WO2009044294A2 - Means and methods for the treatment of cataract and presbyopia - Google Patents

Abstract

The present invention relates to an inhibitor of a protein, wherein said protein (i) comprises or consists of the amino acid sequence of SEQ ID NOs: 2 or 4, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NOs: 1 or 3; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 25; (iii) is a fragment of the protein according to (i), or has a sequence at least 75% identical with the protein according to (i) or with said fragment of the protein according to (i), and exhibits NADPH oxidase activity; or (iv) is a fragment of the protein according to (ii) or has a sequence at least 75% identical with the protein according to (ii) or with said fragment of the protein according to (ii) and stabilizes said protein exhibiting NADPH oxidase activity; and/or an inhibitor of a nucleic acid, wherein said nucleic (v) comprises or consists of the nucleic acid sequence of SEQ ID NO: 1 or 3, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 2 or 4; (vi) comprises or consists of the nucleic acid sequence of SEQ ID NO: 25, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 26; (vii) is a fragment of the nucleic acid according to (v), or has a sequence at least 75% identical with the nucleic acid according to (v) or with said fragment of the nucleic acid according to (v), and encodes a protein exhibiting NADPH oxidase activity; or (viii) is a fragment of the nucleic acid according to (vi), or has a sequence at least 75% identical with the nucleic acid according to (vi) or with said fragment of the nucleic acid according to (vi), and encodes a protein stabilizing said protein exhibiting NADPH oxidase activity; for treatment and/or prevention of cataract and/or presbyopia. The sequences define NADPH oxidases or a subunit of an NADPH oxidase, respectively. A preferred inhibitor is a siRNA. Also provided are a method of treating cataract and presbyopia, pharmaceutical compositions, medical uses and diagnostic uses of compounds of the invention.

Description

DESCRIPTION

MEANS AND METHODS FOR THE TREATMENT OF CATARACT AND

PRESBYOPIA

BACKGROUND OF THE INVENTION

This application claims priority of U.S. provisional patent application No. 60/944,218 filed on June 15, 2007 which is hereby incorporated by reference in its entirety.

Cataract (for reviews see Heitmancik and Kantorow, 2004; Spector, 2000) formation is an opacification of the ocular lens and is the leading course of blindness in the world. It occurs in the elderly or in individuals with particular risk factors (e.g., diabetes mellitus). Cataract leads to severe visual impairment and loss of independence and life quality. Cataract surgery is at present the only curative treatment of cataract, yet, it is not a problem-free solution. 30-50% of patients develop posterior capsule opacification in the years following cataract surgery. Other complications following cataract surgery include retinal detachment (-0.8%); corneal edema (-0.9%), and complications as serious as endophtalmitis (-0.1%). Given the fact that cataract surgery is the most frequently performed surgery in the elderly (e.g., over 1.3 million cataract operations per year in the U.S.) this becomes an important public health problem. Thus, alternative solutions, in particular drugs that might stop or at least slow down the course of the disease are urgently needed.

The pathogenesis of cataract formation is complex and also depends on the specific form of cataract. However there are some elements that are probably common to many different types of cataract. One of them are reactive oxygen species (ROS). This can be directly observed in the rare cases of cataract in patients subjected to hyperbaric oxygen. While in the latter case, the source of the oxidative stress is clear, this is much less clear for other types of cataract. Dysfunction of mitochondrial respiration leading to ROS generation has been typically implied in age-associated ROS-dependent diseases. However, more recently a novel family of RO S -generating NADPH oxidases and its possible relationship with age-associated diseases has received increased attention, see for example Zekry et al. (2003). As opposed to other sources of ROS, NOX enzymes purposefully produce large amounts of ROS. Their physiological function lies in host defense, redox-sensitive biosynthetic processes and redox-sensitive signalling mechanisms. Yet, under pathological conditions, ROS generation by these enzymes can lead to tissue damage and development of age-associated disease.

Presbyopia affects virtually 100% of the population by the age of 50. The affected persons lose their ability for accommodation and thus for focusing near objects. The exact reason for the development of presbyopia is unknown, and many hypotheses have been proposed. Recent results suggest that one of the relevant causes of presbyopia is an increased stiffness of matrix proteins of lens and/or capsule (Koopmans et al, 2003, Heys et al, 2004).

Stiffness of matrix proteins is typically caused by crosslinking of matrix proteins through reactive oxygen species. Thus, cataract and presbyopia appear to be two closely related reactions of matrix proteins to oxidative stress (McGinty and Truscott, 2006).

Over the last decade, it has been proven that the expression of superoxide-generating NADPH oxidases is not restricted to phagocytes. Beside the well-known catalytic subunit of the phagocyte NADPH oxidase, gp91^^ox/NOX2 (for review see Babior et al, 2002), six other superoxide-producing enzymes have been identified in mammals (Bokoch and Knaus, 2003; Lambeth, 2002; Bedard and Krause, 2007). For most NOX and DUOX enzymes, a predominant tissue localization has been described, e.g. colon epithelium for NOXl (Suh et al, 1999 ; Banfi et al, 2000), kidney cortex for N0X4 (Geiszt et al, 2000), lymphoid organs and testis for N0X5 (Banfi et al, 2001), and the thyroid gland for DUOXl and DU0X2 (De Deken et al, 2000 ; Caillou et al, 2001). For N0X3, very low level expression in the embryonic kidney (Kikuchi et al, 2000) has been found. More recently, expression of N0X3 in the inner ear has been demonstrated (Banfi, Malgrange et al, 2004).

Our knowledge of the activation mechanisms of members of the NOX/DUOX family varies considerably among individual enzymes. NOXl and gp9F^Aα7NOX2 are subunit- dependent enzymes that need to assemble with an activator subunit (NOXAl and \)61^phox, respectively) and an organizer subunit (NOXOl and p47^^ox, respectively) to generate superoxide (Babior, 1999; Banfi et al, 2003 ; Geiszt et al, 2003 ; Takeya et al, 2003). N0X5, DUOXl and DU0X2, on the other hand, have N-terminal Ca²⁺-binding motifs (EF- hand domains), and so far one of them, N0X5, has been shown to be activated by increased Ca²⁺ concentration (Banfi, Tirone et al, 2004). The N-terminal EF hand region of N0X5 has to interact with the N0X5 C-terminus for protein activation. Melittin can bind to the N- terminus of NOX5, thereby interfering with the binding of the N-terminus to the C-terminus and inhibiting enzyme activity (Banfϊ, Tirone et ah, 2004).

A recent publication raises the possibility that NOX enzymes are involved in ROS generation by epithelial cells of the ocular lense (Rao et ah, 2004). However no identification of such enzymes has been performed. While introducing a distinction between phagocytic and non-phagocytic NADPH oxidases, the authors fail to identify a specific non-phagocytic NADPH oxidase in the lens. Rather, immunological and PCR data are provided indicating the presence in the eye lens of non-catalytic NADPH oxidase subunits and of gp91phox, which is the NADPH oxidase also occurring in phagocytes.

In view of the limited understanding of processes leading to cataract and presbyopia, the technical problem underlying the present invention was therefore the provision of means and methods for treatment of cataract and presbyopia.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to an inhibitor of a protein, wherein said protein (i) comprises or consists of the amino acid sequence of SEQ ID NOs: 2 or 4, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NOs: 1 or 3; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 25; (iii) is a fragment of the protein according to (i), or has a sequence at least 75% identical with the protein according to (i) or with said fragment of the protein according to (i), and exhibits NADPH oxidase activity; or (iv) is a fragment of the protein according to (ii) or has a sequence at least 75% identical with the protein according to (ii) or with said fragment of the protein according to (ii) and stabilizes said protein exhibiting NADPH oxidase activity; and/or an inhibitor of a nucleic acid, wherein said nucleic (v) comprises or consists of the nucleic acid sequence of SEQ ID NO: 1 or 3, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 2 or 4; (vi) comprises or consists of the nucleic acid sequence of SEQ ID NO: 25, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 26; (vii) is a fragment of the nucleic acid according to (v), or has a sequence at least 75% identical with the nucleic acid according to (v) or with said fragment of the nucleic acid according to (v), and encodes a protein exhibiting NADPH oxidase activity; or (viii) is a fragment of the nucleic acid according to (vi), or has a sequence at least 75% identical with the nucleic acid according to (vi) or with said fragment of the nucleic acid according to (vi), and encodes a protein stabilizing said protein exhibiting NADPH oxidase activity; for treatment and/or prevention of cataract and/or presbyopia.

The term "inhibitor" designates a compound lowering the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be inhibited is lowered, (ii) the translation of the mRNA encoding the protein to be inhibited is lowered, (iii) the protein performs its biochemical function with lowered efficiency in presence of the inhibitor, and (iv) the protein performs its cellular function with lowered efficiency in presence of the inhibitor. Class (iv) includes compounds which do not necessarily directly bind to the target molecule, but still interfere therewith, for example by binding to and/or inhibiting the function or inhibiting expression of members of a pathway which comprises the target molecule. The term "target molecule" refers to the protein and nucleic acids, respectively, as defined herein above.

Compounds falling in class (i) include compounds interfering with the transcriptional machinery and/or its interaction with the promoter of said gene and/or with expression control elements remote from the promoter such as enhancers. Compounds of class (ii) comprise antisense constructs and constructs for performing RNA interference well known in the art (see below). Compounds of class (iii) interfere with molecular function of the protein to be inhibited, in case of an NADPH oxidase with its enzymatic activity. Accordingly, active site binding compounds, in particular compounds capable of binding to the active site of any NADPH oxidase, are envisaged. More preferred are compounds specifically binding to an active site of NADPH oxidase. Also envisaged are compounds binding to or blocking substrate binding sites of NADPH oxidase. Class (iv) includes compounds which do not necessarily directly bind to NADPH oxidase, but still interfere with NADPH oxidase activity, for example by binding to and/or inhibiting the function or inhibiting expression of members of a pathway which comprises NADPH oxidase. These members may be either upstream or downstream of NADPH oxidase within said pathway.

In a further preferred embodiment, the inhibitor is a low molecular weight compound.

Low molecular weight compounds are compounds of natural origin or chemically synthesized compounds, preferably with a molecular weight between 100 and 1000, more preferred between 200 and 750, and even more preferred between 300 and 600. Low molecular weight compounds are particularly preferred when said compounds inhibit the molecular function of the protein as defined above.

The efficiency of the inhibitor can be quantitized by comparing the level of activity in the presence of the inhibitor to that in the absence of the inhibitor. For example, as an activity measure may be used: the change in amount of mRNA formed, the change in amount of protein formed, the change in amount of substrate converted or product formed, and/or the change in the cellular phenotype or in the phenotype of an organism.

In a preferred embodiment, the level of activity is less than 90%, more preferred less than 80%, 70%, 60% or 50% of the activity in absence of the inhibitor. Yet more preferred are inhibitors lowering the level down to less than 25%, less than 10%, less than 5% or less than 1% of the activity in absence of the inhibitor. The term "inhibitor" refers to compounds which partially or completely abolish activity of the target molecule. Inhibitors completely abolishing activity include compounds which irreversibly bind the target molecule.

It is furthermore preferred that said inhibitor is specific for the protein or nucleic acid as defined above. In particular, it is preferred that said inhibitor is specific for the NOX proteins as defined in the main embodiment or the nucleic acids encoding them as defined in the main embodiment, respectively, as opposed to the remainder of NOX proteins and NOX subunits as described herein above. Specificity may be determined in comparative assays the skilled person will consider without further ado. For example, the effect of an inhibitor on cells expressing a protein as defined above may be compared to the effect of said inhibitor on cells expressing, for example, N0X2. The effect of the inhibitor is quantified by means of measuring ROS production as detailed in Example 1. As controls, ROS production of the respective cells is determined in absence of said inhibitor. A stronger reduction of ROS production upon addition of the inhibitor to cells expressing a protein as defined above as compared to reduction of ROS production upon addition of the inhibitor to cells expression N0X2 is indicative of an inhibitor specific for the protein as defined above. Specificity of the inhibitors of the invention for the proteins or nucleic acids as defined above as compared to non-NOX proteins and nucleic acids encoding such non-NOX proteins may involve determination of the activity of said non-NOX proteins in presence and absence, respectively, of said inhibitor. Suitable assays for the activity of said non-NOX proteins will depend on the particular type of non-NOX proteins to be assayed and will be contemplated by the skilled person without further ado.

The term "activity" refers to the biological activity of the recited proteins and includes NADPH oxidase activity, i.e., the capability to oxidize NADPH and concomitantly reduce molecular oxygen to yield superoxide. NADPH oxidase activity may be determined and quantified by means and methods known in the art and described herein below; see, for example, Example 1. The term "activity" furthermore includes the stabilizing effect exerted on NADPH oxidases by p22phox as well as fragments and homologues thereof according to the invention. The stabilizing activity of these homologues and fragments of p22phox may be determined, for example, by measuring the protein amount of NADPH oxidase in a cell, the cell (i) having the p22phox being knocked out and (ii) being transformed with the above defined nucleic acid encoding said fragment or homologue if p22phox. A comparable amount of NADPH oxidase in said cell on the one side and a wild type cell on the other side is indicative of homologues or fragments, respectively, of p22phox exhibiting stabilizing activity on said NADPH oxidase.

The term "NADPH oxidase" comprises any NADPH oxidase. It includes NOX enzymes such as NOXl, N0X2, N0X3, N0X4 and N0X5 as well as DUOX enzymes such as DUOXl and DU0X2. The inventors for the first time demonstrated high-level expression of the NADPH oxidase N0X4 in the ocular lens and significant expression of N0X5 in the ocular lens. Thereby, proteins in the human or animal body which are part of the pathogenesis of cataract and presbyopia and furthermore suitable as targets for therapeutic intervention in cataract and presbyopia are provided. At the same time, said proteins and variants thereof according to the main embodiment are targets for the method of identifying a inhibitor of the invention, i.e., vehicles for the identification of lead compounds. Such lead compounds in turn may be inhibitors of any member of the family of NADPH oxidases.

The term "cataract" according to the invention refers to the opacification of the ocular lens and embraces age-related cataract and diabetes-induced cataract. Cataract is an important pathology not only humans as detailed in the introductory part of this specification, but also in animals, in particular in dogs.

The term "protein" recited above extends to homologues having at least 75% sequence identity. Analogously, the term "nucleic acid" includes homologues having at least 75% sequence identity. Preferably, the protein sequence identity level is 80% or 85%, more preferred 90% or 95%, and yet more preferred 96%, 97%, 98% or 99%. Independently, preferred nucleic acid sequence identity levels are 80% or 85%, more preferred 90% or 95%, and yet more preferred 96%, 97%, 98% or 99%.

The term "presbyopia" according to the invention relates to the loss of the ability for accommodation and thus for focusing near objects the incidence of which increases with age.

The sequences set forth in SEQ ID NOs: 2 and 4 are the protein sequences of human N0X4 and N0X5, respectively. The sequences set forth in SEQ ID NOs: 1 and 3 are the corresponding nucleic acid sequences. The sequences shown in SEQ ID NOs: 25 and 26 are the nucleic acid encoding human p22phox and the encoded protein sequence, respectively.

Without being bound by a specific theory, p22phox is considered as a protein being capable of binding to NOX proteins leading to their stabilization in an in vivo context such as within a cell or an organism. p22phox is a membrane protein. A monomeric N0X4 or N0X5 protein, as opposed to a heterodimer formed by N0X4 and p22phox, or N0X5 and p22phox, respectively, is characterized by an increased likelihood of being degraded by the proteasome.

In addition to inhibitors of N0X4, N0X5 and p22phox and of homologues and fragments thereof as defined herein above, it is envisaged that inhibitors of N0X2, NOXOl, NOXAl, p47phox and p67phox, of fragments thereof and of homologues thereof with at least 75% sequence identity, may also be used for treating cataract and/or presbyopia. Said fragments and homologues of N0X2 exhibit NADPH oxidase activity. Said fragments and homologues of NOXO 1 and p47phox function as organizing subunits of a protein exhibiting NADPH oxidase activity. Said fragments and homologues of NOXAl and p67phox function as activator subunits of a protein exhibiting NADPH oxidase activity.

Also provided by the present invention is the use of an inhibitor of a protein as defined in the main embodiment and/or of an inhibitor of a nucleic acid as defined in the main embodiment for the preparation of a pharmaceutical composition for the treatment and/or prevention of cataract and/or presbyopia.

Further provided is a method of treating a disease selected from the group consisting of cataract and presbyopia comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of the protein as defined in the main embodiment and/or of an inhibitor of a nucleic acid as defined in the main embodiment.

Another embodiment of the invention is a method of treating a patient suffering from or at risk of developing a cataract and/or presbyopia comprising administering to the patient a composition comprising one or more of the inhibitors according to the invention in a therapeutically effective dose thereby treating the patient.

An aspect of the present invention relates to methods of treating a subject afflicted with or at risk of developing a disease selected from the group consisting of cataract and presbyopia, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of a nucleic acid wherein said nucleic acid is selected from any of: (i) the nucleic acid sequence of SEQ ID NO: 1, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 2;(ii) the nucleic acid sequence of SEQ ID NO: 3, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 4;(iii) comprises or consists of the nucleic acid sequence of SEQ ID NO: 25, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 26; (iv) is a fragment of the nucleic acid according to (i), or has a sequence at least 75% identical with the nucleic acid according to (i) or with said fragment of the nucleic acid according to (i), and encodes a protein exhibiting NADPH oxidase activity; (v)is a fragment of the nucleic acid according to (ii), or has a sequence at least 75% identical with the nucleic acid according to (ii) or with said fragment of the nucleic acid according to (ii), and encodes a protein stabilizing said protein exhibiting NADPH oxidase activity;(vi) is a fragment of the nucleic acid according to (iii), or has a sequence at least 75% identical with the nucleic acid according to (iii) or with said fragment of the nucleic acid according to (iii), and encodes a protein stabilizing said protein exhibiting NADPH oxidase activity.

Other aspects of the present invention relate to methods of treating a subject afflicted with or at risk of developing a disease selected from the group consisting of cataract and presbyopia the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of a protein , wherein said protein is selected from the group consisting of: (i) amino acid sequence of SEQ ID NOs: 2, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NOs: l;(ii) comprises or consists of the amino acid sequence of SEQ ID NOs: 4, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NOs: 3;(iii) comprises or consists of the amino acid sequence of SEQ ID NO: 26, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 25; (iv) is a fragment of the protein according to (i), or has a sequence at least 75% identical with the protein according to (i) or with said fragment of the protein according to (i), and exhibits NADPH oxidase activity; or(v) is a fragment of the protein according to (ii) or has a sequence at least 75% identical with the protein according to (ii) or with said fragment of the protein according to (ii) and stabilizes said protein exhibiting NADPH oxidase activity;(vi) is a fragment of the protein according to (iii) or has a sequence at least 75% identical with the protein according to (iii) or with said fragment of the protein according to (iii) and stabilizes said protein exhibiting NADPH oxidase activity;for treatment and/or prevention of cataract and/or presbyopia.

In other aspects, the invention relates to use of a compound binding to the nucleic acid set forth in any one of SEQ ID NOS: 1, 3, 25 for the preparation of a medicament for the treatment of cataract and/or presbyopia and/or an individual's disposition or risk to develop cataract and/or presbyopia, wherein said compound is a polynucleotide complementary to said nucleic acid and at least 15 nucleotides in length.

In other aspects, the present invention relates to methods of identifying a drug or a lead compound for developing a drug, said drug being for the treatment and/or prevention of cataract and/or presbyopia, the method comprising the steps of contacting a test compound with the protein set forth in any one of SEQ ID NOS :2, 4, 26 under conditions allowing binding of said test compound to said protein; optionally determining whether said test compound binds to said protein; and determining whether (ca) said test compound, upon contacting in step (a); or (cb) said test compound, upon binding in step (b) inhibits the activity of said protein, said activity being either NADPH oxidase activity or the stabilizing of a protein exhibiting NADPH oxidase activity. Said protein may be comprised in a membrane preparation or a cell transfected with a nucleic acid set forth in any one of SEQ ID NOS: 1, 3, 25

In yet another aspect, the present invention relates to methods of identifying a drug or a lead compound for developing a drug, said drug being for the treatment and/or prevention of cataract and/or presbyopia, the method comprising the steps of contacting a test compound with the nucleic acid set forth in any one of SEQ ID NOS:1, 3, 25 under conditions allowing binding of said test compound to said nucleic acid; optionally determining whether said test compound binds to said nucleic acid; and determining whether (ca) said test compound, upon contacting in step (a); or (cb) said test compound, upon binding in step (b) inhibits the expression of said nucleic acid. Determining in step (c) may comprise quantifying the expression level of said nucleic acid. Said protein or the protein encoded by said nucleic acid may be comprised in a non-human animal (e.g., a rodent, mouse, rat etc.). Determining in step (c) may comprise quantifying reactive oxygen species (ROS) production, and/or quantifying the opacity of the eye lens of said animal. The method may further comprise the step of formulating said inhibitor with a pharmaceutically acceptable carrier. Prior to said formulating, the affinity, specificity and/or pharmacological properties of the inhibitor may be optimized and/or clinical trials may be performed with said inhibitor or the optimized inhibitor. For the inhibitors, methods, and/or uses of the present invention, the cataract may be a diabetes-induced cataract or age-related cataract.

In a preferred embodiment of the inhibitor of the invention, the use of the invention, or the method of the invention, said inhibitor is selected from the group consisting of (a) an siRNA, an antisense nucleic acid, or a ribozyme binding specifically said nucleic acid; (b) an antibody, aptamer, or a fragment or derivative thereof binding specifically said protein; (c) a iodonium derivative such as diphenylene iodonium (DPI), di-2-thienyliodonium or iodonium biphenyl; (d) a naphthoquinone such as plumbagin; (e) a substituted catechol such as apocynin; (f) a statin such as lovastatin; (g) a compound selected from the group consisting of 4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF); vanillin; 4-nitroguaiacol; imidazole; pyridine; disulfϊram; penicillamine; Cibacron Blue; arylazido-NADP+; quercetin; esculetin; adenosine; auraptene; 2-benzyloxybenzaledehyde; gliotoxin; the peptides gp9 Ids-tat and PR- 39; trifluoperazine; triphenyltin chloride; reactive oxygen species (ROS) scavengers such as tempol and ebselen; PKC inhibitors such as Rottlerin; and TPCK; and (h) palladium derivatives and platinum derivatives such as cisplatin and hexachloroplatinate.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures show:

Figure 1: NOX expression in lens epithelial cells. Expression of NOXl, N0X2,

N0X4 and N0X5 in lens epithelial cells from 4 patients operated for cataract. The cells removed during cataract operations were put into RNAlater at -20⁰C until RNA extraction could be done. RNA extraction was carried out with RNAeasy microkit from Qiagen and the small amount of RNA obtained (less than 50 ng) was transcribed by Sensiscript to obtain cDNA. PCR for all NOX enzymes were carried out and GAPDH was used as a house keeping gene to control that the cDNA was sufficient to do PCR. Similar results were obtain with samples from 6 additional patients.

Figure 2: Lenses from mice were analyzed by RT-PCR for expression of four different NOX enzymes (30 cycles). Only N0X4 was expressed. Control (Ctrl) cDNAs were colon (NOXl), spleen (N0X2), inner ear (N0X3), and kidney (N0X4).

Figure 3: Histological analysis of cornea and lens in 1 control and 2 diabetic animals. Differences in the lenses of hyperglycaemic and normal mice are subtle: i) the presence of nuclear cells in the subepithelial region indicate an aberrant migration from the germinal zone, ii) swelling of the fibers of the lens. The cornea is not affected.

DETAILED DESCRIPTION OF THE INVENTION

A particularly preferred inhibitor is an siRNA. The term "small interfering RNA" (siRNA), sometimes known as short interfering RNA or silencing RNA, refers to a class of generally short and double-stranded RNA molecules that play a variety of roles in biology and, to an increasing extent, in treatment of a variety of diseases and conditions. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene (see, e.g., Zamore Nat Struct Biol 2001, 8(9):746-50; Tuschl T. CHEMBIOCHEM. 2001, 2:239-245; Scherr and Eder, Cell Cycle. 2007 Feb;6(4):444-9; Leung and Whittaker, Pharmacol Ther. 2005 Aug;107(2):222-39; de Fougerolles et ah, Nat. Rev. Drug Discov. 2007, 6: 443-453). The present invention encompasses siRNA which comprises deoxy- and ribonucleotides nucleotides having one or more modifications on the phosphate backbone, sugar moity and or base moiety.

Such siRNAs are generally 18-27 nt long, generally comprising a short (usually 19- 21-nt) double-strand of RNA (dsRNA) with or without 2-nt 3' overhangs on either end. Each strand can have a 5' phosphate group and a 3' hydroxyl (-OH) group or the phosphate group can be absent on one or both strands. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. In this context, other structures than those described above are also envisaged, provided they are capable of interfering with gene expression. Preferably, the double-stranded part has a length of about 12 to about 50 base pairs in length. The siRNA of the invention may either have overhanging sequences of up to 10 bases, preferably not more than 5 bases in length at either end or at one end, or may be blunt-ended. Also preferred is that the complementarity to the target gene extends over the entire length of the double-stranded part. The region which is complementary to the target gene is at least 12 bases, preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23 or more bases in length. The siRNA of the invention may be fully complementary to the target gene. Alternatively, the siRNA may comprise up to 5%, 10%, 20% or 30% mismatches to the target gene. Furthermore, siRNAs and also antisense RNAs can be chemically modified e.g. on the backbone including the sugar residues. Preferred modifications of the siRNA molecules of he invention include linkers connecting the two strands of the siRNA molecule. Chemical modifications serve inter alia to improve the pharmacological properties of siRNAs and antisense RNAs such as in vivo stability and/or delivery to the target site within an organism. The skilled person is aware of such modified siRNAs as well as of means and methods of obtaining them, see, for example, Zhang et ah, Curr Top Med Chem. 2006;6(9):893-900; Manoharan, Curr Opin Chem Biol. 2004 Dec;8(6):570-9.

Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important tool for gene function and drug target validation studies as well as for therapeutic intervention which is envisaged here. The siRNAs of the present invention are capable of reducing or blocking expression of the proteins and nucleic acids defined herein above.

The term "antisense nucleic acid" is known in the art and refers to a nucleic acid which is complementary to a target nucleic acid, here to a nucleic acid according to the main embodiment. An antisense molecule according to the invention is capable of interacting with, more specifically hybridizing with said nucleic acid. By formation of the hybrid, expression of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et ah, Cancer Res. (1991) 51 :2897-2901; Aboul-Fadl, Curr Med Chem. 2005; 12:2193-214; Pirollo et al, Pharmacol Ther. 2003; 99:55-77). Ribozymes are enzymes consisting of or comprising a catalytically active ribonucleic acid which in turn comprises a sequence complementary to a sequence in the target nucleic acid. Ribozymes specifically cleave the target nucleic acid, such as the (pre)-mRNA of a gene, the consequence being reduction or repression of expression. The techniques underlying said repression of expression are well known in the art (see, e.g., EP-Bl 0 291 533, EP-Al 0 321 201, EP-A2 0 360 257). Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke et al. (Methods in Cell Biology (1995) 50:449-460). Ribozymes according to the invention are capable of cleaving nucleic acids defined in the main embodiment.

Said antibody, which is monoclonal antibody, polyclonal antibody, single chain antibody, or fragment thereof that specifically binds said peptide or polypeptide also including bispecifϊc antibody, synthetic antibody, antibody fragment, such as Fab, a F(ab₂)', Fv or scFv fragments etc., or a chemically modified derivative of any of these (all comprised by the term "antibody"). Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kόhler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art. Furthermore, antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the peptide or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in WO89/09622. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit.

The term "monoclonal" or "polyclonal antibody" (see Harlow and Lane, (1988), loc. cit.) also relates to derivatives of said antibodies which retain or essentially retain their binding specificity. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region.

The term "scFv fragment" (single-chain Fv fragment) is well understood in the art and preferred due to its small size and the possibility to recombinantly produce such fragments.

Preferably, the antibody, aptamer, fragment or derivative thereof according to the invention specifically binds the target protein, (poly)peptide or fragment or epitope thereof whose presence or absence is to be monitored.

The term "specifically binds" in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (poly)peptides of similar structures. The term "(poly)peptide includes peptides as well as polypeptides and proteins. Cross-reactivity of a panel of antibodies etc. under investigation may be tested, for example, by assessing binding of said panel of antibodies etc. under conventional conditions (see, e.g., Harlow and Lane, (1988), loc. cit.) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides. Only those antibodies that bind to the (poly)peptide/protein of interest but do not or do not essentially bind to any of the other (poly)peptides which are preferably expressed by the same tissue as the (poly)peptide of interest, are considered specific for the

(poly)peptide/protein of interest and selected for further studies in accordance with the method of the invention.

In a particularly preferred embodiment, said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.

The term "humanized antibody" means, in accordance with the present invention, an antibody of non-human origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been replaced by CDRs of an antibody of human origin having a desired specificity. Optionally, the non-human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human antibody. Methods for the production of humanized antibodies are described in, e.g., EP-Al 0 239 400 and WO90/07861.

The term "aptamer" as used herein refers to DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms. A database of aptamers is maintained at http://aptamer.icmb.utexas.edu/; see also Nucleic Acids Research, 2004, Vol. 32, Database issue D95-D100.

Further preferred inhibitors are listed below.

The present invention also relates to a method of identifying a drug or a lead compound for developing a drug, said drug being for the treatment and/or prevention of cataract and/or presbyopia, the method comprising the steps of (a) contacting a test compound with the protein as defined in the main embodiment under conditions allowing binding of said test compound to said protein; (b) optionally determining whether said test compound binds to said protein; and (c) determining whether (ca) said test compound, upon contacting in step (a); or (cb) said test compound, upon binding in step (b) inhibits the activity of said protein, said activity being either NADPH oxidase activity or the stabilizing of a protein exhibiting NADPH oxidase activity.

The term "lead compound" designates a compound which is a drug candidate and which may require chemical modifications in order to optimize its pharmacological properties and eventually become a drug to be formulated as a drug. Methods of optimization are known in the art and further detailed below.

The optional determination of binding test compounds in step (b) relates to any biophysical binding assay, which may be used to identify binding test molecules prior to performing the functional assay with the binding test molecules only. Suitable biophysical binding assays are known in the art and comprise fluorescence polarization (FP) assay, fluorescence resonance energy transfer (FRET) assay and surface plasmon resonance (SPR) assay. Step (b) is particularly advantageous if said biophysical assay is more amenable to high throughput than the functional assay. Step (c) relates to the above mentioned functional assay. Determining whether a test compound, or a binding test compound, inhibits the expression of a target protein may be accomplished by measuring the expression level.

In a more preferred embodiment, the expression level to be determined is the protein expression level. The skilled person is aware of methods for the quantitation of proteins.

Amounts of purified protein in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular protein in a mixture rely on specific binding, e.g. of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise immunohistochemistry (in situ) and surface plasmon resonance. Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies.

In a further preferred embodiment said protein is comprised in a membrane preparation. Membrane preparations according to the invention may be membrane fractions obtained, for example, by centrifugation upon cell disruption. Alternatively, said membrane preparation is obtained by reconstituting the protein(s) according to the main embodiment with membrane- or micelle-forming amphiphilic lipids.

In a further preferred embodiment said protein is comprised in a cell transfected with a nucleic acid as defined in the main embodiment. This embodiment relates to a cellular screen.

Also provided by the present invention is a method of identifying a drug or a lead compound for developing a drug, said drug being for the treatment and/or prevention of cataract and/or presbyopia, the method comprising the steps of (a) contacting a test compound with the nucleic acid as defined in the main embodiment under conditions allowing binding of said test compound to said nucleic acid; (b) optionally determining whether said test compound binds to said nucleic acid; and (c) determining whether (ca) said test compound, upon contacting in step (a); or (cb) said test compound, upon binding in step (b) inhibits the expression of said nucleic acid.

In a preferred embodiment, determining in step (c) comprises quantifying the expression level of said nucleic acid. In a more preferred embodiment, the expression level to be determined is the mRNA expression level. Methods for the determination of nucleic acid expression levels, including mRNA expression levels, are known in the art and comprise Real Time PCR, Northern blotting and hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for transcripts encoding proteins of the NADPH oxidase family.

In a further preferred embodiment of the method of the invention, said protein or the protein encoded by said nucleic acid is comprised in a non-human animal. This embodiment relates to an in vivo screen. While less amenable to high throughput, the in vivo screen offers the advantage of the assessment of the disease state of the non-human animal. For the purpose of providing a model system, diabetes may be induced in non-human animal such as mouse or rat using streptozotocin (see also Example 3). The diabetes induced thereby leads to cataract formation within weeks.

Quantitation of the inhibition of the activity of the protein as defined in the main embodiment may be effected by quantifying the reactive oxygen species production. Accordingly, determining in step (c) preferably comprises quantifying reactive oxgen species (ROS) production. Methods of quantifying ROS are known in the art and comprise the peroxidase-dependent luminol-amplified chemiluminescence technique (also referred to as luminol-amplified chemiluminescence) which is further exemplified in Example 1 enclosed herewith.

The term "reactive oxygen species (ROS)" is well known in the art and includes oxygen ions, oxygen containing free radicals, inorganic peroxides and organic peroxides. Reactive oxygen species are highly reactive due to the presence of unpaired valence shell electrons. The formation of ROS in the eye, in particular the excessive formation thereof, as a consequence of the activity of N0X4 and/or N0X5 and/or of the presence of p22phox, may lead to the development or aggravation of cataract and/or presbyopia.

Accordingly, in a more preferred embodiment, determining in step (c) involves quantifying the opacity of the eye lens of said animal. In other words, the envisaged in vivo screen may use lens opacification as a read-out. Lens opacification is to be monitored by direct ophtalmoscopy, slitlamp examination and Scheimpflug photography. Alternatively or in addition, monitoring may be effected ex vivo by direct inspection of the ocular lens after sacrifice of the animal (Hedge et ah, 2003).

In a further preferred embodiment, the method of the invention further comprises the step of formulating said inhibitor with a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

More preferred, and prior to said formulating, the affinity, specificity and/or pharmacological properties of the inhibitor are optimized and/or clinical trials are performed with said inhibitor or the optimized inhibitor.

Methods for the optimization of the pharmacological properties of compounds identified in screens, generally referred to as lead compounds, are known in the art and comprise a method of modifying a compound identified as a lead compound to achieve: (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophilic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof; said method optionally further comprising the steps of the above described methods.

The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch-Analysis and Related Approaches," VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche

Apotheker Zeitung 140(8), 813-823, 2000).

Individuals to be selected for said clinical trials comprise healthy individuals, individuals with a disposition or at risk to develop cataract and/or presbyopia, and patients suffering from cataract and/or presbyopia. Cataract is understood to comprise age-related cataract and diabetes-induced cataract.

Moreover, the present invention also relates to a pharmaceutical composition comprising (a) an siRNA, an antisense nucleic acid, or a ribozyme binding specifically the nucleic acid defined in the main claim; (b) an antibody, aptamer, or a fragment or derivative thereof binding specifically the protein defined in the main embodiment; (c) a iodonium derivative such as diphenylene iodonium (DPI), di-2-thienyliodonium or iodonium biphenyl; (d) a naphthoquinone such as plumbagin; (e) a substituted catechol such as apocynin; (f) a statin such as lovastatin; (g) a compound selected from the group consisting of 4-(2- Aminoethyl)-benzenesulfonyl fluoride (AEBSF); vanillin; 4-nitroguaiacol; imidazole; pyridine; disulfϊram; penicillamine; Cibacron Blue; arylazido-NADP+; quercetin; esculetin; adenosine; auraptene; 2-benzyloxybenzaledehyde; gliotoxin; the peptides gp9 Ids-tat and PR- 39; trifluoperazine; triphenyltin chloride; ROS scavengers such as tempol and ebselen; PKC inhibitors such as Rottlerin; and TPCK; and/or (h) palladium derivatives and/or platinum derivatives such as cisplatin and hexachloroplatinate.

Further preferred inhibitors according to the invention or pharmaceutically active agents comprised in the pharmaceutical composition according to the invention are provided in the list below. It is understood that certain compounds listed below may exert their effects by inhibiting p22phox in addition to or instead of acting on N0X4, N0X5 and their active fragments and homologues. L Inhibitors of oxidase translocation

Apocynin (4-hydroxy-3-methoxy-acetophenone): NADPH oxidase inhibitor: ortho- methoxy substituted catechol; isolated from medicinal herb Picroria kurroa; described inter alia in Meyer JW et al, 1999. Described therapeutic effects are: anti-inflammatory action; effect on hypertension (Manning et al, 2003, Beswick et al, 2001).

AEBSF: 4-(2-Aminoethyl)-benzenesulfonyl fluoride; also available as 4-(2-N- methylaminoethyl) benzene sulfonyl fluoride hydrochloride; sulfonylfluoride group essential for inhibitory activity (Diatchuk et al., 1997); available at Merck.

Vanillin (4-Hydroxy-3-methoxybenzaldehyde) and 4-nitroguaiacol (2- methoxyphenol) (Holland et al., 2000).

Quinones and naphthoquinones, in particular plumbagin: a naphtoquinone derived from Plumbago Zeylanica (Chitrak, an indian medicinal plant). Anticarcinogenic and antiatherosclerotic action and inhibitory effect on N0X4 in HEK293, LN229 and trans fected COS cells described in Ding et al, 2005. Available at Indian Institute of Chemical Technology, Hyderabad, India.

II. Flavoprotein inhibitors

Diphenyleneiodonium (DPI).

Di-2-thienyliodonium.

Iodonium biphenyl (IBP): Described, inter alia in Doussiere et al, 1999; available at Aldrich.

Iodonium derivatives act as arylating agents and directly and irreversibly inhibit NOX enzymes. III. Heme ligands

Sulfur containing L-Arginine Derivatives as described in Ichimori et al, 1999.

Imidazole: described in Holland, 2000.

Pyridine: described in Holland, 2000.

IV. Thiol reagents

Disulfϊram: Described in Holland, 2000.

Penicillamine: Described in Holland, 2000.

V. NADPH analogues

Cibacron Blue, arylazido NADP+ and further NADPH analogues as described in Doussiere et al 1986.

VI. Redox inhibitors

Quercetin (Sakane et al, 1984; Holland, 2000).

Esculetin (Ozaki et al, 1986 ; Holland, 2000).

VII. Other inhibitors

Adenosine : Described in Swain et al, 2003.

Auraptene: A Citrus coumarin (Murakami et al, 1997; Murakami et al, 2000).

CCYIa: 2-benzyloxybenzaldehyde: Inhibits superoxide generation in neutrophils (Wang et al, 2003); exhibits anti-inflammatory activity; synthesised by Chang et al, 1971. Gliotoxin: Metabolite of pathogenic fungi (Aspergillus and Candida spp). Implicated in infectious pathways. Exhibits immunosupressive action and antitumor activity. Inhibits activation process of N0X2 (Yoshida et ah, 2000) and the assembly of the enzyme (Tsunawaki et ah, 2004). Directly inhibits electron transport of flavocytochrome b558 before, but not after oxidase activation in a cell free system (Nishida et al, 2005). Available from Sigma.

Peptide gp9 Ids-tat: Competitive inhibitor of NADPH assembly (Rey et α/.2001, Jacobson et al, 2003).

Peptide PR-39: Pro line -rich antibacterial peptide inhibiting phagocyte NADPH oxidase activity by binding to Src homology domain 3 of p47phox (Shi et al, 1996).

Statins: Inhibitors of HMG-CoA. Decrease plasma cholesterol. Block rac-1 dependent activation of NADPH oxidases (Maack et al. 2003). Inhibit myristoylation of rac. Preferred statins include lovastatin.

Trifluoperazine: Inhibitor of PKC/calmodulin; prevents the activation of NADPH oxidases (Seifert and Scachtele, 1988, Holland et al, 2000).

Triphenyltin chloride: Organotin compound; inhibitor of superoxide production by PMA-stimulated PMN (Matsui et al, 1983); TPTCI available from Tokyo Kasei Chemical Co.

Other inhibitors of superoxide production: ROS scavengers such as Tempol (SOD mimetic) and Ebselen (glutathione peroxydase mimetic); PKC inhibitors such as Rottlerin, TPCK and inhibitors of MAP kinase.

The present invention for the first time provides inhibitors of proteins exhibiting NADPH oxidase activity or of nucleic acids encoding proteins exhibiting NADPH oxidase activity for the purpose of treating presbyopia and/or cataract. Accordingly, it is deliberately envisaged that inhibitors according to the invention include known NADPH oxidase inhibitors. Analogously, it is deliberately envisaged that pharmaceutical compositions of the invention comprise known NADPH oxidase inhibitors. The same applies to medical uses according to the invention. Such known NADPH oxidase inhibitors include the compounds described in following patent applications and patents, respectively, the contents of which is incorporated by reference: WO2006033965, WO2006020912, RU2254573, WO2005105130, WO2005119251, WO2005112931, CA2517416, WO2004089412, JP2005168329, WO2004011670, EP1509618, WO03055473, WO02079224, JP2003201255, WO0230453, WO0179467, GB2368012, WO0120001, RU2194077, WO9912539, WO9801120, CA2309881, US5902831, EP0914821, GB2285047, CA2129235, WO9503819, JP7313180, US4954445, WO0057871, WOOl 17533, US7049396, EP1598354, WO2006102114, WO2006009975, WO2005101968, WO2005103297, WO2005099721, WO2005080378, JP2005232059, EP1616576, EP1603543, EP1592429, WO2004045556, US2004063648, US2004259816, EP1525315, WO2004009531, WO2004005267, EP1482034, EP1505068, EP1499742, EP1536819, EP1448127, WO2003037829, EP1435984, EP1281962, EP1281963, WO2002031189, WO2002030453, EP1303308, EP1453522, EP1299558, WO2001089517, EP1265996, WO2001046468, EPl 171112, EPl 140077, EP1410798, WO2000027423, WO9801120, EP861070, EP914821, WO9632129, EP784471, EP750630, WO9503819, EP551662, EP727987, WO9117763, and EP396434.

Pharmaceutical compositions comprising one of these compounds as well as pharmaceutical compositions comprising the combination of a plurality of any of the compounds mentioned above are envisaged.

The pharmaceutical and diagnostic compositions of the invention may furthermore comprise one or more agents that enhance the penetration into the anterior chamber of the eye. Suitable agents enhancing the penetration of a formulation into the anterior chamber of the eye are known in the art.

The pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The compositions may also be administered directly to the target site, e.g., by biolistic delivery (also referred to as "gene gun") to an external or internal target site. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

The pharmaceutical compositions and diagnostic compositions of the invention as well as the uses for the preparation of pharmaceutical or diagnostic compositions and methods of diagnosing according to the invention further detailed below are envisaged for human individuals or patients on the one side, and, on the other side, for veterinary use, preferably in dogs.

In a more preferred embodiment of the pharmaceutical composition or the use of the invention, said pharmaceutical composition are eye drops.

Advantageously, the formulation as eye drops allows the topical administration, thereby avoiding or substantially avoiding systemic effects. Alternatively or in addition, the pharmaceutical composition may be formulated for injection.

Also provided is a method of diagnosing cataract and/or presbyopia and/or an individual's disposition or risk to develop cataract and/or presbyopia, comprising the steps of: (a) determining (a) polymorphism(s) in a N0X4 and/or N0X5 gene and/or cDNA in a sample obtained from said individual; and (b) associating said polymorphism(s) with a disease state or disposition state. Preferably, said sample is a blood sample. Preferably said N0X4 and N0X5 cDNA (or equivalently mRNA), respectively, comprise or consist of the sequence set forth in SEQ ID NO: 1 or 3.

The term "polymorphism", or "nucleotide polymorphism" refers to the occurrence of one or more different nucleotides or bases at a given location on a chromosome. Usually, polymorphisms are distinguished from mutations based on their prevalence. Sometimes a threshold of 1% prevalence in a population of individuals is considered for separating polymorphisms (more frequent) from mutations (less frequent). A single nucleotide polymorphism (SNP) is a polymorphism of a single nucleotide or base. The SNP database maintained at NCBI (www.ncbi.nlm.nih.gov/SNP/; see also, for example, Nucleic Acids Res., Jan 2001; 29: 308-311) divides SNPs into SNPs in the proximity of a known locus and such that are 5' further away than 2 kb from the most 5' feature of a gene and 3' further away than 500 bases from the most 3' feature of a gene. SNPs in the proximity of a known locus are further divided into SNPs occurring at an mRNA location and such that do not. SNPs occurring at an mRNA location comprise coding and non-coding SNPs.

It is understood that the term "polymorphism(s) in a NOX4 and/or NOX5 gene and/or cDNA" embraces polymorphisms in exons, introns and regulatory regions such as promoters.

Polymorphisms in exons may be determined or analysed using genomic DNA or cDNA (or equivalently mRNA). Polymorphisms in introns or regulatory regions such as promoters may be determined or analysed using cDNA (or equivalently mRNA).

Said associating of polymorphism(s) with a disease state or disposition state refers to classifying of individuals and patients. The term "classifying" refers to the assignment of individuals or patients to two or more groups or classes. In other words, individuals, previously unclassified, get labelled by their respective class. The assigned class label may refer to parameters used for classification, e.g. polymorphisms, or may refer to parameters not used for classification because their values are not known beforehand, e.g. fast or slow response to therapy. In the first case, class discovery methods, e.g. clustering may be applied, whereas in the second case predictive classification methods are used. Classification may be done manually by a trained person or by a computer program provided with the values of the parameters used for class distinction. Patients have to give informed consent. Data have to be handled and kept secret in accordance with national laws.

Specific polymorphisms to be used in the method of diagnosing comprise the following SNP in the N0X4 gene: rs317139 (SEQ ID NO: 5) and the following SNPs in the N0X5 gene: rsl6952684, rsl2907196, rs7168025, rs7167318, rs3743091, rs2277553, rs2277552 (SEQ ID NOs: 6 to 12).

The present invention also provides the use of a compound binding to the protein defined in the main embodiment for the preparation of a diagnostic composition for the diagnosis of cataract and/or presbyopia and/or an individual's disposition or risk to develop said cataract and/or said presbyopia, wherein said compound is selected from the group consisting of (a) an antibody, aptamer, or a fragment or derivative thereof binding specifically said protein; (b) a iodonium derivative such as diphenylene iodonium (DPI), di-2- thienyliodonium or iodonium biphenyl; (c) a naphthoquinone such as plumbagin; (d) a substituted catechol such as apocynin; (e) a statin such as lovastatin; (f) a compound selected from the group consisting of 4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF); vanillin; 4-nitroguaiacol; imidazole; pyridine; disulfϊram; penicillamine; Cibacron Blue; arylazido- NADP+; quercetin; esculetin; adenosine; auraptene; 2-benzyloxybenzaledehyde; gliotoxin; the peptides gp9 Ids-tat and PR-39; trifluoperazine; triphenyltin chloride; ROS scavengers such as tempol and ebselen; PKC inhibitors such as Rottlerin; and TPCK; and (g) palladium derivatives and platinum derivatives such as cisplatin and hexachloroplatinate.

Alternatively, said compound to be used for the preparation of a diagnostic composition may bind to a protein which is part of the pathway the protein defined in the main embodiment belongs to.

Also envisaged is the use of a compound binding to a nucleic acid defined in the main embodiment for the preparation of a diagnostic composition for the diagnosis of cataract and/or presbyopia and/or an individual's disposition or risk to develop cataract and/or presbyopia, wherein said compound is a polynucleotide complementary to said nucleic acid and at least 15 nucleotides in length. This embodiment is directed to oligonucleotide probes for the detection of genomic DNA or mRNA. With regard to genomic DNA, also the detection and distinction of polymorphisms is envisaged. Preferably, said polynucleotide is at least 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500 or more nucleotides in length. Complementarity extends over the entire length of said polynucleotide or at least over a length of 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100 or more nucleotides.

The term "polynucleotide" as used herein refers to a condensation polymer of nucleotides. The term "polynucleotide" according to the invention includes oligonucleotides, wherein oligonucleotides are condensation oligomers of about 30 or less nucleotides.

It is understood that the term "polynucleotide" also includes analogs and derivatives of naturally occurring polynucleotides, said analogs or derivatives preferably being selected from the group consisting of peptide nucleic acids, phosphorothioate nucleic acids, phosphoramidate nucleic acids, 2'-O-methoxyethyl ribonucleic acids, morpholino nucleic acids, hexitol nucleic acids and locked nucleic acids. Also envisaged are chimeric molecules comprising two or more of the above.

For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog. The monomeric units for the corresponding derivatives of adenine, guanine, thymine and cytosine are available commercially (for example from Perceptive Biosystems). PNA is a synthetic DNA-mimic with an amide backbone in place of the sugar- phosphate backbone of DNA or RNA. As a consequence, certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. Furthermore, they are stable under acidic conditions and resistant to proteases. Their electrostatically neutral backbone increases the binding strength to complementary DNA as compared to the stability of the corresponding DNA-DNA duplex. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T_m) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15-mer duplex. Thereby discrimination between perfect matches and mismatches is improved. For its uncharged nature, PNA also permits the hybridisation of DNA samples at low salt or no-salt conditions, since no inter-strand repulsion as between two negatively charged DNA strands needs to be counteracted. As a consequence, the target DNA has fewer secondary structures under hybridisation conditions and is more accessible to probe molecules.

Preferably, said compound to be used in diagnostic applications is detectably labelled.

More preferred, said diagnosis to be performed involves imaging of the human or animal body.

In a preferred embodiment of the method or the use of the invention, said animal is a rodent. More preferred, said rodent is mouse or rat.

In a preferred embodiment of the method or the use of the invention, said cataract is diabetes-induced cataract.

In a further preferred embodiment, said cataract is age-related cataract. EXAMPLES

The following examples illustrate the invention but should not be construed as being limiting.

Example 1

Measurement of ROS generation

ROS generation is measured in primary lens epithelial cells from mouse (healthy) or human (cataract epxlants) or immortalized lens epithelial cells. Alternatively, cells transformed with the protein according to the invention are used. Extracellular hydrogen peroxide release is measured by Amplex Red. Briefly, detatched cells (50 000) are plated in 0.2 mL volume of HBSS containing 20 μM Amplex Red and 0.1 U/ml horseradish peroxidase, and where stimuli or inhibitors are optionally present. The plate is kept at 37°C and fluorescence readings are taken every minute for 40-60 minutes with excitation and emission wavelengths of 550 nm and 600 nm, respectively, in a BMG Fluostar microplate reader. Intracellular superoxide production is measured by a quantitative nitro blue tetrazolium (NBT) assay. Cells are plated in 12 well plates, washed in HBSS and incubated with NBT (1.6 mg/ml) in HBSS at 37°C for 45 min. After fixation in 100% methanol and a wash in methanol, the formazan precipitates are dissolved by addition of 560 μl 2M KOH and 480 μl DMSO. The amount of reduced NBT is quantified by determination of OD at 630 nm. Photographs are taken before fixation, using a Nikon

Coolpix CCD camera (Nikon) mounted on an Axiovert SlOO microscope (Zeiss) using a IOOX objective.

Example 2

Expression of NOX enzymes in the ocular lens

Lens explant material was obtained from patients after cataract operation. Using this material, mRNA extraction and subsequent RT-PCR was performed. The cells removed during cataract operations were put into RNAlater at -20⁰C until RNA extraction could be done. RNA extraction was carried out with RNAeasy microkit from Qiagen and the small amount of RNA obtained (less than 50 ng) was transcribed by Sensiscript to obtain cDNA. PCR for all NOX enzymes were carried out and GAPDH was used as a house keeping gene to control that the cDNA was sufficient to do PCR. The following primer sequences have been used: SEQ ID NOs: 13 and 14 for hNOXl, SEQ ID NOs: 15 and 16 for hN0X2, SEQ ID NOs: 17 and 18 for hN0X3, SEQ ID NOs: 19 and 20 for hN0X4, SEQ ID NOs: 21 and 22 for hN0X5, and SEQ ID NOs: 23 and 24 for GAPDH. Examples of PCR results are shown in Fig. 1. Results from ten patients are shown in Table 1 below.

Table 1. RT PCR was performed in a total of 10 patient samples obtained at three different dates (28.04.04; 26.05.04; 22.09.04). GAPDH is a house keeping enzyme which was used to control for the presence and the quality of the mRNA.

We consistently found high level expression of N0X4 and N0X5 in the lense material. Indeed, only 1 out of 10 patients did not clearly express the two enzymes (see Patient 9, Table 1). N0X2 was also observed, but more inconsistently and we tend to attribute the positive PCR results for N0X2 to contamination with blood cells, rather than a genuine expression of N0X2. These findings suggest that NOX enzymes are an important source of ROS within the ocular lens and they also suggest that NOX-targeted drugs provide a therapeutic approach for the treatment and prevention of cataract.

Further Analysis of the expression of NOX enzymes in the ocular lens of mice

The study of gene expression in the ocular lens of mice is technically difficult, because of the small size of the lenses, as well as the fact that this materiel is difficult to homogenize for mRNA extraction. However, the inventors were able to study the expression of NOX enzymes in the ocular lens of mice. Results are shown in Figure 2. These results demonstrate that N0X4 is expressed in the mouse lens. Note that as opposed to humans (that also express N0X5 in the lens), rodents do not possess a N0X5 gene. These results are also important as N0X4 is already expressed in the lens before cataract development. Thus, N0X4 expression can be considered as the cause of cataract formation, rather than simply its consequence.

Example 3

Use of siRNA and NOX inhibitors to prevent cataract formation and presbyopia

Animal model: The inventors have opted for the diabetic mouse model because this closely models the human situation and also because this allows the performance of proof of principal experiments in knock-out mice.

The mouse model was prepared as follows: streptozotocin (200 mg/kg) was administered to induce diabetes in C57BL/6 mice; only mice that were hyperglycaemic (blood glucose >300 mg/dl) were considered diabetic (i.e. 70 % of the streptozotocin-treated mice).

The mice were regularly followed by ophthalmic examination performed by an experienced ophthalmologist. All diabetic mice developed a mild cataract (score value: 1 out of 3) within 3 weeks of hyperglycaemia. After 3 weeks, mice were sacrificed and lens explants were analyzed by histology (Figure 3).

The results of the histological analysis corroborate the results obtained by the clinical examination and show that hyperglycemic mice develop a mild cataract wtihin 3 weeks of hyperglycemia.

Using this mouse model, NOX inhibitors such as inhibitory RNAs (siRNA, antisense, ribozyme) are either applied as eye-drops to the cornea or through injection into the anterior chamber. REFERENCES

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Claims

1. A method of treating a subject afflicted with or at risk of developing a disease selected from the group consisting of cataract and presbyopia, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of a nucleic acid, wherein said nucleic acid is selected from any of

(a) the nucleic acid sequence of SEQ ID NO: 1, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 2;

(b) the nucleic acid sequence of SEQ ID NO: 3, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 4; (c) comprises or consists of the nucleic acid sequence of SEQ ID NO: 25, or encodes a protein comprising or consisting of the sequence of SEQ ID NO: 26;

(d) is a fragment of the nucleic acid according to (a), or has a sequence at least 75% identical with the nucleic acid according to (a) or with said fragment of the nucleic acid according to (a), and encodes a protein exhibiting NADPH oxidase activity;

(e) is a fragment of the nucleic acid according to (b), or has a sequence at least 75% identical with the nucleic acid according to (b) or with said fragment of the nucleic acid according to (b), and encodes a protein stabilizing said protein exhibiting NADPH oxidase activity; or (f) is a fragment of the nucleic acid according to (c), or has a sequence at least

75% identical with the nucleic acid according to (c) or with said fragment of the nucleic acid according to (c), and encodes a protein stabilizing said protein exhibiting NADPH oxidase activity.

2. A method of treating a subject afflicted with or at risk of developing a disease selected from the group consisting of cataract and presbyopia the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of a protein , wherein said protein is selected from the group consisting of

(a) amino acid sequence of SEQ ID NOs: 2, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NOs: 1; (b) comprises or consists of the amino acid sequence of SEQ ID NOs: 4, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NOs: 3;

(c) comprises or consists of the amino acid sequence of SEQ ID NO: 26, or is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID

NO: 25;

(d) is a fragment of the protein according to (a), or has a sequence at least 75% identical with the protein according to (a) or with said fragment of the protein according to (a), and exhibits NADPH oxidase activity; or (e) is a fragment of the protein according to (b) or has a sequence at least 75% identical with the protein according to (b) or with said fragment of the protein according to (b) and stabilizes said protein exhibiting NADPH oxidase activity;

(f) is a fragment of the protein according to (c) or has a sequence at least 75% identical with the protein according to (c) or with said fragment of the protein according to (c) and stabilizes said protein exhibiting NADPH oxidase activity; for treatment and/or prevention of cataract and/or presbyopia.

3. The method of claim 1, wherein said inhibitor of a nucleic acid is selected from the group consisting of an siRNA, an antisense nucleic acid, or a ribozyme binding said nucleic acid.

4. The method of claim 2 wherein said inhibitor of a protein is selected from the group consisting of

(a) an antibody, aptamer, or a fragment or derivative thereof binding specifically said protein; (b) a iodonium derivative such as diphenylene iodonium (DPI), di-2- thienyliodonium or iodonium biphenyl; (c) a naphthoquinone such as plumbagin;

(d) a substituted catechol such as apocynin;

(e) a statin such as lovastatin; (f) a compound selected from the group consisting of 4-(2-Aminoethyl)- benzenesulfonyl fluoride (AEBSF); vanillin; 4-nitroguaiacol; imidazole; pyridine; disulfiram; penicillamine; Cibacron Blue; arylazido-NADP+; quercetin; esculetin; adenosine; auraptene; 2-benzyloxybenzaledehyde; gliotoxin; the peptides gp9 Ids-tat and PR-39; trifluoperazine; triphenyltin chloride; ROS scavengers such as tempol and ebselen; PKC inhibitors such as Rottlerin; and TPCK; and

(g) palladium derivatives and platinum derivatives such as cisplatin and hexachloroplatinate.

5. A pharmaceutical composition comprising

(a) an siRNA, an antisense nucleic acid, or a ribozyme binding specifically the nucleic acid set forth in any one of SEQ ID NOS: 1, 3, 25

(b) an antibody, aptamer, or a fragment or derivative thereof binding specifically the protein set forth in any one of SEQ ID NOS :2, 4, 26;

(c) a iodonium derivative such as diphenylene iodonium (DPI), di-2- thienyliodonium or iodonium biphenyl;

(d) a naphthoquinone such as plumbagin;

(e) a substituted catechol such as apocynin; (f) a statin such as lovastatin;

(g) a compound selected from the group consisting of 4-(2-Aminoethyl)- benzenesulfonyl fluoride (AEBSF), vanillin, 4-nitroguaiacol, imidazole, pyridine, disulfiram, penicillamine, Cibacron Blue, arylazido-NADP+, quercetin, esculetin, adenosine, auraptene, 2-benzyloxybenzaledehyde, gliotoxin, the peptides gp9 Ids-tat and PR-39, trifluoperazine, triphenyltin chloride, a ROS scavenger, tempol, ebselen, a PKC inhibitor, Rottlerin, and TPCK; and/or

(h) palladium derivatives and/or platinum derivatives such as cisplatin and hexachloroplatinate; and a pharmaceutically acceptable carrier or excipient.

6. The methods of any one of claims 1 or 2 or the pharmaceutical composition of claim 5, wherein said pharmaceutical composition are eye drops.

7. Use of a compound binding to the nucleic acid set forth in any one of SEQ ID NOS: 1 , 3, or 25 for the preparation of a medicament for the treatment of cataract and/or presbyopia and/or an individual's disposition or risk to develop cataract and/or presbyopia, wherein said compound is a polynucleotide complementary to said nucleic acid and at least 15 nucleotides in length.

8. Use of a compound binding to the protein set forth in any one of SEQ ID NOS :2, 4, 26 for the preparation of a medicament for the treatment of cataract and/or presbyopia and/or an individual's disposition or risk to develop cataract and/or presbyopia, wherein said compound is selected from the group consisting of

(a) an antibody, aptamer, or a fragment or derivative thereof binding specifically said protein;

(b) a iodonium derivative such as diphenylene iodonium (DPI), di-2- thienyliodonium or iodonium biphenyl;

(c) a naphthoquinone such as plumbagin;

(d) a substituted catechol such as apocynin;

(e) a statin such as lovastatin;

(f) a compound selected from the group consisting of 4-(2-Aminoethyl)- benzenesulfonyl fluoride (AEBSF); vanillin; 4-nitroguaiacol; imidazole; pyridine; disulfiram; penicillamine; Cibacron Blue; arylazido-NADP+; quercetin; esculetin; adenosine; auraptene; 2-benzyloxybenzaledehyde; gliotoxin; the peptides gp9 Ids-tat and PR-39; trifluoperazine; triphenyltin chloride; ROS scavengers such as tempol and ebselen; PKC inhibitors such as Rottlerin; and TPCK; and

(g) palladium derivatives and/or platinum derivatives such as cisplatin and hexachloroplatinate.

9. Use of a compound binding to the protein set forth in any one of SEQ ID NOS :2, 4, 26 for the preparation of a diagnostic composition for the diagnosis of cataract and/or presbyopia and/or an individual's disposition or risk to develop cataract and/or presbyopia, wherein said compound is selected from the group consisting of (a) an antibody, aptamer, or a fragment or derivative thereof binding specifically said protein;

(b) a iodonium derivative such as diphenylene iodonium (DPI), di-2- thienyliodonium or iodonium biphenyl; (c) a naphthoquinone such as plumbagin;

(d) a substituted catechol such as apocynin;

(e) a statin such as lovastatin;

(f) a compound selected from the group consisting of 4-(2-Aminoethyl)- benzenesulfonyl fluoride (AEBSF); vanillin; 4-nitroguaiacol; imidazole; pyridine; disulfiram; penicillamine; Cibacron Blue; arylazido-NADP+; quercetin; esculetin; adenosine; auraptene; 2-benzyloxybenzaledehyde; gliotoxin; the peptides gp9 Ids-tat and PR-39; trifluoperazine; triphenyltin chloride; ROS scavengers such as tempol and ebselen; PKC inhibitors such as

Rottlerin; and TPCK; and (g) palladium derivatives and/or platinum derivatives such as cisplatin and hexachloroplatinate.

10. Use of a compound binding to the nucleic acid set forth in any one of SEQ ID NOS: 1, 3, 25 for the preparation of a diagnostic composition for the diagnosis of cataract and/or presbyopia and/or an individual's disposition or risk to develop cataract and/or presbyopia, wherein said compound is a polynucleotide complementary to said nucleic acid and at least 15 nucleotides in length.

11. The use of claim 9 or 10, wherein said compound is detectably labelled.

12. The use of claim 11, wherein said diagnosis to be performed involves imaging of the human or animal body.

13. A method of identifying a drug or a lead compound for developing a drug, said drug being for the treatment and/or prevention of cataract and/or presbyopia, the method comprising the steps of (a) contacting a test compound with the protein set forth in any one of SEQ ID NOS :2, 4, 26 under conditions allowing binding of said test compound to said protein;

(b) optionally determining whether said test compound binds to said protein; and (c) determining whether (ca) said test compound, upon contacting in step (a); or

(cb) said test compound, upon binding in step (b) inhibits the activity of said protein, said activity being either NADPH oxidase activity or the stabilizing of a protein exhibiting NADPH oxidase activity.

14. The method of claim 13, wherein said protein is comprised in a membrane preparation.

15. The method of claim 13, wherein said protein is comprised in a cell transfected with a nucleic acid set forth in any one of SEQ ID NOS: 1, 3, or 25.

16. A method of identifying a drug or a lead compound for developing a drug, said drug being for the treatment and/or prevention of cataract and/or presbyopia, the method comprising the steps of

(a) contacting a test compound with the nucleic acid set forth in any one of SEQ ID NOS: 1, 3, 25 under conditions allowing binding of said test compound to said nucleic acid;

(b) optionally determining whether said test compound binds to said nucleic acid; and

(c) determining whether (ca) said test compound, upon contacting in step (a); or (cb) said test compound, upon binding in step (b) inhibits the expression of said nucleic acid.

17. The method of claim 16, wherein determining in step (c) comprises quantifying the expression level of said nucleic acid.

18. The method of claim 17, wherein said protein or the protein encoded by said nucleic acid is comprised in a non-human animal.

19. The method of any one of claims 13 to 18, wherein determining in step (c) comprises quantifying reactive oxygen species (ROS) production.

20. The method of any one of claims 13 to 18, wherein determining in step (c) comprises quantifying the opacity of the eye lens of said animal.

21. The method of any of claims 13 to 20, wherein the method further comprises the step of formulating said inhibitor with a pharmaceutically acceptable carrier.

22. The method of claim 21, wherein, prior to said formulating, the affinity, specificity and/or pharmacological properties of the inhibitor are optimized and/or clinical trials are performed with said inhibitor or the optimized inhibitor.

23. The inhibitor, method, or use of any of the preceding claims, wherein said cataract is diabetes-induced cataract or age-related cataract.