BR112018009582B1 - BIFUNCTIONAL PEG-CONTAINING MOLECULES FOR USE IN INHIBITING CATARACTS AND PRESBYOPIA - Google Patents

Abstract

BIFUNCTIONAL PEG-CONTAINING MOLECULES FOR USE IN THE INHIBITION OF CATARACTS AND PRESBYOPIA. Described herein are bifunctional molecules for use in a method of inhibiting or reversing the progression of cataract formation or presbyopia in an eye, wherein the bifunctional molecule comprises an amine, succinimide, carboxylic acid, isocyanate, isothiocyanate, sulfonyl chloride, substituted or unsubstituted aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate, Nhydroxysuccinimide ester, imidoester, epoxide or fluorophenyl ester, covalently linked to a molecular bristle which is a polyethylene glycol. Both presbyopia and cataracts are caused by aggregation of soluble crystalline lens proteins called crystallins.

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[1] This application claims priority to US Provisional Application 62/254,863, filed November 13, 2015, which is incorporated herein by reference in its entirety.

FIELD OF DISCLOSURE

[2] The present disclosure relates to compositions and methods for inhibiting or reversing the progression of age-related changes in the lens of an eye.

BACKGROUND OF THE INVENTION

[3] The lens of the eye is a transparent structure that is suspended immediately behind the iris, and which brings light rays to a focus on the retina. The lens contains soluble and insoluble proteins; together, they constitute 35% of the wet weight of the lens. In a young, healthy lens, soluble proteins, commonly called lens proteins, constitute 90% of the lens proteins. During the aging process, the lenses of the lens form insoluble aggregates, which, at least in part, are responsible for the decreased deformability of the lens nucleus, which characterizes presbyopia, the loss of the eye's ability to change focus to see objects. Upcoming. The formation of insoluble lens aggregates in presbyopia is believed to be an early stage in the formation of age-related cataracts.

[4] Cataracts are defined as cloudiness or opacification in the lens of the eye. As an individual ages, cataracts form as the crystalline lenses present in the lens are converted into aggregates, resulting in increased opacity of the lens. Specifically, there is a progressive decrease in the concentration of the soluble chaperone, α-crystallin, in human lens nuclei with age, as it is incorporated into high molecular weight, insoluble protein aggregates. The presence of aggregates compromises the health and function of the lens, and when left untreated, cataracts can lead to substantial vision loss or even blindness. Currently, the most common treatment for cataracts is surgery.

[5] Crystallins are structural proteins most highly expressed in the lens fiber cells of the vertebrate eye. Crystallins are divided into two subfamilies: α-crystallins (αA and αB), which are members of the superfamily of small heat shock proteins, also functioning as structural proteins and molecular chaperones; and the evolutionarily linked superfamily of β- and -crystallins that function primarily as structural proteins in the lens and contribute to the transparency and refraction properties of the lens structure. In addition to their role in the development of cataracts, α-crystallin and αB-crystallin have been implicated in neurodegenerative diseases such as Alexander disease, Creutzfeldt-Jacob disease, Alzheimer's disease, and Parkinson's disease.

[6] Patent application US 2008/0227700 describes the disaggregation of proteins using peptides with chaperone activity as a therapeutic treatment. Specifically, aB-peptides were used to disaggregate pH-induced β-crystalline aggregates, measured by light scattering. Providing a continuous supply of alpha-crystallins into the lens is challenging. What is needed are suitable alternative methods for crystalline disaggregation for inhibition and/or reversal of cataracts and presbyopia.

SUMMARY OF THE INVENTION

[7] In one aspect, an ophthalmic composition comprises a bifunctional molecule, the bifunctional molecule comprising an amine, succinimide, carboxylic acid, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate , substituted or unsubstituted N-hydroxysuccinimide ester, imidoester, epoxide or fluorophenyl ester covalently linked to a molecular bristle, wherein the molecular bristle is a polyethylene glycol with 1 to 3 oxyethylene groups; an alkoxy-polyethylene glycol with 1 to 3 alkoxyethylene groups, or an aryloxypolyethylene glycol with 1 to 3 aryloxyethylene groups.

[8] In another aspect, a method of inhibiting or reversing the progression of cataract formation, presbyopia, or age-related degeneration of a crystalline lens in an eye comprises contacting the eye with a cataract-inhibiting effective amount of a ophthalmic composition comprising a bifunctional molecule, the bifunctional molecule comprising an amine, succinimide, carboxylic acid, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester , substituted or unsubstituted epoxide or fluorophenyl ester, covalently linked to a molecular bristle, wherein the molecular bristle is a polyethylene glycol having 1 to 3 oxyethylene groups, an alkoxy-polyethylene glycol having 1 to 3 alkoxyethylene groups, or an aryloxypolyethylene glycol having 1 to 3 aryloxyethylene groups.

[9] In another aspect, an ophthalmic composition further comprises at least one second bifunctional molecule comprising an amine, succinimide, carboxylic acid, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate , substituted or unsubstituted N-hydroxysuccinimide ester, imidoester, epoxide or fluorophenyl ester, covalently linked to a second molecular bristle, wherein the second molecular bristle is a linear or branched polyethylene glycol having 4 to 200 oxyethylene, alkoxyethylene or aryloxyethylene groups, poly (2-hydroxypropyl)methacrylamide (HPMA); poly(2-hydroxyethyl)methacrylate (HEMA), a layer (2-oxazoline), poly(m-phosphocholine), poly-lysine or polyglutamic acid, the second molecular bristle having a molecular weight of 150 to 8000.

BRIEF DESCRIPTION OF THE DRAWINGS

[10] Figure 1 shows modalities of functional groups for the bifunctional molecules disclosed here. In each structure, R is the molecular bristle.

[11] Figure 2 shows the measurement of the g2-1 correlation function plotted against the delay time (latency time) for the 30° scattering angle for unmodified human D-crystallin protein.

[12] Figure 3 shows the CONTIN weight analysis of various relaxation times for unmodified human crystalline yD-protein.

[13] Figure 4 shows data analysis for human D-crystallin protein using dynamic light scattering theory.

[14] Figure 5 shows the correlation of the measured g2-1 function plotted against the delay time (latency time) for the scattering angle of 30° for the CA(PEG)4 modified human D-crystallin protein .

[15] Figure 6 shows the CONTIN weight analysis of various relaxation times for unmodified human D-crystallin, modified with CA(PEG)4.

[16] Figure 7 shows data analysis for the CA(PEG)4 modified D-crystalline protein using dynamic light scattering theory.

[17] Figure 8 shows the correlation of the measured g2-1 function plotted against the delay time (latency time) for the scattering angle of 30° for the CA(PEG)1 modified human yD-crystallin protein.

[18] Figure 9 shows the CONTIN weight analysis of various relaxation times for unmodified, CA(PEG)1-modified D-crystalline human protein.

[19] Figure 10 shows data analysis for human D-crystallin protein modified with CA(PEG)1 using dynamic light scattering theory.

[20] Figure 11 shows the correlation of the measured g2-1 function plotted against the delay time (latency time) for the scattering angle of 30° for the CA(PEG)2 modified human D-crystallin protein .

[21] Figure 12 shows the CONTIN weight analysis of various relaxation times for unmodified human D-crystallin, modified with CA(PEG)2.

[22] Figure 13 shows data analysis for human D-crystallin protein modified with CA(PEG)2 using dynamic light scattering theory.

[23] Figure 14 shows the correlation of the measured g2-1 function plotted against the delay time (latency time) for the scattering angle of 30° for the CA(PEG)3 modified human D-crystallin protein .

[24] Figure 15 shows the CONTIN weight analysis of various relaxation times for the unmodified human γD-crystallin protein modified with CA(PEG)3.

[25] Figure 16 shows data analysis for human yD-crystalline protein modified with CA(PEG)3 using dynamic light scattering theory.

[26] The above-described and other characteristics will be appreciated and understood by those skilled in the technical field from the following detailed description, drawings and attached claims.

DETAILED DESCRIPTION OF THE INVENTION

[27] Disclosed herein are methods of disaggregating/preventing the formation of a crystalline aggregate comprising contacting the crystalline aggregate with a composition comprising a bifunctional molecule in an amount sufficient to disaggregate and/or prevent the formation of the crystalline aggregate. One skilled in the art recognizes that although the bifunctional molecules disclosed herein are generally described as -crystallin charge masking agents, they are also expected to further disaggregate/prevent aggregation of -crystallin protein. Further disclosed are methods of inhibiting or reversing the progression of cataract formation in an eye comprising contacting the eye with an effective cataract-inhibiting amount of an ophthalmic composition comprising the bifunctional molecules as described herein. Also disclosed are methods for inhibiting or reversing the progression of presbyopia in an eye comprising contacting the eye with an effective presbyopia-inhibiting amount of an ophthalmic composition comprising the bifunctional molecules described herein.

[28] The inventor used techniques such as dynamic light scattering to study the aggregates formed by -crystallines in solution. Both β- and -crystallins are highly stable structural proteins, comprising four Greek key motifs in two domains. While β-crystallins form dimers as well as hetero- and homo-oligomers, -crystallins are monomeric in the eye. Furthermore, while β-crystallins exhibit a repulsive force in solution, -crystallins exhibit an attractive interaction attributed to non-specific interactions with protein or water. It has also been hypothesized that thiol modifications cause -crystalline aggregates to form in solution.

[29] The human -crystallin family contains five members, the A-D crystallins and the -S crystallin. A-D crystallins are expressed early in development and are found mainly in the lens nucleus; C and D- crystalline are more prevalent. The unfolding and refolding of -D crystallin in vitro has been shown to lead to increased protein aggregation due to the lack of stability of the refolded protein.

[30] Without sticking to theory, it is believed that the aggregation of -crystallin is an electrostatic and hydrophobic phenomenon, with electrostatic forces dominating. Adding the heat shock proteins αA and αB-crystallin disrupts the aggregation of -crystallin. A -crystallin charge masking agent that can disrupt electrostatic interactions can replace the chaperone activity of α-crystallin and prevent/reduce the size of -crystallin aggregate.

[31] Administration of bifunctional molecules (e.g., -crystallin charge masking agents) can be used to treat diseases and/or conditions resulting from -crystallin aggregation such as cataracts and presbyopia. As used herein, a cataract is an opacity of the lens of the eye caused by altered protein interactions in the lens. Protein interactions include protein misfolding as well as protein-protein interactions such as aggregation. Presbyopia is the impairment of vision due to advancing years or old age. Symptoms of presbyopia include decreased ability to focus on close objects, eye strain, difficulty reading small print, fatigue when reading or looking at a bright screen, difficulty seeing clearly up close, less contrast when reading print, need for more light clear and more direct reading, need to hold reading material further away to see clearly, and headaches, especially headaches when using near vision. Individuals who suffer from presbyopia may have normal vision, but the ability to focus on nearby objects is at least partially lost over time, and these individuals begin to need glasses for tasks that require close vision, such as reading. Presbyopia affects almost everyone over the age of 40 to a greater or lesser extent.

[32] In the method of inhibiting the progression of cataract formation in an eye, the eye may already contain one or more developing or fully developed cataracts before coming into contact with the bifunctional molecules. Consequently, the method can be used to inhibit the formation of other cataracts in the eye, or to inhibit the formation of mature cataracts from developing cataracts already present in the eye. Alternatively, the eye may be free of any developing or fully developed cataracts before being contacted with the bifunctional molecules.

[33] In the method of reversing the progression of cataract formation in an eye, at least partial to complete reversal of cataracts in the eye is achieved by contacting the eye with a bifunctional molecule as disclosed herein.

[34] Similarly, in the method of inhibiting the progression of presbyopia in an eye, the individual may already be experiencing one or more symptoms of presbyopia before the eye is contacted with the bifunctional molecule. Consequently, the method can be used to reduce the progression of the symptom(s) experienced or to inhibit the formation of additional symptoms of presbyopia. Alternatively, the eye may be free of any symptoms of presbyopia before coming into contact with the bifunctional molecule.

[35] In the method of reversing the progression of presbyopia in one eye, at least partial to complete reversal of the symptoms of presbyopia in the eye is achieved by contacting the eye with a bifunctional molecule as disclosed herein.

[36] As used herein, a -crystallin charge masking agent is a molecule suitable for interfering with electrostatic protein-protein interactions, such as electrostatic protein-protein interactions of -crystallin, which lead to the aggregation of -crystallin . In one embodiment, the masking agent is not a polypeptide. γ-crystallin charge masking agents prevent -crystallin aggregates from forming and/or reduce the size of preformed aggregates.

[37] In a specific embodiment, the -crystallin charge masking agent is a bifunctional molecule containing a functional group covalently linked to a molecular bristle. The bifunctional molecule interacts with charges on -crystallin molecules, such as positively charged lysine and arginine residues and negatively charged glutamate and aspartate residues. The molecular bristle is a hydrophilic, water-soluble species that provides distance between the -crystalline molecules, preventing aggregation. Without sticking to theory, it is believed that the bifunctional molecule interacts with the protein and effectively places the molecular bristle on the protein, and the functional group can be expelled during the reaction. The molecular bristle, which can interact with crystallins via covalent, hydrophobic or ionic interactions, prevents the aggregation of -crystallin molecules. Without being bound by theory, it is believed that the bifunctional molecules described here can also act as inhibitors of the interaction with β-crystallin.

[38] Exemplary functional groups (which may be leaving groups or reactive groups, for example) include, by way of example, an amine, succinimide, carboxylic acid, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, azide substituted or unsubstituted acyl, anhydride, fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester, epoxide or fluorophenyl ester. Figure 1 shows embodiments of functional groups in which the R group is the molecular bristle. Without sticking to theory, when the bifunctional molecule contains COOH, water comes out when it reacts with the amine group of a protein. Without sticking to theory, when the bifunctional molecule contains N-hydroxysuccinimide, water is not released. In an NH2 reaction, water leaves when the NH2 reacts with a COOH group on the protein.

[39] Exemplary molecular bristles include linear or branched polyethylene glycols having one to three oxyethylene groups, modified polyethylene glycols such as alkoxy- and aryloxy polyethylene glycols having one to three oxyethylene, alkoxyethylene or aryloxyethylene groups. In a specific embodiment, the bifunctional -crystallin charge masking agent is

[40] In one embodiment, the ophthalmic composition further comprises at least one second bifunctional molecule, the at least second bifunctional molecule comprising an amine, succinimide, carboxylic acid, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, carbodiimide, azide substituted or unsubstituted acyl, anhydride, fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidoester, epoxide or fluorophenyl ester covalently linked to a second molecular bristle, wherein the second molecular bristle is a polyethylene glycol, an alkoxy-polyethylene glycol or a alkoxypolyethylene glycol with 4 to 200 oxyethylene, alkoxyethylene or aryloxyethylene groups; poly(2-hydroxypropyl) methacrylamide (HPMA); poly(2-hydroxyethyl)methacrylate (HEMA), a poly(2-oxazoline), poly(m-phosphocholine), poly-lysine or polyglutamic acid, the second molecular bristle having a molecular weight of 150 to 8000.

[41] The bifunctional molecule and the at least second bifunctional molecule can be present in any weight ratio, such as 1:99 to 99:1.

[42] In one embodiment, the bifunctional molecules described herein are also useful in treating diseases related to protein misfolding, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. In a specific embodiment, the bifunctional molecules are administered as oral compositions in the treatment of diseases related to protein misfolding.

[43] An advantage of the -crystalline charge masking agents described herein is that they are expected to be effective in the presence of -crystalline post-translational modifications, including, for example, transamidation, oxidation, modified and dysfunctional connections, and high concentrations of inorganic and organic ions, such as Ca+2.

[44] Bifunctional molecules are placed in contact with the eye to inhibit cataract progression and/or reduce existing cataracts or to inhibit and/or reduce the symptoms of presbyopia. As used herein, the term “eye contact” encompasses methods of directly applying the bifunctional molecules to the eye. In the method described above, suitable means known to those skilled in the art can be used to contact the eye with the bifunctional molecules. Examples of such methods include, but are not limited to, the compound being injected into the eye, being dripped or sprayed into the eye, applied in the form of an ophthalmic device, applied by iontophoresis, or otherwise applied topically to the eye.

[45] As used herein, the term “effective cataract-inhibiting amount” means an amount that will inhibit the progression or formation of cataracts in an eye or will inhibit the progression or formation of mature cataracts from developing cataracts already present in the eye. The effective cataract-inhibiting amount of the -crystalline charge masking agent will depend on several factors known to those skilled in the art. Such factors include, but are not limited to, the size of the eye, the number and progression of any fully developed or developing cataracts already present in the eye, and the mode of administration. The effective cataract-inhibiting amount will also depend on whether the pharmaceutical composition is to be administered once, or whether the pharmaceutical composition is to be administered periodically over a period of time. The time period can be any number of days, weeks, months, or years. In one embodiment, the effective cataract-inhibiting amount of the γ-crystallin charge masking agent, specifically the bifunctional molecules described herein, is about 0.001 g to about 0.1 g. More specifically, the effective cataract-inhibiting amount may be from about 0.01 g to about 0.05 g. Exemplary amounts of the bifunctional molecules in the ophthalmic compositions are 0.00005 wt % to 50 wt %, or more, for example, more specifically 0.01 wt % to 10 wt %, and more specifically 1 wt % to 3 % by weight.

[46] As used herein, the term “effective presbyopia-inhibiting amount” means an amount that will reduce a symptom of presbyopia in an eye or inhibit the progression of additional symptoms of presbyopia in the eye. The effective presbyopia-inhibiting amount of the γ-crystalline charge masking agent will depend on several factors known to those skilled in the art. Such factors include, but are not limited to, the size of the eye, the number and type of symptoms already present in the individual, and the mode of administration. The effective cataract-inhibiting amount will also depend on whether the pharmaceutical composition is to be administered once, or whether the pharmaceutical composition is to be administered periodically over a period of time. The time period can be any number of days, weeks, months, or years. In one embodiment, the effective presbyopia-inhibiting amount of the β-crystallin charge masking agent, specifically the bifunctional molecules described herein, is about 0.001 g to about 0.1 g. Specifically, the effective presbyopia-inhibiting amount may be from about 0.01 g to about 0.05 g. Exemplary amounts of the bifunctional molecules in the ophthalmic compositions are 0.00005 wt % to 50 wt %, or more, for example, more specifically 0.01 wt % to 10 wt %, and more specifically 1 wt % to 3 % by weight.

[47] As used herein, the term “ophthalmic composition” refers to a pharmaceutically acceptable formulation, delivery device, mechanism or system suitable for administration to the eye. The term “ophthalmic compositions” includes, but is not limited to, solutions, suspensions, gels, ointments, sprays, depot devices, or any other type of formulation, device, or mechanism suitable for short- or long-term delivery of charge masking agents. from crystallin, for example, bifunctional molecules, to the eye. In contrast to oral formulations, for example, ophthalmic compositions exhibit specific technical features associated with their application to the eyes, including the use of pharmaceutically acceptable ophthalmic vehicles that avoid the induction of various reactions such as, for example, irritation of the conjunctiva and cornea, closure of the eyelids, tear secretion and painful reactions. Specific ophthalmic compositions are advantageously in the form of ophthalmic solutions or suspensions (i.e., eye drops), ophthalmic ointments or ophthalmic gels containing crystalline charge masking agents, e.g., bifunctional molecules. Depending on the particular form selected, the compositions may contain various additives, such as buffering agents, isotonizing agents, solubilizers, preservatives, viscosity increasing agents, chelating agents, antioxidant agents, antibiotics, sugars and pH regulators.

[48] Examples of preservatives include, but are not limited to, chlorobutanol, sodium dehydroacetate, benzalkonium chloride, pyridine chlorides, phenethyl alcohols, parahydroxybenzoic acid esters, benzethonium chloride, dihalogenated hydrophilic copolymers of ethylene and dimethyl ethyleneimine, mixtures thereof and the like. Viscosity increasing agents can be selected, for example, from methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, carboxymethylcellulose, chondroitin sulfate and their salts. Suitable solubilizers include, but are not limited to, polyoxyethylene hydrogenated castor oil, polyethylene glycol, polysorbate 80 and polyoxyethylene monostearate. Typical chelating agents include, but are not limited to, sodium edetate, citric acid, salts of diethylenetriamine pentaacetic acid, diethylenetriaminepentamethylenephosphonic acid, and stabilizing agents such as sodium edetate and sodium hydrogen sulfite.

[49] Exemplary buffers include, but are not limited to, borate buffers, phosphate buffers, carbonate buffers, acetate buffers, and the like. The buffer concentration in ophthalmic compositions can vary from about 1 mM to about 150 mM or more, depending on the particular buffer chosen.

[50] As used herein, the term “vehicle” is intended to include a carrier, diluent or excipient suitable for ophthalmic use. “Excipient” refers to an ingredient that provides one or more bulk, satisfactory processing characteristics, helps control the rate of dissolution, and otherwise provides additional desirable characteristics to the compositions. In particular, the excipients are selected in such a way that the ophthalmic composition does not trigger a secretion of tears that will carry away the active ingredient. Acceptable excipients are well known to those skilled in the art, who know how to select them depending on the desired formulation.

[51] In one embodiment, the -crystalline charge masking agent is administered in the form of an ophthalmic device, such as a contact lens or a tear punctum patch. Suitable ophthalmic devices include biocompatible devices with a corrective, cosmetic or therapeutic quality.

[52] In one embodiment, the -crystalline charge masking agent can be adhered, incorporated or associated with a contact lens, optionally as a sustained release composition. Contact lenses can be produced using known materials, for example, without limitation, hydrogels, silicone hydrogels, silicone elastomers and gas permeable materials such as polymethyl methacrylate (PMMA), methacrylic acid ester polymers, copolymers of oligosiloxanylalkyl monomers (meth)acrylate/methacrylic acid and the like. Specific examples of water-containing soft ophthalmic lens materials include those described in U.S. Pat. US No. 5,817,726, 2-hydroxyethyl methacrylate polymers as described in U.S. Pat. US No. 5,905,125, ophthalmic lens materials as described in European Patent Application No. 781,777, the hydrogel lens that is coated with a lipid layer in advance as described in U.S. Pat. No. 5,942,558; all incorporated here by his teachings on contact lenses. Generally used contact lenses, such as hard or rigid corneal-type lenses, and gel, hydrogel or soft-type lenses that are produced from the above known materials, can be used.

[53] It is common in the contact lens industry to characterize contact lenses into two main categories; conventional and silicone hydrogels. Conventional-based hydrogels began as poly(hydroxyethyl methacrylate) (poly-HEMA) and evolved into copolymers of poly-HEMA with other hydrophilic moieties such as n-vinylpyrrolidone (nVP), acrylamide, dimethylacrylamide, and methacrylated phosphorylcholine. Polyvinyl alcohol lenses can also be used.

[54] Silicone hydrogels (SiH) typically consist of copolymers of methacrylated or met(acrylamide) silicone monomers, prepolymers or macromers with typical conventional hydrogel monomers. Examples of silicone monomers include, without limitation, trisubstituted, tetrasubstituted, alkyl terminated, methacrylated polydimethylsiloxane (PDMS) and block copolymers of silicone and hydrophilic monomers. ABA triblock copolymers are common where group A is a hydrophilic block and group B is the monomeric silicone block. In addition to methacrylates, other reactive groups include vinyl, acrylamide, or any other reactive group capable of chain polymerization. Cross-linking and polymerization can also be achieved through step growth and other polymerization methods using monomers with at least bi-functionality. An example of step-growth polymerization is the reaction of a hydroxyl group with a carboxylic acid group in two amino acids or from terephthalic acid and ethylene glycol.

[55] Plasma-based coating methods are commonly used on silicone hydrogels, including plasma oxidation and plasma coatings.

[56] An extended release -crystalline charge masking agent composition can be produced, for example, by incorporating, associating or adhering to the contact lens the -crystalline charge masking agent composition in accordance with the known methods for producing contact lenses with extended release drugs as described in US Patent No. 5,658,592; 6,027,745; WO2003/003073; US-2005-0079197, incorporated herein by its teachings on contact lenses and extended release, or by other appropriate means. Specifically, without limitation, the contact lens can be produced by adhering the -crystalline charge masking agent to a portion of a finely divided or gel extended release agent such as polyvinylpyrrolidone, sodium hyaluronate and the like. Furthermore, sustained release can be produced by forming a reservoir of the α-crystalline charge masking agent composition, such as by producing a contact lens from a member that forms a front surface of the lens and a member that forms a posterior surface of the lens.

[57] In one embodiment, the charge masking agent can be inserted into the aqueous or vitreous as a sustained release injection.

[58] In one embodiment, the -crystallin charge masking agent is administered into a tear punctum plug. As used herein, the term lacrimal punctum plug refers to a device of suitable size and shape for insertion into the inferior or superior lacrimal canaliculus of the eye through, respectively, the inferior or superior lacrimal punctum.

[59] In one embodiment, the -crystallin charge masking agent is administered by iontophoresis. Iontophoresis is a technique that uses a small electrical charge to deliver a medicine or other chemical through the skin.

[60] In one embodiment, the ophthalmic composition is administered using ultrasound intensification.

[61] The invention is further illustrated by the following non-limiting examples:

Examples Methods

[62] The γD- and S-crystallin DNA sequences are in the pQe1 plasmids that were provided by King Labs at the Massachusetts Institute of Technology (Cambridge, MA). The  -crystallin protein sequences contain a 6x N-terminal histidine tag (its tag) for purification purposes. The plasmids were transformed into a cloning-competent cell line to create additional plasmid DNA. The plasmids were subsequently transformed into an expression-competent cell line for protein synthesis (E. coli TAM 1 cells (Active Motif. Carlsbad, CA)).

[63] -crystallin plasmid DNA was chemically transformed into E. coli M15pRep cells for protein synthesis. 1 L cultures were grown for protein purification. The -crystallin protein was purified by Ni affinity chromatography. The N-terminal His tag contained in -crystallin proteins preferentially binds to the Ni column. The bound protein can be eluted with an imidazole gradient that competitively binds Ni, releasing the purified protein. The purification procedure is brief (as before): the protein is loaded onto the column and washed first with 20 mM (0% B), then 35 mM (10% B), then 60 mM (20% B) imidazole. The protein is then eluted in 150 mM (57% B) imidazole. Purity was confirmed by SDS PAGE gel electrophoresis and fast protein liquid chromatography (FPLC). Chemical modification protocol: 1) A batch of purified protein was buffer exchanged in PBS pH = 6.8 using a HiTrap® desalting column. (2) Concentration (A280) was measured before adding CA(PEG)n. (3) 100 mg of CA(PEG)n was added to a 3 to 5 ml batch of the above protein. (4) The mixture was placed on a shaker at 4 °C at 250 rpm overnight.

Example 1: Dynamic light scattering of unmodified D- crystalline protein

[64] First, an unmodified D- crystalline protein solution at a concentration of 0.1 mg/ml was studied by DLS. The correlation of the measured g2 function is plotted against the delay time (latency time) is given for the 30° scattering angle in Figure 2. The CONTIN analysis of the weight of various relaxation times is given in Figure 3 for angles of scattering ranging from 30° to 60°. It is clear that there are two populations in the solution, one corresponding to the aggregates and one corresponding to the non-aggregated protein. Analysis of these data using dynamic light scattering theory, as presented in Figure 4, shows that the corresponding hydrodynamic radius of rotation is respectively.

Example 2: Dynamic light scattering of crystalline D protein modified with CA(PEG)4

[65] The results corresponding to Example 1 for modifications by CA(PEG)4 are given in Figures 5-7, with the result that the aggregate is absent and the size of the non-aggregated protein is 2 nm. The concentration is 0.9 mg/ml protein and 33 mg/ml CA(PEG)4. CA(PEG)4 is

Example 3: Dynamic light scattering of the protein Dcrystallin modified with CA(PEG)1

[66] The results corresponding to Example 1 for modifications by CA(PEG)1 are given in Figures 8-10, with the result that the aggregate is absent and the size of the non-aggregated protein is 2 nm. The concentration is 0.9 mg/ml protein and 33 mg/ml CA(PEG)1. CA(PEG)1 is

Example 4: Dynamic light scattering of crystalline D protein modified with CA(PEG)2

[67] The results corresponding to Example 1 for modifications by CA(PEG)2 are given in Figures 11-13, with the result that the aggregate is absent and the size of the non-aggregated protein is 2 nm. The concentration is 0.4 mg/ml protein and 15 mg/ml CA(PEG)2. CA(PEG)2 is

Example 5: Dynamic light scattering of crystalline D protein modified with CA(PEG)3

[68] The results corresponding to Example 1 for modifications by CA(PEG)3 are given in Figures 14-16, with the result that the aggregate is absent and the size of the non-aggregated protein is 2 nm. The concentration is 0.3 mg/ml protein and 11 mg/ml CA(PEG)3. CA(PEG)3 is

[69] Conclusion: All CA(PEG)1-4 are effective agents in completely preventing aggregation of the yD-crystalline protein, as determined by dynamic light scattering.

[70] The term “substituted”, as used herein, means that any one or more hydrogens on the designated atom or group is replaced by a substituent, provided that the normal valence of the designated atom is not exceeded. Exemplary substituents include, independently, one or more alkyl groups, phenyl groups, cycloalkyl groups, halogens, halogenated alkyl groups and others, and/or combinations thereof.

[71] As used herein, an "alkyl group", alone or as part of a larger moiety (alkylamine, alkoxy and the like) is preferably a straight or branched chain saturated aliphatic group having 1 to about 12 carbon atoms, e.g. example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl, or a saturated cycloaliphatic group having 3 to about 12 carbon atoms. The alkyl groups may be optionally independently substituted, such as with groups selected from C1-C12 alkyl, C6-C14 aryl, alkoxy, amine, esters such as COO(R), acetates such as -O-CO-(R), ethers such as -CO(R) and -O(C1-C12 alkyl), -COOH, halogens (preferably, F, Cl, Br or I), fluorinated or perfluorinated C1C12 alkyl, -OH, S(R), -SiO( R2), -Si(R3) and the like, wherein each R is independently a C1-C12 alkyl, C3-C10 cycloalkyl or C6-C14 aryl group.

[72] The term “cycloalkyl”, as used herein, means saturated cyclic hydrocarbons, that is, compounds in which all ring atoms are carbons. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[73] As used herein, an "aryl", alone or as part of a larger moiety (e.g., diarylamine) is a carbocyclic aromatic group, preferably including 622 carbon atoms. Examples include, but are not limited to, carbocyclic groups such as phenyl, naphthyl, biphenyl and phenantryl, and may optionally be independently substituted, such as with groups selected from C1-C12 alkyl, C6-C14 aryl, alkoxy, amine, esters such as COO(R), acetates such as -O-CO-(R), ethers such as -CO(R) and -O(C1-C12 alkyl), -COOH, halogens (preferably F, Cl, Br, or I), fluorinated or perfluorinated C1-C12 alkyl, -OH, S(R) and -Si(R3) and the like, wherein each R is independently a C1-C12 alkyl, C3C10 cycloalkyl or C6-C14 aryl group.

[74] The terms “alkoxy”, as used herein, mean an “alkyl-O-” group, where alkyl is defined above. Examples of an alkoxy group include methoxy or ethoxy groups.

[75] As used herein, the term amine includes NH2 as well as alkylamines, arylalkylamines and arylamines, which are secondary or tertiary alkyl or arylalkyl amino groups, wherein the alkyl groups and aryl groups are as defined above. The attachment point of alkylamine, arylamine, or arylalkylamine is at the nitrogen. Examples include methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, dipropylamine, ethylpropylamine, ethylhexylamine, cyclohexylamine, phenethylamine, phenylisopropylamine and the like. Alkylamines, arylalkylamines and arylamines are not limited, and may be, for example, C1-C20 alkylamines, C6-C22 arylalkylamines, C6-C22 arylamines.

[76] The terms “one” and “ones” do not denote a quantity limitation, but denote the presence of at least one of the referenced items. The term “or” means “and/or”. The terms “comprising”, “having”, “including” and “containing” should be interpreted as open terms (i.e. “including, but not limited to”). All tracks published here are inclusive and combinable.

[77] Embodiments are described herein, including the best modes known to the inventors. Variations of such embodiments will become apparent to those skilled in the art upon reading the foregoing description. It is expected that one skilled in the art will utilize such variations as appropriate, and it is expected that the disclosed modes will be practiced differently than specifically described herein. Accordingly, all modifications and equivalents of the subject matter recited in the appended claims are included to the extent permitted by applicable law. Furthermore, any combination of the above-described elements in all their possible variations is contained unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims (9)

1. Ophthalmic composition characterized by comprising a bifunctional molecule, wherein the bifunctional molecule is 2. Ophthalmic composition, according to claim 1, the ophthalmic composition being characterized by being an eye drop. 3. Ophthalmic composition according to claim 1, the ophthalmic composition being characterized by being in the form of an ophthalmic solution, an ophthalmic suspension, an ophthalmic ointment, a spray or an ophthalmic gel. 4. Ophthalmic composition according to claim 3, the ophthalmic composition being characterized by including a buffering agent, an isotonizing agent, a solubilizer, a preservative, a viscosity increasing agent, a chelating agent, an antioxidant agent, a antibiotic, a sugar, a pH regulator or a combination thereof. 5. Ophthalmic composition according to claim 1, the ophthalmic composition being an ophthalmic device. 6. Ophthalmic composition according to claim 5, characterized by the fact that the ophthalmic device is in the form of a contact lens or a punctal plug. 7. Ophthalmic composition according to claim 1, the ophthalmic composition being characterized by being a sustained release composition. 8. Use of a composition as defined in any one of claims 1 to 7, characterized in that it is for the preparation of a medicament for inhibiting or reversing the progression of cataract formation, presbyopia or age-related degeneration of a crystalline lens in an eye . 9. Use, according to claim 9, characterized by the fact that said ophthalmic composition is administered by means of drip, spray, injection, iontophoresis or ultrasound intensification.