Background of the Invention

This invention relates to methods of treatment and/or prevention of corneal scarring or haze and to compositions and articles of manufacture used for the treatment and/or prevention of corneal scarring or haze, which involve the application of compositions to the eye prior to, during, and/or after irradiation and/or ophthalmic surgery.

In recent years, the feasibility of using ophthalmic lasers to ablate corneal tissue has been proven. Current applications include the removal of corneal scars (phototherapeutic keratectomy) and the correction of re¬ fractive error (photorefractive keratectomy). Photoablation of corneal tissue can be achieved using, e.g., neodymium:YAG (Nd:YAG), carbon dioxide (CO ₂ ), and ultraviolet-emitting (excimer) lasers. Methods for conducting ophthalmic surgery using an excimer laser are described, e.g., in U.S. Patent Nos. 4,665,913 and 4,732,148, which disclose methods and devices for achieving a predetermined refractive correction by ablation of corneal tissue.

Although current evidence indicates that laser corneal procedures are generally highly effective, a reported complication is the post- operative phenomenon of corneal haze. This opacification of the corneal stroma and/or Bowman's layer is an artifact that was not previously reported in conjunction with ophthalmic surgery prior to the introduction of laser photoablation of the cornea. Neither the mechanism of corneal

haze formation nor the exact composition of the haze is known. Many experts regard corneal haze formation to be the chief barrier remaining to the widespread acceptance of laser photoablation of the cornea for refractive and therapeutic indications. Corneal haze has been shown to occur after both phototherapeutic keratectomy (to remove corneal opacities or irregularities) [W.C.S. Wu et al., "Corneal Wound Healing After 193-nm Excimer Laser Keratectomy," Arch. Opthalmol. 109 (1991 ), 1426-1432] and photorefractive keratec¬ tomy (to changes refractive error) [T. Seiler, "Photorefractive keratec- tomy: European Experience," in Color Atlas/Text of Excimer Laser

Surgery: The Cornea, ed. F.B. Thompson and P.J. McDonnell, Igaku- Shoin Medical Publishers, Inc., New York, 53-62, 1993. Seiler has found that the intensity of this haze is sufficient to result in loss of best- corrected visual acuity of less than or equal to 6 diopters in less than 1 % of patients treated for myopia and may occur in up to 9% of patients treated for myopia of greater than 6 diopters.

Histology of corneas that have been treated with the excimer laser shows keratocytes, collagen, and other macromolecules within the treated area. See, e.g., F. Fantes et al., "Wound Healing After Excimer Laser Keratomileusis (Photorefractive Keratectomy) in Monkeys," Arch.

Ophthalmol. 108 (1990), 665-675. Fagerholm et al. [T.D. Fitzsimmons et al., "Hyaluronic Acid in the Rabbit Cornea After Excimer Laser Super¬ ficial Keratectomy," Invest. Ophthalmol. Vis. Sci. 33 (1992) 301 1-3016] concluded that hyaluronic acid was a major component of the haze based on animal experiments, while others found a variety of collagens, non- collagenous proteins, and other macromolecules within the treated areas. The corneal haze begins to appear a few weeks after the surgery, generally is maximal between 4 and 12 weeks after the surgery, and may then begin to decrease in intensity. In animal experiments, it has been shown that the intensity of this haze is related to the smoothness of the post-operative surface of the cornea [M. Campos et al., "Corneal Wound

Healing After Excimer Laser Ablation: Effects of Nitrogen Gas Blower," Ophthalmology, 99 (1992), 893-897], to the depth of the ablation [E. Shieh et al., "Quantitative Analysis of Wound Healing After Cylindrical and Spherical Excimer Laser Ablations," Ophthalmology, 99 (1992), 1050-1055], and to the shape of the edge of the ablation profile [Ibid.].

When the haze first appears, histologic examination reveals a hγpercellu- lar connective tissue, including keratocytes, newly synthesized collagen, and ground substance. The thickness of this newly deposited material has been measured quantitatively, and the thickness is related to the amount of corneal tissue initially ablated with the excimer laser [Ibid.].

In U.S. Patent Nos. 5,124,392 and 4,939,135, a method of treat¬ ing corneal haze produced by exposure of the eye to laser irradiation has been described using agents referred to as "wound healing modulators."

Summary of the Invention The present invention relates to methods of preventing and/or treating, e.g., inhibiting, reducing or lessening, corneal haze, corneal scarring, and/or other related corneal and eye conditions, especially those which occur after laser surgery to the eye. The invention also especially relates to methods of preventing, inhibiting, treating, and/or reducing other eye conditions which result from the same type of injury produced by laser irradiation. The invention also relates to compositions and articles of manufacture which can be used to prevent, reduce, inhibit and/or treat the above-mentioned conditions, as well as other corneal and eye conditions, e.g., which result from injury by laser irradiation. The present invention also relates to a novel use of compounds administered before and/or after eye injury, e.g., excimer surgery, which can inhibit the formation of mature collagen in the eye and therefore treat corneal scarring haze or scarring, e.g., by modifying the structure of collagen, e.g., in its secondary, tertiary, quantenary, or triple helix structure, slowing the rate of collagen secretion, accelerating intracellular

degradation of collagen, and/or inhibiting the biosynthesis of collagen entirely.

The methods and compositions involve, e.g., modifying the struc¬ ture of collagen by administering a compound which is incorporated into the collagen molecule, preferably a proline analog, most preferably cis- hydroxyproline (CHP), in an amount which is effective in preventing, re¬ ducing, and/ or treating corneal haze, scarring, or other corneal conditions which result from corneal injury, wounding, or damage, especially of the type caused by laser irradiation. The administration of such a structure- modifying compound, preferably a proline analog, can inhibit and/or dis¬ rupt the formation of the structure of collagen, e.g., its triple helix structure. The administration of such a structure-modifying compound can also slow the rate of collagen secretion from the cell and/or accel¬ erate its intracellular degradation rate. The present invention also relates to an eye dropper, ocular insert, or lens comprising a topical, ophthalmic composition, comprising an amount of a compound as described above, e.g., which modifies the structure of collagen, effective in treating corneal scarring or haze when administered topically to the eye, and essentially free of any separate compound having angiostatic activity, e.g., as required in U.S. Pat. No.

5,021 ,404.

The present invention also relates to a method of treating and/or preventing corneal scarring or haze produced as a result of laser surgery of a patient comprising, administering to said patient a compound which inhibits the formation of mature collagen in an amount which is effective in treating and/or preventing said corneal scarring or haze.

The present invention also relates to a method of treating and/or preventing corneal scarring or haze produced as a result of laser surgery of a patient, comprising administering to the patient an amount of a proline analog compound which is effective in treating and/or preventing said corneal scarring or haze.

The present invention also relates to an eye dropper comprising a topical, ophthalmic composition, comprising a compound which modifies the structure of collagen in an amount effective for treating and/or preventing corneal scarring or haze when administered topically to the eye, and essentially free of any additional compound having angiostatic activity.

The present invention additionally relates to method of treating and/or preventing corneal scarring or haze in a patient comprising, administering to said patient a compound which inhibits the formation of mature collagen in an amount which is effective in treating and/or preventing said corneal scarring or haze.

According to the present invention, proline analogs can be used to treat an eye exposed to laser irradiation. During collagen formation, a proline hydroxylase can modify L-proline to 4-hydroxy-proline by the addi- tion of a hydroxy group to C ₄ of its side chain R group. The hydroxy is normally added in the trans-configuration. However, if the cis-configura- tion, i.e., CHP, is incorporated into a primary amino acid chain of a colla¬ gen molecule rather than the naturally-occurring L-proline, no further hydroxylation takes place, and the resulting molecule is unable to assume the structure, e.g., its triple helix structure, characteristic of normal collagen. It has been reported that when CHP and other proline analogs are incorporated into collagen molecules instead of the normal proline, abnormal collagen is formed which is easily degraded and thus sup¬ presses accumulation of collagen. All discussion herein of the theory of operation of this invention is presented for information only and is not intended to limit the invention in any way. See, e.g, for a discussion of the effects of CHP and other proline analogs on collagen formation, "The cis-Hydroxyproline Induced Inhibition of Collagen Formation" by J.C. Javitt, Senior Thesis, Princeton University, 1978. Other proline analogs that can be used according to the present in¬ vention include, e.g., those wherein the hydroxy group in CHP is replaced

by a halogen (e.g., CI, Br, F, I), NO ₂ , CN, C,. _β -alkyl (e.g., methyl, ethyl, etc.), C,. _e -alkoxy (e.g., methoxy, ethoxy, etc.), and the like, it also being possible for proline to be substituted by more than one such substituent in any position. Proline analogs that can be administered according to the present invention also include, e.g., L-azetidine-2-carboxylic acid; D,L-

3,4-dehydroproline; thioproline; etc.

The present invention also relates to other compounds which be¬ come incorporated into collagen and which causes the resultant collagen molecule to lose all or possess less ability to produce haze or scarring. Such agents can be incorporated into the structure of collagen analagous- ly to CHP during translation of the collagen molecule or can be incor¬ porated into collagen after translation or during and/or after glycosylation of the collagen molecule.

CHP and the other analogs are known per se and/or can be pre- pared conventionally.

The formation of mature collagen in the eye can also be inhibited by administering compounds which modify its structure, e.g., its secon¬ dary or triple helix structure, by inhibiting proline hydroxylase, such as by using a,a dipyridyl, by inhibiting lysine hydroxylase, or by inhibiting normal collagen cross-linking such as by using beta-aminoproprionitrile.

Compounds can also be administered which inhibit the biosynthesis of collagen entirely. The amount of such a compound administered is effective in preventing, reducing, and/ or treating corneal haze, scarring, or other corneal conditions which result from corneal injury, wounding, or damage, especially of the type caused by laser irradiation. Other compounds are described, e.g., in U.S. Pat. No. 5,021 ,404.

Scarring and/or hazing due to laser irradiation which is treatable by this invention can be caused by any mechanism (e.g., the subsequent process of healing of tissue injured by the irradiation). Applicable laser irradiation includes that produced by IR lasers (e.g., CO ₂ lasers), espe¬ cially UV (e.g., excimer) or even shorter wavelength lasers.

Any kind of resultant scarring or hazing in the eye can be treated, including, e.g., any tissue opacification or tissue growth in or adjacent to the cornea (or other eye components), which is non-endogenous and which has a transmission and/or refractive index in the visible light region which is different from that of the endogenous tissue which it replaces or to which it is adjacent, especially the stromal haze reported in association with excimer laser photorefractive keratectomy.

Effective amounts of agents per this invention, including proline analogs or mixtures of agents, will vary depending upon the route admin- istered and the nature of the condition which is to be treated or pre¬ vented. Precise amounts for a given set of circumstances can be rou¬ tinely determined in view of the guidance of this application and normal considerations of patient condition, age, health, etc., and, e.g., can be bioequivalent to effective amounts of CHP. Typical amounts of CHP to be administered, e.g., topically, include, e.g., from about 1 to 1000 mg/ml, preferably from about 5 to 150 mg/ml, more preferably from about 10 to 100 mg/ml. However, effective amounts can also be lower or higher to achieve the desired beneficial effect. It can be administered topically, e.g., as eye drops, e.g., in 4 separate applications per day of typically one or two drops of such solutions.

The ability and amount of a compound according to the present invention to treat, prevent, reduce, and/or inhibit the above-mentioned eye conditions can be routinely determined, e.g., as described in the example disclosed herein and/or by in vitro and in vivo measurements of collagen properties, biosynthesis, degradation, and secretion as determined conventionally.

Administration according to the present invention can be through any route which is conventionally or typically used to administer active agents ocularly, as long as the desired beneficial effect is achieved. For example, a composition may be administered orally, topically, intra- orbitally, parenterally, by surgical implantation, etc. The choice of a route

can be according to the physician's discretion, depending on the nature of the eye condition, and the age, health, and other attributes of the patient that may be considered relevant. Preferably, the agent is admin¬ istered topically as an ointment, hydrogel, eye-drop, e.g., one to a few (2-3) drops per dose can be instilled in the eye, e.g., 1 to 6 times a day.

The compositions can also be applied via a collagen shield, contact lens, ophthalmic insert, an eye dropper, or other solid matrix placed on the ocular surface. Such shields, lenses or matrices can provide for slow or sustained release of the compositions as well as serving as a protective environmental barrier. See, for example, U.S. Patent No. 4,442,089.

Administration of the agent of this invention can be carried out be¬ fore, during and/or after laser eye surgery. The administration can be alone or in combination, either sequentially (before or after) or simulta¬ neously, with other active agents. For example, CHP can be admini- stered with steroids, growth factors, basement membrane components, anti-inflammatory agents, antibiotics, immunomodulators, cell adhesion modulators, aldose reductase inhibitors, anti-oxidants, anti-microbial agents, anti-viral agents, alpha-adrenergic, beta-adrenergic, or other agents as desired. These and other agents and/or treatments include those described in, e.g., U.S. Patent Nos. 4,442,089; 4,939,135;

4,985,417; 5,100,874; 5,021 ,404; 5,124,392; 5,135,919. The other active agent can be administered in the dosages in which it is conven¬ tionally used, or in an amount effective in combination with a compound of this invention as can be determined routinely. See, e.g., Remington's Pharmaceutical Sciences, Eighteenth Edition, Mack Publishing Company,

1990.

A composition of the present invention, e.g., a topical composition, can further comprise conventional additives, including, e.g., cyclodextrin, amorphous β-cyclodextrin, such as hydroxy-propyl- ?-cyclodextrin preser- vatives, corneal storage medium, solvents, viscosity formers, antimicrobial agents, tissue compatibility enhancers, (e.g., cholesterol),

salts, buffers, surfactants, and other conventional compounds typically employed in formulations for use, e.g., in the eye. Regarding use of cyclodextrans see copending application Serial No. 08/000,935, filed on

January 5, 1993. The solutions of the invention can also be sterile or non-sterile as desired. These commonly employed additives are fully conventional and are described, e.g., in the Physician's Desk References of Ophthalmology.

The compounds useful according to the present invention are also effective to treat and prevent scars in the eye caused by wounding, inva- sive activity, or other types of surgery especailly those which produce injuries analogous to those caused by lasers as discussed above.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, there- fore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following example, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight. The entire disclosures of all applications, patents, and publications, cited above and below, are hereby incorporated by reference.

Example

A solution of cHyp at a concentration of 10 mg/ml was formulated in a vehicle of 40% hydroxy-propyl- ?-cyclodextrin in normal saline. Two New Zealand white rabbits were prepared for excimer laser photoablation using standard laboratory practice. A VISX 20/20 excimer laser was calibrated according to manufacturer's instructions, and photorefractive keratectomy was performed in both eyes. The ablation was performed at a fluence of 160 mJ/cm ² , a 6 mm optical zone, and 1 10 μm central depth. The anticipated refractive correction was 10 diopters. Following laser treatment, one drop of the cHyp solution was applied to the right eye of each rabbit four times a day for four weeks. The left eye was treated with the cyclodextrin vehicle but no cHyp. The animals were examined daily for a period of six weeks. Starting on the third day following laser treatment, corneal haze could be seen to develop in both eyes. However, it was of considerably smaller magnitude in the eye treated with cHyp. This difference in degree of corneal haze was still apparent at six weeks when the experiment was halted.

The preceding example can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding example.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.