Cross-linking agents and associated methods

Abstract

To provide an ophthalmic composition that increases corneal lysyl oxidase activity in an eye.SOLUTION: An ophthalmic composition or dosage form can include a therapeutically effective amount of a cross-linking agent, and a pharmaceutically acceptable carrier. The composition or dosage form can be used to treat an ophthalmic condition by administering a therapeutically effective amount of the composition to an eye of a subject during a treatment period.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 305,227, filed March 8, 2016, which is incorporated herein by reference. [Background technology]

[0002] Many ophthalmic conditions can result in corneal thinning and biomechanical weakening. In some cases, the shape of the cornea can be altered, leading to astigmatism, light sensitivity, and other complications. Such conditions can include keratoconus, corneal ulcers, corneal ectasia, pellucid peripheral degeneration, and the like. Summary of the Invention [Problem to be solved by the invention]

[0003] For example, keratoconus (KCN) is a progressive disorder associated with structural changes in the corneal collagen makeup, which can lead to corneal thinning and tears in Bowman's layer and Descemet's membrane. The disease typically manifests in the teenage years, when the cornea becomes more conical, resulting in irregular astigmatism, progressive myopia, corneal thinning, and subsequent poor vision. The prevalence of KCN varies and depends on genetic, geographic, racial, gender, and diagnostic considerations, with the most commonly cited prevalence rate being 50 per 100,000. The severity of KCN is often assessed by minimal corneal thickness, recessive-dominant or interocular asymmetry, maximal keratometry, or corneal hysteresis. [Brief explanation of the drawings]

[0004] [Figure 1A] FIG. 1A is a graph showing increased lysyl oxidase activity in corneal fibroblasts in response to Cu treatment. [Figure 1B] FIG. 1B is another graph showing increased lysyl oxidase activity in corneal fibroblasts in response to Cu treatment. [Figure 2A] FIG. 2A illustrates a chromatograph showing an increase in lysinonorleucine cross-links in keratoconus in response to Cu treatment. [Figure 2B] FIG. 2B illustrates a chromatograph showing that there are no detectable lysinonorleucine cross-links in untreated keratoconus. [Figure 2C] FIG. 2C illustrates a chromatograph showing lysinonorleucine cross-links in normal cornea. [Figure 3A] FIG. 3A illustrates a chromatograph showing an increase in histidino-hydroxylysinonorleucine cross-links in keratoconus in response to Cu treatment. [Figure 3B] FIG. 3B illustrates a chromatograph showing that histidino-hydroxylysinonorleucine cross-links are minimal in untreated keratoconus. [Figure 3C] FIG. 3C illustrates a chromatograph showing histidino-hydroxylysinonorleucine cross-links in normal cornea. [Figure 4] FIG. 4 is a graph showing the increase in corneal strength in response to Cu treatment. [Figure 5] FIG. 5 is a chart showing the improvement in corneal biomechanics in response to Cu treatment. [Figure 6] FIG. 6 is a chart showing the reduction in corneal diopter measurements in response to Cu treatment. [Figure 7A] FIG. 7A is an image of a stained cornea after 6 weeks of Cu treatment. [Figure 7B] FIG. 7B is an image of the stained retina / choroid after 6 weeks of Cu treatment.

[0005] These drawings are provided to illustrate various aspects of the present invention and are not intended to be limiting in terms of dimensions, materials, configurations, arrangements, or proportions unless otherwise limited by the appended claims. DETAILED DESCRIPTION OF THE INVENTION

[0006] Although the following detailed disclosure contains many specifics for purposes of illustration, those skilled in the art will recognize that many variations and modifications to the following details can be made and are considered to be included herein. Accordingly, the following embodiments are described without any loss of generality to, and without imposing limitations on, any claims that are written. It should also be understood that the terminology used herein is for the purpose of disclosing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0007] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells.

[0008] In this disclosure, the terms "comprises," "comprising," "containing," and "having," and the like, may have the meaning assigned to such terms in U.S. patent law and may also mean "includes," "including," and the like, and are generally construed as open-ended terms. The terms "consisting of" and "consists of" are closed-ended terms and include the components, structures, steps, or the like specifically recited with such terms, and pursuant to U.S. patent law. "Consisting essentially of" and "consists essentially of" have the meaning generally assigned to such terms by U.S. patent law. In particular, such terms are generally closed-ended terms, except to permit the inclusion of additional items, materials, components, steps, or elements that do not materially affect the basic and novel characteristics or function of the item with which they are used. For example, minor elements that are present in a composition but do not affect the properties or characteristics of the composition would be permissible if they are present under the "consisting essentially of" phraseology even though they are not explicitly recited in the list of items following a term such as "consisting essentially of." It is understood that when open-ended terms such as "comprising" or "including" are used in the written disclosure, direct support should be given to the "consisting essentially of" phraseology, as well as the "consisting of" phraseology, as if expressly stated, and vice versa.

[0009] The terms "first," "second," "third," "fourth," and the like in the disclosure and appended claims, if present, are used to distinguish between similar elements and not necessarily to disclose a particular order or chronological order. It should be understood that, under appropriate circumstances, all terms so used are interchangeable, such that the embodiments disclosed herein are operable, for example, in other orders than those illustrated or otherwise disclosed herein. Similarly, if a method is disclosed herein as having a series of steps, the order of such steps presented herein is not necessarily the only order in which such steps may be performed, and certain of the steps described may potentially be excluded and / or certain other steps not disclosed herein may potentially be added to the method.

[0010] The term "conjugated," as used herein, is defined as being directly or indirectly connected chemically, mechanically, electronically, or non-electronically. Objects disclosed herein as being "adjacent" to one another may be in physical contact with one another, in close proximity to one another, or in the same general area or region as one another, as appropriate for the context in which the phrase is used. Occurrences of the phrases "in one embodiment" or "in one aspect" herein do not necessarily all refer to the same embodiment or aspect.

[0011] As used herein, the terms "therapeutic agent," "active agent," and the like, are used interchangeably and may refer to an agent that can have a useful or beneficial effect on a subject when administered to the subject in an appropriate or effective amount. In one aspect, the therapeutic agent or active agent can be a copper-containing compound.

[0012] As used herein, an "effective amount" of a drug is an amount sufficient to accomplish the drug's specialized task or desired function. A "therapeutically effective amount" of a composition, drug, or agent refers to a non-toxic but sufficient amount of the composition, drug, or agent to result in treating or preventing a condition for which the composition, drug, or agent is known to be effective. It is understood that various biological factors can affect the ability of a substance to perform its intended task. Thus, an "effective amount" or "therapeutically effective amount" may, in some cases, depend on such biological factors. Furthermore, while achievement of a therapeutic effect can be measured by a physician, veterinarian, or qualified medical professional using assessments known in the art, it is recognized that individual variations and response to treatment may make achievement of a therapeutic effect a somewhat subjective determination. Determination of an effective or therapeutically effective amount is well within the ordinary skill in the art of pharmaceutical sciences and medicine. See, for example, Meiner and Tonascia, "Clinical Trials: Design, Conduct, and Analysis," Monographs in Epidemiology and Biostatistics, Vol. 8 (1986).

[0013] As used herein, a "dosing regimen," or a "regimen," such as a "therapeutic dosing regimen," or a "prophylactic dosing regimen," refers to how, when, how much, and for how long a dose of an active ingredient or composition can or should be administered to a subject to achieve an intended treatment or effect.

[0014] As used herein, the terms "treat," "treatment," or "treating" refer to the administration of a therapeutic agent to a symptom-free or symptomatic subject. In other words, "treat," "treatment," or "treating" may reduce, alleviate, or eliminate symptoms associated with a condition present in a subject, or may be prophylactic (i.e., preventing the condition or reducing the occurrence of the condition in a subject). Such prophylactic treatment may also be referred to as preventing a condition.

[0015] As used herein, the terms "formulation" and "composition" are used interchangeably and refer to a mixture of two or more compounds, elements, or molecules. In some embodiments, the terms "formulation" and "composition" can be used to refer to a mixture of one or more active agents with a carrier or other excipient. A composition can be in almost any physical state, including a solid, liquid (i.e., solution), or gas. Furthermore, the term "dosage form" can include one or more formulations or compositions provided in a format for administration to a subject. For example, an injectable dosage form would be a formulation or composition that is adapted to be administered by injection.

[0016] As used herein, a "subject" refers to an animal. In one embodiment, the animal is a mammal. In another embodiment, the mammal can be a human.

[0017] As used herein, the term "substantially" refers to a complete or nearly complete degree or level of an action, property, characteristic, state, structure, item, or result. For example, an object that is "substantially" enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The precise acceptable degree of deviation from absolute perfection may in some cases depend on the specific context. Generally speaking, however, the proximity to perfection would be such that one would have the same overall result as if absolute and total perfection had been achieved. The use of "substantially" is equally applicable in its negative connotation to refer to the complete or nearly complete absence of an action, property, characteristic, state, structure, item, or result. For example, a composition "substantially free" of particles would be completely devoid of particles, or so nearly completely devoid of particles that the effect would be the same as if the particles were completely absent. In other words, a composition "substantially free" of a component or element may still actually contain such an item, so long as there is no measurable effect of such an item.

[0018] As used herein, the term "about" is used to provide flexibility for the endpoints of numerical ranges by allowing a given value to be "slightly above" or "slightly below" its endpoints. Unless otherwise stated, the use of the term "about" following a specific number or numerical range should be understood to also provide support for such numerical term or range without the term "about." For example, for convenience and brevity, a numerical range of "about 50 angstroms to about 80 angstroms" should also be understood to provide support for the range "50 angstroms to 80 angstroms." Furthermore, it should be understood that support for the actual numerical value is provided even when the term "about" is used in conjunction with it. For example, a recitation of "about" 30 should be interpreted as providing support not only for values ​​slightly above and slightly below 30, but also for the actual numerical value 30.

[0019] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may, for convenience, be presented in common lists. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, unless otherwise indicated, no individual member of such a list should be construed as being de facto equivalent to any other member of the same list solely based on its presentation in a common group.

[0020] Concentrations, amounts, and other numerical data may be expressed or presented in range format herein. It should also be understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limitations, but also every individual numerical value or subrange, as if each numerical value or subrange within that range were expressly recited. By way of example, a numerical range of "about 1 to about 5" should be interpreted not only to include the explicitly recited values ​​of about 1 to about 5, but also to include each individual value and subrange within the recited range. Thus, this numerical range includes individual values ​​such as 2, 3, and 4, as well as subranges such as 1-3, 2-4, and 3-5, etc., as well as 1, 2, 3, 4, and 5, respectively.

[0021] This same principle applies to ranges reciting only one numerical value as a minimum or maximum value. Moreover, such interpretation should apply regardless of the breadth or nature of the range disclosed.

[0022] References throughout this specification to an "example" mean that the particular feature, structure, or characteristic disclosed in connection with that example is included in at least one embodiment. Thus, appearances of the phrase "in an example" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0023] Example embodiment Below, an initial overview of invention embodiments is provided, and then specific embodiments are disclosed in more detail. This initial overview is intended to help the reader more quickly understand technical concepts, but is not intended to identify key or essential features thereof, nor is it intended to limit the scope of the claimed subject matter.

[0024] In one example, an ophthalmic composition or dosage form is disclosed herein. The ophthalmic composition or dosage form may include a cross-linking agent, such as a copper-containing agent, in an amount sufficient to increase lysyl oxidase activity in the subject's eye or otherwise increase cross-linking in the subject's cornea. The composition or dosage form may further include a pharmaceutically acceptable carrier. In some examples, the dosage form may be an ophthalmic composition formulated as a topical eye drop and contained in a container adapted to dispense the composition dropwise in a drop volume of about 5 μl to about 50 μl. In another embodiment, a method for using such a composition or dosage form is disclosed. The method may include administering a therapeutically effective amount of the composition or dosage form disclosed herein for a treatment period.

[0025] As will become more apparent from the various embodiments and examples provided herein, the compositions, dosage forms, and methods disclosed in this disclosure may increase corneal lysyl oxidase activity in the eye of a subject, may increase corneal tissue strength in the eye of a subject, and may provide many other benefits as disclosed herein.

[0026] With this in mind, the compositions, dosage forms, and methods may be used to treat a variety of ophthalmic indications. For example, various ophthalmic conditions can cause corneal thinning, corneal biomechanical weakening, and other corneal complications. Such conditions may include corneal ulcers, marginal ulcerative keratitis, corneal melting, Persido's peripheral degeneration, Therrien's peripheral degeneration, neurotrophic keratitis, corneal ectasia (e.g., after refractive surgery), keratoconus, and the like, or combinations thereof. This disclosure discloses compositions, dosage forms, and methods for treating these conditions, similar conditions, or combinations thereof. However, for the sake of brevity, specific reference will be made throughout to the exemplary condition, keratoconus. This focus on keratoconus is for illustrative purposes only and is not intended to be limiting in any way.

[0027] In light of this, keratoconus (KCN) is a corneal disease characterized by the gradual thinning and ectasia of the noninflammatory stroma, scarring, and eventual rupture of Descemet's membrane. The incidence of KCN depends on genetic, geographic, racial, gender, and diagnostic considerations, with the most commonly cited incidence being 50 per 100,000. The severity of KCN depends on the biomechanical strength of the cornea and is often assessed by the thinnest corneal thickness. However, this criterion is being supplanted by emerging parameters such as posterior corneal surface protrusion, corneal hysteresis, and collagen cross-links per unit corneal thickness. This latter measurement takes into account racial and gender differences in population-specific corneal collagen content.

[0028] The outcomes of KCN are as variable as its epidemiology, ranging from mild to severe astigmatism. However, treatment options are surprisingly limited. While most mild cases of KCN can be corrected using glasses or soft contact lenses, patients very often require toric lenses or hard contact lenses as the disease progresses. Ultimately, one in five patients will require surgery, most commonly deep lamellar keratoplasty or penetrating keratoplasty. Such procedures incur substantial costs and carry significant risks of intraoperative and postoperative complications (e.g., bleeding, scarring, cataract formation). Therefore, more effective preoperative treatment options are desperately needed.

[0029] Intacs (clear, crescent-shaped plastic polymer pieces) have been used as a treatment of choice for mild to moderate keratoconus. These intrastromal corneal rings are inserted as a minimally invasive procedure and have a 74% success rate in restoring best-corrected visual acuity of 20 / 20. However, costs and risks (neovascularization, channel buildup, secondary surgery, etc.) are still limiting.

[0030] Methods to enhance collagen cross-linking have also been attempted. Corneal collagen cross-linking (CXL) therapy is a well-tolerated technique in which riboflavin drops are applied above or below the corneal epithelium, and then ultraviolet A light is used to release free oxygen radicals to strengthen collagen bonds. This procedure has been shown to significantly slow or halt the progression of KCN. Although this technique results in minimal side effects (high-energy UVA light and excessively high concentrations of free oxygen radicals can cause toxicity), the cost remains a barrier for many patients.

[0031] The present disclosure discloses alternative compositions, formulations, and methods for enhancing collagen cross-linking using cross-linking agents, such as copper-containing agents, calcium-containing agents, magnesium-containing agents, silver-containing agents, aluminum-containing agents, zinc-containing agents, iron-containing agents, and other suitable cross-linking agents. Some specific, but non-limiting, examples of cross-linking agents may include acai extract, decorin, copper(II) sulfate, or combinations thereof. In some examples, the cross-linking agent may be or include any divalent or polyvalent ion or compound suitable for inducing or promoting cross-linking in the cornea. In some examples, the cross-linking agent may be or include a metal ion, such as, for example, an alkaline earth metal, a transition metal, a post-transition metal, or a combination thereof. In some examples, the cross-linking agent may be or include a cation. In some specific examples, the cross-linking agent may be or include a divalent metal ion, such as copper, magnesium, iron, zinc, or the like. However, for the sake of brevity and clarity, specific reference will be made throughout this disclosure to copper-containing agents as examples of suitable cross-linking agents. This focus on copper-containing agents is for illustrative purposes only and is not intended to be limiting in any way.

[0032] Copper is a cofactor for the enzyme lysyl oxidase (LOX), which forms various types of collagen cross-links. The single nucleotide polymorphism rs1800449 in LOX increases the genetic risk of KCN. Furthermore, KCN tissue from patients exhibits lower LOX activity. Therefore, copper deficiency can result in reduced collagen content in the tissue. As such, copper supplementation with copper-containing salts, compounds, chelates, or their analogs can be used to increase collagen binding. Such supplementation can improve the biomechanical properties of diseased corneas by increasing lysyl oxidase activity. Because low LOX activity in the cornea can contribute to the development of KCN and copper supplementation can improve collagen cross-links, topical copper treatment may provide a noninvasive and relatively cost-effective tool for preventing the progression of KCN.

[0033] Therefore, the ophthalmic compositions or dosage forms disclosed herein may contain a cross-linking agent, such as a copper-containing agent, in an amount sufficient to increase lysyl oxidase activity or otherwise increase corneal cross-linking in the subject's eye. Various copper-containing agents, such as copper-containing salts, compounds, chelates, or analogs thereof, may be used. Non-limiting examples of copper salts may include copper sulfate, copper carbonate, copper acetate, copper chloride, copper bromide, copper fluoride, copper nitrate, copper iodide, copper perchlorate, copper molybdate, copper thiocyanate, copper tartrate, copper tetrafluoroborate, copper selenide, copper pyrophosphate, analogs thereof, or combinations thereof. Other suitable copper carriers may include GHK-copper, tetraamine copper sulfate, copper-histidine, copper-glycinate, copper-gluconate, analogs thereof, or combinations thereof.

[0034] Therefore, the copper-containing agent can be any copper-containing agent that can provide a therapeutically effective amount of bioavailable copper. The therapeutically effective amount can be sufficient to increase corneal lysyl oxidase activity in the eye, such that collagen cross-linking is increased compared to collagen cross-linking before treatment. This can also increase the biomechanical strength of the cornea compared to the biomechanical strength before treatment. Furthermore, this can reduce the diopter of the cornea in the treated eye compared to the diopter before treatment.

[0035] The therapeutically effective amount of the copper-containing agent can be based on the amount of copper retained by the copper-containing agent. In some examples, the copper-containing agent can provide a copper level lower than about 0.05 mg / ml, about 0.02 mg / ml, about 0.005 mg / ml, or about 0.0002 mg / ml, but still effective for increasing lysyl oxidase activity. It can be important to maintain copper levels low enough to avoid copper-induced toxicity while maintaining a sufficient amount of bioavailable copper to increase lysyl oxidase activity.

[0036] Therefore, the therapeutically effective amount of the copper-containing agent can be determined based on the type of delivery vehicle, the type of copper-containing formulation, the desired duration of delivery, and the like. For example, depending on how the composition is formulated, the composition may contain a copper amount of about 0.0001 mg / ml to about 5 mg / ml or about 50 mg / ml. In other examples, the composition may contain a copper amount of about 0.0005 mg / ml to about 0.03 mg / ml, about 0.01 mg / ml to about 5 mg / ml, or about 0.001 mg / ml to about 0.005 mg / ml. In some additional examples, the composition may contain a copper amount of about 0.0001 mg / ml to about 0.05 mg / ml, about 0.00025 mg / ml to about 0.015 mg / ml, about 0.0005 mg / ml to about 0.000075 mg / ml, or about 0.0008 mg / ml to about 0.0011 mg / ml. Thus, in some instances, a therapeutically effective amount can be defined as the amount of copper contained in a composition. For example, an amount of about 0.0016 mg / ml of copper(II) sulfate anhydrous provides a composition with a copper content of about 0.00064 mg / ml. This is because, although the molecular weight of copper(II) sulfate is about 159.6 g / mol, only about 63.5 g / mol, or about 40%, of the agent is copper itself. Therefore, a therapeutically effective amount can be determined based on the copper content provided by the copper-containing agent, rather than the amount of the copper-containing agent itself. Alternatively, 0.0018 mg / ml of copper(II) acetate anhydrous provides a composition with a copper content of about 0.00063 mg / ml.

[0037] Alternatively, the therapeutically effective amount can be defined as the wt% of the copper-containing agent in the composition. Again, depending on how the composition is formulated, the therapeutically effective amount of the copper-containing agent can be about 0.00005 wt% to about 5 wt%, about 10 wt%, or about 15 wt%. In some examples, the therapeutically effective amount of the copper-containing agent can be about 0.05 wt% to about 15 wt%, about 0.01 wt% to about 10 wt%, or about 0.005 wt% to about 5 wt%. In other examples, the therapeutically effective amount of the copper-containing agent can be about 0.0001 wt% to about 0.0005 wt%, about 0.0001 wt% to about 0.0002 wt%, about 0.0002 wt% to about 0.0003 wt%, or about 0.0003 wt% to about 0.0004 wt%. In yet another example, the therapeutically effective amount of the copper-containing agent may be from about 0.001 wt% to about 0.01 wt%, or from about 0.003 wt% to about 0.008 wt%. In yet another example, the therapeutically effective amount of the copper-containing agent may be from about 0.01 wt% to about 0.1 wt%, or from about 0.03 wt% to about 0.08 wt%. It is noted that these weight percentages are calculated based on copper(II) sulfate anhydrous. Therefore, if an alternative copper-containing agent is utilized, the weight percentages may be converted accordingly.

[0038] However, a specific amount of a copper-containing agent in a composition does not necessarily mean that all of the copper content will be bioavailable upon administration. Copper bioavailability may vary to some extent with each individual copper-containing agent, as well as with pH and other compositional factors. Therefore, the therapeutically effective amount of a copper-containing agent may also be adjusted based on the copper bioavailability for a specific copper carrier, pH, formulation, or the like.

[0039] The copper-containing agent may also be administered with a therapeutically effective amount of a second active or therapeutic agent. Such additional agents may include riboflavin, rose bengal, hydroxylysine, analogs thereof, or combinations thereof. Furthermore, multiple different copper-containing agents, or multiple different cross-linking agents, may be administered simultaneously, such as a copper-containing formulation in combination with another non-copper-containing cross-linking agent, e.g., a zinc-, magnesium-, iron-, or silver-containing agent.

[0040] The copper-containing formulation may be provided in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be formulated in various ways to deliver the copper-containing agent. Non-limiting examples may include a solution, suspension, gel, hydrogel, thermoresponsive gel, subconjunctival injection, depot, film, contact lens, pledget, or the like. In one particular example, the composition may be an ophthalmic eye drop. In some examples, the composition may be formulated as a copper-eluting contact lens, such as a soft lens, a toric lens, a hard lens, a scleral lens, the like, or a combination thereof.

[0041] Depending on how the composition is formulated, pharmaceutically acceptable carriers can include a variety of excipients. For example, pharmaceutically acceptable carriers can include solubilizing agents, tonicity adjusting agents, pH adjusting agents, thickening or gelling agents, polymers or polymer matrices, preservatives, water, and the like, as well as mixtures thereof.

[0042] Non-limiting examples of solubilizing agents can include phosphate buffered saline (PBS), Dulbecco's PBS, Alsever's solution, Tris buffered saline (TBS), water, or balanced salt solutions (BSS) such as Hank's BBS, Earle's BBS, Gray's BBS, Puck's BBS, Simm's BBS, Tyrode's BBS, BBS Plus, Ringer's lactate solution, normal saline (i.e., 0.9% saline), 1 / 2, the like, or combinations thereof. The solubilizing agent can be present in various amounts in a pharmaceutically acceptable carrier. In one embodiment, the solubilizing agent can have a concentration in the carrier of from about 10 wt%, about 20 wt%, about 30 wt%, about 40 wt%, or about 50 wt%, to about 80 wt%, about 90 wt%, about 95 wt%, about 97 wt%, about 99 wt%, or about 99.9999 wt%.

[0043] Non-limiting examples of tonicity agents include the solubilizers listed above, as well as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, dextrose, glycerin, propylene glycol, ethanol, trehalose, analogs thereof, or combinations thereof. The tonicity agent can be used to provide an appropriate tonicity for the formulation. In one embodiment, the tonicity of the formulation is about 250 to 350 milliosmoles per liter (mOsm / L). In another embodiment, the tonicity of the formulation is about 270 to 330 mOsm / L. The tonicity agent can be present in various amounts in a pharmaceutically acceptable carrier. In one embodiment, the tonicity agent can have a concentration in the carrier of about 0.1 wt%, about 0.5 wt%, or about 1 wt%, to about 2 wt%, about 5 wt%, or about 10 wt%.

[0044] Non-limiting examples of pH adjusting agents include numerous acids, bases, and combinations thereof, such as hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like. The pH adjusting agent can be used to provide an appropriate pH to the formulation. In one embodiment, the pH can be from about 5.5 to about 8.5. In one embodiment, the pH can be from about 5.8 to about 7.8. In another embodiment, the pH can be from about 6.5 to about 7.8. In yet another embodiment, the pH can be from about 7.0 to about 7.6. The pH adjusting agent can be present in various amounts in a pharmaceutically acceptable carrier. In one embodiment, the pH adjusting agent can have a concentration in the carrier of from about 0.01 wt%, about 0.05 wt%, about 0.1 wt%, or about 0.5 wt% to about 1 wt%, about 2 wt%, about 5 wt%, or about 10 wt%.

[0045] Non-limiting examples of thickening or gelling agents may include glycerol, propylene glycol, polyethylene glycol, polyvinyl alcohol, cellulose derivatives (such as methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, and the like), ethyl vinyl alcohol, hyaluronic acid, the like, or combinations thereof. The thickening or gelling agent may be present in a pharmaceutically acceptable carrier in various amounts. In one embodiment, the thickening or gelling agent may have a concentration in the carrier of about 0.01 wt%, about 0.05 wt%, about 0.1 wt%, or about 0.5 wt%, to about 2 wt%, about 8 wt%, about 15 wt%, or about 30 wt%.

[0046] Non-limiting examples of polymers that can be used to prepare the polymer matrix for the film can include biodegradable or non-biodegradable polymers. Non-limiting examples of polymers or polymer combinations can include poly(methyl methacrylate), hydroxyethyl methacrylate, polysiloxane, poly(lactic-co-glycolic acid) (varying ratios of lactic acid glycolide content to end groups such as acid or ester ends), polyvinyl alcohol, polyvinyl acetate, ethylene vinyl acetate, polyethylene glycol, polylactic acid, polyglycolic acid, hydroxypropyl methylcellulose, hydroxypropyl cellulose, carboxymethylcellulose, croscarmellose, polycaprolactone, hyaluronic acid, albumin, sodium chloride block copolymers thereof, salts thereof, analogs thereof, or combinations thereof. Certain copolymers, such as polylactic acid-polyglycolic acid block copolymer (PLGA), polyglycolic acid-polyvinyl alcohol block copolymer (PGA / PVA), hydroxypropyl methylcellulose (HPMC), polycaprolactone-polyethylene glycol block copolymer, croscarmellose, and the like, can be particularly effective for biodegradable matrices, if desired.

[0047] In some examples, the composition may include a thermoresponsive polymer. Non-limiting examples of thermoresponsive polymers include poly(N-isopropylacrylamide), poly[2-(dimethylamino)ethyl methacrylate], hydroxypropyl cellulose, poly(vinylcaprolactam), polyvinyl methyl ether, polyethylene oxide, polyhydroxyethyl methacrylate, ABCBA-type pentablock polymers, chitosan, analogs thereof, or combinations thereof. Such thermoresponsive polymers may be functionalized to bind or bind to specific copper-containing agents within a wide temperature range and release the copper-containing agent upon a change in the temperature of the surrounding environment, such as by placing the composition in contact with the eye, applying a heat source to the eye after administration of the composition, or the like.

[0048] Non-limiting examples of preservatives may include benzalkonium chloride (BAK), cetrimonium chloride, sodium perborate, ethylenediaminetetraacetic acid (EDTA) and its various salt forms, chlorobutanol, and the like. The preservative may be present in a pharmaceutically acceptable carrier in various amounts. In one embodiment, the preservative may have a concentration in the carrier of about 0.001 wt%, about 0.005 wt%, about 0.01 wt%, or about 0.05 wt%, to about 0.1 wt%, about 0.25 wt%, about 0.5 wt%, or about 1 wt%.

[0049] In one particular example, the pharmaceutically acceptable carrier may be formulated as ophthalmic drops and may comprise BSS in an amount of about 50 wt% to about 99.9999 wt%. In another particular example, the pharmaceutically acceptable carrier may be formulated as ophthalmic drops and may comprise artificial tears (i.e., Refresh Tears®, Genteal®, Oasis Tears®, and the like).

[0050] Regardless of how the ophthalmic composition is formulated, the ophthalmic composition can be used as an ophthalmic dosage form for administering a therapeutically effective dose of a copper-containing agent. In some examples, the ophthalmic dosage form can provide about 0.0005 μg to about 0.5 μg of copper per administration event. In still other examples, the ophthalmic dosage form can provide about 0.006 μg to about 0.06 μg, about 0.01 μg to about 0.03 μg, or about 0.016 μg to about 0.044 μg of copper per administration event. In still other examples, the ophthalmic dosage form can provide about 0.0005 μg to about 5 μg of copper per administration event. In still other examples, the ophthalmic dosage form may provide about 0.001 μg to about 2 μg, about 0.006 μg to about 0.24 μg, about 0.01 μg to about 0.12 μg, or about 0.016 μg to about 0.18 μg of copper per administration event. It is noted that not all of the copper provided by the dosage form is necessarily bioavailable, although in some instances it may be.

[0051] In some instances, the ophthalmic dosage form may be used in an effective dosing regimen to provide a therapeutically effective amount of the copper-containing agent, which may include administering the ophthalmic dosage form once a day, twice a day, three times a day, four times a day, or more times a day.

[0052] In still other examples, the ophthalmic dosage form can be formulated to biodegrade and provide controlled or sustained release of the copper-containing agent over a predetermined period of time. In still other examples, the ophthalmic dosage form can be formulated to provide controlled or sustained release of the copper-containing agent from a non-biodegradable matrix. In examples such as these, the dosage form can be formulated to release the copper-containing agent over a period of hours, days, or weeks, as desired. In some specific examples, the dosage form can be formulated to deliver about 0.005 mcg to about 250 mcg of copper per week. In still other examples, the dosage form can be formulated to deliver about 0.008 mcg to about 200 mcg, about 0.01 mcg to about 150 mcg, or about 0.1 mcg to about 100 mcg per week. Furthermore, the dosage form can generally be formulated to have zero-order drug release kinetics.

[0053] The dosage form may be held or stored in a container as a premixed composition ready for administration without further dilution or adjustment. In some embodiments, a single container may hold a volume or amount of the composition sufficient for a single dose. In yet other instances, a single container may hold a volume or amount of the composition sufficient for multiple doses.

[0054] Any number of suitable containers may be used. In one embodiment, the container may be an amber container. In some examples, the container may be made of glass, polypropylene, polyethylene, polycarbonate, polyvinyl chloride, analogs thereof, or combinations thereof. In some examples, the container may have a capacity of about 0.5 ml to about 50 ml. In some examples, the container may have a capacity of about 1 ml to about 30 ml, about 5 ml to about 20 ml, or about 3 ml to about 15 ml. In one embodiment, the container may hold a single dose of the therapeutic composition or dosage form. In another embodiment, the container may hold multiple doses of the therapeutic composition or dosage form.

[0055] In some examples, about 0.005 mg to about 1 mg of the copper-containing agent may be included in the container. In yet other examples, about 0.01 mg to about 0.5 mg of the copper-containing agent may be included in the container. In some examples, about 0.001 mg to about 0.5 mg of copper may be included in the container. In some examples, about 0.005 mg to about 0.2 mg of copper may be included in the container.

[0056] In some specific examples, the dosage form may be a topical ophthalmic dosage form formulated as eye drops and contained in a container adapted to dispense the composition dropwise in a drop volume of about 5 μl to about 50 μl. When the composition is formulated as eye drops, in some examples, the container includes an adapted nozzle or tip through which the composition is dispensed. As such, the container may generally be collapsible to dispense the composition. However, in some cases, air may be sucked back into the container after the composition is dispensed, which may cause contamination of the composition. In some examples, the nozzle or tip may include a valve mechanism, a filter, the like, or a combination thereof to prevent or minimize the introduction of bacteria or other foreign matter into the container. Furthermore, as previously discussed, the container may be adapted to dispense the ophthalmic composition dropwise. For example, the container may be adapted to dispense the ophthalmic composition in a drop volume of about 5 μl to about 50 μl, such as about 15 μl, about 20 μl, about 25 μl, about 30 μl, about 35 μl, about 40 μl, about 45 μl, or about 50 μl. In some specific examples, the drop volume may be about 15 μl to about 40 μl, about 5 μl to about 30 μl, about 20 μl to about 30 μl, about 25 μl to about 35 μl, or about 30 μl to about 40 μl.

[0057] An administration mechanism, such as a syringe, eye dropper, or other mechanism, may further be included in the dosage form. In further embodiments, suitable packaging may be used to provide the composition, container, and instructions for their use, and optionally the administration mechanism, in a single, unified system.

[0058] The compositions or dosage forms disclosed herein may also be used in methods for treating ophthalmic conditions. Such methods may include administering a therapeutically effective amount of the composition or dosage form to the eye of a subject for a treatment period. The methods may be used to treat any of the conditions previously mentioned herein and similar ophthalmic conditions.

[0059] In one example, the composition or dosage form can be administered one to four times per day per eye in need thereof, with the dosage at each time point being about 5 μl to about 50 μl, about 5 μl to about 30 μl, about 20 μl to about 30 μl, about 25 μl to about 35 μl, or about 30 μl to about 40 μl.

[0060] The duration of treatment can depend on numerous factors, such as the type and severity of the condition, the age of the subject at the time of diagnosis, and the like. Generally, the duration of treatment can range from about one month to chronic treatment, depending on the circumstances. For example, in some cases, young children may receive chronic treatment, while older children and teenagers (e.g., 10-18 years old) may receive treatment for two to three years, young adults (e.g., 18-30 years old) may receive treatment for one to two years, and adults 30 years old or older may receive treatment for about six months to one year. Thus, in some instances, the duration of treatment can range from about one month to chronic treatment. In yet other instances, the duration of treatment can be from about one month, about three months, or about six months to about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, or until the condition is satisfactorily resolved.

[0061] In some instances, the ophthalmic composition may be administered as eye drops. In yet other instances, the eye drops may be administered as a subconjunctival injection. In yet other instances, the eye drops may be administered in the form of a topical film or contact lens. In some instances, the topical film or contact lens may be configured to biodegrade over time to provide a controlled and sustained release of the copper-containing agent.

[0062] Generally, the methods disclosed herein can increase collagen cross-links in the cornea compared to an untreated eye. More specifically, the methods can increase lysinonorleucine cross-link density, histidinyl-hydroxylysinonorleucine cross-link density, or both, as well as other collagen cross-links associated with the cornea, compared to an untreated eye. Furthermore, the methods disclosed herein can reduce corneal centripetal strain by at least about 10%, 25%, or 50% compared to an untreated cornea. Furthermore, the methods disclosed herein can reduce corneal diopters in keratoconus compared to an untreated keratoconus. [Example]

[0063] Example 1 - Copper-containing active agents can increase corneal lysyl oxidase activity. Cultured corneal stromal cells from normal and keratoconus corneas (n = 3 each) in 10% FBS DMEM were exposed to BSS control or 0.0016 mg / mL CuSO4 in balanced salt solution (BSS), then filtered through a 0.25 μ filter. The conditioned culture medium was subjected to a peroxidase-coupled fluorometric activity assay for LOX.

[0064] Tissues were harvested under sterile technique in a surgical environment. Tissue samples were stored in Optisol solution in 10 cm tissue culture dishes. Corneal tissue was suspended in 15% fetal bovine serum (FBS) DMEM / F12 with penicillin / streptomycin. The epithelium and Descemet's membrane were mechanically removed under a stereomicroscope. The samples were further cut into small pieces with surgical scissors and added to 1 mg / mL collagenase; 10 mL was used per cornea. The sample pieces were seeded into 10 cm tissue culture dishes and placed in a tissue culture incubator at 37°C with 5% CO2. After 5 days, cells were harvested and seeded in 10% FBS DMEM without phenol red.

[0065] LOX enzyme activity in the culture medium was measured using Amplex Red peroxidase-coupled fluorometric assay. Briefly, corneal stromal cells were seeded at 0.2 x 10^6 cells in 2 mL of culture medium in a 6-well plate. After 3 days, the medium was collected for LOX enzyme activity assay. 50 μL of each culture medium was placed in a black 96-well plate. Then, 50 μL of 2x assay buffer (2.4 M urea, 100 mM sodium borate (pH 8.2), 20 mM 1,5-diaminopentane, 20 μM Amplex Red, and 2 units / mL horseradish peroxidase) was added. In a parallel assay, we added 500 μM aminopropiononitrile (BAPN), which can completely quench LOX activity. Fluorescence of oxidized Amplex Red was recorded every 10 minutes using a fluorescence plate reader. After subtracting background fluorescence, the fluorescence intensity was plotted against the incubation time (FIGS. 1A and 1B).

[0066] Based on peroxidase-coupled fluorometric lox activity assays and as illustrated in Figures 1A-1B, copper was found to dramatically increase lox activity in normal and keratoconus fibroblasts, suggesting that copper may increase collagen cross-linking by enhancing lox activity in keratoconus. [Example]

[0067] Example 2 - Copper-containing active agents can increase collagen cross-linking in the cornea. Sample preparation: Each human keratoconus was divided into two halves. One half was treated with 0.0016 mg / ml CuSO4 in Optisol solution for one week, while the other half was left untreated. After one week, each keratoconus was stored at -80°C for later analysis.

[0068] Sample extraction: Corneal samples were washed with PBS pH 7.4, powdered in liquid nitrogen, and crushed in a freezer mill. Corneal samples were demineralized using 0.5 M EDTA in 0.05 M Tris buffer pH 7.4 at 4°C for 48 h. The insoluble fraction was washed twice with distilled water by centrifugation at 5000 g for 20 min at 47°C and then frozen dry. The frozen dry samples were hydrolyzed in vacuum at 110°C for 24 h using constantly boiling double-distilled 6 N HCl. The hydrolysate was neutralized with 2.5 N NaOH and dissolved in distilled water. Lysinonorleucine (LNL) and histidinohydroxylysinonorleucine (HHL) were analyzed separately by HPLC.

[0069] HPLC Method for Lysinonorleucine (LNL): Amino acid analysis is an important application area in protein chemistry and food analysis. Many different applications exist in this field. Automated precolumn derivatization followed by reversed-phase LC has become a useful procedure for efficient analysis due to its ease of use and simple technique. For rapid and sensitive analysis of lysinonorleucine (LNL), an Agilent 1290 Infinity LC System was used with automated precolumn derivatization. The column used for this method was an Agilent ZORBAX Eclipse Plus RRHD C18 (3.0 mm x 50 mm, 1.8 μm) and a mobile phase consisting of Solvent A: 10 mM disodium phosphate + 10 mM sodium tetraborate pH 8.2 and Solvent B: ACN / MeOH / HO (45:45:10).

[0070] HPLC method for histidinohydroxylysinonorleucine (HHL): 25 ml of protein hydrolysate sample (total amino acid concentration less than 3 mM) was added to 50 μl of 1.5 mM Ne-methyl-L-lysine hydrochloride (Mly) in 0.26 M borate buffer, pH 9.5, and 50 μl of acetone in a 1.5 ml reaction vessel. Then, 25 μl of FMOC-Cl reagent (6 mM FMOC-Cl in acetone) was added, and the mixture was allowed to react for 2 minutes at room temperature. The reaction mixture was extracted twice with 350 ml of pentane to remove excess FMOC-Cl reagent. The aqueous phase was subjected to chromatography.

[0071] Corneal cross-links are tissue-specific. The major cross-link generated in the human cornea is lysinonorleucine (LNL). LNL is the building block for subsequent polyvalent cross-links, such as histidinyl-hydroxylysinonorleucine (HHL). Normal and keratoconic human corneas were treated with 0.0016 mg / ml CuSO4 for 1 week. HPLC analysis was then performed to measure the levels of LNL (Figures 2A-2C) and HHL (Figures 3A-3C).

[0072] Indeed, a significant increase in LNL and HHL levels was observed in copper-treated corneas. As illustrated in Figure 2A, LNL cross-links were observed in human keratoconus treated with CuSO4 as disclosed above. In contrast, as illustrated in Figure 2B, no LNL cross-links were observed in keratoconus not treated with CuSO4. Figure 2C illustrates the presence of LNL cross-links in normal corneas.

[0073] Similarly, as illustrated in Figure 3A, HHL cross-links are also observed in human keratoconus treated with CuSO4 as disclosed above. In contrast, as illustrated in Figure 3B, negligible levels of HHL cross-links were observed in keratoconus not treated with CuSO4. Figure 3C illustrates that HHL cross-links in normal corneas are at higher levels than those observed in keratoconus not treated with CuSO4.

[0074] Thus, CuSO4 treatment clearly provided increased cross-linking of both LNL and HHL in keratoconus. [Example]

[0075] Example 3 - Treatment with copper-containing agents increases corneal strength. Corneal centripetal strain measurements were performed on human cadaver corneas and rabbit corneas. Human cadaver corneas (n = 2 pairs) were cultured as previously described in Example 1. The treatment group was immersed in 0.0016 mg / ml CuSO4 for 2 weeks. The specimens contained a small portion of sclera on both ends. Mineral oil was applied to the surface of the sclera-cornea-sclera strip to minimize tissue dehydration. The rabbit corneas were divided into four groups. Two groups were treatment groups that received either 0.0025 mg / ml CuSO4 three times a day or 0.0025 mg / ml CuSO4 once a day. Two groups served as controls that received only BSS.

[0076] The specimen was coupled between a motor and a transducer, which applied a controlled pressure of 5-30 mmHg, and the resulting centripetal strain was measured. The specimen length between the two gripping jaws was approximately 10 mm. Geometric information, including specimen width and thickness, was entered into the Rheometrics System Analyzer (RSA) control panel. Specimen thickness was measured using an ultrasonic pachymeter (DGH 550 Pachymeter; DGH Technology, Exton, PA).

[0077] The results show a significant increase in stiffness and a decrease in centripetal strain after copper sulfate treatment (Figure 4). The average peak centripetal strain induced by an ocular pulse at 20 mmHg in treated corneas was approximately 1.5 times lower than that of untreated corneas. This represents an approximately 50% increase in corneal strength, indicating that cross-linking results in a stiffer corneal response. Furthermore, as illustrated in Figure 5, treatment with CuSO4 also provided corneal biomechanics comparable to those seen with laser cross-linking. [Example]

[0078] Example 4 - Rabbit Corneal Topography Corneal distortion in specific regions was monitored by corneal topography. Corneal topography results are expressed in diopters. New Zealand white rabbits were administered 0.0025 mg / ml copper sulfate (CuSO4) once daily and three times daily. Additionally, New Zealand white rabbits were administered a 0.025 mg / ml high-concentration sample once daily during the treatment period. Three different controls were administered: no eye drops, BSS, or artificial tears. Each of the six groups included six study subjects. Topography images were taken before treatment and weekly for five weeks. Diopter measurements were at the mean K value and 3 mm. These values ​​were compared with diopter measurements in laser-crosslinked corneas one month and one year later.

[0079] As can be seen in Figure 6, there was a greater increase in diopter measurements in the treatment group compared to the control group. Furthermore, one month of treatment with CuSO4 provided diopter measurements similar to those observed after one year of laser cross-linking. [Example]

[0080] Example 5 - In Vivo Safety of Copper Eye Drops Copper sulfate (CuSO4) solution was prepared at a concentration of 0.0025 mg / ml and administered to rabbits three times a day (T1D) or once a day (QD). Anterior segment evaluation was performed using a slit lamp biomicroscope and a Heidelberg Spectralis anterior segment optical coherence tomography (AS-OCT). On day 1, week 1, week 4, and week 6, two rabbits (n = 4 eyes) were sacrificed for copper levels in ocular tissues and compared with control rabbits (no eye drops). Aqueous humor, vitreous, retina, cornea, lens, and blood samples were analyzed for copper ions using inductively coupled plasma mass spectrometry (ICP-MS).

[0081] Rabbits (n = 2) were sacrificed after 42 days by intravenous injection of 0.3 mg / kg euthanasia solution, and the eyes were enucleated for histological evaluation and assessment of signs of inflammation, tissue damage, scarring, and fibrosis. The anterior segments of the eyes were fixed in 10% formaldehyde-glutaraldehyde solution, dehydrated in a graded alcohol series, embedded in paraffin, and sectioned with a microtome. Sections were stained with appropriate stains (hematoxylin and eosin and Masson Trichrome).

[0082] Treatment with Cu had no effect on electroretinogram (ERG) measurements. Furthermore, no toxicity was observed in any of the dissected ocular tissues. Furthermore, although laser cross-linking can induce corneal opacity for up to approximately one year, no corneal opacity was observed. These results are further illustrated in Figures 7A-7B. Figure 7A illustrates an image of the cornea stained with hematoxylin and eosin after 6 weeks of Cu treatment. Figure 7B illustrates an image of the retina / choroid stained with hematoxylin and eosin after 6 weeks of Cu treatment.

[0083] It should be understood that the above disclosed methods are only illustrative of some embodiments of the present invention. Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention, and the appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been disclosed above with specificity and detail in connection with what are presently considered to be the most practical and preferred embodiments of the invention, it will be apparent to those skilled in the art that variations can also be made without departing from the principles and concepts set forth herein.

Claims

1. An ophthalmic composition, A therapeutically effective amount of copper perchlorate, A pharmaceutically acceptable carrier, An ophthalmic composition having the following properties.

2. An ophthalmic composition according to Claim 1, wherein the ophthalmic composition contains copper in an amount of about 0.0001 mg / ml to about 50 mg / ml.

3. An ophthalmic composition according to Claim 2, wherein the ophthalmic composition contains copper in an amount of about 0.01 mg / ml to about 5 mg / ml.

4. An ophthalmic composition according to Claim 1, wherein the ophthalmic composition contains copper perchlorate in an amount of about 0.00005 wt% to about 15 wt%.

5. An ophthalmic composition according to claim 4, wherein the ophthalmic composition contains copper perchlorate in an amount of about 0.01 wt% to about 0.1 wt%.

6. An ophthalmic composition according to any one of claims 1 to 5, wherein the pharmaceutically acceptable carrier comprises at least one of an isotonic agent, a solubilizer, a thickener, a polymer, a buffer, a pH adjuster, and water.

7. An ophthalmic composition according to any one of claims 1 to 5, wherein the pharmaceutically acceptable carrier comprises an isotonic agent selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, dextrose, glycerin, propylene glycol, ethanol, trehalose, and combinations thereof.

8. An ophthalmic composition according to claim 7, wherein the isotonic agent comprises glycerin.

9. An ophthalmic composition according to any one of claims 1 to 5, wherein the ophthalmic composition has a tensile strength of about 250 mOsm / L to about 350 mOsm / L.

10. An ophthalmic composition according to any one of claims 1 to 5, wherein the ophthalmic composition has a pH of about 5.5 to about 8.

5.

11. An ophthalmic composition according to any one of claims 1 to 5, wherein the ophthalmic composition comprises substantially the copper perchlorate and the pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier comprising water and at least one of an isotonic agent and a pH adjuster.

12. An ophthalmic composition according to any one of claims 1 to 5, wherein the ophthalmic composition is formulated as one of a solution, suspension, gel, hydrogel, thermoresponsive gel, depot, film, and contact lens.

13. An ophthalmic composition according to any one of claims 1 to 5, wherein the ophthalmic composition is formulated as a solution for administration as an ophthalmic eye drop.

14. A topical ophthalmic preparation comprising the ophthalmic composition according to any one of claims 1 to 5, wherein the ophthalmic composition is formulated as eye drops and is contained in a container adapted for dispensing the ophthalmic composition in a dropper manner at a drop volume of about 5 μl to about 50 μl.

15. A topical ophthalmic preparation according to claim 14, wherein the container is adapted to dispense the ophthalmic composition in a drop volume of about 15 μl to about 40 μl.

16. A topical ophthalmic preparation according to claim 14, wherein the preparation provides about 0.0005 μg to about 5 μg of copper per drop of the ophthalmic composition.

17. A topical ophthalmic preparation according to claim 14, wherein the container includes a nozzle or tip from which the ophthalmic composition is dispensed, and the nozzle or tip includes at least one of a valve mechanism and a filter.

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