WO1994005322A1 - Agent reducteur de l'activite des plasmines pour le traitement d'une plaie resultant de l'ablation superficielle de la cornee et procede de depistage associe - Google Patents

Agent reducteur de l'activite des plasmines pour le traitement d'une plaie resultant de l'ablation superficielle de la cornee et procede de depistage associe Download PDF

Info

Publication number
WO1994005322A1
WO1994005322A1 PCT/US1993/008334 US9308334W WO9405322A1 WO 1994005322 A1 WO1994005322 A1 WO 1994005322A1 US 9308334 W US9308334 W US 9308334W WO 9405322 A1 WO9405322 A1 WO 9405322A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmin
agent
wound
ablation
regression
Prior art date
Application number
PCT/US1993/008334
Other languages
English (en)
Inventor
Chris P. Lohmann
John Marshall
Original Assignee
Summit Technology, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB9218731A external-priority patent/GB2271507A/en
Application filed by Summit Technology, Inc. filed Critical Summit Technology, Inc.
Priority to AU48470/93A priority Critical patent/AU4847093A/en
Publication of WO1994005322A1 publication Critical patent/WO1994005322A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans

Definitions

  • This invention relates to a plasmin activity reducing agent useful for reducing or preventing visual refractive regression following a wide area superficial ablation wound to the cornea. Such a wound may be created, e.g., by a surgical procedure.
  • the invention also relates to screening and monitoring methods.
  • the healing of wounds involves complex interactions between two major systems controlling the degradation and removal of damaged tissue and the synthesis of cellular and extracellular elements.
  • these systems are targeted on the degradation and synthesis of collagen and the glycosaminoglycan ("GAG") matrix.
  • the first system is the plasminogen-activator/plasmin system, which is involved in the proteolysis, e.g., degradation and removal, of damaged extracellular materials by plasmin. Through this proteolytic activity, plasmin also modulates the growth of normal tissue and tissue damaged by trauma, infection, or inflammation.
  • the second system is the activated keratocyte system, which is involved in the replacement of damaged collagen by the synthesis of new collagen and the collagen matrix of GAGs.
  • the first healing phase involves the removal of cell debris and damaged extracellular materials, e.g., the GAGs which form a matrix that supports the collagen of the cornea, by the proteolytic activity of the plasminogen-activator/plasmin system.
  • the second healing phase involves the repair of partially damaged tissues.
  • the third phase involves the replacement of severely damaged extracellular materials by newly synthesized GAGs and collagen, which is regulated by the activated keratocyte system, and the replacement of severely damaged cells by new epithelial cells which migrate to the wound site on a temporary fibrin/ fibronectin matrix.
  • This cell proliferation is enhanced by growth factors, e.g., epidermal growth factor. Plasmin regulates re- epithelialization in this phase.
  • Proteolytically active plasmin is a serine protease derived from plasminogen, which is found in nearly all body fluids and tissues.
  • the precursor plasminogen is activated by plasminogen-activators to become plasmin.
  • Plasmin degrades many matrix proteins, such as GAGs, fibronectin, and laminin, and can activate other enzymes such as pro-collagenase and macrophage elastase.
  • Fibronectin and laminin are important proteins in the extracellular matrix and facilitate cell healing by connecting the epithelium to the underlying tissue.
  • fibronectin is an adhesive cell-surface glycoprotein which mediates cross-linking of collagen and epithelial cell migration, and laminin promotes cell adhesion.
  • Plasminogen-activators are classified in two main groups: tissue type plasminogen-activator ("tPA”) , and urokinase type plasminogen-activator (“uPA”) .
  • tPA tissue type plasminogen-activator
  • uPA urokinase type plasminogen-activator
  • the concentration of plasmin in the tear fluid of healthy, unwounded eyes is very low or zero, typically below 0.2 ⁇ g/ml of tear fluid.
  • higher concentrations of plasmin e.g., up to 100 ⁇ g/ml, are associated with various ocular surface wounds, disorders, or irritations.
  • mechanical wounding of the cornea e.g., by keratectomy, or exposure to allergens, induces an increase in plasmin and a short-term decrease in plasminogen-activator in the tears.
  • Increased plasmin levels are also associated with " icrobial ulcers of the cornea, corneal epithelial defects caused by contact lens wear or vernal keratoconjunctivitis, recent mechanically produced wounds, and lesions caused by corrosive chemicals.”
  • bacterial ulcers of the cornea corneal epithelial defects caused by contact lens wear or vernal keratoconjunctivitis, recent mechanically produced wounds, and lesions caused by corrosive chemicals.
  • plasminogen-activators are normal components of tear fluid. However, like plasmin, higher activities of these plasminogen-activators also occur as wound healing progresses.
  • the normal level of plasminogen activator is about 2.0 ⁇ 0.6 IU/ml, the level immediately after wounding is about 0.3 ⁇ 0.1 IU/ml, and the level during subsequent healing is about 2.1 ⁇ 0.3 IU/ml.
  • the normal activation of the plasminogen- activator/plasmin system in corneal wound healing may therefore be evidenced by plasmin and plasminogen- activator levels in tear fluid. Van Setten, et al., Current Eve Res. , 8.:1293-1298 (1989).
  • Tervo et al. describe treating patients having corneal defects with topical aprotinin, a serine protease, to inhibit plasmin, to protect fibrinogen and laminin. In the context of such defects, treatment with aprotinin results in enhanced corneal resurfacing and healing (at p. 155-157) .
  • Tervo et al. used aprotinin to treat non-healing corneal ulcers and other persistent epithelial lesions. The authors state that "aprotinin therapy is a valuable adjunct in the treatment of epithelial defects, and that monitoring of plasmin levels in the tears provides useful information on the state of corneal healing and selection and timing of medical therapy in these eyes" (at p. 151) .
  • the process of corneal wound healing is of critical importance in all ophthalmic methods of correcting myopia, astigmatism, etc. by surgical procedures.
  • the cornea is cut or ablated, i.e., wounded, to alter or smooth the surface curvature of the cornea and thereby change, and hopefully improve, the visual acuity of the patient. If the wound created by the surgery heals with extensive tissue regrowth or scarring that cause refractive errors, the patient will experience a regression or decrease in refractive power of the cornea, and the improvement gained by the surgery will be diminished or lost.
  • a limited thickness of the cornea is ablated by exposure to laser irradiation, e.g., excimer laser irradiation.
  • laser irradiation e.g., excimer laser irradiation.
  • the laser is used to ablate very thin layers from the remaining corneal tissue to a depth of about 5 to 50 ⁇ m, through Bowman's membrane (thickness about 10 ⁇ m) , and into the stroma (thickness about 500 ⁇ m) , depending on the attempted correction.
  • Re- epithelization which is part of the wound healing process, is normally completed within 72 hours of the surgery.
  • PRK often results in a temporary loss of corneal transparency, often described as corneal scarring or "haze" that typically lasts from three months to a year after surgery.
  • the worst aspect of haze occurs in the first 3 months and is due to activated keratocyte cells that migrate into the stromal collagen to effect repair. After this repair phase, these cells migrate out of the cornea and the scatter or haze due to these cells disappears.
  • a secondary aspect of haze is caused by new collagen and vacuoles between intersected lamellae in the stroma. These vacuoles are filled with cell debris and cause a high level of light scatter, which changes the refractive indices of the cornea and impairs its transparency.
  • the corneal wound created by excimer laser irradiation leaves only a minute amount of damaged tissue at the wound edge.
  • the tissue immediately adjacent the ablated tissue is largely unaffected by the excimer laser ablation.
  • the minute amount of damaged or dead cells a layer on the order of only a few Angstroms thick, is enough to release the factors that trigger wound healing and so-called "wound amplification," i.e., an enlargement of the wound area into those tissues adjacent the wound, which would normally be damaged in a conventional wound.
  • wound healing response is much greater than required by the amount of damaged tissue that exists at the wound site.
  • the term "wide area superficial ablation wound” itieans a wound which is very shallow, on the order of 5 to 150 ⁇ m deep, is associated with minimal or no damage of the tissue immediately adjacent to the ablated tissue, i.e., at the wound edge, and exhibits no inflammation of the adjacent tissue.
  • a wound may be created, e.g, by excimer laser ablation, or by mechanical or chemical ablation.
  • visual refractive regression means any detrimental change in refractive power of the cornea that results from the natural healing process which occurs after a wide area superficial ablation wound is made to the cornea. Such wounds are typically made to change the refractive power and thereby enhance eyesight, but are hampered by the problem of regression.
  • epithelial hyperplasia which is one aspect of regression, results from plasmin's role in the regulation of the growth of normal tissue and re-epithelization, and have found that inhibiting plasmin activity inhibits epithelial hyperplasia. Furthermore, applicants have found that by inhibiting plasmin activity, and thereby impeding the removal of healthy collagen remaining in the cornea, the replacement of the ablated collagen (Type IV and Type V) with new atypical Type III and VII collagen, which is another aspect of regression, is also inhibited. Ideally, no Type III or VII collagen should be added to the eye to "heal" the wide area superficial ablation wound.
  • a tear fluid plasmin level greater than 0.2 ⁇ g/ml indicates the likelihood of postoperative visual regression in a prospective patient for any surgical procedure which creates a wide area superficial ablation wound.
  • high pre-operative plasmin levels are a contraindication for a surgical procedure which creates a wide area superficial ablation wound, e.g., excimer laser PRK.
  • high levels of plasminogen and plasminogen-activator are also an indication of potential postoperative visual regression.
  • proteolytic agent means plasmin, plasminogen, and/or plasminogen-activator ("PA”) .
  • the invention also features a method of monitoring tear fluid from a patient for proteolytic agents after a wide area superficial ablation wound has been created on the anterior surface of the cornea, for the risk of post- wound visual refractive regression, by collecting a sample of tear fluid from the patient after the ablation wound has been created, and determining the level of a proteolytic agent in the sample as an indicator of potential post-wound visual regression.
  • the ablation wound may be the result of a photo-, mechanical, or chemical ablation procedure, e.g., photorefractive keratectomy.
  • the tested proteolytic agent is plasmin
  • a level of plasmin greater than 0.2 ⁇ g/ml of tear fluid is an indictor of potential post-operative visual regression.
  • the tested proteolytic agent is plasminogen-activator
  • a level of plasminogen-activator greater than about 1.5 IU/ml of tear fluid is an indictor of potential post ⁇ operative visual regression.
  • the method also includes testing the level of plasminogen in the tear fluid.
  • Applicants have also developed a pharmaceutical treatment for reducing, in an eye of a patient, visual refractive regression associated with a wide area superficial ablation wound of the anterior corneal surface, by administering to the eye a plasmin activity reducing agent.
  • plasmin activity reducing agent means any inhibitor of plasmin, plasminogen, or PA.
  • the term includes all such "inhibitors” regardless of whether they degrade, inactivate, disrupt the synthesis of, or otherwise render ineffective, plasmin, plasminogen, or PA, as long as they ultimately reduce or eliminate plasmin activity.
  • a plasiin activity reducing agent may include any combination of plasmin, plasminogen, and/or PA inhibitors and any other drug that may have a therapeutic effect on the eye.
  • the plasmin activity reducing agent may include a steroid or other drug to inhibit inflammation or infection.
  • the plasmin activity reducing agent may be a plasmin inhibitor, e.g., aprotinin, or a plasminogen inhibitor or plasminogen-activator inhibitor, e.g., plasminogen-activator inhibitor-2.
  • the plasmin activity reducing agent may be administered topically to the cornea prior to, during, or after the occurrence of the ablation wound, or may be administered in a series of administrations after a wide area superficial ablation wound has been created.
  • the superficial ablation wound may be the result of a photo-, mechanical, or chemical ablation procedure, e.g., photorefractive keratectomy.
  • the ablation wound may be the result of a procedure to change the curvature of the cornea a desired degree to produce a refractive correction, or a procedure to produce corneal smoothing without substantial change to the refractive properties of the eye.
  • the pharmaceutical treatment may also include administering to the eye an anti-inflammatory drug, e.g., a steroid, in combination with the plasmin activity reducing agent.
  • an anti-inflammatory drug e.g., a steroid
  • the invention also features a plasmin activity reducing agent useful for the pharmaceutical treatment described herein, and a plasmin activity reducing agent for use in the manufacture of a medicament for use in this pharmaceutical treatment.
  • the treatment may involve the administration of the plasmin activity reducing agent to the surface of the cornea prior to, during, or after, the occurrence of the ablation wound, and this ablation wound may be the result of a photo-, mechanical, or chemical ablation procedure.
  • Fig. 1 is a graph showing the average plasmin level in tear fluid of patients before and after a wide area superficial corneal ablation procedure.
  • Fig. 2 is a graph showing the average pre- operative plasmin level in patients compared to the refractive change three months after surgery.
  • Fig. 3 is a graph showing mean change in refraction in patients over time after surgery.
  • Fig. 4 is a graph showing mean change in refraction in steroid (solid line) and placebo (dotted line) treated patients over time after a -3.0 diopter correction.
  • Fig. 5 is a graph showing mean change in refraction in steroid (solid line) and placebo (dotted line) treated patients over time after a -6.0 diopter correction.
  • the amount of visual regression that a patient develops varies greatly between individual patients and the amount of ablation or attempted correction, and the severity of regression typically increases with the degree of correction attempted. For example, a -6.0 diopter correction typically results in little regression, whereas any correction above -7.0 diopters causes at least a 0.5 diopter regression. Certain patients are also more susceptible to regression even with lower order corrections or removal of very thin layers of corneal tissue.
  • the screening test of the invention i.e., pre- operatively measuring tear fluid levels of plasmin, plasminogen, and/or plasminogen-activator, is used to determine whether a patient is susceptible to regression, especially in those cases in which the attempted correction is over -6.0 diopters. Based on the test results, the patient is counseled whether to attempt the surgery at all, or whether to use the pharmaceutical treatment for reducing visual regression before, during, and/or after the surgical procedure to alleviate the regression.
  • Tear fluid samples are collected from one eye of each prospective patient.
  • a sample of 10 ⁇ l of tear fluid may be collected by capillary action using either two blunted 5 ⁇ l glass microcapillary tubes or a single 10 ⁇ l tube (Brand, FRG) .
  • the glass tube is held at 10 to 30 degrees over the horizontal axis and at 10 to 40 degrees to the surface of the lower fornix.
  • the tip of the tube is brought into contact with the tear fluid meniscus and then slowly moved on the inferior fornix from the middle towards the lateral canthus. All tear fluid samples are immediately frozen on dry ice and stored at -70°C until the plasmin concentration can be determined.
  • Plasmin, plasminogen, and plasminogen-activator ("PA") levels in tear fluid of potential candidates for a wide area ablation wound to the cornea may be measured pre-operatively, or monitored postoperatively, by various techniques.
  • the plasmin and PA levels in tear fluid may be measured as follows (p. 4) :
  • Tear fluid was collected into a glass capillary.
  • Proteolytic activity using an 8 ⁇ l specimen of tear fluid, was measured by the radial caseinolysis procedure (Saksela, Anal. Bioche . , 111:276-282 (1981)), using agarose gel and bovine milk casein as substrate.
  • Human plasmin (20 casein units per mg; Kabi Diagnostica, Sweden) was used as standard. The results are expressed as micrograms of plasmin-like activity per ml tear fluid.
  • the advantages of this assay include, the small sample volume needed (minimum 5 ⁇ l) , small intra-assay variation ( ⁇ 5%) and sensitivity (0.1 ⁇ g plasmin per ml).
  • Plasminogen activator levels were determined according to Saksela (1981) using plasminogen- containing casein-agarose gels and urokinase (50,000 Ploug units mg; Calbiochem) as standard. Rabbit antibodies to human plasminogen and albumin (DAKO, Copenhagen) were used in the identification.
  • plasmin levels are proportional to the radial displacement of degraded casein which is determined by staining the gel with Amido black (1 mg/ml in 2% acetic acid) . Concentrations are determined by direct comparison with the plasmin standards. The proteolytic activity is then confirmed to be human plasmin in a further series of samples in which the gels are reacted with monoclonal human plasmin antibodies (anti-pig 1) .
  • Tear fluid plasmin levels can also be measured by various standard immunofluorescence techniques that can easily be adapted to detect plasmin in tear fluid.
  • plasmin, plasminogen, and/or PA levels are above certain threshold levels, e.g., above 0.2 ⁇ g/ml for plasmin, and above 1.5 IU/ml for plasminogen-activator, the candidate is informed of an increased risk of postoperative visual regression. If the patient chooses to undergo the surgery anyway, a plasmin activity reducing agent is applied to the eye before, during, and/or after the wide area superficial ablation procedure.
  • the ablation procedure may be performed without the ancillary pharmaceutical treatment, but the patient's tear fluid levels are carefully monitored for proteolytic agents during the postoperative healing process, and the plasmin activity reducing agent is applied at the first increase of plasmin, plasminogen, or PA in the tear fluid.
  • Tear fluid samples were collected pre-operatively from one eye each of 21 patients. Samples were taken immediately befoire the PRK procedure, and then 15 minutes and 1 week after the ablation wound was created by the procedure. Relatively high pre-operative levels of plasmin were detected in only 3 of the 21 patients. The mean value for the pre-wound plasmin level of the group, including all the zero or negligible values and the three elevated values, was 4.3 ⁇ g/ml. Fig. 1 shows that by fifteen minutes excimer laser ablation, the average plasmin level was almost 60 ⁇ g/ml. Plasmin was found in the tear fluids of all 21 patients.
  • Post-wound i.e., postoperative
  • values were higher than pre-wound values in every patient, and gave a mean value of 38.2 ⁇ g/ml.
  • plasmin levels decreased significantly in the tear fluid samples with a mean value of 8.3 ⁇ g/ml at one week postoperatively.
  • the wide area superficial ablation wound described above may be created with a laser, e.g., an excimer laser, or by various mechanical or chemical techniques.
  • Excimer laser PRK may be carried out with a Summit
  • This laser can be adjusted to have a fixed pulse repetition rate of 10 Hz and a fixed radiant exposure of 180 mJ/cm 2 at the cornea.
  • the maximal ablation diameter is preferably adjusted to about 4.0 to
  • lasers and wavelengths may also be used to create a wide area superficial ablation wound of the cornea.
  • very high repetition rate, short pulsed, infrared lasers should also create wide area superficial ablation wounds of the cornea.
  • a near-infrared wavelength of about 2.9 microns is the maximum wavelength that can be absorbed by water and is suitable to create a wide area superficial ablation wound.
  • Optimal wavelengths for lasers are 2.7 to 2.9 microns in the infrared region and 193 nanometers in the vacuum ultraviolet.
  • Mechanical ablation may be carried out by freezing the cornea and removing, e.g., grinding away, very thin layers of tissue with a high speed rotating burr.
  • mechanical ablation can be achieved by rotating or oscillating a sharpened knife edge tangentially to the top of the cornea to scrape away very thin layers of corneal tissue as described in Kilmer et al., U.S. Patent No. 5,063,942, which is incorporated herein by reference.
  • Chemical ablation to create a wide area superficial ablation wound of the cornea may be carried out by applying an alkaline substance to the anterior surface of the cornea in a carefully controlled manner to limit damage to tissue adjacent to the ablated tissue to a few nanometers.
  • Chemicals such as ammonium, sodium hydroxide, and calcium hydroxide may be used to degrade the corneal tissue, and may be applied to the cornea by standard techniques.
  • Patients who undergo a surgical procedure that creates a wide area superficial ablation wound of the cornea may experience subsequent visual regression, depending on their individual reaction to the procedure and the attempted level of correction.
  • these patients may be treated with a plasmin activity reducing agent that inhibits plasmin activity in the cornea.
  • This treatment is carried out independently of the ablation procedure and may be performed before, during, or after the ablation, depending on the individual patients' needs. These needs may be determined, for example, by the screening test described above.
  • the purpose of the plasmin activity reducing agents of the invention is to decrease or eliminate plasmin activity in the cornea. This can be achieved by degrading the plasmin, plasminogen, and/or PA, or by rendering them inactive or ineffective.
  • Suitable plasmin inhibitors include aprotinin (20,000 IU/ml Trasylol®, Bayer) , ⁇ -antitrypsin, ⁇ 2 -antiplasmin, ⁇ 2 -macroglobulin, and monoclonal antibodies to plasmin.
  • Aprotinin is a polypeptide derived from animal organs and acts as a serine proteinase, and is the presently preferred inhibitor for use in the present invention.
  • Alpha 2 -antiplasmin operates by binding to the lysine active site which controls the interaction of plasmin with fibrin, thereby inactivating the plasmin.
  • Plasminogen-activator inhibitors include PAI-1, which is derived from endothelial cells, and PAI- 2, which is derived from placental cells. Monoclonal antibodies that bind to PA, or to plasminogen, are also suitable for carrying out the treatment of the invention if they bind in such a way as to prevent any activation of the plasminogen by the PA.
  • PAI-2 is the preferred PAI for use in the present invention.
  • the plasmin activity reducing agent may include any combination of plasmin inhibitors and/or PA inhibitors, and may be combined with any other drug that has a therapeutic effect on the eye.
  • the plasmin activity reducing agent may be combined with an antibiotic, e.g., chloramphenicol, a non-steroid anti- inflammatory drug, e.g., flurbiprofen, or an anti- inflammatory steroid, e.g., dexamethasone, prednisolone, fluorometholone, or others as described, e.g., in Robertson et al., U.S. Patent No. 4,939,135, which is incorporated herein by reference.
  • an antibiotic e.g., chloramphenicol
  • a non-steroid anti- inflammatory drug e.g., flurbiprofen
  • an anti- inflammatory steroid e.g., dexamethasone, prednisolone, fluorometholone
  • Steroids inhibit the keratocyte invasion of the collagen in the stroma which causes inflammation.
  • the combined use of steroids and aprotinin, or other plasmin inhibitors should create a synergistic effect that substantially prevents the replacement of the ablated collagen by Types III and VII atypical collagen.
  • the collagen replacement aspect of the third phase of wound healing should be substantially limited so as to avoid visual regression.
  • the plasmin activity reducing agent e.g., aprotinin (Bayer, Leverkusen, FRG)
  • aprotinin (Bayer, Leverkusen, FRG)
  • Aprotinin is diluted from stock preparations in sterile saline or commercially obtained wetting agent (e.g., Liquifilm Tears; Allergan) to achieve a concentration of 20 or 40 IU/ml, and is administered in 1-2 drops (50 ⁇ l each) per eye, 4 times per day during waking hours for the first two weeks after surgery.
  • any combination of plasmin, plasminogen, and/or PA inhibitors, and other therapeutic agents may be used in, or in combination with, the plasmin activity reducing agent of this invention. If the plasmin activity reducing agent is applied before the ablation wound is created, it should be applied in the same concentrations as described above, and the single application, or last in a series of applications, should be no more than a few hours prior to the surgery, so that the plasmin activity is effectively inhibited at the time the wound is created.
  • the aprotinin treated patients showed an initial small overcorrection (of about +1.0 diopters) at one week, followed by rapid stabilization at the desired refractive correction by 6 to 12 weeks.
  • This initial overcorrection i.e., hyperopic shift
  • This initial hyperopic shift is thought to be caused by edema of the cornea due to disturbed water relationships arising after ablation.
  • This initial hyperopic shift is common to all PRK patients, but was significantly smaller in patients treated with aprotinin.
  • the plasmin inhibitor aprotinin helps reduce this edema because less correal tissue is removed.
  • Untreated patients typically show a marked overcorrection (e.g., +2.0 to +3.0 diopters) followed by 3 to 5 months of regression to a stable, typically myopic correction.
  • PRK patients were also measured objectively for the time course and magnitude of anterior stromal haze for both reflected and scattered light using a digital video system which includes a Charge Coupled Device (CCD)-camera fixed to a slit-lamp microscope and connected via a frame grabber to a computer.
  • CCD Charge Coupled Device
  • This device measures haze by measuring corneal light scattering and can discriminate between reflected and scattered light.
  • the device and its method of operation are described in Lohmann et al., Refr. Corneal Surg.. :114-121 (March/April 1992) , which is incorporated herein by reference.
  • the aprotinin treated group of patients exhibited significantly less haze and a reduction in the period of time that vision was degraded by scattered light compared to the control group of patients.
  • Two groups of patients were treated with PRK to receive a correction of -3.0 (57 patients) and -6.0 (56 patients) diopters, respectively.
  • some patients were treated only with a steroid, dexamethasone (no plasmin inhibitor) , at a dosage of 1 drop 5 times per day for the first two months. This was followed by a stepwise reduction in the number of applications such that no medication was given after the end of the third month, i.e., 4 times per day for the next two weeks, 3 times per day for the next week, and 2 times per day for the last week.
  • the remaining patients received the same treatment regimen, but with a placebo containing neither steroids nor aprotinin.
  • the patients receiving the - 3.0 diopter correction showed a hyperopic shift in the first two weeks after surgery of about 2.0 diopters greater than the desired correction in the steroid treated group (solid line), and about 1.0 diopter greater in the placebo treated group (dotted line) . Both groups then regressed through the first three months after surgery.
  • the steroid treated group stabilized at about 1.0 diopter below the desired correction at three months and improved only very slightly by six months.
  • the placebo treated group regressed to a point more than 1.0 diopter below the desired correction at three months, but then improved gradually to almost the same level as the steroid treated group, i.e., almost 1.0 diopter below the desired correction.
  • the patients receiving the - 6.0 diopter correction showed a hyperopic shift in the first two weeks after surgery of more than 3.0 diopters greater than the desired correction in the steroid treated group (solid line), and about 2.0 diopters greater in the placebo treated group (dotted line) . Both groups then regressed through the first three months after surgery.
  • the steroid treated group regressed to about 2.0 diopters below the desired correction at three months and regressed slightly more by six months.
  • the placebo treated group regressed to a point more than 3.0 diopters below the desired correction at three months, but then improved gradually to about 3.0 diopters below the desired correction.
  • Two of the rabbits were used in the histological study described below to determine the presence and distribution of plasminogen-activator in the wounded area.
  • One was treated postoperatively with a steroid solution (no aprotinin) and the other was treated postoperatively with aprotinin for four days using the dosage regimens described below. These two animals were sacrificed four days after surgery. In the remaining six animals, three different postoperative regimens were employed with two rabbits in each set. All six animals had a basic postoperative regimen consisting of topically applied antibiotics (chloramphenicol) twice a day over one week.
  • the first set was a control which had no further medication.
  • aprotinin 40 IU/ml was administrated topically 1 drop 4 times a day over a two week period.
  • a similar regimen was employed for aprotinin, but was supplemented by topically applied steroids, e.g., prednisolone acetate (Allergan), and a non-steroid anti-inflammatory, e.g., flurbiprofen (Allergan) at a dosage of 1 drop 12 times per day for the first week, followed by 5 times per day for the first two months. This was followed by a stepwise reduction in the number of applications such that no medication was given after the end of the third month. After four months of observations, the rabbits treated with aprotinin exhibited limited clinical signs of haze.
  • Both eyes of two rabbits were enucleated immediately after death and the corneas were removed and immediately fixed at 4°C in 96% ethanol for 1 to 2 hours, rehydrated, and immersed overnight in 0.1 M phosphate- buffered saline (PBS), pH 7.4, containing 25% w/v sucrose.
  • PBS phosphate- buffered saline
  • the corneas were hemisected with a razor blade quench frozen by rapid immersion in partially frozen isopentane precooled in liquid nitrogen.
  • the frozen samples were mounted on a microtome chuck before being sectioned in a Reichart Frigocut. Sections of about 10 ⁇ to 15 microns in thickness were mounted on gelatinized slides and dried for at least 30 minutes and stored in a cryostat.
  • uPA urokinase type PA
  • uPA-PAI-1 and -2 complexes Polyclonal goat anti-human urokinase (uPA-PAI-1 and -2 complexes, Cambio, UK) served as the primary antibodies and were incubated on the samples overnight at 4°C.
  • Fluorescein isothiocyanate- conjugated rabbit anti-goat IgG (Sigma, UK) served as the secondary (labelled) antibody and was applied to the sample and incubated for 1/2 to 1 hour at room temperature. Control sections omitted either the primary or secondary antibodies. Samples were viewed on a Leitz fluorescence microscope under UV illumination.
  • the enucleated eyes were immediately immersed in a fixative solution of 2.5% glutaraldehyde buffered in 0.1M sodium cacodylate containing 10 mg/ml calcium chloride with a final pH of 7.4. After 5 minutes in this solution, the corneas were isolated by a circumferential incision and replaced in the initial fixative solution for a period of one hour.
  • the corneal specimens were hemisected through the area of ablation and were processed for light (LM) and transmission electron microscopy (TEM) . These specimens were post-fixed for two hours in 2.0% osmium tetroxide buffered in 0.2 M sodium cacodylate before being dehydrated through an ascending series of alcohol concentrations and embedded in Araldite via epoxy-propane.
  • Sections for LM were cut at 1 ⁇ m with glass knives using an ultramicrotome (Reichart, FRG) and stained with toluidine blue.
  • Sections for TEM were cut ultrathin with diamond knives on a Reichart ultramicrotome and stained with lead citrate and uranyl acetate before examination in an AEI 801 transmission electron microscope.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Agent réducteur de l'activité des plasmines utilisable pour réduire la régression réfringente visuelle de l'÷il suite à une plaie résultant de l'ablation superficielle d'une zone étendue de la surface cornéenne antérieure, par administration locale à l'÷il dudit agent réducteur de l'activité des plasmines, par exemple, un inhibiteur de plasmine tel que l'aprotinine. Une telle régression réfringente se déclare souvent chez des malades soumis à des opérations chirurgicales ayant pour but d'améliorer l'acuité visuelle chez le patient et provoquant une telle plaie. L'invention se rapporte également à un procédé d'essai préopératoire d'un échantillon lacrymal d'un patient potentiel, afin d'établir le risque de régression visuelle postopératoire, résultant d'une opération qui provoque une plaie d'ablation superficielle présentant une zone étendue sur la cornée, par détermination du niveau de l'agent protéolytique dans l'échantillon comme indicateur de la régression visuelle postopératoire potentielle.
PCT/US1993/008334 1992-09-04 1993-09-03 Agent reducteur de l'activite des plasmines pour le traitement d'une plaie resultant de l'ablation superficielle de la cornee et procede de depistage associe WO1994005322A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU48470/93A AU4847093A (en) 1992-09-04 1993-09-03 A plasmin activity reducing agent for treating a superficial corneal ablation wound and related screening method

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9218731.9 1992-09-04
GB9218731A GB2271507A (en) 1992-09-04 1992-09-04 Compositions containing plasmin activity inhibitors
US94296792A 1992-09-10 1992-09-10
US07/942,967 1992-09-10

Publications (1)

Publication Number Publication Date
WO1994005322A1 true WO1994005322A1 (fr) 1994-03-17

Family

ID=26301544

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/008334 WO1994005322A1 (fr) 1992-09-04 1993-09-03 Agent reducteur de l'activite des plasmines pour le traitement d'une plaie resultant de l'ablation superficielle de la cornee et procede de depistage associe

Country Status (2)

Country Link
AU (1) AU4847093A (fr)
WO (1) WO1994005322A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0795333A2 (fr) * 1996-03-15 1997-09-17 Senju Pharmaceutical Co., Ltd. Substance régulatrice du TGF-bêta pour le traitement de la myopie ou l'hypermétropie
WO1998029069A2 (fr) * 1997-01-02 1998-07-09 Allergan Sales, Inc. Procede de modification de la refringence d'un oeil
WO1999049887A1 (fr) * 1998-04-01 1999-10-07 Biotech Australia Pty. Limited Utilisation d'inhibiteurs de la protease pour traiter les blessures cutanees
AU2003213546B2 (en) * 1998-04-01 2006-06-29 Pai-2 Pty Limited Use of protease inhibitors for treating skin wounds
US10377818B2 (en) 2015-01-30 2019-08-13 The Board Of Trustees Of The Leland Stanford Junior University Method of treating glioma

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4939135A (en) * 1988-10-03 1990-07-03 Alcon Laboratories, Inc. Pharmaceutical compositions and methods of treatment to prevent and treat corneal scar formation produced by laser irradiation
WO1991007983A1 (fr) * 1989-11-27 1991-06-13 Huhtamäki Oy Protection de structures intra-oculaires

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4939135A (en) * 1988-10-03 1990-07-03 Alcon Laboratories, Inc. Pharmaceutical compositions and methods of treatment to prevent and treat corneal scar formation produced by laser irradiation
WO1991007983A1 (fr) * 1989-11-27 1991-06-13 Huhtamäki Oy Protection de structures intra-oculaires

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ACTA OPHTHALMOLOGICA, Volume 65, issued February 1987, SALONEN et al., "Plasmin in Tear Fluid of Patients with Corneal Ulcers: Basis for New Therapy", pages 3-12. *
ACTA OPHTHALMOLOGICA, Volume 70, Supplement, issued 1992, TERVO et al., "Experience with Plasmin Inhibitors", pages 47-52. *
ARCHIVES OF OPHTHALMOLOGY, Volume 108, No. 12, issued December 1990, BOISJOLY et al., "Topical Fibronectin and Aprotinin for Keratectomy Wound Healing in Rabbits", pages 1758-1763. *
HEALING PROCESSES OF THE CORNEA, Chapter 12, issued 1989, TERVO et al., "Aprotinin for Inhibition of Plasmin on the Ocular Surface: Principles and Clinical Observations", pages 151-163. *
INVESTIGATIVE OPHTHALMOLOGY AND VISUAL SCIENCE SUPPLEMENT, THE ASSOCIATION FOR RESEARCH IN VISION AND OPHTHALMOLOGY INCORPORATED, No. 10, issued April/May 1986, TERVO et al., "A New Therapy to Promote Corneal Healing", page 53. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0795333A2 (fr) * 1996-03-15 1997-09-17 Senju Pharmaceutical Co., Ltd. Substance régulatrice du TGF-bêta pour le traitement de la myopie ou l'hypermétropie
US6010699A (en) * 1996-03-15 2000-01-04 Senju Pharmaceutical Co., Ltd. Method for controlling axial length of the eye
EP0795333A3 (fr) * 1996-03-15 2002-05-02 Senju Pharmaceutical Co., Ltd. Substance régulatrice du TGF-bêta pour le traitement de la myopie ou l'hypermétropie
WO1998029069A2 (fr) * 1997-01-02 1998-07-09 Allergan Sales, Inc. Procede de modification de la refringence d'un oeil
WO1998029069A3 (fr) * 1997-01-02 1998-10-08 Allergan Inc Procede de modification de la refringence d'un oeil
WO1999049887A1 (fr) * 1998-04-01 1999-10-07 Biotech Australia Pty. Limited Utilisation d'inhibiteurs de la protease pour traiter les blessures cutanees
AU2003213546B2 (en) * 1998-04-01 2006-06-29 Pai-2 Pty Limited Use of protease inhibitors for treating skin wounds
US10377818B2 (en) 2015-01-30 2019-08-13 The Board Of Trustees Of The Leland Stanford Junior University Method of treating glioma

Also Published As

Publication number Publication date
AU4847093A (en) 1994-03-29

Similar Documents

Publication Publication Date Title
Hovanesian et al. Surgical techniques and adjuvants for the management of primary and recurrent pterygia
Lama et al. Antifibrotics and wound healing in glaucoma surgery
Bian et al. Differential effects of dexamethasone and doxycycline on inflammation and MMP production in murine alkali-burned corneas associated with dry eye
Hersh et al. Topical nonsteroidal agents and corneal wound healing
Fagerholm Wound healing after photorefractive keratectomy
Nagpal et al. Phototherapeutic keratectomy
CA2423595A1 (fr) Activateur du plasminogene permettant d'eviter un trouble corneen et sous-epithelial apres une intervention chirurgicale de correction de la vision au laser
US6444791B1 (en) Methods and compositions for the treatment of keratoconus using protease inhibitors
Kenyon Decision-making in the therapy of external eye disease: noninfected corneal ulcers
Setten et al. Plasmin and plasminogen activator activities in tear fluid during corneal wound healing after anterior keratectomy
Holopainen et al. Altered expression of matrix metalloproteinases and their tissue inhibitors as possible contributors to corneal droplet formation in climatic droplet keratopathy
Mahdy et al. Safety and efficacy of fibrin glue versus vicryl sutures in recurrent pterygium with amniotic membrane grafting
Amano et al. Corneal endothelial changes after excimer laser photorefractive keratectomy
Tervo et al. Tear fluid plasmin activity after excimer laser photorefractive keratectomy.
Virtanen et al. Tear fluid plasmin activity of dry eye patients with Sjögren's syndrome
WO1994005322A1 (fr) Agent reducteur de l'activite des plasmines pour le traitement d'une plaie resultant de l'ablation superficielle de la cornee et procede de depistage associe
AU752950B2 (en) Remedies for corneal epithelium disturbance
GB2271507A (en) Compositions containing plasmin activity inhibitors
Al-Mohaimeed Effect of prophylactic mitomycin C on corneal endothelium following transepithelial photorefractive keratectomy in myopic patients
Busin et al. The effect of collagen cross-linkage inhibitors on rabbit corneas after radial keratotomy.
Csutak et al. Plasminogen activator inhibitor in human tears after laser refractive surgery
JPH08110337A (ja) 角膜病変の診断方法
Čejková et al. Histochemical changes in the rabbit cornea and plasmin activity in the tear fluid during contact lens wear. Favourable influence of protease inhibitors (aprotinin, PC5, elastatinal)
Csutak et al. Plasminogen activator activity and inhibition in rabbit tears after photorefractive keratectomy
Wu et al. Determination of the nerve growth factor level in the central cornea after LASIK and Epi-LASIK treatment in a rabbit model system

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA JP KR

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA