Crosscrossing agents and related methods
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- UNIV OF UTAH RES FOUND
- Filing Date
- 2021-08-09
- Publication Date
- 2026-08-05
Smart Images
Figure 0007900903000001 
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Abstract
Description
Technical Field
[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 305,227, filed Mar. 8, 2016, which is hereby incorporated herein by reference in its entirety.
Background Art
[0002] Many ophthalmic conditions can lead to corneal thinning and biomechanical weakening of the cornea. In some cases, the shape of the cornea can be altered, causing astigmatism, light sensitivity, and other complications. Such conditions can include keratoconus, corneal ulcers, corneal ectasia, pellucid marginal degeneration, and the like.
Summary of the Invention
Problems to be Solved by the Invention
[0003] For example, keratoconus (KCN) is a progressive disorder associated with structural changes in the corneal collagen composition that can lead to corneal thinning and breaks in Bowman's layer and Descemet's membrane. Generally, the disease presents symptoms in the teens of life, where the cornea becomes more conical, resulting in irregular astigmatism, progressive myopia, corneal thinning, and subsequently poor vision. The prevalence of KCN varies and depends on genetic, geographic, ethnic, gender, and diagnostic considerations, with the most cited prevalence being 50 per 100,000. The severity of KCN is often evaluated by the minimum corneal thickness, inferior-superior or interocular asymmetry, maximum keratometry, or corneal hysteresis.
Brief Description of the Drawings
[0004] [Figure 1A] FIG. 1A is a graph showing an increase in lysyl oxidase activity in corneal fibroblasts in response to Cu treatment. [Figure 1B] FIG. 1B is another graph showing an increase in lysyl oxidase activity in corneal fibroblasts in response to Cu treatment. [Figure 2A] Figure 2A illustrates a chromatogram showing increased lisinonorleucine cross-linking in keratoconus in response to Cu treatment. [Figure 2B] Figure 2B illustrates a chromatogram showing the absence of detectable lisinonorleucine cross-linking in untreated keratoconus. [Figure 2C] Figure 2C illustrates a chromatogram showing lisinonorleucine cross-linking in a normal cornea. [Figure 3A] Figure 3A illustrates a chromatogram showing increased histidino-hydroxylysinonorleucine cross-linking in keratoconus in response to Cu treatment. [Figure 3B] Figure 3B illustrates a chromatogram showing minimal histidino-hydroxylysinonorleucine cross-linking in untreated keratoconus. [Figure 3C] Figure 3C illustrates a chromatogram showing histidino-hydroxylysinorleucine cross-linking in a normal cornea. [Figure 4] Figure 4 is a graph showing the increase in corneal strength in response to Cu treatment. [Figure 5] Figure 5 is a chart showing the improvement in corneal biomechanics in response to Cu treatment. [Figure 6] Figure 6 is a chart showing the decrease in corneal diopter measurements in response to Cu treatment. [Figure 7A] Figure 7A shows an image of a stained cornea after 6 weeks of copper treatment. [Figure 7B] Figure 7B shows an image of the stained retina / choroid after 6 weeks of copper treatment.
[0005] These drawings are provided to illustrate various aspects of the present invention and are not intended to limit the scope of dimensions, materials, configurations, setups, or proportions, unless otherwise limited by the appended claims. [Modes for carrying out the invention]
[0006] The following details contain many specifics for illustrative purposes, but those skilled in the art will recognize that many variations and modifications to the following details are possible and may be considered included herein. Accordingly, the following embodiments are described without loss of any universality to any of the claims described, and without imposing any limitations on any of the claims described. It should also be understood that the terms used herein are for the purpose of disclosing specific embodiments only and are not intended to be restrictive. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.
[0007] In this specification and the attached claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "cells" includes multiple such cells.
[0008] In this disclosure, “comprises,” “comprising,” “containing,” and “having,” and similar terms, may have the meanings assigned to them under U.S. patent law, and may also mean “includes,” “including,” and similar terms, and are generally interpreted as open terms. The terms “consisting of” and “consists of” are closed terms, including any components, structures, steps, or similar items specifically listed with such terms, as well as those subject to U.S. patent law. “consisting essentially of” and “consists essentially of” have the meanings generally assigned to them under U.S. patent law. In particular, such terms are generally closed terms, with the exception of allowing the inclusion of additional matters, materials, components, steps, or elements that do not substantially affect the fundamental and novel features or functions of the matter used in connection with them. For example, trace elements present in a composition but not affecting its properties or characteristics may be acceptable if they exist under the "consisting essentially of" usage, even if they are not explicitly listed in the list of items following a term such as "consisting essentially of." When open terms such as "comprising" or "including" are used in the described disclosure, it is understood that the "consisting essentially of" usage, as well as the "consisting of" usage, should be given direct support as if they were explicitly stated, and vice versa.
[0009] The terms “first,” “second,” “third,” “fourth,” and similar terms in the disclosed and appended claims are used to distinguish between similar elements, if any, and are not necessarily used to disclose a particular order or chronological order. Where appropriate, it should be understood that any terms used in this way are interchangeable, so as to enable the embodiments disclosed herein to operate, for example, in an order different from that illustrated or otherwise disclosed herein. Similarly, where a method is disclosed herein having a set of steps, the order of such steps presented herein is not necessarily the only order in which such steps can be performed, and certain steps among those described may be potentially excluded, and / or certain other steps not disclosed herein may potentially be added to the method.
[0010] As used herein, the term “conjugated” is defined as being directly or indirectly linked 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 appropriate to the context in which the phrase is used. Occurrences of the phrases “in one embodiment” or “in one aspect” herein do not necessarily refer to the same embodiment or aspect.
[0011] As used herein, the terms “therapeutic agent,” “activator,” and similar terms may refer to drugs that, when used interchangeably and administered to a subject in an appropriate or effective amount, may be useful or have a beneficial effect on that subject. In one embodiment, the therapeutic agent or activator may be a copper-containing compound.
[0012] As used herein, “effective dose” of a drug is the amount sufficient to achieve the drug’s specialized task or desired function. “Therapeutic effective dose” of a composition, drug, or agent refers to the amount of that composition, drug, or agent that is nontoxic but sufficient to treat or prevent a condition for which the composition, drug, or agent is known to be effective. It is understood that various biological factors can influence a substance’s ability to perform its intended task. Therefore, “effective dose” or “therapeutic effective dose” may, in some cases, depend on such biological factors. Furthermore, while the achievement of therapeutic effects may be measured by physicians, veterinarians, or qualified medical professionals using assessments known in the art, it is recognized that individual variations and responses to treatment can make the achievement of therapeutic effects a somewhat subjective determination. The determination of effective doses or therapeutic effective doses is well within the normal art in the fields of pharmaceutical science and medicine. For example, see Meiner and Tonascia, "Clinical Trials: Design, Conduct, and Analysis," Monographs in Epidemiology and Biostatistics, Vol. 8 (1986).
[0013] As used herein, “medication regimen,” “therapeutic drug regimen,” or “prophylactic drug regimen” refers to how, when, how much, and for how long a dose of an active ingredient or composition may or should be administered to a subject in order to achieve the intended treatment or effect.
[0014] As used herein, the terms “to treat,” “treatment,” or “treatment process” refer to the administration of a therapeutic agent to a subject who is asymptomatic or exhibiting symptoms. In other words, “to treat,” “treatment,” or “treatment process” may reduce, alleviate, or eliminate, or be preventative (i.e., prevent the symptoms or reduce their occurrence in the subject), a condition and associated symptoms present in the subject. Such preventative treatment may also be called prevention of 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" may be used to refer to a mixture of one or more active agents with a carrier or other excipient. A composition can take on substantially any physical state, including a solid, liquid (i.e., a solution), or gas. Further, the term "dosage form" can include one or more formulations or compositions provided in a form for administration to a subject. For example, an injectable dosage form would be a formulation or composition that is adapted for administration by injection.
[0016] As used herein, "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 an action, property, characteristic, state, structure, item, or result that is complete or nearly complete in degree or extent. For example, an object that is "substantially" enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact acceptable degree of deviation from absolute completeness may in some cases depend on the particular context. Generally speaking, however, the near-completeness would be such that the same overall result would be obtained as if absolute and total completeness had been achieved. The use of "substantially" is equally applicable in the negative sense to refer to a complete or nearly complete lack of an action, property, characteristic, state, structure, item, or result. For example, a composition that "substantially" lacks particles would either completely lack particles or lack particles to such an extent that the effect would be the same as if it completely lacked particles. In other words, a composition that "substantially" lacks a component or element may actually still contain such an item as long as there is no measurable effect of such an item.
[0018] As used herein, the term "about" is used to provide flexibility to the endpoints of a numerical range by allowing that a given value can be "slightly above" or "slightly below" its endpoints. Unless otherwise stated, it should also be understood that the use of the term "about" with respect to a specific number or numerical range provides support for such numerical terms or ranges without the term "about". For example, for the sake of convenience and brevity, it should also be understood that the numerical range of "from about 50 angstroms to about 80 angstroms" provides support for the range of "from 50 angstroms to 80 angstroms". Further, it should be understood that in this specification, support is provided for the actual numerical value even when the term "about" is used in conjunction therewith. For example, a detailed description 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] Multiple items, structural elements, components, and / or materials used herein may be presented, for convenience, in the form of a common list. However, these lists should be interpreted as if each member of the list is individually identified as a separate and distinct member. Thus, unless otherwise indicated, no individual member of such a list should be construed as being in fact equivalent to any other member of the same list based solely on its presentation in the common group.
[0020] In this specification, concentrations, quantities, and other numerical data may be expressed or presented in the form of ranges. It should also be understood that such range forms should be interpreted flexibly to include not only the numbers used merely for convenience and brevity and thus explicitly detailed as limitations of the range, but also all of the individual numbers or subranges within that range, as if each number or subrange within that range were explicitly detailed. For example, the numerical range "about 1 to about 5" should be interpreted to include not only the explicitly detailed values about 1 to about 5, but also the individual values and subranges within the indicated 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, and further, 1, 2, 3, 4, and 5, respectively.
[0021] This same principle applies to ranges that detail only one numerical value as the minimum or maximum value. Furthermore, such interpretation should apply regardless of the breadth or nature of the disclosed range.
[0022] References to “examples” throughout this specification mean that any particular feature, structure, or property disclosed in connection with such example is included in at least one embodiment. Accordingly, the appearance of the phrase “in an example” in various places throughout this specification does not necessarily all refer to the same embodiment.
[0023] Example of an embodiment A preliminary overview of the embodiments of the invention is provided below, after which specific embodiments are disclosed in more detail. This preliminary overview is intended to help the reader more quickly grasp the technical concepts, but it is not intended to identify its main or essential features, nor is it intended to limit the scope of the claims.
[0024] In one example, an ophthalmic composition or dosage form is disclosed herein. The ophthalmic composition or dosage form may contain a cross-crosslinking agent, such as a copper-containing agent, in an amount sufficient to increase lysyl oxidase activity in the eye of a subject or otherwise increase cross-crosslinking in the cornea of the subject. The composition or dosage form may further contain 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 for dispensing the composition in a droppery manner at a droppery 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 of use may include administering a therapeutically effective amount of the composition or dosage form disclosed herein during a treatment period.
[0025] As will become clearer from the various embodiments and examples provided herein, the compositions, dosage forms, and methods disclosed herein may increase corneal lysyl oxidase activity in the eyes of subjects, increase corneal tissue strength in the eyes of subjects, and provide many other benefits disclosed herein.
[0026] Based on this, the composition, dosage form, and method may be used to treat a variety of ophthalmic indications. For example, various ophthalmic conditions can cause corneal thinning, biomechanical weakening of the cornea, and other corneal complications. Such conditions may include corneal ulcers, marginal ulcerative keratitis, corneal lysis, peripheral degeneration of Pellucid, peripheral degeneration of Tellien, neurotrophic keratitis, corneal ectasia (e.g., after refractive surgery), keratoconus, and analogues thereof, or combinations thereof. This disclosure discloses compositions, dosage forms, and methods for treating these conditions, analogues, or combinations thereof. However, for the sake of brevity, specific references to keratoconus as an example condition will be made throughout. This focus on keratoconus is for illustrative purposes only and is not intended to be limiting in any way.
[0027] Based on this, keratoconus (KCN) is a corneal disease characterized by non-inflammatory, progressive thinning and ectasia of the stroma, scarring, and eventual rupture of Descemet's membrane. The frequency of KCN depends on genetic, geographical, racial, gender, and diagnostic considerations, with the most commonly cited frequency 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 superseded by newly emerging parameters such as corneal posterior protrusion, corneal hysteresis, and collagen cross-crosslinking per unit corneal thickness. The last measure takes into account racial and gender differences in population-specific corneal collagen content.
[0028] The outcomes of keratoconjunctival cornea (KCN) are as variable as their epidemiology, ranging from mild to severe astigmatism. However, treatment options are surprisingly limited. Most mild KCN can be corrected with glasses or soft contact lenses, but very often patients require annular lenses or hard contact lenses as the disease progresses. Ultimately, one in five patients will require surgery, most commonly deep lamellar corneal transplantation or full-thickness corneal transplantation. Such procedures are considerably costly and carry a significant risk of intraoperative and postoperative complications (bleeding, scarring, cataract formation, etc.). Therefore, there is a desperate need for more effective preoperative treatment options.
[0029] For mild to moderate keratoconus, Intax (a clear, crescent-shaped plastic polymer piece) has been used as a treatment option. These stromal corneal rings are inserted as a minimally invasive procedure and have a 74% success rate in restoring a maximum corrected visual acuity of 20 / 20. However, the cost and risks (neovascularization, channel deposition, secondary surgery, etc.) are also limited.
[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 to the surface or under the corneal epithelium, followed by the use of ultraviolet A light to release free oxygen radicals to strengthen collagen binding. This procedure has been shown to significantly slow or halt the progression of KCN. While this technique has 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] This disclosure discloses alternative compositions, dosage forms, and methods for enhancing collagen cross-crosslinking using cross-crosslinking 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-crosslinking agents. Some specific but non-limiting examples of cross-crosslinking agents may include acai extract, decoin, copper(II) sulfate, or combinations thereof. In some examples, the cross-crosslinking agent may be, or include, any divalent or polyvalent ion or compound suitable for inducing or promoting cross-crosslinking in the cornea. In some examples, the cross-crosslinking agent may be, or include, a metal ion such as, alkaline earth metals, transition metals, post-transition metals, or combinations thereof. In some examples, the cross-crosslinking agent may be, or include, a cation. In some specific examples, the cross-crosslinking agent may be, or include, a divalent metal ion such as, copper, magnesium, iron, zinc, or analogues thereof. However, for the sake of brevity and clarity, specific references to copper-containing agents will be made throughout this disclosure as examples of suitable cross-crosslinking agents. This focus on copper-containing agents is for illustrative purposes only and is not intended to be restrictive in any way.
[0032] Copper is a cofactor for lysyl oxidase (LOX), an enzyme that forms various types of collagen cross-links. The single nucleotide polymorphism rs1800449 of LOX increases the genetic risk of KCN. Furthermore, KCN tissue in patients exhibits lower LOX activity. Therefore, copper deficiency can lead to a decrease in collagen content in tissues. Copper supplementation, such as copper-containing salts, compounds, chelates, or analogues thereof, can be used to increase collagen binding. Such supplementation can improve the biomechanical properties of diseased corneas by increasing lysyl oxidase activity. Since low LOX activity in the cornea may contribute to the development of KCN, and copper supplementation can improve collagen cross-linking, topical copper therapy may offer a non-invasive and relatively cost-effective tool for preventing the progression of KCN.
[0033] Accordingly, the ophthalmic compositions or dosage forms disclosed herein may contain cross-crosslinking agents, such as copper-containing agents, in an amount sufficient to increase lysyl oxidase activity or otherwise increase corneal cross-crosslinking in the eye of the subject. Various copper-containing agents may be used, such as copper-containing salts, compounds, chelates, or analogues thereof. 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, analogues thereof, or combinations thereof. Other suitable copper carriers include GHK-copper, tetraamine copper sulfate, copper-histidine, Copper glycinate This may include copper-gluconic acid, analogues 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. This therapeutically effective amount may be sufficient to increase corneal lysyl oxidase activity in the eye, such that collagen cross-linking increases compared to pre-treatment collagen cross-linking. This may also increase the biomechanical strength of the cornea compared to pre-treatment biomechanical strength. Furthermore, this may decrease corneal diopters in the treated eye compared to pre-treatment diopters.
[0035] The therapeutically effective dose of a copper-containing agent may depend on the amount of copper retained by the agent. In some cases, a copper-containing agent may provide copper levels lower than approximately 0.05 mg / ml, 0.02 mg / ml, 0.005 mg / ml, or 0.0002 mg / ml, but still effective in increasing lysyl oxidase activity. It may be important to maintain a sufficient amount of bioavailable copper to increase lysyl oxidase activity while keeping copper levels low enough to avoid copper-induced toxicity.
[0036] Therefore, the therapeutically effective dose of a copper-containing agent can be determined based on the type of delivery medium, the type of copper-containing formulation, and the desired duration of delivery. For example, depending on how the composition is formulated, the composition may contain copper amounts ranging from about 0.0001 mg / ml to about 5 mg / ml or about 50 mg / ml. In other examples, the composition may contain copper amounts ranging from about 0.0005 mg / ml to about 0.03 mg / ml, from about 0.01 mg / ml to about 5 mg / ml, or from about 0.001 mg / ml to about 0.005 mg / ml. In some additional examples, the composition may contain copper amounts ranging from about 0.0001 mg / ml to about 0.05 mg / ml, from about 0.00025 mg / ml to about 0.015 mg / ml, from about 0.0005 mg / ml to about 0.000075 mg / ml, or from about 0.0008 mg / ml to about 0.0011 mg / ml. Therefore, in some examples, the therapeutically effective dose can be defined as the amount of copper contained in the composition. For example, about 0.0016 mg / ml of anhydrous copper(II) sulfate 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. Thus, the therapeutically effective dose can be determined based on the copper content provided by the copper-containing agent rather than the amount of the copper-containing agent itself. As an alternative example, 0.0018 mg / ml of anhydrous copper(II) acetate provides a composition with a copper content of about 0.00063 mg / ml.
[0037] Alternatively, the therapeutically effective dose 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 dose of the copper-containing agent can range from about 0.00005 wt% to about 5 wt%, about 10 wt%, or about 15 wt%. In some examples, the therapeutically effective dose of the copper-containing agent can range from about 0.05 wt% to about 15 wt%, from about 0.01 wt% to about 10 wt%, or from about 0.005 wt% to about 5 wt%. In other examples, the therapeutically effective dose of the copper-containing agent can range from about 0.0001 wt% to about 0.0005 wt%, from about 0.0001 wt% to about 0.0002 wt%, from about 0.0002 wt% to about 0.0003 wt%, or from about 0.0003 wt% to about 0.0004 wt%. In further examples, the therapeutically effective dose of a copper-containing agent may be approximately 0.001 wt% to 0.01 wt%, or approximately 0.003 wt% to 0.008 wt%. In yet another example, the therapeutically effective dose of a copper-containing agent may be approximately 0.01 wt% to 0.1 wt%, or approximately 0.03 wt% to 0.08 wt%. It should be noted that these weight percentages are calculated based on anhydrous copper(II) sulfate. Therefore, if an alternative copper-containing agent is used, the weight percentage may be converted accordingly.
[0038] However, the presence of a specific amount of copper in a composition does not necessarily mean that all of that copper will be bioavailable upon administration. The bioavailability of copper can vary to some extent depending on the individual copper-containing agent, as well as on pH and other compositional factors. Therefore, the therapeutically effective dose of a copper-containing agent may also be adjusted based on the bioavailability of copper with respect to a particular copper carrier, pH, formulation, or analogue thereof.
[0039] Copper-containing agents may also be administered together with a therapeutically effective dose of a secondary activator or therapeutic agent. Such additional agents may include riboflavin, rose bengal, hydroxylysine, their analogues, or combinations thereof. Furthermore, multiple different cross-crosslinking agents may be administered simultaneously, such as multiple different copper-containing agents, or copper-containing formulations in combination with other non-copper-containing cross-crosslinking agents, such as zinc-containing agents, magnesium-containing agents, iron-containing agents, or silver-containing agents.
[0040] Copper-containing formulations may be provided in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be formulated in a variety of ways to deliver the copper-containing formulation. Non-limiting examples may include solutions, suspensions, gels, hydrogels, thermoresponsive gels, subconjunctival injections, depots, films, contact lenses, cotton threads, or analogues thereof. 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, annular lens, hard lens, scleral lens, analogues thereof, or a combination thereof.
[0041] Depending on how the composition is formulated, a pharmaceutically acceptable carrier may contain a variety of excipients. For example, a pharmaceutically acceptable carrier may contain solubilizers, isotonic agents, pH adjusters, thickeners or gelling agents, polymers or polymer matrices, preservatives, water, and analogues thereof, as well as mixtures thereof.
[0042] Non-limiting examples of solubilizers may include phosphate-buffered saline (PBS), Dulbecco's PBS, Alsever's solution, Tris-buffered saline (TBS), water, or equilibrium salt solutions (BSS) such as Hank's BBS, Earle's BBS, Grey's BBS, Puck's BBS, Simm's BBS, Tyrode's BBS, BBS Plus, Ringer lactate solution, ordinary saline (i.e., 0.9% saline), 1 / 2, analogues thereof, or combinations thereof. Solubilizers may be present in varying amounts in pharmaceutically acceptable carriers. In one embodiment, the solubilizer may have concentrations in the carrier ranging 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 isotonic 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, their analogues, or combinations thereof. The isotonic agent may be used to provide the appropriate tonicity of the formulation. In one embodiment, the tonicity of the formulation is about 250 to 350 milliosmoles / liter (mOsm / L). In another embodiment, the tonicity of the formulation is about 270 to 330 mOsm / L. The isotonic agent may be present in a variety of amounts in a pharmaceutically acceptable carrier. In one embodiment, the isotonic agent may be present in the carrier at concentrations ranging from 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 adjusters may include a number of acids, bases, and combinations thereof, such as hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, and analogues thereof. The pH adjuster may be used to provide an appropriate pH for a formulation. In one embodiment, the pH may be from about 5.5 to about 8.5. In another embodiment, the pH may be from about 5.8 to about 7.8. In yet another embodiment, the pH may be from about 6.5 to about 7.8. In yet another embodiment, the pH may be from about 7.0 to about 7.6. The pH adjuster may be present in a variety of amounts in a pharmaceutically acceptable carrier. In one embodiment, the pH adjuster may be present in the carrier at concentrations ranging 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 thickeners or gelling agents may include glycerol, propylene glycol, polyethylene glycol, polyvinyl alcohol, cellulose derivatives (such as methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, and their analogues), ethyl vinyl alcohol, hyaluronic acid, their analogues, or combinations thereof. The thickener or gelling agent may be present in a variety of amounts in a pharmaceutically acceptable carrier. In one embodiment, the thickener or gelling agent may be present in the carrier at concentrations ranging from 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 may be used to prepare polymer matrices for films may include biodegradable or non-biodegradable polymers. Non-limiting examples of polymers or polymer combinations may include poly(methyl methacrylate), hydroxyethyl methacrylate, polysiloxane, poly(lactic acid-co-glycolic acid) (different ratios of lactate glycolide content to terminal groups such as acid or ester endpoints), 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, analogues 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 their analogues, may 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 may include poly(N-isopropylacrylamide), poly[2-(dimethylamino)ethyl methacrylate], hydroxypropylcellulose, poly(vinyl caprolactam), polyvinyl methyl ether, polyethylene oxide, polyhydroxyethyl methacrylate, ABCBA-type pentablock polymers, chitosan, analogues thereof, or combinations thereof. Such thermoresponsive polymers may be functionalized to bind to or conjugate a specific copper-containing agent over a wide temperature range, releasing its copper-containing formulation when the temperature of the surrounding environment is changed, such as when the composition is placed in contact with the eye, when a heat source is applied to the eye after administration of the composition, or similar actions.
[0048] Non-limiting examples of preservatives may include benzalkonium chloride (BAK), cetrimonium, sodium perborate, ethylenediaminetetraacetic acid (EDTA) and its various salt forms, chlorobutanol, and analogues thereof. Preservatives may be present in a variety of amounts in a pharmaceutically acceptable carrier. In one embodiment, the preservative may be present in the carrier at concentrations ranging from 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 a specific example, the pharmaceutically acceptable carrier may be formulated as an ophthalmic drop and may contain BSS in an amount of approximately 50 wt% to approximately 99.9999 wt%. In another specific example, the pharmaceutically acceptable carrier may be formulated as an ophthalmic drop and may contain artificial tears (i.e., Refresh Tears®, Genteal®, Oasis Tears®, and similar products).
[0050] Regardless of how the ophthalmic composition is formulated, it can be used as an ophthalmic dosage form for administering a therapeutically effective dose of copper-containing material. In some examples, the ophthalmic dosage form may provide about 0.0005 μg to about 0.5 μg of copper per administration event. In yet other examples, the ophthalmic dosage form may 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 yet another example, the ophthalmic dosage form may provide about 0.0005 μg to about 5 μg of copper per administration event. In other examples, ophthalmic dosage forms may provide approximately 0.001 μg to 2 μg, 0.006 μg to 0.24 μg, 0.01 μg to 0.12 μg, or 0.016 μg to 0.18 μg of copper per administration event. It should be noted that not all copper provided by these dosage forms is necessarily bioavailable, although it can be in some cases.
[0051] In some cases, ophthalmic dosage forms may be used in effective dosing regimens to provide a therapeutically effective amount of copper-containing agent. Such effective dosing regimens may include administering the ophthalmic dosage form once, twice, three, four times, or more times daily.
[0052] In further examples, ophthalmic dosage forms may be formulated to biodegrade and provide controlled or sustained release of copper-containing agents over a predetermined period. In yet another example, ophthalmic dosage forms may be formulated to release copper-containing agents controlled or sustainedly from a non-biodegradable matrix. In these and other examples, the dosage form may be formulated to release copper-containing agents over a period of hours, days, or weeks as desired. In some specific examples, the dosage form may be formulated to deliver approximately 0.005 mcg to approximately 250 mcg of copper per week. In yet another example, the dosage form may be formulated to deliver approximately 0.008 mcg to approximately 200 mcg, approximately 0.01 mcg to approximately 150 mcg, or approximately 0.1 mcg to approximately 100 mcg per week. Furthermore, the dosage form may generally be formulated to have a zero-order drug-release kinetic.
[0053] The dosage form may be held or stored in a container as a premixed composition that can be administered immediately without further dilution or adjustment. In some embodiments, a single container may hold a volume or amount sufficient for a single dose. In yet another embodiment, a single container may hold a volume or amount sufficient for multiple doses.
[0054] Numerous suitable containers can be used. In one embodiment, the container may be amber in color. 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 cases, the container may contain approximately 0.005 mg to 1 mg of copper-containing agent. In other cases, the container may contain approximately 0.01 mg to 0.5 mg of copper-containing agent. In some cases, the container may contain approximately 0.001 mg to 0.5 mg of copper. In some cases, the container may contain approximately 0.005 mg to 0.2 mg of copper.
[0056] In some specific cases, the dosage form may be a topical ophthalmic dosage form formulated as eye drops and contained in a container adapted to dispense the composition in a dropper-by-dropper manner in a dropper volume of about 5 μl to about 50 μl. When the composition is formulated as eye drops, in some cases the container includes an adapted nozzle or tip from which the composition is dispensed. As such, the container may generally be foldable for dispensing the composition. However, in some cases, after the composition has been dispensed, air may be drawn back into the container, which can cause foreign matter contamination of the composition. In some cases, to prevent or minimize the introduction of bacteria or other foreign matter into the container, the nozzle or tip may include a valve mechanism, a filter, an analogue thereof, or a combination thereof. Furthermore, as previously discussed, the container may be adapted to dispense the ophthalmic composition in a dropper-by-dropper manner. For example, the container may be adapted to dispense the ophthalmic composition in drop volumes ranging from approximately 5 μl to approximately 50 μl, such as approximately 15 μl, approximately 20 μl, approximately 25 μl, approximately 30 μl, approximately 35 μl, approximately 40 μl, approximately 45 μl, or approximately 50 μl. In some specific examples, the drop volumes may be approximately 15 μl to approximately 40 μl, approximately 5 μl to approximately 30 μl, approximately 20 μl to approximately 30 μl, approximately 25 μl to approximately 35 μl, or approximately 30 μl to approximately 40 μl.
[0057] A dosage form may further include a mechanism of administration, such as a syringe, eye dropper, or other mechanism. In further embodiments, appropriate packaging may be used to provide the composition, container, and instructions for their use, as well as optionally the mechanism of administration, 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 during a treatment period. Such methods may be used to treat any of the conditions and similar ophthalmic conditions previously mentioned herein.
[0059] In one example, the composition or dosage form may be administered at one to four time points per day for each eye requiring it. The dosage at each time point may be approximately 5 μl to 50 μl, approximately 5 μl to 30 μl, approximately 20 μl to 30 μl, approximately 25 μl to 35 μl, or approximately 30 μl to 40 μl.
[0060] The duration of treatment can depend on numerous factors, including the type and severity of the condition, the patient's age at diagnosis, and similar conditions. Generally, the duration of treatment can range from about one month to chronic treatment, depending on the circumstances. For example, in some cases, younger children may receive chronic treatment, while older children and teenagers (e.g., 10-18 years) may receive treatment for two to three years, young adults (e.g., 18-30 years) may receive treatment for one to two years, and adults aged 30 or older may receive treatment for about six months to one year. Thus, in some cases, the duration of treatment can range from about one month to chronic treatment. In other cases, the duration of treatment can range from about one month, three months, or six months to about twelve months, eighteen months, twenty-four months, thirty months, thirty-six months, or until the condition is satisfactoryly resolved.
[0061] In some cases, the ophthalmic composition may be administered as eye drops. In other cases, the eye drops may be administered as subconjunctival injections. In yet another case, the eye drops may be administered in the form of topical films or contact lenses. In some cases, the topical films or contact lenses may be configured to biodegrade over time to provide controlled and sustained release of the copper-containing agent.
[0062] In general, the methods disclosed herein can increase collagen cross-linking in the cornea compared to an untreated eye. More specifically, the methods can increase lisinonorleucine cross-linking density, histidinyl-hydroxylisinonorleucine cross-linking density, or both, as well as other collagen cross-linking associated with the cornea, compared to an untreated eye. Furthermore, the methods disclosed herein can reduce corneal afferent strain by at least about 10%, 25%, or 50% compared to an untreated cornea. Furthermore, the methods disclosed herein can reduce corneal diopter in keratoconus compared to an untreated keratoconus. [Examples]
[0063] Example 1 - Copper-containing activators 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 0.0016 mg / mL CuSO4 in BSS control or equilibrium salt solution (BSS), and then filtered through a 0.25 μm filter. The prepared culture media were subjected to peroxidase-conjugated fluorescence quantitative activity analysis for LOX.
[0064] Tissue was collected under sterile conditions 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 containing penicillin / streptomycin. 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 in 10 cm tissue culture dishes and placed in a tissue culture incubator at 37°C with 5% CO2. After 5 days, cells were collected and seeded in 10% FBS DMEM without phenol red.
[0065] LOX enzyme activity in culture medium was measured using peroxidase-conjugated fluorescence quantitative analysis with Amplex Red. Briefly, corneal stromal cells were seeded in 6-well plates at 0.2 x 10^6 cells with 2 mL of culture medium. After 3 days, the medium was collected for LOX enzyme activity analysis. 50 μL of each culture medium was placed in a black 96-well plate. It was then placed in 50 μL of 2x analytical 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). As a parallel analysis, we added 500 μM aminopropion nitrile (BAPN), which can completely eliminate LOX activity. Fluorescence of Amplex Red oxide was recorded every 10 minutes using a fluorescence plate reader. After subtracting background fluorescence, fluorescence intensity was plotted against incubation time (Figures 1A and 1B).
[0066] Based on peroxidase-coupled fluorescence quantitative analysis of lox activity, and as illustrated in Figures 1A-1B, copper dramatically increased lox activity in normal and keratoconus fibroblasts. This suggests that copper may increase collagen cross-linking by increasing lox activity in keratoconus. [Examples]
[0067] Example 2 - Copper-containing surfactants can increase collagen cross-linking in the cornea. Sample preparation: Each human keratoconus was divided into two equal parts. One part was treated with 0.0016 mg / ml CuSO4 in Optisol solution for one week, while the other part was left untreated. After one week, each keratoconus was stored at -80°C for subsequent analysis.
[0068] Sample Extraction: Corneal samples were washed with PBS pH 7.4, powdered in liquid nitrogen, and crushed in a freezer mill. The corneal samples were demineralized with 0.5 M EDTA in 0.05 M Tris buffer pH 7.4 at 4°C for 48 hours. The insoluble fraction was washed twice with distilled water by centrifugation at 5000 g at 47°C for 20 minutes, and then dried and frozen. The dried and frozen samples were hydrolyzed at 110°C for 24 hours in vacuum using continuously boiling redistilled 6N HCl. The hydrolysates were neutralized with 2.5 N NaOH and dissolved in distilled water. Lisinonorleucine (LNL) and histidinohydroxylisinonorleucine (HHL) were analyzed separately by HPLC.
[0069] HPLC Method for Lisinonorleucine (LNL): Amino acid analysis is an important application area in protein chemistry and food analysis. Many different applications exist in this field. Automated pre-column derivatization and subsequent reverse-phase LC have become useful procedures for efficient analysis due to their ease of use and simplicity. For rapid and highly sensitive analysis of lisinonorleucine (LNL), the Agilent 1290 Infinity LC System was used in conjunction with automated pre-column derivatization. The column used for this method was Agilent ZORBAX Eclipse Plus RRHD C18 (3.0 mm x 50 mm, 1.8 μm), and the mobile phase consisted of Solvent A: 10 mM disodium phosphate + 10 mM sodium tetraborate pH 8.2 and Solvent B: ACN / MeOH / H2O (45:45:10).
[0070] HPLC method for histidinohydroxylysinonorleucine (HHL): 25 ml of protein hydrolysate sample (total amino acid concentration less than 3 mM) was mixed with 50 μl of 1.5 mM Ne-methyl-L-lysine hydrochloride compound (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 reacted at room temperature for 2 minutes. 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-crosslinking is tissue-specific. The main cross-crosslinking produced in human corneas is lisinonorleucine (LNL). LNL is a fundamental component for subsequent polyvalent cross-crosslinking, such as histidinyl-hydroxylisinonorleucine (HHL). Normal and keratoconus human corneas were treated with 0.0016 mg / ml CuSO4 for one week. HPLC analysis was then performed to measure the levels of LNL (Figure 2A-2C) and HHL (Figure 3A-3C).
[0072] In fact, significant increases in LNL and HHL levels were observed in copper-treated corneas. As illustrated in Figure 2A, LNL cross-linking was observed in human keratoconus treated with CuSO4 as disclosed above. In contrast, as illustrated in Figure 2B, LNL cross-linking was not observed in keratoconus not treated with CuSO4. Figure 2C illustrates the presence of LNL cross-linking even in normal corneas.
[0073] Similarly, as illustrated in Figure 3A, HHL cross-crossings are also observed in human keratoconus treated with CuSO4 as disclosed above. In contrast, as illustrated in Figure 3B, negligible levels of HHL cross-crossings were observed in keratoconus not treated with CuSO4. Figure 3C illustrates that HHL cross-crossings in normal corneas are at a higher level than those observed in keratoconus not treated with CuSO4.
[0074] Therefore, CuSO4 treatment clearly provided an increase in cross-crosslinking of both LNL and HHL in keratoconus. [Examples]
[0075] Example 3 - Treatment with a copper-containing agent increases corneal strength. Corneal afferent strain measurements were performed on human cadaveric corneas and rabbit corneas. Human cadaveric 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 samples contained small portions of sclera at both ends. Mineral oil was applied to the surface of the sclera-cornea-sclera fragment to minimize tissue dehydration. Rabbit corneas were divided into four groups. Two groups were treated with CuSO4 0.0025 mg / ml three times a day or CuSO4 0.0025 mg / ml once a day. Two groups served as controls, receiving only BSS.
[0076] The sample was conjugated between a motor and transducer to which a control pressure of 5–30 mmHg was applied, and the resulting centripetal strain was measured. The sample length between the two grip jaws was approximately 10 mm. Geometric information, including the sample width and thickness, was entered into the Rheometrics System Analyzer (RSA) control panel. Sample thickness was measured using an ultrasonic pachymeter (DGH 550 Pa-chette2; DGH Technology, Exton, PA).
[0077] The results show a significant increase in stiffness and a decrease in afferent strain after copper sulfate treatment (Figure 4). The mean peak afferent strain induced by an ocular pulse at 20 mmHg in the treated cornea was approximately 1.5 times lower than that in the untreated cornea. This indicates an approximately 50% increase in corneal strength and suggests that cross-crosslinking results in a stiffer corneal response. Furthermore, as illustrated in Figure 5, treatment with CuSO4 also provided biomechanics comparable to those observed with laser cross-crosslinking. [Examples]
[0078] Example 4 - Rabbit corneal topography Corneal strain in specific regions was monitored by corneal topography. Corneal topography results are expressed in diopter values. 0.0025 mg / ml copper sulfate (CuSO4) was administered to New Zealand white rabbits once daily and three times daily. In addition, a high-concentration sample of 0.025 mg / ml was administered to New Zealand white rabbits once daily during the treatment period. Three different controls were administered: no eye drops, BSS, or artificial tears. Each of the six groups contained six subjects. Topographic images were taken before treatment and weekly for five weeks. Diopter measurements were taken at mean K values and 3 mm. These values were compared to diopter measurements in laser-cross-crossed corneas at one month and one year.
[0079] As shown in Figure 6, a significant increase in diopter measurements was observed in the treatment group compared to the control group. Furthermore, one month of treatment with CuSO4 provided diopter measurements similar to those observed in laser cross-linking one year later. [Examples]
[0080] Example 5 - Safety of copper eye drops in vivo 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 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 their ocular tissue and compared to control rabbits (no eye drops). Aqueous humor, vitreous humor, retina, cornea, lens, and blood samples were analyzed for copper ions using inductively coupled plasma mass spectrometry (ICP-MS).
[0081] The rabbits (n=2) were euthanized 42 days later by intravenous injection of 0.3 mg / kg euthanasia solution, and their eyes were excised for histological evaluation and assessment of signs of inflammation, tissue damage, scarring, and fibrosis. The anterior portion of the eye was fixed in a 10% formaldehyde-glutaraldehyde solution, dehydrated in a stepwise series of alcohols, embedded in paraffin, and sectioned with a microtome. The sections were stained with appropriate stains (hematoxylin and eosin, as well as Masson Trichrome).
[0082] There was no effect of Cu treatment on electroretinogram (ERG) measurements. Furthermore, no toxicity was observed in any dissected ocular tissue. Moreover, although laser cross-crosslinking 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 images of the cornea stained with hematoxylin and eosin after 6 weeks of Cu treatment. Figure 7B illustrates images of the retina / choroid stained with hematoxylin and eosin after 6 weeks of Cu treatment.
[0083] It should be understood that the methods of disclosure above are merely illustrative of some embodiments of the present invention. Numerous modifications and alternative procedures can be devised by those skilled in the art without departing from the essence and scope of the present invention, and the appended claims are intended to cover such modifications and procedures. Thus, although the present invention is disclosed above with specificity and detail in relation to what is now considered the most practical and preferred embodiment 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 eye drop composition, An eye drop composition comprising a substantially pharmaceutically acceptable carrier and an activator, wherein the activator is copper in an amount of about 0.001 mg / ml to about 0.1 mg / ml.
2. The composition according to claim 1, wherein the copper is provided by a copper-containing agent selected from a copper salt, a copper covalent complex, or a combination thereof.
3. The composition according to claim 2, wherein the copper-containing agent is selected from the group consisting of copper sulfate, copper carbonate, copper acetate, copper chloride, copper gluconate, copper bromide, copper fluoride, copper nitrate, copper iodide, copper perchlorate, copper molybdate, copper thiocyanate, copper tartrate, copper tetrafluoroborate, copper selenide, copper pyrophosphate, GHK-copper, copper-histidine, copper-glycinate, and combinations thereof.
4. The composition according to claim 3, wherein the copper-containing agent is copper sulfate.
5. The composition according to claim 3, wherein the copper-containing agent is copper perchlorate.
6. A composition according to any one of claims 3 to 5, wherein the composition comprises about 0.001 wt% to about 0.1 wt% of the copper-containing agent.
7. The composition according to claim 6, wherein the composition comprises about 0.01 wt% to about 0.1 wt% of the copper-containing agent.
8. The composition according to claim 6, wherein the composition comprises about 0.001 wt% to about 0.01 wt% of the copper-containing agent.
9. A composition according to any one of claims 1 to 8, wherein the composition has a tensile strength of about 270 mOsm / L to about 350 mOsm / L.
10. A composition according to any one of claims 1 to 9, wherein the composition has a pH of about 5.5 to about 7.8.