractive keratoplasty is featured which prevents or inhibits subsequent loss of refractive effect that is caused by thermally induced corneal epithelial apoptosis.
  • the method comprises applying thermal energy to the cornea such that the energy increases sub-epithelial stromal temperature in at least one selected location sufficient to induce hydrothermal collagen fibrillar shrinkage and induce refractive changes, and cooling the corneal epithelium prior to, subsequent to, or concurrent with the application of thermal energy, so that any heat generated by the energy to the cornea that radiates to the epithelium is unable to increase the epithelial temperature in at least some epithelial cells to a level sufficient to induce apoptosis so that fewer epithelial cells die than would without cooling.
  • the thermal energy used is preferably photothermal coherent energy or radiofrequency conductive energy, and the cooling is preferably achieved by spraying a cryogenic solution onto the corneal surface or placing a cooled corneal contact device onto the cornea.
  • the corneal contact device is preferably fabricated of a material having thermal mass sufficient to remain cooler than ambient temperature for the duration of thermal energy application and which transmits about 100% of mid-infrared irradiation, such as sapphire or quartz.
  • the corneal device can be pre-cooled or cooled in place on the cornea by spraying a pharmaceutically acceptable cryogen upon it, such as 1,1,1,2 tetrafluorethane.
  • the corneal contact device is fabricated with a base radius of curvature flatter than the apical radius of curvature of the comea upon which it is placed, so that it remains in close contact with the cornea and water does not pool beneath it.
  • This method comprises interrupting at least one step in the stromal remodeling response of the cornea elicited by the refractive procedure by providing an effective amount of a composition that modulates the step, where the interrupted step results in inhibition of epithelial apoptosis, inhibition of keratocyte apoptosis, inhibition of keratocyte necrosis, inhibition of keratocyte proliferation and migration into the wounded cornea, inhibition of glycosaminoglycans synthesis, or inhibition of collagenesis.
  • the refractive procedure used is preferably ablative refractive keratoplasty or thermal refractive keratoplasty.
  • the step inhibited can be the cascade associated with Connective Tissue Growth Factor (CTGF) or biologically active fragments thereof, and can be an antibody or fragment thereof, a nucleic acid or other molecule which inhibits expression of the gene or protein, or one which blocks a receptor of CTGF.
  • CTGF Connective Tissue Growth Factor
  • the composition can be an antibody/fragment, chemical compound, or nucleic acid that inhibits the accumulation of heat shock protein-70 and result in an inhibition of collagen assembly to its properly folded state, or inhibits the effects of C- proteinase in stromal remodeling, or the effects of prolyl hydroxylase.
  • Preferred inhibitors of prolyl hydroxylase include FG-1648 and FG-041 (proprietary compounds of Fibrogen, Inc., San Francisico, CA).
  • An advantage of this method lies in that it disrupts one or more phases of the wound repair cascade pharmacologically without disrupting all phases of fibrosis and wound repair, thus selectively preventing specific causes of loss of refractive effect.
  • Another featured method of preventing or inhibiting a loss of refractive effect in a patient who is undergoing or has undergone a refractive keratoplasty procedure comprises applying an effective amount of a composition that creates or restores cross-links between stromal lamellae, where the cross-links increase the stability of the corneal stroma and cause it to hold the corneal shape induced by the refractive keratoplasty procedure longer than it would have if the composition had not been applied.
  • Compositions of the method preferably create or restore hydrogen bonds, electrostatic forces, or covalent bonds between lamellar fibers.
  • a preferred composition is glycerol, which requires no heat or light activation to induce cross-links.
  • compositions include photoactivatable substances, and when these are used, the method further comprises applying photoirradiation to the cornea after application of the composition to activate the composition and create the cross-links.
  • a preferred photoactivatable composition is riboflavin, which is activated by light energy in the ultraviolet spectrum.
  • Other preferred compositions are activated by heat to form cross-links.
  • compositions for preventing or inhibiting refractive regression of the cornea following a refractive keratoplasty procedure where the composition comprises an agent which inhibits the deposition of collagen matrix in response to keratocyte apoptosis or necrosis and an excipient capable of combining with the agent and carrying it across the corneal epithelial barrier and into the corneal stroma, and which is physiologically compatible with the eye.
  • the composition does not affect keratocyte proliferation or migration.
  • the composition further comprises a topical ocular insert which serves as a reservoir and delivery device for the agent.
  • Preferred inserts include pleggets, polymer contact lenses soaked in the agent, collagen shields impregnated with the agent, and liposomes, flexible capsules, or flexible wafers containing the agent, or other membrane or reservoir systems.
  • the agent and excipient are preferably efficacious and bioavailable in doses practical to administer to the eye, and are non-toxic and physiologically compatible with the eye.
  • the agent is an antibody, a small molecule, or a nucleic acid, more preferably an antibody against CTGF or a fragment thereof, an antibody against prolyl hydroxylase or a fragment thereof, an antibody against hsp-70 or a fragment thereof, or an antibody against C- Proteinase or a fragment thereof.
  • compositions for preventing or inhibiting refractive regression of the cornea following a refractive keratoplasty procedure which comprises an agent that creates or restores intermolecular cross-links between lamellar fibers in the corneal stroma, and an excipient capable of combining with the agent and carrying it across the corneal epithelial barrier and into the corneal stroma, and that is physiologically compatible with the eye.
  • the agent comprises glycerol.
  • the composition further comprises a topical ocular insert which serves as a reservoir and delivery device for the agent.
  • Preferred devices include pleggets, polymer contact lenses soaked in the agent, collagen shields impregnated with the agent, and liposomes, flexible capsules, or flexible wafers containing the agent, or other membrane or reservoir systems.
  • the agent and excipient are preferably efficacious and bioavailable in doses practical to administer to the eye, and are non-toxic and physiologically compatible with the eye.
  • the agent is an antibody, a small molecule, or a nucleic acid
  • the invention also features a method of measu ⁇ ng a change in comeal ⁇ gidity comp ⁇ sing applying a first weight to a cornea using an indentation tonometry device to obtain a first tonomet ⁇ c scale reading, applying a second weight of different value to the cornea using the indentation tonometry device to obtain a second tonomet ⁇ c scale reading, wherein the difference between the first and the second tonomet ⁇ c scale readings is indicative of a first particular comeal ⁇ gidity then waiting for a selected time or for a selected event to transpire, and re-applying the first weight to the cornea using the indentation tonometry device to obtain a third tonomet ⁇ c scale reading, and re-applying the second weight to the cornea using the indentation tonometry device to obtain a fourth tonomet ⁇ c scale reading, where the difference between the third and the fourth tonomet ⁇ c scale readings is indicative of a second particular comeal ⁇ gi
  • a different embodiment of the comeal ngidity measure involves applying an indentation tonometry device to a cornea to obtain a first reading, on a tonometnc scale, applying an applanation tonometer to the cornea to obtain a second reading, of intraocular pressure in rnrnHg, where the difference between the first and the second readings is indicative of a first particular comeal ⁇ gidity
  • the indentation tonometry device is re-apphed to the cornea to obtain a third reading, on the tonomet ⁇ c scale
  • the applanation tonometer is re-applied to the cornea to obtain a fourth reading, of intraocular pressure in mmHg, where the difference between the third and the fourth readings is indicative of a second particular comeal rigidity
  • the difference between the first particular comeal rigidity and the second particular comeal ⁇ gidity is indicative of a change in comeal ⁇ gidity
  • Figure 1 is a graph showing the overlapping phases of wound repair, where (A) is the inflammation stage (early and late), (B) is re-epithehazation and granulation tissue formation, and (C) is matrix formation and remodeling. Collagen accumulation begins shortly after the onset of granulation tissue formation.
  • Figure 2 is a schematic of the formation of hydroxylated procollagen from nonhydroxylated procollagen catalyzed by prolyl hydroxylase.
  • Figure 3 is a schematic of the formation of collagen fibers following the cleavage of the C and N terminals of the procollagen molecule by C-Proteinase and N-Proteinase, respectively.
  • Figure 4 illustrates the manner in which hydrogen bonds form between ⁇ chains in the tropocollagen molecule and provide stability to the triple helical structure. Heat above 60 °C will denature collagen and cause the collapse of the helical configuration and the separation of individual ⁇ chains into random coil configuration. The intramolecular cross link is unaffected and the two chains remain linked in the denatured state.
  • Figure 5 shows the intermolecular cross-linking between collagen molecules.
  • the cross-link is shown as bonding a lysine in the amino terminal of a torpocollagen molecule with a hydroxylysine near the carboxyl terminal of another tropocollagen molecule.
  • the proposed reactions leading to stable intermolecular cross-links are the proposed reactions leading to stable intermolecular cross-links.
  • Figure 6 shows intramolecular cross-linking. At the top is shown amino acid sequences of the amino ends of the three peptide chains of the tropocollagen molecule. A cross-link between lysines in an ⁇ l and the ⁇ 2 chain is shown. Postulated steps in the formation of this cross-link are shown in the lower figure.
  • Figure 7a-7c show various methods of instilling therapeutics into the eye, such as by irrigation (7a), periodic irrigation via drops (7b), and aerosol spraying (7c).
  • Figure 8a-8d demonstrates application of dmg reservoirs such as soft contact lenses (8a), pledgets (8b), sponges (8c), flexible capsules, wafers, or other membrane or reservoir systems (8d).
  • dmg reservoirs such as soft contact lenses (8a), pledgets (8b), sponges (8c), flexible capsules, wafers, or other membrane or reservoir systems (8d).
  • Figure 9 illustrates subconjunctival injection.
  • Figure 10 details a method for evaluating comeal rigidity and a protocol to indicate the success of remedial cross-linking of the stroma.
  • Figure 11 schematically depicts the cellular events associated with various refractive modalities.
  • the wound and wound repair responses affected by the methods and compositions of the invention include the epithelial and keratocyte apoptosis and necrosis, and the disruption of the stromal lamellae of the cornea.
  • a novel method of thermal quenching during refractive procedures that induce thermal damage is disclosed that reduces the apoptosis associated with thermal injury, thus reducing the subsequent wound healing response.
  • This method alone or in combination with the wound healing inhibition and remedial cross-linking methods disclosed herein, is designed to reduce the amount of refractive regression experienced by patients who have undergone refractive procedures.
  • An important component of invention is concerned with regression accompanying laser and radiofrequency conductive refractive keratoplasty.
  • the biological responses to trauma and the wound repair cascade following these thermal refractive procedures are becoming better understood, as is their relationship to refractive regression ( Figure 1 1).
  • Apoptosis and direct cellular necrosis induces the wound repair response in each case. Damage to the stabilizing cross-links also occurs with each method. These processes are induced and integrated during the various techniques for refractive keratoplasty and tend to reverse the desired effects generated by the refractive process.
  • thermal effects are an important means of correcting the anterior comeal slope and refractive properties. Because coupled radiative- thermal effects are present during and after pulsed i ⁇ adiation by laser or radiofrequency energy, determination of the optical and/or the thermal properties of the test material can be complex. For example, if the irradiation of the tissue is repeated before the tissue completely cools, the elevation of the tissue temperature is additive When, however, the repetition rates are fast, the pathophysiological effects of pulsed laser irradiation can be essentially like those produced by continuous wave lasers The pathologic changes of thermal injury can be placed mto temperature-time catego ⁇ es in increasing order of seventy The tissue response to injury or the wound repair response thus also vanes
  • PPTR pulsed photothermal radiometry
  • Photothermal effects are produced within the target tissue when, by means of the approp ⁇ ate laser exposure parameters, the radiant energy exceeds the threshold required for tissue modification
  • the photothermal changes tngger a biological response which culminates in a complex sequence of events within the madiated tissue These changes may be only represented by a phase transition or may proceed to tissue destruction with a wound healing response and new tissue synthesis In any case, the definitive change will be determined by the magnitude of the thermal response, or the "thermal history" of the tissue.
  • PPTR is a non-contact method that uses a rapid acting infrared detector to measure the temperature changes induced in a test mate ⁇ al exposed to pulsed radiation Heat generated as a result of light absorption by subsurface chromophores in the test mate ⁇ al diffuse to the surface and results in increased infrared emission levels at the surface
  • a PPTR signal that represents the time evolution of temperature near the test mate ⁇ al's surface
  • Collagen phase transition from the tightly wound triple helical form to the relaxed contracted form begins as the tissue temperature reaches about 55° C. If a sufficient volume of tissue does not reach that state, comeal front surface radius changes do not occur. If the temperature exceeds 68° C, collagen denaturation proceeds to destruction. The contracted state then regresses, as does the desired refractive comeal recurvature.
  • PPTR is a novel method, when combined with infrared pulsed laser irradiation, to determine objective patient-specific laser treatment conditions.
  • infrared pulsed laser irradiation When combined with front surface cooling or the conduction of heat away from the front surfaces (discussed below), it provides an even more refined ability to determine and extraordinarly control the laser generated thermal profile within the comeal tissues.
  • Superficial tissue cooling processes may be either static as in the form of a simple heat sink, or dynamic as in the addition of an evaporative cooling agent.
  • PPTR permits patient-specific determinations with or without additional cooling capabilities.
  • thermal monitoring system embodies the use of a rapid acting thermal sensor that is focused upon the delivery site of the laser.
  • the steady state tissue temperature is obtained and integrated with the known or experimentally determined thermal gradient between the surface and the subsurface target tissue after sub-threshold radiant exposure.
  • Computing electronics within the laser system can automatically determine the appropriate dosimetry.
  • This system can be applied to any coherent or non-coherent light source, as well as to radiofrequency energy sources used to perform any beneficial treatment within the human body that is dominated by thermal or photothermal biological mechanisms.
  • Conductive Keratoplasty causes secondary keratocyte apoptosis immediately beneath the irradiated sites, a novel dynamic cooling process is presented to protect the epithelium from damage. This procedure is refe ⁇ ed to as "thermal quenching.” A similar procedure has been used in aesthetic dermatology to protect the epidermis while an infrared laser achieves thermal denaturation of the proteins in the papillary and reticular dermis to reduce rhytides or superficial cutaneous wrinkles. A modification of this procedure is disclosed that allows the cooling method to be used in refractive keratoplasty.
  • this technology can be used to pre-cool a sapphire contact lens or lens , which would transmit 100% of the coherent thermal energy to the stroma while the contact lens acts as a heat sink to conduct heat away from the superficial cornea, thus preventing thermal trauma and the subsequent apoptotic keratocyte death.
  • This process is a combination static-dynamic cooling process which can be temporally integrated with the laser irradiation.
  • This keratocyte destruction precipitates a wound repair process culminating in the synthesis of glycosaminoglycans (GAG's) and collagen. It is desirable to inhibit this process before it is precipitated. Conduction of heat away from the comeal epithelial surfaces will prevent the epithelial destruction during thermal refractive keratoplasty. It is the epithelial damage which causes the epifhelial-stromal interaction precipitating the apoptotic keratocyte death seen in laser refractive keratoplasty.
  • the process of thermal quenching is applicable to refractive keratoplasty, as discussed above.
  • Various methods of thermal quenching are efficacious and include both passive and dynamic cooling of the comeal epithelial surface.
  • Static cooling may be accomplished by means of a comeal device which transmits the IR radiation used in the thermal keratoplasty, such as 2 micron THC: YAG irradiation, but has enough thermal mass to serve as an efficient heat-sink to the comeal epithelium.
  • a comeal device which transmits the IR radiation used in the thermal keratoplasty, such as 2 micron THC: YAG irradiation, but has enough thermal mass to serve as an efficient heat-sink to the comeal epithelium.
  • Such materials include sapphire and quartz.
  • the cornea is best served if the comeal device has a base curve parallel to or flatter than the flattest radius of the cornea. Pre-cooling of the device can be done.
  • Dynamic cooling or thermal quenching can be accomplished by spraying the cornea with a cryogen, such as 1,1,1,2 tetrafluoroethane (R 134a according to the National Institute of
  • the epithelium is rendered hypo hermic, thus protecting it from destruction as the photothermal energy is transmitted through and absorbed by the universal chromophore, water, within the stroma.
  • procedures such as LASIK are performed, comeal stroma is lathed away, leaving portions of the cornea thinner or reduced, and a certain amount of necrotic debris.
  • Thermal procedures cause keratocyte death via apoptosis and/or necrosis.
  • the cornea's response is to try and correct the insult and return to its original state by initiating a wound healing cascade of events and stromal remodeling.
  • Wound repair is an integration of dynamic interactive processes. In the absence of intervention, these wound repair processes follow a specific time sequence and can be temporally categorized into three phases: (A) inflammation, (B) tissue formation, and (C) tissue remodeling. These phases of wound repair are not mutually exclusive, but rather overlap in time (FIG. 1), and are roughly as follows.
  • platelets secrete growth factors that initiate granulation tissue formation
  • monocytes change into reparative macrophages which secrete growth factors
  • fibroplasia consists of granulation tissue components (fibroblasts and extracellular matrix (ECM)).
  • cytokines stimulate fibroblasts to migrate and switch their major role to protein (collagen) synthesis
  • fibroblasts participate in ECM remodeling, e.g., by depositing fibronectin as a second-order provisional matrix (the appearance of fibronectin, then collagen in healing wounds is consistent with fibronectin serving as a template for collagen fibril organization).
  • hyaluron is a major component of early granulation tissue, promoting cellular movement.
  • proteoglycans are secreted by the ECM and mature fibroblasts; their function in the ECM is to regulate collagen fibrillogenesis.
  • collagen fibrillar deposition into the wound site peaks around 7 to 14 days and provides the structural support.
  • myofibroblasts differentiate and cause tissue contraction by means of dynamic linkages between actin bundles and ECM.
  • FIG. 9 collagen remodeling during the transition of granulation tissue to mature scar is dependent upon both continued collagen synthesis and collagen catabolism controlled by a variety of enzymes.
  • Figure 1 shows these wound repair processes plotted as a logarithmic function of time. The phases of wound repair overlap considerably with one another. Inflammation is divided into early and late phases denoting neutrophil-rich and mononuclear cell-rich infiltrates, respectively. Wound contraction begins after granulation tissue is well established. Collagen accumulation begins sharply after the onset of granulation formation.
  • Apoptosis differs from cellular necrosis, in which the cell contents including the proteolytic enzymes are released into the surrounding area and stimulate an inflammatory response and further damage.
  • Apoptosis is a programmed cell death process in which the cells die in a complex and controlled way with minimal collateral tissue damage or inflammation.
  • Apoptosis occurs when the keratoplasty procedure is accompanied by injury to the comeal epithelium, during which various growth factors or cytokines are released from the injured epithelial cells. Among these growth factors are interleukin-1 (IL-1), ⁇ and ⁇ , and FAS ligands. The cytokines diffuse into the stroma and, thereby, bind to specific receptors located on the cellular surface of the stromal keratocytes and fibroblast like cells.
  • IL-1 interleukin-1
  • ⁇ and ⁇ and FAS ligands.
  • the cytokines diffuse into the stroma and, thereby, bind to specific receptors located on the
  • Interleukin- l ⁇ binds to IL-1 receptor type 1 (keratocytes) thus modulating epithelial- stromal communications. This leads to keratocyte disappearance beneath the epithelial injury site. The increased levels of IL-la and IL-lb induce apoptotic death of keratocytes in the stroma.
  • FAS ligand is a membrane-bound protein, which also induces apoptotic cell eath in cells expressing the FAS receptor. All keratocytes to a depth of 50% of the stroma (200 microns) or more may die, depending upon the type of epithelial injury.
  • the morphologic changes in the stromal apoptotic keratocytes include chromatin shrinkage and fragmentation, cell shrinkage and cellular blebbing of apoptotic bodies.
  • stroma is essential for the proper refraction of light though the cornea, the elimination of keratocytes induced by comeal epithelial trauma can result in stromal haze such as that observed after various refractive keratoplasty procedures. It now appears that these cytokine systems are inter-related. Without wishing to be bound by a mechanism, evidence shows that the IL-1 release from the damaged epithelial cells facilitates expression of FasL mRNA and protein at the time that cell death in response to IL-l ⁇ is noted. It appears that IL-1 may trigger autocrine suicide of keratocytes by induction of FAS ligand in cells already expressing the FAS receptor. It thus, appears that several systems may mediate keratocyte apoptosis in response to the epithelial wounding.
  • HGF hepatocyte growth factor
  • KGF keratinocyte growth factor
  • protein levels all of which aid in the wound remodeling.
  • HGF and KGF both stimulate epithelial cell proliferation.
  • Concu ⁇ ent stimulation of proliferation and inhibition of differentiation in comeal epithelial cells can produce thickening of the comeal epithelium and hyperplasia.
  • Epithelial hyperplasia has been shown to be another factor that leads to regression of refractive effect after PRK (see, e.g., Spadea et al, J. Refractive Surgery, 16: 133-139 (2000); Salchow et al J.
  • apoptotic cells The destruction and removal of the apoptotic cells stimulates the migration of new keratocytes from the posterior and peripheral stroma to the site of injury in the anterior stroma. These repopulating keratocytes differ in appearance from the apoptotic cellsin that they have dark nuclei and prominent intracellular organelles, and are indistinguishable from fibroblasts in other parts of the body.
  • activated keratocytes are associated with the increased stromal collagen synthesis and collagen disorganization accompanying stromal remodeling (correlating with the increased haze as seen in excimer photorefractive keratectomy) and the regression of effect or comeal topographic hysteresis as observed in all refractive surgical procedures.
  • Keratocyte apoptosis is, therefore, an initiating event in the wound healing response after the epithelial removal in PRK and/or thermal damage in photothermal keratoplasty and radiofrequency keratoplasty. While somewhat diminished in laser-assisted in situ keratomileusis (LASIK), there is also a significant wound healing response within the stroma with the level of regression after LASIK varying from essentially zero to as much as 50% in individual cases. Interference with one or more steps in the apoptosis triggering mechanism, or with the migration of new keratocytes into the recently remodeled comea, can prevent the deleterious keratocyte and/or collagenous matrix proliferation that leads to regression.
  • LASIK laser-assisted in situ keratomileusis
  • the wound repair-related regression in LASIK appears to be more the result of cellular necrosis inducing collagenesis and stromal remodeling than the result of cellular apoptosis and epithelial hyperplasia (as the epithelial damage in this procedure results only from the laser microkeratome).
  • Cellular necrosis (not apoptosis) results from the ablation of the stroma.
  • the necrotic cell's appearance differs from apoptotic cells and is characterized by the rapid loss of membrane function and abnormal permeability. There is early disruption of organelles and irreversible damage to mitochondria.
  • Standard, art-known H & E staining characteristics can be used for identification of necrosis and TUNEL stain can be used to identify apoptotic nuclei with their characteristic DNA fragmentation.
  • Nuclear DNA fragmentation occurs relatively early in the process of apoptosis, preceding significant disruption of the nuclear membrane, but it is a relatively late event in necrosis, occurring when there is already severe nuclear and/or cellular membrane disruption.
  • the TUNEL stain labels fragmented DNA and is not specific for either apoptosis or necrosis. Therefore, the presence of apoptotic cell death is determined by identifying cells stained by TUNEL that have an intact nuclear membrane. In contrast, necrotic tissue will stain positively in the TUNEL assay and display a dismpted nuclear membrane.
  • TUNEL to identify fragmented DNA, in conjunction with H & E staining of the nuclear membrane, provides that necrosis and apoptosis can be reliably distinguished, allowing for the separate analysis of each.
  • the stress response Immediately after a sudden increase in temperature, all cells increase production of a certain class of molecules. When first observed some 35 years ago, this phenomenon was called the “heat shock response.” It is now commonly referred to as the stress response and the expressed molecules as stress proteins. These proteins are far more than just defensive molecules. Throughout life of a cell, many of these proteins participate in essential metabolic processes, including the pathways by which other cellular proteins are synthesized and assembled. Some stress proteins appear to orchestrate the activities of molecules that regulate cell growth and differentiation. The stress proteins do play an active role in cellular defense. For example, the stress response plays an important role in the ability of animals to withstand brief exposures to high temperatures. The stress response facilitates the identification and removal of denatured protein from the traumatized cell.
  • a highly inducible heat shock protein, hsp-70 accumulates inside the nucleolus after heat shock.
  • the nucleolus manufactures ribosomes, the organelles on which proteins are synthesized. After heat shock, cells stop making ribosomes and their nucleolus becomes awash in denatured ribosomal particles, hsp-70 recognizes denatured intracellular proteins and restores them to their correctly folded, biologically active shape.
  • hsp 70-related proteins There is a family of hsp 70-related proteins. All of them share certain properties, including a high affinity for adenosine triphosphate (ATP). All of these related proteins, with one exception, are present in cells growing under normal conditions, yet in cells experiencing metabolic stress, they are synthesized at much higher levels.
  • ATP adenosine triphosphate
  • BiP immunoglobulin binding protein
  • BiP helps to orchestrate the early events associated with protein secretion.
  • BiP seems to act as a molecular overseer of quality control, allowing properly folded proteins to enter the secretory pathway but holding back those unable to fold correctly.
  • hsp- 10 and hsp-60 are also essential to protein folding and assembly.
  • the hsp-60 molecule consists of two seven-member rings stacked one atop the other. This large structure appears to serve as a "work-bench" onto which unfolded proteins bind and acquire their final three dimensional structure.
  • the folding process is extremely dynamic and involves a series of binding and release events. Each requires energy, which is provided by the enzymatic splitting of ATP, and the participation of hsp- 10 molecules. Through multiple rounds of binding and release, the protein undergoes conformational changes that take it to a stable, properly folded state.
  • hsp-60 and the hsp-70 families work together to facilitate protein maturation.
  • the new polypeptide, still bound to its hsp-70 escort is transferred to a form of hsp-60, on which folding of the protein and its assembly with other protein components commences.
  • Hsp-60 and hsp-70 act as "molecular chaperones.” Although the molecules do not convey the information for the folding or assembly of proteins, they do ensure that those processes occur quickly and with high fidelity. They expedite self-assembly by reducing the possibility that a maturing protein will head down an inappropriate folding pathway.
  • Heat-denatured proteins like newly synthesized and unfolded proteins, would therefore represent targets to which hsp-60 and hsp-70 can bind.
  • the levels of available chaperones drop and begin to limit the ability of the cell to produce new proteins. The cell somehow senses this reduction and responds by increasing the synthesis of new stress proteins that serve as molecular chaperones.
  • a rise in the expression of stress proteins may also be a requirement for the ability of cells to recover from a metabolic insult. If heat or other metabolic insults irreversibly denatures many cellular proteins, the cell will have to replace them. Raising the levels of those stress proteins that act as molecular chaperones will help facilitate the synthesis and assembly of new proteins. In addition, higher levels of stress proteins may prevent the thermal denaturation of other cellular proteins.
  • the magnitude of the resulting stress response appears to correlate with the relative severity of damage sustained. This has focused the utility of using the changes in stress protein levels as markers for tissue and organ injury. Our pre-clinical studies to develop an animal model for photo-thermal trauma has utilized this principle in determining appropriate markers of laser-induced trauma.
  • Stromal remodeling is one of the important factors in refractive regression
  • the wound healing process that results in the remodeling is a complex cascade of events which, in most cases, is hematogenous in o ⁇ gm
  • An exception to this is the response to injury in the comea, which is an avascular tissue
  • Fibroblasts differentiate and are guided by a chemotactic gradient into the provisional mat ⁇ x- filled wound space
  • An active proteolytic system also cleaves a way for the fibroblast migration
  • the fibroblast After the fibroblast has migrated into the wound, it gradually switches its major function to synthesis of great quantities of collagen in response to TGF- ⁇ Once an abundant collagen matnx is deposited in the wound, the fibroblasts cease collagen production, despite the continuing expression of TGF- ⁇ Signals responsible for down-regulating fibroblast proliferation and matrix synthesis may include factors such as gamma-interferon and the collagen matnx itself (suppressing both fibroblast proliferation and fibrobtastic collagen synthesis) A complex interaction and feedback control between cells-cytokines/growth factors-enzymes-matnx is what controls the production of collagen
  • Hyaluronic acid (a glycosaminoglycan or GAG) appears to promote cellular migration in early granulation tissue and as it becomes hydrated, it promotes expansion of the interstitial spaces, allowing more cell recruitment and proliferation in these areas
  • TGF- ⁇ The role of TGF- ⁇ in scar formation is not well understood, but this is where certain p ⁇ or art methods to control the fibrotic response have been directed These methods are very non-specific TGF- ⁇ suppresses the proliferation of epithelial cells, which, by suppressing re- epithelia zation of denuded basement membrane, may potentiate scar ⁇ ng and fibrosis that appear to be initiated in the absence of efficient re-epithelialization; and this growth factor has quite profound inhibitory activities on cells on the immune system. Hence, by suppressing the accumulation and/or activation of T-cell sub-population, TGF- ⁇ may also indirectly potentiate tissue scarring and fibrosis.
  • Fibrosis takes many forms and is the result of diverse causes.
  • each of the many fibrotic diseases and disorders involves an excessive deposition of collagen and the accumulation of scar tissue.
  • Scar tissue is composed of dense and inelastic collagen fibers.
  • the formation of scar tissue causes the distortion and loss of normal histology and function. Regardless of the underlying cause, the cascade resulting in fibrotic tissue is the same.
  • Currently available pharmacologically based treatments for fibrotic disorders are not very effective in preventing the pathological progression of fibrosis.
  • recent advances in cell and cytokine biology have brought a new understanding of the molecular events underlying tissue fibrosis and reveal promising new therapies.
  • This invention takes advantage of these new developments to remedy the consequences of biological response to trauma in ocular pathology, not particularly with respect to scar formation, but rather with a view to limiting and/or preventing refractive regression of the comea.
  • Tissue engineering studies have resulted in the development of selective and focused compounds which inhibit collagen repair mechanisms, and human neutralizing antibodies which block the fibrogenic cascade.
  • Targets for this inhibition of effect include: (1) Heat Shock Protein 70 (hsp-70), (2) fibrogenic cytokines (CTGF), (3) intra-cellular collagen synthesis enzymes, (prolyl hydroxylase) and (4) extra-cellular collagen fibrillar assembly enzymes (C-proteinase). Encouraging preclinical data on these programs has validated the molecular targets.
  • hsp-70 Heat Shock Protein 70
  • CTGF fibrogenic cytokines
  • intra-cellular collagen synthesis enzymes (prolyl hydroxylase)
  • C-proteinase extra-cellular collagen fibrillar assembly enzymes
  • Growth factors are a class of secreted polypeptides that stimulate target cells to proliferate, differentiate and organize in developing tissues. The action of growth factors is dependent upon their binding to specific receptors which stimulate a signaling event within the cell. Examples of well-studied growth factors include platelet-derived growth factor
  • PDGF insulin-like growth factor
  • IGF-1 insulin-like growth factor
  • TGF- ⁇ transforming growth factor beta
  • EGF transforming growth factor alpha
  • FGF fibroblast growth factor
  • PDGF is a cationic, heat-stable protein found in the alpha-granules of circulating platelets and is known to be a mitogen and a chemotactic agent for connective tissue cells such as fibrolasts and smooth muscle cells. Because of the activities of this molecule, PDGF is believed to be a major factor in normal wound healing and may contribute to such diseases as atherosclerosis and fibrotic diseases.
  • PDGF is a dimeric molecule consisting of an A chain and a B chain. The chains form heterodimers or homodimers, and all combinations isolated to date are biologically active.
  • PDGF-like proteins share both immunological and biological activities with PDGF and can be blocked with antibodies specific to PDGF. These new growth factors likely play a significant role in the normal development, growth and repair of human tissue.
  • Therapeutic agents derived from these molecules may be useful in augmenting normal or impaired growth processes involving connective tissues in certain clinical states, e.g., wound healing. When these growth factors are involved pathologically in diseases, therapeutic developments from these proteins may be used to Control or ameliorate uncontrolled tissue growth.
  • Various cell types produce and secrete PDGF and PDGF-related molecules.
  • CTGF Connective Tissue Growth Factor
  • CTGF stimulates matrix production and the proliferation of cells.
  • CTGF does not substitute for TGF- ⁇ , which appears to prepare fibroblasts for responding to CTGF, but can supplement the action of TGF- ⁇ in inducing cell growth.
  • CTGF is a heparin binding growth factor which may be involved in the activation and tissue storage of TGF- ⁇ (Kothapalli at al, Cell Growth & Differentiation, 1997).
  • the TGF- ⁇ response element in the CTGF promoter confers the CTGF gene's responsiveness to TGF- ⁇ (Grotendorst et al, Cell Growth & Differentiation, 1996). Cells exposed to TGF- ⁇ produce CTGF, and this response can be used in a high throughput screen for compounds that block this pathway.
  • CTGF binds to at least two classes of receptors, small molecule agonists or antagonists can be developed using screening methods known in the art, and CTGF's activity modulated at the receptor-binding step in the cascade.
  • the C-terminal fragments have the ability to stimulate cell proliferation, while the N- terminal region stimulates matnx production
  • Antibodies specific to biologically active fragments of CTGF are far more specific in their antagonistic activities Because of these specific functions, control of refractive regression can be achieved by carefully controlling either or both proliferation or matnx production In this way, comeal wound healing and healthy cell turnover is promoted without the accompanying stromal remodeling that promotes regression
  • CTGF receptors have been isolated and identified and the signal transduction pathways activated by these receptors have been characte ⁇ zed Small molecules which block the induction of CTGF have also been identified It appears that these compounds have the ability to block collagen production in cells exposed to fibrogenic cytokines They will thus also have the ability to be used to inhibit refractive regression, since reduced collagen production will allow the refractive changes induced by keratoplasty to persist intact without stromal remodelling
  • nucleotide sequence complementary to DNA or RNA sequences encoding CTGF or portions thereof, or CTGF promotor or other regulatory sequences may be used to prevent induction of CTGF
  • Methods known in the art to produce and implement antisense therapeutics are within the scope of the invention, and are applicable to inhibit CTGF or any other component of the molecular cascade discussed herein
  • Antisense therapy directed to any step that inhibits apoptosis, necrosis, keratocyte migration or proliferation, or collagenesis is included in the invention
  • Collagens make up about 30% of the total body proteins. They are the major structural proteins that hold cells together and determine tissue and organ architecture. In fibrotic disorders, collagen molecules are synthesized and secreted in excess, and the resulting fibrotic scar tissue is comprised of a strong cross-linked collagen scaffold. Attention has been focused upon key enzymes required for collagen formation and deposition as a target for inhibition of the formation and accumulation of collagen which leads to fibrotic scar.
  • Prolyl-4-hydroxylase is an intracellular enzyme required for the synthesis and formation of all the 20 known types of collagen, therefore representing an important target for anti-fibrotic therapies.
  • P4Hase works inside the cell by modifying polypeptide chains to allow them to form stable triple-he ⁇ eal structures.
  • Prolyl hydroxylase catalyzes the hydroxylation of prolyl residues to 4-hydroxyproline during post-translational modification of collagen. The formation of hydroxyproline enables the molecule to form the stable triple helical conformation typical of collagen (FIG. 2).
  • FIG. 2 Hundreds of thousands of compounds have been screened in high throughput screening (HTS) for assaying diverse organic compounds against this target.
  • a recombinant expression system has been established to produce large quantities of prolyl hydroxylase. Effective inhibition of the activities of this enzyme prevents the assembly of all types of collagen. Novel compounds that inhibit prolyl hydroxylase have been identified, and these have been tested in secondary screens to determine efficacy, toxicity and potency. Additional compounds have been developed from promising leads by focused combinational chemistry.
  • Selected compounds have been tested for oral and topical administration, and the compounds have been found to be active and effective in both forms. Safety has been demonstrated in 14-week administration. In these pre-clinical tests, functional improvement was also demonstrated.
  • Collagen synthesis has been blocked using a novel inhibitor identified as FG-041 in an animal model. Scar formation has been prevented and wound healing and tissue regenaration has been improved. The effect is maintained on withdrawal of the dmg after local administration. In other studies, the drug was well tolerated and showed good pharmacodynamics after oral dmg delivery.
  • Another compound, FG- 1648, a water-soluble inhibitor of prolyl hydroxylase has been shown to be effective by means of topical administration in a pig model for dermal wound scarring. The safety and efficacy of these compounds make them promising therapeutics for topical ophthalmic administration for use in regression therapy. Their short term use would inhibit regression via stromal remodeling, but would avoid the side effects of long-term suppression of collagen formation that could impair comeal health.
  • C-proteinase is the enzyme that then coverts the procollagen into collagen, a necessary step in the formation of types I, II, and III collagen by removing the carboxy terminal domain of the procollagen (FIG. 3).
  • C- Proteinase is an important target for inhibition in refractive regression due to its selective effects on formation of types I and III collagens. It converts fibrillar collagens to collagen by removing the carboxyl terminal globular domain, thereby enabling assembly into larger collagen fibrils. By blocking the action of C-proteinase, procollagens can not assemble into collagen fibrils. As a consequence, C-proteinase inhibitors can block the proliferative effects of types I and II collagen that induce refractive regression, without affecting the formation of other types of collagen.
  • C-Proteinase Three alternative forms of C-Proteinase have been identified, including bone mo ⁇ hogenic protein, or BMP-1, M-tolloid and M-tolloid-like protein (see, e.g., US Patent 5,939,321). Two methods have been created for the blocking of these enzymes, monoclonal antibodies and small molecule compounds. It is understood for this and all other antibody therapeutics discussed herein, that polyclonal antibodies or active fragments (e.g., FAb) may be used.
  • Inhibitors identified to date have shown good selectivity in blocking the C-Proteinase at 200 to 1000 fold lower levels than matrix metalloproteinases MMP- 1, 9 and 13.
  • MMP-9 matrix metalloproteinase-9
  • C-Proteinase inhibition should correlate well with the disappearance of MMP-9 in the ophthalmic model and thus provides a sensitive method of monitoring intervention during anti-regression therapy.
  • the methods of the invention can also make use of the following drugs as alternatives for the prevention of refractive regression: 1. Interferon- ⁇ 2. 3 hydroxypyridine-2-carboxamidoesters
  • calcium channel blockers chosen from nifedipine, hydropyridine, verapamil, colbalt chloride, and biologically acceptable cobalt salts
  • polypeptide including a sequence of at least about 5 amino acids corresponding substantially to an amino acid sequence from within the 33kD fragment of the A chain of fibronectin, within the G domain of the A chain of laminin, or within the NCI domain of the ⁇ 2 chain of the type IV collagen.
  • the essential features of the primary structure of collagen include the presence of glycine as every third residue and the presence of the amino acids hydroxyproline and hydroxylysine. Proline and hydroxyproline together account for approximately one-third of the amino acids present. There is evidence that the collagen molecule is stabilized by one or more disulfide cross-links in addition to hydrogen bonding.
  • the individual peptide chains of collagen molecules are twisted into a right-handed helix.
  • This helical twist is the result of the presence of large numbers of amino acid residues, proline, and hydroxyproline. Because one side of the five-sided ring structure of these amino acides constitutes one part of the peptide backbone of the molecule, free rotation of the chain cannot occur at these sites and the backbone of these chains are bent at these points.
  • the net result of the presence of these residues is that the chain of amino acids is forced into the helical configuration.
  • the three peptide chains must be held together in some fashion to make a molecule. When first assembled, the three chains are held together by relatively weak forces. The molecule thus formed, before it has become polymerized into fibrils and fibers, is referred to as "tropocollagen.”
  • the term collagen usually refers to the polymeric aggregation of a number of tropocollagen units to form a long polymeric chain. Covalent cross-links can form among the three chains.
  • the spatial a ⁇ angement of three chains in the molecule also is unique to collagen.
  • the ⁇ chains are right-handed helices, and the three helices are then twisted into a left-handed "super helix.”
  • the entire structure is held together by hydrogen bonds (FIG. 4).
  • Hydrogen bonds are formed between a hydrogen atom and a strongly electronegative atom such as F. O, or N.
  • a hydrogen attached to an amino group or a hydroxyl group may form a bond within adjacent oxygen derived from a carboxyl group.
  • Hydrogen bonds are quite weak and be broken by mild heat, such as that which is generated by laser thermal or conductive radiofrequency keratoplasty. When this happens, the helical structure of the molecule is destroyed and individual chains separate as randomly coiled structures. In the case of collagen, this gentle heating leads to hydrogen bond mpture and denaturation of the collagen. This process is inherent in the thermal keratoplasty methods and results in collagen fibrillar shrinkage or contraction. If the thermal profile is uncontrolled and exceeds the thermal shrinkage temperature of collagen (Ts), collagen is reduced to parent gelatin and loses all structural integrity.
  • Ts thermal shrinkage temperature of collagen
  • the solubility of collagen undergoes change. Collagen is no longer soluble in water when so aggregated but can be solubilized in neutral salt solutions. Initially the forces holding chains of tropocollagen together are electrostatic. Salt serves to neutralize the electrostatic forces holding the molecules together and thus solubilizes collagen. As intermolecular cross-links form, collagen losses its salt solubility, but can be solubilized in dilute acid, such as 0.5 M acetic acid.
  • the major intermolecular covalent cross-links that form are aldimines which hydolyze in acid solution. As these aldimine bonds become reduced or substitutions occur across the double bond, collagen fibrils become completely insoluble, even in acid.
  • a model of the collagen fibril based upon physical chemistry, describes the micro fibril of collagen as held together by covalent cross-links and the bundles of microfibrils are "cemented" together to form fibrils by protein polysaccarides of glycoproteins.
  • each succeeding chain is distorted or twisted a bit more because of increasing surface of the fibril. This introduces a stress into the polymer and it requires force to maintain this stressed position. This force is provided by ionic or charge attraction between bonding sites in the tropocollagen chain.
  • Maturation of collagen can be defined as the process by which the fragile, soluble fibers of collagen change into strong, insoluble fibers and how they proceed from a disorganized, random, and not very useful a ⁇ angement to an oriented, organized structure providing mechanical strength to a tissue.
  • LTK requires heat to reduce the hydrogen bonds in order to obtain efficacious comea shape change by means of collagen fibrillar shrinkage. Hydrogen bonds are quite weak and can be broken by mild heating. When this happens, the helical stmcture of the molecule is destroyed and individual chains separate as randomly coiled structures. In the case of collagen, mild heating, which leads to hydrogen bond mpture, produces denaturation; parent gelatin is the resulting product. The absence of the triple-helical formation of the molecule leads to extreme fragility of the structural integrity.
  • the hydrothermal shrinkage of the collagen molecule occurs within a small temperature range, from 60 to 70 C, and is attributed to the cleaving of the internal stabilizing cross-links. Exceeding this temperature range will further damage these cross-links and culminate in comeal lamellar instability.
  • the fibroblast or keratocyte in the case of the cornea, is the cell primarily responsible for the synthesis of collagen. When a wound occurs, the fibroblast is among the first cells to appear. Amino acids are picked up by specific transfer RNAs and inco ⁇ orated into growing peptide chains within these cells and become collagen precursors or procollagen. Collagen chains exist as a helical coil a ⁇ anged parallel to each other. The alpha chains are right-handed helices, and three helices are then twisted into a left-handed "super- helix.” This entire stmcture is held-together by hydrogen bonds. Hydrogen bonds (FIG. 4) are formed between a hydrogen atom and a strongly electro-negative atom.
  • a hydrogen attached to an amino group or a hydroxyl group may form a bond with an adjacent oxygen derived from a carboxyl group. Hydrogen bonds are quite weak and can be broken by mild heat. When this happens, the helical stmcture of the molecule unwinds and the collagen molecule contracts.
  • comeal stroma consists of collagen fibers of uniform diameter gathered into bundles or comeal lamellae. Each successive lamellar layer has collagen fibrils oriented at right angles to the ones above and below it. Each fiber is imbedded in a characteristic matrix, or ground substance.
  • the possible stabilization of collagen fibrils through covalent bonding with glycoprotein to collagen also plays a role in the physical properties of the collagen fibril and may regulate the size attained by the fibril.
  • Regressive modification of the desired refractive change following thermal keratoplasty is directly related to these biochemical alterations.
  • the introduction of cross- linking agents at the time of, or subsequent to, thermal injury would hasten the increase in tensile strength of the comea and would provide stability to the resultant refractive modification.
  • tissue bioadhesives for sutureless closures of wounds and in the development of contact lens material, lens or comeal implant material, a wet occlusive bandage, patch graft, implant material to replace silicone in cosmetic plastic surgery, artificial joint lining material and as a dmg delivery mechanism.
  • a biodegradable collagen shield saturated with this photosensitive agent can also be utilized followed by photoactivation by short-wave UV or visible photonic radiation.
  • Unique to this method is the fact that while one end of the bifunctional reagent form peptide-like bonds with the collagen amino acid side chains, the other end remains unbound until photoactivation. This end is then converted to a highly reactive compound called a "nitrene” or a "carbene”, which in turn bonds with an amino acid side chain of tissue collagen.
  • the concentration of the cross-linking reagent mixtures used in this application may vary between 5 ⁇ M and 250 mM dissolved in a biologically compatible solvent such as, but not limited to, DMSO.
  • Photoactivation of the reagents can be achieved within a wave length range of 220 nanometers to 310 nanometers. The duration of the photoactivation will vary depending upon the cross-linker used.
  • Photoactivation depending on the wavelength and time required, may cause additional comeal epithelial trauma. It may, therefore, be preferable to utilize cross-linking agents, which do not require photoactivation.
  • a prefe ⁇ ed cross-linker of the invention is glycerine or glycerol. Glycerol is the simplest trihydric alcohol, with the formula CH 2 OHCHOHCH 2 OH.
  • glycerol is prefe ⁇ ed for the pure chemical, but the commercial product is usually called glycerin. It is widely distributed in nature in the form of its esters, called glycerides. The glycerides are the principal constituents of the class of natural products known as fats and oils. It is completely soluble in water and alcohol and has a very low toxicity in mammals. It has properties that allow it to stabilize lamellar stmctures without the need to photoactivate it. Because it requires no potentially damaging photoactivation, has very low toxicity, and is a common additive to food items, it provides the advantage of an effective, non-toxic, inexpensive cross- linking reagent that is very suitable for human use.
  • the anti-fibrotic and cross-linking dmgs and reagents can be instilled in several different ways but these agents must penetrate the physical barrier that the comea presents. Dmgs appear to penetrate the comea by diffusion and the rate of diffusion parallels the dmg concentration.
  • the comeal epithelium is the first ba ⁇ ier and dmgs must enter this layer rapidly or be washed away. Cell membrane lipids, which are present within the five layers of the epithelium, limit drug penetration.
  • the epithelium contains hydrophilic constituents, which also retard dmg penetration.
  • the dmgs For there to be clinical significance in the restoration of stromal lamellar stability by means of reagents or dmgs, the dmgs must be presented to the comea in a "bioavailable" manner.
  • the bioavailability of a drug product is defined as the percentage of the reagent that is absorbed.
  • the other important parameter to be considered is the pharmokinetics of the dmg in the patient's tissues. Many factors will alter each parameter and will result in the choice of method or means of administration of the reagent of choice.
  • the excipients or inactive compounds present in the dmg such as diluants, lubricants, binders and the like, which are important determinants of bioavailability. Variations in dissolution rate are also important.
  • the pH of the dmg solution determines whether it is the ionized or non-ionized form. The non-ionized form, being more lipid soluble, is better able to penetrate the comeal epithelium. Viscosity will affect the drug- comeal contact and, therefore, its bioavailability.
  • Surfactants or detergents may be used to increase the solubility of dmgs that are hydrophobic. Osmotics may be added to adjust the tonicity of the ophthalmic solutions to that of the tears.
  • Lacrimal volume will affect the concentration of the dmg, but reflex tearing can be minimized by the prior administration of a local anesthetic and dmg abso ⁇ tion can be increased by altering the comeal epitheliun. Eye drop size and/or punctal occlusion will result in greater reagent concentration being available.
  • reagents or dmgs are presented to the comea by one of three modes of administration: 1) Topical administration of solutions, suspensions, ointments, powders, particulates, and the like, by periodic or continuous irrigation (FIGS. 7a and 7b), and aerosol spraying (FIG. 7c), iontophoresis, or the like.
  • dmg reservoirs such as pledgets (usually, squares of pressed cotton), or sponges soaked with a drug (FIGS. 8b and 8c), soft contact lenses soaked in a reagent (FIG.8a), polymer dmg delivery systems, collagen shields soaked in the dmg
  • FIG. 8a liposomes, flexible capsules or wafers, or other membrane or reservoir systems (FIG. 8d), and
  • Solutions and aqueous suspensions are the pharmaceutical forms most widely used to administer dmgs that must be active on the eye surface or in the eye after passage through the comea or conjunctiva.
  • various dosage forms have been developed over the years.
  • soluble inserts undergoing gradual dissolution/or surface erosion
  • insoluble inserts e.g., medicated contact lenses such as OcusertTM, etc.
  • gels e.g., GelriteTM
  • liposomal and dmg delivery via nanoparticles emulsion, suspension, etc.
  • ointment See Edman, BIOPHARMACEUTICS OF OCULAR DRUG DELIVERY, CRC Press, 1993.
  • Suspensions require shaking well before administration to ensure the particles are well dispersed in the suspension solution.
  • FIG. 7b or continuous irrigation, such as by reagent reservoir 40 and irrigation tube 42 in FIG. 7a, may be useful.
  • periodic irrigation may be achieved by the use of eyedropper 44, having reagent reservoir 46, and pressure bulb 48, to deliver reagent to the eye 10.
  • periodic irrigation can be achieved by similar periodic drip systems, either manual or automated.
  • continuous irrigation may be achieved by use of reagent reservoir 40 which feeds reagent, either by gravity or controlled feeding, to irrigation tube 42, and thereby to the eye 10.
  • continuous irrigation can be achieved by similar continuous reagent supply systems, either manual or automated.
  • aerosols may be employed, such as by use of a reagent reservoir 50 and aerosol device 52, including reagent access tube 54, of FIG. 7c.
  • Periodic or continuous irrigation, or aerosol spraying should be used to i ⁇ igate, or to spray the eye, respectively, with reagent for about five minutes or until the reagent has sufficiently penetrated the comeal epithelium.
  • the reagent should be applied before the blink reflex.
  • means can be employed to inhibit the blink reflex and, thus, facilitate the delivery of reagent by any of these systems. Iontophoresis is largely of historical significance to aid the penetration of ionizable dmgs having limited lipid solubility.
  • This technique works by placing one electrode upon the comea usually imbedded within a scleral contact lens in the presence of a dmg solution.
  • the other electrode can be placed anywhere else on the body.
  • the electrode, the cu ⁇ ent, the topical anesthetic, the hypertonic solution, and the non-physiologic pH-aided dmg penetration may cause some comeal epithelial damage. If the epithelium is breached, the cu ⁇ ent alters the steady state of the stroma and, therefore, the Ts (thermal shrinking temperature), thus adding another variable that may pertubate the results.
  • a syringe 64 of FIG. 7 is used to penetrate the subconjunctiva, the "mucous membrane" of the eye which lines the inner surface of the eyelids and is reflected over the fore part of the sclera and the comea, and to deliver a reagent to the subconjunctival tissue.
  • Syringe 64 includes needle 66, typically a 25 gauge, reagent reservoir 68, and plunger 70 (shown in part) of FIG. 9.
  • the needle 66 is placed adjacent the conjunctiva, as schematically illustrated by the dashed line of FIG.
  • plunger 70 is depressed to deliver reagent from the reagent reservoir 68 to the subconjunctival tissue.
  • the injection is usually directed to the perilimbal portion of the subconjunctiva.
  • Pledgets 58 of FIG. 8b or sponges 60 of FIG. 8c are soaked (as indicated by a droplet) with a reagent and then placed inside one or both lids, adjacent the comea 11 of FIG 8a, for about five minutes or until the reagent sufficiently penetrates the comea 11.
  • Pledger 58 may be composed of a fibrous material, such as cotton or cellulose, as illustrated by the curved and overlapping lines in the interior of pledger 58 of FIG. 8b.
  • Sponge 60 may be composed of known sponge materials, such as cellulose, its sponge-like characteristics being illustrated by the void, and non-void spaces in the interior of the sponge 60 of FIG. 8c. While pledger 58 is often a cotton square, when placed adjacent the comea 1 1 in the same manner as described below with respect to the contact lens 56 of FIG. 8a, it substantially conforms to the shape of the comea 1 1. Similarly, sponge 60 substantially conforms to the shape of the comea 1 1 when it is placed adjacent the comea 1 1 in the same manner. For simplicity, pledget 58 and sponge 60 are shown in two dimensions, it being understood that pledger 58 and sponge 60 have substantially the three- dimension configuration of a contact lens 56 of FIG. 8a.
  • dmgs contained within a membrane device 62 of FIG. 8d and placed within the conjunctival sac produce a more even release of dmg than any topical system.
  • Membrane device 62 is left in the conjunctival sac for about five minutes or until the dmg or reagent sufficiently penetrates the comea 1 1.
  • These membrane devices 62 may take the form of flexible capsules, shaped as semi-circular wafers, which are placed in one or both eyelids (usually the lower eyelid), although other forms are contemplated. While the lipophilic or hydrophilic nature of the membrane, the pore size, and the membrane thickness may mitigate the rate of dmg release, such membrane system may have advantages over other modalities.
  • Liposomes which are synthetic phospholipid vesicles, absorb to the comeal epithelium cell membrane and transfer dmg directly. Liposomes are generally described in published international patent application under the Patent Cooperation Treaty, International Publication No. WO 86/03938, of inventors John H. Crowe and Lois M. Crowe (International Filing Date of Jan. 8, 1986), on pages 1 and 2. The entire disclosure of these pages is inco ⁇ orated herein by this reference. The primary limitations of liposomes are their limited binding power to the epithelium and their expense.
  • Hydrophilic soft contact lenses 56 of FIG. 8a have been used as dmg reservoirs with success.
  • Polymer dmg delivery systems while effective, are generally more rigid than soft contact lenses 56.
  • Particularly prefe ⁇ ed are cross-linked collagen shields 56, also shown of FIG. 8a, which are shaped like contact lenses 56 and are used to both promote comeal epithelial healing and to provide drug delivery. Because they conform to the shape of the comea 11, multiple base curves are not required. They are also biodegradable and thus provide protection to the comea during the post-laser recovery period after the dmg is dissipated and do not require removal. As schematically shown in FIG.
  • a pre-soaked (as indicated by a droplet) contact lens 56 or collagen shield 56 which is shaped to conform substantially to the comea 11 is placed (as indicated by the pair of parallel direction a ⁇ ows) in contact with the comea 11 of an eye 10 of a patient.
  • the patient's eyelids are closed over the shield for about five minutes or until the reagent sufficiently penetrates the comea 11.
  • the ideal system combines the administration of topical anesthetic, such as proparacaine, with the efficient administration of reagent by means of a collagen shield. Topical anesthetic is used to breach the epithelial barrier and to provide, simultaneously desensitization for the laser exposure.
  • Presenting topical anesthesia by this method provides protracted desensitization for comfort following the laser exposure and, by way of the collagen shield, protects the surface epithelium preventing denudation.
  • the reagent further provides the intermolecular stabilization necessary of the comeal stroma following the destabilizing laser treatment.
  • ophthalmic dmg administration devices and methods including mucoadhesive polymers, particulates, liposomes, ocular iontophoresis, ocular films, ocular inserts and comeal collagen shields
  • mucoadhesive polymers, particulates, liposomes, ocular iontophoresis, ocular films, ocular inserts and comeal collagen shields are described in A.K. Mitra, Ophthalmic Drug Delivery Systems, 58 Dmgs and the Pharmaceutical Sciences Series (February 1993). Fundamentals and applications of controlled dmg delivery systems are further provided in JR. Robinson and V.H.L. Lee, Controlled Drug Delivery, 29 Dmgs and the Pharmaceutical Sciences Series (2 nd Edition).
  • the comeal collagen shield is an ophthalmologic product available from such companies as Chiron and Bausch & Lomb.
  • the shield resembles a translucent contact lens and is fabricated from bovine or porcine collagen tissue, which resembles the collagen molecule of the human eye.
  • EXAMPLE 1 Biochemical method to maintain the structural integrity of the eye following photothermal keratoplasty surgery performed to correct ametropia.
  • Methods Culture human comeal epithelial collagen-producing cells and determine the optimal cross-linking agent dose; preferably without inducing necrosis or apoptosis.
  • the minimal cross-linking agent concentration, light exposure (visible and laser) and intensity required to activate the agents to induce cross-links will further be determined.
  • the growth protocol for human comeal equivalents i.e., containing the epithelium, stroma, and endothehum described in Science. 286:2169-272. 1999, herein inco ⁇ orated by reference, can be used.
  • Standard immunofluorescent methods to confirm riboflavin or other agent remains adherent to cells (e.g., primary anti-riboflavin antibodies + appropriate secondary)
  • Treat isolated, intact pig or rabbit eyes in accordance with the methods determined in aim 1 above.
  • the eyes are subsequently dissected and mounted for standard immunohistochemical analyses.
  • the basic tissue stmcture and cellular organization from control and treated eyes is compared and documented under light microscopy.
  • the cross-linking agent localization is then determined to ensure that collagen cross-links do not occur outside of the surgical site.
  • Comeal rigidity has been defined as the resistance offered by the envelope of the globe (comea) to a change in intra-ocular volume, this resistance being manifested as a change in intra-ocular pressure (IOP). It thus associates changes in volume with the pressure in the eye. It is related, in fact, to the distensibility of the globe and not to the usual connotation associated with "rigidity” or "inflexibility.”
  • the ocular rigidity is reduced following intra-ocular surgery. It is raised when the globe is immersed in cross-linking reagents, such as formaldehyde, and varies with the state of hydration and temperature. Alterations in ocular rigidity of enucleated eyes is observed when immersed in solutions of different osmolarities and rigidity is increased when there is a fall in water content; the converse has been observed when the tissues were hydrated.
  • the determination of ocular rigidity involves altering the volume of the ocular contents by a known amount and measuring the accompanying change in intra- ocular pressure (IOP).
  • IOP intra- ocular pressure
  • Tonometric methods of estimating the rigidity differ merely in that the change in volume consists of a displacement of fluid from one part of the eye to another when the indentation tonometer (Schiotz) is applied and the comea is indented.
  • "Differential tonometry” is one method whereby different degrees of indentation are made by applying different weights to the instmment. This tensile force varies with the elasticity of the comeo- sclera and has a component tangential to the walls of the globe; at the site of the comeal indentation it therefore acts outwards and upwards, tending to raises the indenting plunger integrated within the indentation tonometer.
  • a combination of applanation tonometry with indentation tonometry provides an improvement upon differential tonometry.
  • the principle of the method is the same as for differential tonometry, but the Goldmann applanation tonometer displaces a negligible amount of fluid so that the initial value is independent of ocular rigidity. This is the best available method for measuring the rigidity of the living human eye using readily available devices.
  • remedial cross-linking reagents should then be instilled by the method of choice.
  • the tonometric measurement protocol should then be repeated in 24 hours thus allowing remedial cross-linking to take place.
  • Increased comeal rigidity (or the reciprocal finding of decreased comeal distensibility) determined by means of differential tonometry will be observed by reduced indentibility of the lower weighted tonometric instmment when compared with the greater weight as determined by the scale units and coincident estimated IOP (FIG. 10).
  • This combination method merely substitutes applanation tonometric readings for that of the greater weighted indentation tonometer. A similar comparison is made.
  • step (1) the indentation tonometer displaces a normal volume of intra-ocular fluid in steady-state.
  • step (2) LTK damages rigidity-causing collagenous cross-linking (especially the heat-labile H+ bonds) allowing greater indentation of tonometer plunger, and in step (3), chemical-induced remedial collagenous cross-linking yields normal tonometer plunger indentation confirming re-establishment of pre-laser rigidity.
  • H & E staining shows marked swelling of the stroma that resolves by 4 hours with less swelling in the stromal matrix as a function of time.
  • the epithelium has sloughed and is replaced by proliferating adjacent epithelium.
  • the stroma is devoid of cells with PMN's invading through the epithelium into the stroma.
  • HSP70 antibody staining showed a definite staining pattern that was maximal at 4 hours and then gone by 48 hours adjacent to the laser treated areas. This provides a good marker for early changes.
  • Standard H & E staining and TUNEL stains can be used to distinguish between necrosis and apoptosis. This is desirable in order to determine the specific inhibitory effects of various interventions in the comeal traumatic model.
  • Heplock catheters were placed in the ear vein, and approximately lcc of ketamine hydrochlonide (10 mg/kg) and xylazine (3 mg/kg) diluted 1 :3 with sterile salt solution was injected IV. A sterile drop of Ophthaine anesthetic was placed in the eye.
  • Corneas were removed, rinsed in BSS and cut into 4 pieces (see below). 8) One central piece was placed into OCT and frozen in liquid nitrogen.
  • Tissue is processed using TUNEL assay and immunochemistry with antibodies to specific proteins involved in the apoptosis pathway.
  • Other tissue is prepared for TEM.

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Abstract

L'invention concerne des procédés destinés à prévenir ou empêcher la régression de la cornée provoquée par le remodelage du stroma de la cornée consécutif à l'apoptose ou à la nécrose des cellules épithéliales ou kératocytaires cornéennes, chez des patients subissant ou ayant subi une kératoplastie de réfraction. L'invention concerne encore des procédés et compositions qui empêchent l'apoptose des cellules épithéliales et consistent à refroidir la cornée pendant la chirurgie de réfraction thermique, à créer ou restaurer des liaisons croisées entre les fibres lamellaires du stroma, et à interrompre au moins une étape de la réponse au remodelage du stroma, notamment à inhiber l'apoptose, la prolifération et la migration des kératocytes, et à inhiber la collagenèse. L'invention concerne encore des procédés additionnels, lesquels consistent à mesurer la rigidité cornéenne afin d'évaluer la perte de rigidité consécutive à la chirurgie de réfraction ou à restaurer cette rigidité à la suite d'une intervention thérapeutique visant à restaurer les liaisons croisées lamellaires.
PCT/US2000/013600 1999-06-04 2000-05-15 Prevention de la regression dans la keratoplastie de refraction Ceased WO2000074648A2 (fr)

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US15277399P 1999-09-03 1999-09-03
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US15358999P 1999-09-13 1999-09-13
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US8348935B2 (en) 2008-01-23 2013-01-08 Avedro, Inc. System and method for reshaping an eye feature
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US8366689B2 (en) 2008-09-30 2013-02-05 Avedro, Inc. Method for making structural changes in corneal fibrils
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