24 Current Drug Delivery, Biomarkers of Oxidative Stress ... · 26 Current Drug Delivery, 2014,...

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Biomarkers of Oxidative Stress and Cataract. Novel Drug Delivery Thera-peutic Strategies Targeting Telomere Reduction and the Expression of Telomerase Activity in the Lens Epithelial Cells with N-Acetylcarnosine Lubricant Eye Drops: Anti-Cataract which Helps to Prevent and Treat Cataracts in the Eyes of Dogs and other Animals

Mark A. Babizhayev1,2* and Yegor E. Yegorov3

1Innovative Vision Products, Inc., 3511 Silverside Road, Suite 105, County of New Castle, Delaware, USA 19810; 2Moscow Helmholtz Research Institute of Eye Diseases, Str. Sadovaya-Chernogryazskaya 14/19, Moscow 103064, Russian Federation; 3Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov street, Moscow, 119991, Russian Federation

Abstract: Cataracts in Small Animals are shown to be at least partially caused by oxidative damage to lens epithelial cells (LECs) and the internal lens; biomarkers of oxidative stress in the lens are considered as general biomarkers for life expectancy in the canine and other animals. Telomeres lengths and expressed telomerase activity in canine LECs may serve as important monitors of oxidative damage in normal LECs with documented higher levels of telomerase activity in cata-ractous LECs during cells’ lifespan. Loss of functional telomere length below a critical threshold in LECs of canines dur-ing the effect of UV and chronic oxidative stress or metabolic failure, can activate programs leading to LEC senescence or death. Telomerase is induced in LECs of canines at critical stages of cataractogenesis initiation and exposure to oxidative stress through the involvement of catalytically active prooxidant transition metal (iron) ions. This work documents that transition metal ions (such as, ferrous ions- catalytic oxidants) might induce premature senescence in LECs of canines, telomere shortening with increased telomerase activity as adaptive response to UV light, oxidative and metabolic stresses. The therapeutic treatment with 1% N-acetylcarnosine (NAC) prodrug delivery is beneficial for prevention and dissolution of ripe cataracts in canines. This biological activity is based on the findings of ferroxidase activity pertinent to the dipep-tide carnosine released ophthalmically from NAC prodrug of L-carnosine, stabilizing properties of carnosine on biological membranes based on the ability of the imidazole-containing dipeptides to interact with lipid peroxidation products and reactive oxygen species (ROS), to prevent membrane damage and delute the associated with membrane fragements protein aggregates. The advent of therapeutic treatment of cataracts in canines with N-acetylcarnosine lubricant eye drops through targeting the prevention of loss of functional telomere length below a critical threshold and “flirting” with an indi-rect effect with telomerase expression in LECs of canines during the effects of UV, chronic oxidative stress increases the successful rate of cataract management challenges in home veterinary care.

Keywords: Aging and cataract, biological marker, carnosine, cataract treatment in canines, ferritin, ferroxidase activity, L-carnosine as universal antioxidant, lens epithelial cells, lifespan, N-acetylcarnosine ophthalmic prodrug, oxidative stress, small animals, transition metal (ferrous) ions, telomerase activity, telomere-dependent senescent phenotype, telomere shortening.

Properly trained, a man can be dog's best friend. (Corey Ford)

1. INTRODUCTION

Topical ocular drug administration is the most preferred route for treating conditions affecting the surface of the eye as well as anterior segment diseases; this is mainly due to the rapid and localised drug action and patient acceptability. However, the ocular bioavailability is typically less than 5% from conventional ophthalmic dosage forms such as eye drops (Fig. 1). This is mainly due to the unique anatomical

*Address correspondence to this author at the Innovative Vision Products, Inc., Moscow Division, Ivanovskaya 20, Suite 74 Moscow 127434 Russian Federation; Tel:/Fax: +7(499)977-2387; E-mail: [email protected]

and physiological features of the eye. The pharmacokinetics and constraints of ocular drug absorption are nowadays well understood. It is recognized that the ocular bioavailability of drugs topically applied as eye drops is very poor, due to effi-cient protective mechanisms ensuring the proper functioning of the eye, and to other concomitant factors. As a conse-quence of these mechanisms and factors the rate of loss of drug from the eye can be 500 to 700 times greater than the rate of absorption into the anterior chamber, and 1-5% or less of the drug applied topically as a solution reaches the inner eye. One of the effective pharmaceutical approaches is to provide a controlled and continuous drug release to the sur-face of the eye to compensate drug loss by nasolacrimal drainage and non-productive absorption of the topically applied

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Telomere Lengths, Telomerase Activity in Canine Lens Epithelial Cells Current Drug Delivery, 2014, Vol. 11, No. 1 25

drug [1]. Successful treatment of eye diseases requires effec-tive concentration of drug at the eye for sufficient period of time [2]. Most of the ophthalmic formulation strategies aim at maximizing ocular drug permeability through prolonga-tion of the drug residence time in the cornea and conjunctival sac, as well as minimizing precorneal drug loss. The conven-tional topical ocular drug delivery systems show drawbacks such as increased precorneal elimination and high variability in efficacy. Attempts have been made to overcome these problems and enhance ocular bioavailability by the devel-opment of newer drug delivery systems [3]. This review pro-vides a critical appraisal of the drug delivery strategies that provides controlled and continuous N-acetylcarnosine lubri-cant eye drops drug supply to the surface of the eye targeted for the treatment of cataracts in humans and canines. Carriers used in accordance to the current inventions are typically suitable for topical or general administration, and are for example water, mixtures of water and water-miscible sol-vents, such as C1 - to C7 -alkanols, vegetable oils or mineral oils comprising from 0.5 to 5% by weight hydroxy-ethylcellulose, ethyl oleate, carboxymethylcellulose, poly-vinyl-pyrrolidone and other non-toxic water-soluble poly-mers for ophthalmic uses, such as, for example, cellulose derivatives, like methylcellulose, alkali metal salts of car-boxymethylcellulose, hydroxymethylcellulose, hydroxy-ethylcellulose, methylhydroxypropylcellulose, hydroxy-propylcellulose, chitosan and scleroglucan, acrylates or methacrylates, salts of polyacrylic acid or ethyl acrylate, polyacrylamides, natural products, such as gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum, carrageenin, agar and acacia, starch derivatives, such as starch acetate and hydroxypropyl starch, and also other synthetic products, such as poloxamers, e.g. Poloxamer F127, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid, such as neu-tral Carbopol, or mixtures of those polymers. Preferred carri-ers are water, cellulose derivatives, such as methylcellulose, alkali metal salts of carboxymethylcellulose, hydroxyethyl-cellulose, hydroxypropylmethylcellulose, methylhy-droxypropylcellulose, methylcellulose and hydroxypropyl-cellulose, neutral Carbopol, or mixtures thereof. The concen-tration of the carrier is, for example, from 0.1 to 100000 times the concentration of the active ingredient [4]. Mucoad-hesive polymers, i.e., macromolecules capable of retaining the medication in the precorneal area by establishing phys-icochemical interactions with the mucin layer covering the corneal epithelium are a relatively recent discovery [5]. The following synthetic, semi-synthetic and naturally occurring polymers have been found to possess mucoadhesive proper-ties: carboxymethylcellulose, hydroxypropylcellulose, poly-vinyl alcohol, polyacrylic acid (Carbomer), high molecular weight (>200,000) polyethylene glycols, chitosans, hyaluronic acid, polygalacturonic acid, xyloglucan, etc [4]. Some of these polymers have found their way in lachrymal substitutes for treatment of dry eye conditions, as e.g. Cellu-fresh®, Celluvisc® monodose, Dacriosol®, Refresh® (cellu-lose derivatives); Liquifilm® Tears, Lacrilux®, Hypotears®

(polyvinyl alcohol); Dropstar® TG, Vismed®, Hy-drop®

(hyaluronic acid); Dacriogel®, Viscotirs®(polyacrylic acid); TSP® 0.5 (xyloglucan). Examples of medicated formulations containing polyacrylic acid (Carbopol®) are Fucithalmic®

viscous eye drops (1% fusidic acid, Leo Pharma Inc.) and the previously mentioned Pilopine® HS gel. All of these formu-lations presumably owe their improved ocular retention to the presence of a mucoadhesive polymer. The use of absorp-tion promoters, i.e. of substances facilitating drug penetra-tion through the corneal tissues is a potentially interesting, still little-exploited approach to improve ophthalmic bioavailability [6]. The effect of these substances (surface active agents, calcium chelators) on the cornea is to enhance the permeability of corneal epithelium by altering the cell membranes and loosening the tight junctions between super-ficial cells. Among the promoters that have been investi-gated, sometimes with positive results, the following can be mentioned: benzalkonium chloride, polyoxyethylene glycol lauryl ether (Brij® 35), polyoxyethylene glycol stearyl ether (Brij® 78), polyoxyethylene glycol oleyl ether (Brij® 98), ethylenediaminetetraacetic acid, Na salt (EDTA), digitonin, sodium taurocholate, saponins, Cremophor-EL, etc. Some of the previously mucoadhesive polymers (e.g., chitosans, car-boxypolymethylene, polyvinlypyrrolidone, the latter two cited compounds relevant specifically for the enhancement of N-acetylcarnosine transcorneal permeation) have been found in our studies active also as ocular absorption promot-ers. Unfortunately some agents, while effective, cause tran-sient irritation or produce irreversible damages to corneal tissues. A typical example is benzalkonium chloride, a pre-servative present in a myriad of ophthalmic formulations. This agent, although proved effective as corneal absorption promoter for different ophthalmic drugs, is now viewed with suspect on account of potential eye irritation, and preserva-tive-free, single-use eyedrops are gradually substituting the preserved multi-dose ones. A multi-dose container fitted with a sterilizing filter, allowing to dispense sterile, preserva-tive-free solutions (Abak®) has been recently patented by Thea Laboratories (France) and is used for an artificial tear formulation (FilmAbak® PVP artificial tears).

Even if the introduction of novel ocular dosage forms appears to proceed at a very slow pace, both on consideration of industrial costs and of the many restraints of the site of application, some drugs already in use have been recently revived in new, longer-acting liquid presentations advertised for once-daily application. Some of these dosage forms have been developed in the light of the vast body of knowledge derived from studies on bio/mucoadhesion. The time appears ripe, therefore, for introduction of advanced, more efficient ocular delivery systems. The need for ophthalmic medica-tions is continuously increasing as the populations of the industrialized nations age. The development of new products for treatment of ophthalmic diseases is facing a double chal-lenge: pharmacology and drug delivery. The conventional ocular drug delivery systems like solutions, suspensions, and ointments show drawbacks such as increased precorneal elimination, high variability in efficiency, and blurred vision respectively. In spite of active and continued research and of frequent introduction of novel ophthalmic drugs, ocular drug delivery does not seem to progress at the lively pace typical of e.g. oral, transdermal or transmucosal delivery. In recent years, only few advanced ocular delivery systems have been introduced into the market. The vast majority of existing ocular delivery systems are still «fairly primitive and ineffi-cient».

26 Current Drug Delivery, 2014, Vol. 11, No. 1 Babizhayev and Yegorov

The various approaches that have been attempted to in-crease the bioavailability and the duration of the therapeutic action of ocular drugs can be divided into two categories [7]. The first one is based on the use of sustained drug delivery systems, which provide the controlled and continuous deliv-ery of ophthalmic drugs, such as implants, inserts and col-loids. The second involves maximizing corneal drug absorp-tion and minimizing precorneal drug loss through viscosity and penetration enhancers, prodrugs and colloids. The differ-ent responses of the corneal and conjunctival drug penetra-tions to the absorption promoters may be useful in control-ling the extent and pathway of the ocular and systemic ab-sorptions of instilled peptide based drugs [4-6,8]. The pro-motional effects of absorption promoters on the corneal drug penetration apparently increased with an increase in pene-trant molecular weights, although those on the conjunctival drug penetrations did not depend on the molecular weights. The effects of mucoadhesive ophthalmic carriers and oph-thalmic preservatives as absorption promoters on the in-traocular and systemic absorption of L-carnosine active prin-ciple derived from N-acetylcarnosine peptide prodrug through the ocular route were investigated recently [9]. Of ophthalmic preservatives investigated in the formulation benzyl alcohol, parabens and EDTA showed promoting ef-fects [9]. The different responses of corneal and conjunctival drug penetrations to ophthalmic cellulose carriers and oph-thalmic preservatives may be useful to control the extent and pathway for the ocular and systemic absorptions of instilled peptide drug.

One of the specific ocular drug delivery forms for pep-tides can include in situ- forming hydrogels, that are liquid upon instillation and undergo phase transition in the ocular cul-de-sac to form viscoelastic gel and this provides a re-sponse to environmental changes. In the past few years, an impressive number of novel temperature, pH, and ion in-duced in situ-forming systems have been reported for sustain ophthalmic drug delivery. Each system has its own advan-tages and beneficial characteristics. The following characteristics are required to optimize ocular drug delivery systems. • A good corneal penetration. • A prolonged contact time with corneal tissue. • Simplicity of installation for the patient. • A non-irritative and comfortable form (the viscous solu-

tion should not provoke lachrymation and reflex blink-ing).

• Appropriate rheological properties and concentration of viscolyzer

The sustain ophthalmic drug delivery systems should have the benefits of • Prolonged drug release • Reduced systemic side effects • Reduced number of applications

Fig. (1). Topical administration is generally considered the preferred route for the administration of ocular drugs due to its convenience and affordability. Drugs applied in this manner can be packaged in multiple forms, including solutions, ointments, and suspensions. Corneal ab-sorption is limited by drainage of the instilled solutions, lacrimation, tear turnover, metabolism, tear evaporation, non-productive absorp-tion/adsorption, limited corneal area, poor corneal permeability, binding by the lacrimal proteins, enzymatic degradation, and the corneal epithelium itself. These limitations confine the absorption window to a few minutes after administration and reduce corneal absorption to < 5% [1-13].


• Better patient compliance. • Generally more comfortable than insoluble or soluble

insertion. Less blurred vision as compared to ointment. [10].

The choice of a particular hydrogel depends on its intrin-sic properties and envisaged therapeutic use. The involved professional in the creation of the ophthalmic peptide based drug should consider various temperatures, pH, and ion in-duced in situ- forming polymeric systems used to achieve prolonged contact time of peptide drugs with the cornea and increase their bioavailability [11]. The adhesive bioerodible ocular drug delivery system that provides bioerodible, water-soluble pharmaceutical carriers for ocular (e.g., transcon-junctival or transcorneal) delivery of pharmaceuticals for either systemic or local therapy was recently described [12]. This method is targeted for delivering a pharmaceutical via an ocular surface of a mammal, the method comprising con-tacting the ocular surface of the mammal with a mucoadhe-sive film that comprises: a water-soluble bioadhesive layer to be placed in contact with an ocular surface, the bioadhesive layer including one or more bioadhesive polymers and/or one or more film- forming, water-soluble polymers; a water-soluble non-adhesive backing layer that comprises one or more water-soluble, film-forming, pharmaceutically accept-able polymers; and one or more pharmaceuticals associated with the bioadhesive layer, associated with the non-adhesive layer, or associated with both the bioadhesive and non- adhe-sive layers; wherein the mucoadhesive film is compatible with ocular surfaces; the mucoadhesive film adheres to ocu-lar surfaces; the mucoadhesive film is flexible; and the mu-coadhesive film is water-soluble, biodegradable, and bio-erodible in tear fluids. The employed method describes the one or more film-forming water-soluble polymers comprises an alkyl cellulose or a hydroxyalkyl cellulose. The intro-duced method also is describing the one or more film-forming water-soluble polymers comprises hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxy-propylmethyl cellulose (HPMC), hydroxyethylmethyl cellu-lose (HEMC), or a combination thereof [12].

To summarize the considered items, the main purpose of pharmacotherapeutics is the attainment of an effective drug concentration at the intended site of action for a sufficient period of time to elicit the response. A major problem being faced in ocular therapeutics is the attainment of an optimal concentration at the site of action. Poor bioavailability of drugs from ocular dosage forms is mainly due to the tear production, non-productive absorption, transient residence time, and impermeability of corneal epithelium. The above presented components and carriers in ocular topical drug delivery systems contribute to the following: (1) the barriers that decrease the bioavailability of an ophthalmic peptide based drug and the capacities for their penetration; (2) the objectives in producing optimal formulations; and (3) the approaches currently being used to improve the corneal penetration of a peptide drug molecule and delay its elimina-tion from the eye and subjection to the enzymatic hydrolysis [13]. The focus of the above presented information is on the recent developments in topical ocular drug delivery systems, the rationale for their use, their drug release mechanism, and the characteristic features and limitations of the drug deliv-

ery system. As adjunct, one might consider the attempts to develop various analytical and technical procedures includ-ing the animal models and other biological models required for bioavailability and pharmacokinetic studies. The latter can aid in the design and predictive evaluation of newer pep-tide drug delivery systems. The dosage forms are generally divided into the ones which affect the precorneal parameters, and those that provide a controlled and continuous delivery to the pre- and intraocular tissues. Overall, the systems ap-plicable for the topical ocular drug delivery include: (a) the commonly used dosage forms such as gels, viscosity impart-ing agents, ointments, and aqueous suspensions; (b) the newer concept of penetration enhancers, phase transition systems, use of cyclodextrins to increase solubility of various drugs, vesicular systems, and chemical delivery systems such as the prodrugs; (c) the developed and under-development controlled/continuous drug delivery systems including ocular inserts, collagen shields, ocular films, disposable contact lenses, and other new ophthalmic drug delivery systems; and (d) the newer trends directed towards a combination of drug delivery technologies for improving the therapeutic response of a non-efficacious drug. The practical beneficial implica-tion of the above-mentioned technological suggestions can result in a superior dosage sustain release ocular forms for both topical and intraocular ophthalmic application. In this article, we have been focused on the major types of commonly used optimal N-acetylcarnosine ophthalmic formulations, indicated the generality of their applicability and acceptance, differentiated their characteristics and util-ity, and projected anticipated use and development in the decade to come. This should also serve to put into perspec-tive the discussions of more sophisticated components and elaborations described in this issue. A critical focus of the discussion will include not only optimal structure/ transport properties of N-acetylcarnosine molecule, but also the poten-tial metabolic transformations in surroundings and at the surface of target tissues. This knowledge provides for the potential of site-specific bioactivation of the prodrug, thus enabling the realization of significant improvements in effi-cacy and minimization of side-effect profiles, locally and systemically. Recently, it has been demonstrated that oxidants, includ-ing reactive oxygen species (ROS) induced premature senes-cence in human lens epithelial cells, with accelerated te-lomere shortening and reduced telomerase activity (although many human lenses lack detectable telomerase activity) [14-16]. We have presented the data that human cataractogenesis is characterized by senescence of lens cells, accelerated by oxidative stress-induced DNA damage, inhibition of telom-erase and marked telomere shortening [15,16]. Telomerase in lens epithelial cells in mammals may function in the quies-cent, central lens to maintain telomeres damaged by oxida-tive stress thus preventing accelerated loss of these elements which triggers cell senescence [17]. It remains to be deter-mined if the threat of telomeres attrition and increase in te-lomerase activity in lens epithelial cells (LEC) from catarac-tous lenses of small animals (canines) is a primary dysregu-lation chain that may have a role in the development of cata-racts, or serve as a therapeutic target for N-acetylcarnosine lubricant eye drops treatment for cataract management. In this work, we have focused the center of data analysis on


dogs to demonstrate the efficacy of N-acetylcarnosine pro-drug delivery in the treatment of cataract in dogs and to ana-lyze the data showing that telomeres length and telomerase activity in LEC of canines may serve as a therapeutic target for N-acetylcarnosine prodrug of L-carnosine bioactivated antioxidant eye drop treatment of the lens diseases in small animals. One of the obscure aspects of the carnosine problem is the biological significance of the enzymatic metabolism of carnosine or its derivatives in tissues. Thus, in order to change an antioxidant status, tissue enzymes can modify the NAC prodrug molecule and deacetylation will increase invivo the resistance of lens tissues and its cells to oxidative stress. The topical administration of N-acetylcarnosine as a universal bioactivating antioxidant for vision in the devel-oped and patented lubricant eye drop formulations delivers pure L-carnosine and allows its increased intraocular absorp-tion into the aqueous humor surrounding the lens, thus ena-bling significant improvements in anti-cataract drug efficacy and the minimization of side-effect from either local or sys-temic drug absorption/bioavailability to the eye, and also creates optimization effects in the number of ocular degen-erative age-dependent disorders (Reviewed in Refs. [4,9]). Based on the features of the specific ocular drug delivery form for peptides, a method for prevention or treatment of an eye disease in mammals has been developed and patented, which includes topically applying to a patient in need of the treatment an aqueous ophthalmic composition which in-cludes N-acetylcarnosine, a N-acetylcarnosine derivative or a pharmacologically acceptable salt of N-acetylcarnosine, in combination with an amount of a cellulose compound or a pharmacologically acceptable salt which is unexpectedly and surprisingly effective to increase intraocular absorption of N-acetylcarnosine or L-carnosine or a derivative of L-carnosine, such as anserine or balenine or carcinine, in the aqueous humor. A method for the treatment of an eye dis-ease, comprising topically applying to a mammal in need of

said treatment an aqueous ophthalmic composition compris-ing N-acetylcarnosine, a N-acetylcarnosine derivative or a pharmacologically acceptable salt of N-acetylcarnosine, in combination with carboxymethylcellulose or its pharmaco-logically acceptable salt, in an amount effective to increase intraocular absorption of of L-carnosine or a L-carnosine derivative into the aqueous humor, wherein said N-acetylcarnosine is present in an amount ranging from 0.5% to 2% by weight of the composition is utilized in this work [18]. Free radicals and the peroxidative processes caused by them are believed to be one of the causes of the structural and functional degradations of human and animal tissue, especially lens tissues during aging. Below we concisely consider the aspects of the oxidation related damages during cataractogenesis in dogs and other small animals.

2. SPECIAL FEATURES OF CATARACTOGENESIS IN DOGS

Cataracts are one of the most significant ophthalmologic diseases in veterinary medicine. Cataract refers to the cloudiness in the crystalline lens of the eye, varying from complete to partial opacity. Because the cloudiness of the eye lens prevents light from passing to the retina, a cataract can cause vision loss. It is well understood that dogs are more prone to develop cataracts than other domestic animals (Fig. 2). Cataracts are a leading cause of blindness in dogs with approximately 100 breeds affected by primary heredi-tary forms [19]. Some canine breeds such as the Australian Shepherd exhibit a pronounced tendency toward inherited cataracts [19], and some diseases such as diabetes are also known to cause cataracts owing to a change in the crystalline lens metabolic pathway [20]. Most cases of cataracts are inherited; for instance, Miniature poodles, American cocker spaniel, miniature schnauzer, golden retrievers, Boston terri-ers, and Siberian hyskies are all predisposed to cataracts. The results of the published study [20] suggest that the majority of dogs with diabetes will develop cataracts within 5-6

A B

Fig. (2). (A) Cataract in dog as the common eye condition. Cataracts form when the lens in the eye becomes cloudy. This will lead to re-duced vision. As the condition worsens, vision loss can become significant. There are many different forms and causes of cataract in dogs. They affect all breeds and ages of dogs, but certain types show up more commonly in certain breeds. Dog cataracts can occur for any number of reasons. Genes play a major role as most dogs who develop cataract condition did so because they inherited it. Some puppies are even born with them. Other common causes include diabetes mellitus, trauma, infection, and normal aging. In most situations, cataracts form in both eyes. However, if the condition is caused by an infection or trauma, there may be only one eye affected. (B) The structures of the canine lens are presented.


months from the time of diagnosis of the disease, and that approximately 80% of dogs will develop cataracts within 16 months of diagnosis. Meanwhile, if a dog has diabetes melli-tus-related cataract, one may also observe increased thirst, increased frequency of urination, and weight loss in a dog, along with vision impairment symptoms (Figs. 3 and 4). Despite the large number of breeds affected with heredi-tary cataracts (HC) little is known about the genetics of the condition, and to date only a single gene, HSF4, has been implicated in the development of the disease in dogs. Using DNA samples from almost 400 privately owned Australian Shepherds Mellersh et al. [19] have investigated the associa-tion between the deletion mutation in HSF4 and cataracts in this breed. The authors have revealed that the mutation is significantly associated with cataracts and that a dog carry-ing the mutation is approximately 17 times more likely to develop binocular cataracts than dogs that are clear of the mutation. The data also indicate that additional mutations associated with the development of cataracts are likely to be co-segregating in the Australian Shepherd population [19]. Symptoms of cataract typically relate to the degree of vision impairment (Fig. 3). Dogs with less than 30 percent lens opacity, for example, display little or no symptoms, whereas those with more than 60 percent opacity of the lens may suf-fer from loss of vision or have difficulty seeing in dimly lit areas. Although most cases of cataracts are inherited, the following are other causes and risk factors associated with the condition:

• Diabetes mellitus • Old age • Electrical shock • Inflammation of the eye's uvea (uveitis) • Abnormally low levels of calcium in blood (hypocalce-

mia) • Exposure to radiation or toxic substances (e.g., dinitro-

phenol, naphthalene) Clinical data from 72 dog breeds of varying size and life expectancy were grouped according to breed body mass and tested for prevalence at ages 4 to 5, ages 7 to 10, and lifetime incidence of non-hereditary, age-related cataract [21]. The incidence of age-related cataract was found to be directly related to the relative life expectancies in the breed groups: The smallest dog breeds had a lower age-related cataract prevalence between ages 4 and 5 than mid-size breeds and these, in turn, a lower prevalence than the giant breeds. A similar sequence was evident for ages 7 to 10 and for overall lifetime incidence of age-related cataract. These differences became more significant when comparing small and giant breeds only. Urfer et al. have confirmed the inverse relation-ship between body size and life expectancy in these same sets of dog breeds [21]. The results have shown that body size, life expectancy, and age-related cataract incidence are interrelated in dogs [21]. Oxidative stress on lens compo-nents has been recognized as an important mechanism in the

Fig. (3). Cataract formation is one of the most prevalent eye diseases in the dog population, and in about 60 breeds of dogs the prevalence of cataract exceeds that of the baseline mixed-breed/hybrid group. Most cataracts in dogs are inherited and can occur at any age. The cataract may develop rapidly over weeks, or slowly over years, and occur in one or both eyes. Different breeds of dogs have different characteristics of cataract development. The prevalence of cataract is also influenced by age in most purebred dogs and affects 16.80% of the 7-15+-year-old mixed-breed/hybrid dog population [23]. For the most part cataract is the disruption of the normal arrangement of the lens fibers in the eye, which causes the loss in the transparency of the lens, causing vision loss. A retrospective study of all dogs presented with cataracts to veterinary medical teaching hospitals in North America between 1964 and 2003 was conducted to determine cataract prevalence. The differ-ent decades, breeds, gender, and age at time of presentation with cataract were compared. The prevalence of dogs presented with cataract varied by decade and ranged from 0.95% (1964-73), 1.88% (1974-83), 2.42% (1994-2003), to 3.5% (1984-93). The total number of dogs presented with cataracts over the 40-year period was 39,229. From 1964 to 2003 the prevalence of cataract formation in this patient popula-tion increased by about 255%. Fifty-nine breeds of dogs were affected with cataracts above the baseline prevalence of 1.61% seen in mixed-breed/hybrid dogs. The breeds with the highest cataract prevalence included: Smooth Fox Terrier (11.70%), Havanese (11.57%), Bichon Frise (11.45%), Boston Terrier (11.11%), Miniature Poodle (10.79%), Silky Terrier (10.29%) and Toy Poodle (10.21%). The breeds with the largest number of cataractous dogs during the entire four decades were the Boston Terrier (11.11%), Miniature Poodle (10.79%), American Cocker Spaniel (8.77%), Standard Poodle (7.00%), and Miniature Schnauzer (4.98%) [23].


Fig. (4). Cataract in the diabetic dog. Normally, the lens absorbs glucose from the eye fluids, using most of this for its own energy needs. Some of the excess is converted to another sugar called “sorbitol.” When there is excess sugar in the eye fluids, there is excess sorbitol pro-duced. Sorbitol is not soluble and it draws fluid into the lens which in turn disrupts lens clarity and causes the cataract. Fructose is also pro-duced from the excess glucose and also contributes to this water imbibition. The lens becomes intumescent leading to two important consid-erations: 1. Diabetic cataracts are usually easier to treat surgically because they are softer than other mature cataracts, reducing the length of surgery

and the potential for complications. 2. The relatively rapid swelling of the lens may lead to rupture of the lens capsule (which is non-distensible), with leakage of lens contents

through the capsule, usually at the equator (periphery) of the lens. Massive exposure of lens material to the intraocular immune system may cause a severe inflammatory reaction (phacoclastic uveitis).

The presence of cataracts does not necessarily imply poor diabetic control. Even well controlled dogs still can get cataracts.

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development of cataracts (Reviewed in Ref. [22]). Given that age-related cataract has been shown to be at least partially caused by oxidative damage to lens epithelial cells and the internal lens, it has been suggested that it can be considered not only as a general biomarker for life expectancy in the canine and possibly other species, but also for the systemic damages produced by reactive oxygen species (ROS) [21] (Fig. 5). In this article we investigate the acceptable concept that the specific factors of oxidative stress in canine lens epithe-lial cells (LECs) accelerate telomere shortening and the in-crease in telomerase activity in canine LECs from catarac-tous lenses is a primary dysregulation that may have a role in the development of the canine cataract (Figs. 6 and 7) [17]. Telomerase activity was found in normal canine lens epithe-lial cells in the central, germinative and equatorial regions of the anterior lens capsule at equivalent levels. Similar find-ings were made in feline and murine lens epithelial cells (but not in human LECs), indicating that the presence of telom-erase activity in the lens was not animal species specific [17]. Unexpectedly, telomerase activity and telomere lengths were significantly greater in lens epithelia from canine cata-ractous lenses when compared with normal lenses [17]. Since telomerase activity is associated with an immortal phenotype, the presence of telomerase activity in the LECs may function to prevent conversion to senescence. Telom-erase may function in the quiescent, central lens to maintain telomeres damaged by oxidative stress and ultraviolet light exposure, thereby preventing accelerated loss and attrition of these elements which triggers cell senescence [17] (Fig. 8). Based on the revealed understanding of the leading role of oxidative stress as the established telomere attrition threat major risk factor for cataractogenesis and for compensatory regulation of telomerase, it might be considered that that the therapeutic telomerase-induced telomere length manipula-tions as a target for N-acetylcarnosine lubricant eye drops may have a therapeutic utility for lens tissue engineering and for controlling the molecular mechanisms underlying cata-ractogenesis, prevention and the therapeutic treatment of cataracts in canines .

3. CANINE LENS EPITHELIAL CELLS AND THEIR ROLE IN THE DEVELOPMENT OF CATARACTS. OXIDATIVE STRESS AND CATARACT FORMA-TION

The adult crystalline lens is lined on its anterior surface with a monolayer of lens epithelial cells (LEC) with variable replicative potential. LECs are the parental cells responsible for growth and development of the transparent ocular lens [23-26]. The lens epithelial cells (LECs) are the progenitors of the lens fibers in vivo and undergo a developmental transi-tion into fiber cells of the lens cortex, a process characterized by distinct biochemical and morphologic changes such as the synthesis of crystallin proteins, cell elongation, loss of cellu-lar organelles, and disintegration of the nucleus [25,26] (Figs. 6 and 7). Recently, optimal culture conditions for LECs from the dog were reported, even though proliferation stopped around passage 6 [27]. Canine LEC are typically isolated by mechanical dissection of the canine globe and enzymatic digestion of the lens capsule from fresh lenses.

Isolated capsules and cell suspensions were seeded in laminin-coated culture flasks [27]. Canine LEC proliferated and formed monolayers, which could be passaged and main-tained for approximately 2 weeks. Cells are characterized morphologically and cell lysates examined for expression of protein markers of epithelial origin and differentiation. Ca-nine LEC eventually exhibit morphologic characteristics of epithelial cells when cultured on laminin/lysine coated flasks [27]. Expression of epithelial cell marker, cytokeratin 5, was highest at passage 1 and diminished with increasing passage number. Expression of gamma-crystallin, a protein found only in differentiated lens fiber cells, increased at passage 6. A laminin/lysine-coated surface supported optimal prolifera-tion of canine LEC. Both an initial seeding density of 1 x 105

cells/cm2 and culture in Dulbecco's modified essential media (DMEM) supplemented with 10% FBS supported a doubling time of less than 48 h in canine LEC [28]. It was understood that primary damage to LECs may cause abnormal differen-tiation of epithelial cells to lens fibers, which is eventually expressed as an opacity of lens tissues. The primary canine LECs retain the characteristics of lens epithelial cells prior to passage 6 under the appropriate culture conditions and repre-sent a suitable in vitro model for investigating lens physiology, cataractogenesis and the effects of oxidative stress [24-27]. Oxidative stress is one of the key factors associated with initial cataractous changes in the crystalline lens [22,26,28-31]. Oxidants, such as hydrogen peroxide, dehydroascorbic acid, lipid peroxides can damage the structure and impair the integrity of the LEC by inducing protein disulfide crosslinks, decreased protein solubility, and decreased cellular adhe-sions [28-34] (Fig. 5). After exposure to oxidative stress, the redox set point of the single layer of the lens epithelial cells (but not the remainder of the lens) quickly changes, going from a strongly reducing to an oxidizing environment. Al-most concurrent with this change is extensive damage to DNA and membrane pump systems, followed by loss of epithelial cell viability and death by necrotic and apoptotic mechanisms. The data document that the epithelial cell layer is the initial site of attack by oxidative stress and that in-volvement of the lens fibers follows, leading to cortical cata-ract [31]. The reducing compound glutathione (GSH) exists in an unusually high concentration in the lens where it func-tions as an essential antioxidant vital for maintenance of the tissue's transparency. In conjunction with an active glu-tathione redox cycle located in the lens epithelium and super-ficial cortex, GSH detoxifies potentially damaging oxidants such as H2O2 and dehydroascorbic acid. The published stud-ies have indicated an important hydroxyl radical-scavenging function for GSH in lens epithelial cells, independent of the cells' ability to detoxify H2O2. Depletion of GSH or inhibi-tion of the redox cycle allows low levels of oxidant to dam-age lens epithelial targets such as Na/K-ATPase, certain cy-toskeletal proteins and proteins associated with normal membrane permeability (Reviewed in Ref. [33]). A high level of glutathione is maintained in the LEC; 95% of lens glutathione is typically in the reduced form (GSH) and is thus available for detoxification of potentially damaging oxidants [33,35]. The GSH functioning is medi-ated through a redox cycle located in the LEC and superficial cortex that utilizes glutathione reductase, NADPH, and the


Dehydroascorbic acid

A.

B.

Reduced Glutathione


(Fig. 5) contd….

Fig. (5). Ophthalmic pathology of lens in small animals. A. At laboratories of Innovative Vision Products, Inc. (Delaware, USA) we have determined the oxidants, antioxidant status and biomarkers of oxidative stress of the aqueous humor after extracapsular lens extraction in 20 dogs of various breeds weighing about 10 kg. Samples of aqueous humor were obtained by anterior chamber paracentesis. B. Normal dog lens, H&E stain. Normal lens cortex under relatively good conditions of fixation and preparation. The splits in the tissue with granular material within are artifacts in this case. The reducing compound glutathione (GSH) exists in an unusually high concentration in the lens where it functions as an essential antioxidant vital for maintenance of the tissue's transparency. In conjunction with an active glutathione redox cycle located in the lens epithelium and superficial cortex, GSH detoxifies potentially damaging oxidants such as H2O2 and dehy-droascorbic acid. Recent studies have indicated an important hydroxyl radical-scavenging function for GSH in lens epithelial cells, independ-ent of the cells' ability to detoxify H2O2. Depletion of GSH or inhibition of the redox cycle allows low levels of oxidant to damage lens epithelial targets such as Na/K-ATPase, certain cytoskeletal proteins and proteins associated with normal membrane permeability. The level of GSH in the nucleus of the lens is relatively low, particularly in the aging lens, and exactly how the compound travels from the epithelium to the central region of the lens is not known [33]. A-D. Overall, the eyes of dogs and felines can suffer the effect of oxidative damage due to the etiopathogenesis of some pathological changes related to oxidative stress. This article highlights the role of oxidative stress in the onset and progression of damage in different eye structures in canines, the involvement of the natural antioxidant networks, (such as accumulation of ferritin in the canine lens structures) in protecting and maintaining the homeostasis of the lens, and the potential assessment diagnostic and therapeutic treatment methodologies used in research and in some cases including clinical practice. C. Immature cataract in canine involving 100% of the lens with denser opacities at the level of the cortical suture lines. Although in this case examination of the fundus is impaired, a clear green reflection can be seen from fundic retroil-lumination. D. Hypermature cataract in dog. The picture, taken with slit lamp, shows the wrinkling of the anterior capsule due to lens proteinloss.

C D


Fig. (6). Cataract (incipient), dog, H&E stain; changes are the retained nuclei in the subepithelial fibers. Above is anterior capsule, below lens epithelium is visualized; fissures are artifactitious. The unequivocal (not subject to artifact) cataractous changes are the retained nuclei in the subepithelial fibers; these fibers are mature and should have lost their nuclei. The fissures are artifacts of preparation.

hexose monophosphate shunt to reduce any oxidized glu-tathione (GSSG) [36]. GSH may be important in maintaining protein thiols in the reduced state, thus preventing the forma-tion of high molecular weight protein aggregates which are the basis for light scattering and lens opacification. A second function may be to protect membrane -SH groups that are important in cation transport and permeability. A third func-tional role is to detoxify hydrogen peroxide and other or-ganoperoxides. The glutathione redox cycle is intimately involved in the detoxification of H2O2 and phospholipid hy-droperoxides which are normally present in the aqueous hu-mor [29,36]. It has been demonstrated that thioltransferase has a remarkable resistance to oxidation (H2O2) in cultured human and rabbit lens epithelial cells under oxidative stress conditions when other oxidation defense systems of GSH peroxidase and GSH reductase are severely inactivated. A second repair enzyme, thioredoxin (TRx), which is NADPH-dependent, can dethiolate protein disulfides and thus is an extremely important regulator for redox homeostasis in the cells. Thioredoxin has been recently found in the lens and has been shown to participate in the repair process of oxida-tively damaged lens proteins/enzymes. These two enzymes may work synergistically to regulate and repair thiols in lens proteins and enzymes, keeping a balanced redox potential to maintain the function of the lens [37].

The ability of transparent and cataractous human, rabbit and mice lenses to metabolize hydrogen peroxide in the sur-rounding medium was evaluated [38]. The ability of opaque human lenses to catalyze the decomposition of 10-4 M H2O2was significantly decreased. However, this was reversed by

the addition of GSH to the incubation medium. Incubation of the mice lenses with the initial concentration H2O2 10-4 M led to partial depletion of GSH in normal and cataractous lenses. Human cataractous lenses showed decreased activi-ties of glutathione reductase, glutathione peroxidase (catalyz-ing reduction of organic hydroperoxides including hydroper-oxides of lipids), superoxide dismutase, but no signs of de-pletion in activities of catalase or glutathione peroxidase (utilizing H2O2). The findings indicated an impairment in peroxide metabolism of the mature cataractous lenses com-pared to normal lenses to be resulted from a deficiency of GSH. An oxidative stress induced by accumulation of lipid peroxidation (LPO) products in the lens membranes during cataract progression could be considered as a primary cause of GSH deficiency and disturbance of the redox balance in the lens [38]. In acute studies, excised lenses were exposed to the oxidant tert-butyl hydroperoxide (TBHP) for 0–120 min [34]. TBHP was used because it has been shown to in-duce DNA damage, specifically single strand breaks and oxidised bases, along with cell killing, LPO and redox state alteration [39,40] and apoptosis [41]. Treatment with TBHP significantly reduced glutathione content and glutathione reductase activity, and increased glutathione peroxidase ac-tivity, indicating that TBHP induced oxidative stress in the treated cells. TBHP also induced reduction of cell viability and DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner [41]. Neither alpha-tocopherol (vitamin E) nor ascorbate prevented the accumulation of DNA single-strand breaks caused by TBHP, indicating that these vitamins do not effectively scavenge the TBHP-derived radicals re-sponsible for DNA damage [39].


Fig. (7). (A) Cataract, dog, H&E stain; zone of fibrous metaplasia of epithelium. Upper place: anterior lens capsule, in the middle area of the image is cortex. The unequivocal (not subject to artifact) cataractous change is the zone of epithelial proliferation (fibrous metaplasia). The fissures are artifacts of preparation. Telomere length has been shown to be associated with nutritional status in animal studies [16,172]. (B)Increase of telomere length after administration of the hTERT gene in several cell types including human neural stem cells (presented case studies), fibroblasts or epithelial cells.

Published results indicate that thickness of the anterior lens capsule in dogs increases with age and that this increase in thickness is not significantly different between normal lenses and lenses with cataracts. In addition, epithelial cells

from lenses with cataracts may undergo metaplasia to form plaques composed of fibrous tissue and ectopic basement membrane produced by epithelial cells [42]. Immunohisto-chemically, normal lens epithelial cells and cells within

A.

B.


plaques stained for vimentin. Most cells and some areas of the extracellular matrix within plaques stained for TGF-beta and alpha-smooth muscle-specific actin. Fibronectin and tenascin were also detected in the extracellular matrix [43]. Canine lens capsular plaques are histologically and immuno-histochemically similar to posterior capsule opacification and subcapsular cataracts in humans, which suggests that the canine condition, like the human conditions, is associated with fibrous metaplasia of lens epithelial cells. Transforming growth factor-beta may play a role in the genesis of capsular plaques [43]. As these destructive changes occur, opacification of the lens occurs, resulting in cataract formation [32]. It was reported previously that dietary ascorbate (ASC) delays the development of galactose-induced cataract in guinea pigs compared to the rate which is observed in ASC-deficient animals. Guinea pigs were fed for a period of up to 4 weeks either a normal diet (1 g ASC/kg diet) or a scorbutic diet (< 0.04 g ASC/kg diet) combined with 10% galactose in the drinking water. After 2 weeks, levels of ASC in animals on the scorbutic diet decreased by 95% in the aqueous humor and by 78% in the lens. Hexose monophosphate shunt activ-ity was elevated in lenses of normal galactose-fed animals during the first hour of culture after death whereas lenses of scorbutic galactose-fed animals were not. Consistent with the in vivo findings, galactitol accumulation in dog lens epithe-lial cells exposed to 30 mM galactose was significantly in-hibited by the presence of either ASC or dehydroascorbate (DHA) in the medium. Hexose monophosphate shunt activ-ity in the cells was stimulated to two-and-a-half times its initial level by either 1 mM DHA or 30 mM galactose and slightly more than three-fold by a combination of the two challenges. The results suggest that decreased polyol accu-mulation in the lens epithelium of the normal galactose-fed guinea pig, which has a high level of ASC in the aqueous humor, accounts for the delay in onset of cataract compared to that for the ASC-deficient animal [44]. The molecular chaperones alpha-crystallins are abundant proteins synthesized in the differentiated lens fiber cell cyto-plasm. However, their expression in lens epithelial cells has only been appreciated very recently. Besides their important roles in the refractive and light focusing properties of the lens, alpha-crystallins have been implicated in a number of non-refractive pathways including those involving stress response, apoptosis and cell survival. The most convincing evidence for their importance in the lens epithelium has been shown by studies on the properties of lens epithelial cells from alphaA and alphaB-crystallin gene knockout mice [45]. Alpha B-crystallin, a major lens protein, was induced in pri-mary cultures of dog lens epithelial cells and glomerular endothelial cells when they were grown under conditions of hypertonic stress. With Western blot analysis using a specific alpha B-crystallin antibody, Dasgupta et al have observed a significant increase in the concentration of alpha B-crystallin protein in cells grown for 4-6 days in media supplemented with 150 mM NaCl or 250 mM cellobiose. These supple-ments increased the osmolarity of the medium from 300 to 550-600 mosmol kg-1 [46]. Aldose reductase (AR) mRNA levels increase when dog lens epithelial cells are exposed to hypertonic conditions. Hybridization of mRNA to an AR cDNA, using Northern

and slot blots, showed that AR mRNA is elevated at least fourfold when primary dog lens epithelial cells are grown in media (300 mosmol kg-1) supplemented with 150 mM NaCl (600 mosmol kg-1 final) [47]. Cells grown in media supple-mented with 250 mM sorbitol also showed a substantial in-crease in AR mRNA. These data indicate, as in other cell types, the lens, a target tissue of diabetes, responds to hyper-somotic stress with an induction of AR expression and sug-gests that AR may play a role in intracellular osmotic regula-tion [47]. Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cul-tured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 mM D-galactose or 30 mM D-glucose. These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 microM sorbinil) [48]. The results suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity. Na,K-ATPase activity increases in lens cells exposed to hypertonic stress. To test whether the increase in activity involves stimulation of Na,K-ATPase expression, dog lens epithelial cells were sub-jected to hypertonic stress, and the time course of Na,K-ATPase protein and mRNA response was measured. Eleva-tion of Na,K-ATPase activity in dog lens epithelial cells ex-posed to hypertonic stress was associated with increased expression of Na,K-ATPase subunit mRNAs and was de-pendent on protein synthesis. These results suggest that upregulation of the enzyme activity is the result of an induc-tion of Na, K-ATPase [49]. Bucolo et al. have investigated the effect of monosialo-ganglioside on oxidation-induced changes in organ-cultured rabbit lenses and in cultured dog lens epithelium and human retinal pigment epithelial cells [50]. Exposure of organ-cultured lenses to 0.5 mM hydrogen peroxide for 1 hr in-creased the efflux of 86Rb from intact lenses and loss of myo-inositol from the capsule epithelium. Pretreatment of the lenses with monosialoganglioside significantly reduced the efflux rate of 86Rb and loss of myoinositol. Monosialogan-glioside also prevented morphological changes induced by 0.1 mM hydrogen peroxide in dog lens epithelium and loss of cell viability caused by docosahexaenoic acid in dog lens epithelium and in human retinal pigment epithelial cells. In contrast to the protective effect of monosialoganglioside on permeability and morphological changes in cultured cells, it had no effect against single-strand breaks of DNA in dog lens epithelium resulting from exposure to hydrogen perox-ide, X-ray and UV-B radiation. It appears that this gangli-oside serves as a membrane stabilizer rather than as a free-radical scavenger [50]. Vertebrate lens tissues contain several species of acidic and neutral glycosphingolipids in relatively high amounts. However, the epithelia with capsule from dog and rhesus monkey lenses had a simpler composition and lower content of glycosphingolipids than whole lenses. Gangliosides and neutral glycosphingolipids in monolayer cultures of lens epithelial cells were also different from those in whole lenses [51]. Glycosphingolipid synthesis in lens epithelia is intrinsi-cally different from that in cortical and nuclear fibres, and the expression of Lewis(x) and alpha-galactosyl epitopes in


glycosphingolipids appears to be associated with the differ-entiation of epithelial cells to fibres [51]. Transforming growth factor-beta 2 (TGF-beta 2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid levels of TGF-beta 2 are quite high. Although the sources of ocular TGF-beta are not completely defined, the retinal pigment epithelium, the epithelium of the ciliary body and trabecular meshwork cells all secrete it. In a separate series of experi-ments, Allen et al. have utilized canine lens and rabbit ciliary pigmented epithelial cell cultures to quantitate the in vitrosecretion of TGF-beta 2 [52]. In addition, the effects of aphakia or the presence of cataractous lenses on intraocular fluid TGF-beta 2 levels were determined. TGF-beta 2 accu-mulated in the media bathing lens epithelial cell cultures (0.7 +/- 0.03 ng/ml at day 2) and ciliary pigmented epithelial cell cultures (0.8 +/- 0.06 ng/ml at day 2) in a time-dependent manner. Aqueous humor from aphakic rabbit eyes contained significantly higher levels of TGF-beta 2 than their contra-lateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-beta 2 than normal eyes [52]. The published results suggest that the lens secretes TGF-beta 2 and that the presence and status of the lens may influence intraocular fluid TGF-beta 2 levels [52].

4. PROLIFERATIVE STATUS OF LENS EPITHELIAL CELLS (LECs)

Central LEC are capable of undergoing mitosis, though this occurs in less than 1% of the cells at any given time but increases substantially following traumatic injury [53-59]. Wound healing in the lens in vivo is accompanied by cellular migration and mitosis. The published experiments demon-strate that a highly purified serine protease, thrombin, which is present at the site of lenticular injury in vivo, is capable of inducing mitosis and migration in lens epithelia. The results suggest that thrombin or other exogenous and endogenous serine proteases might contribute to the process of wound healing in the ocular lens [55]. The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Me-chanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [3H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [3H]TdR was applied 5 hr before S phase (9 hr after wound-ing), all the cells were lightly labelled. The small amounts of the label were available to these cells 5 hr after application [56]. The published study to Rakic et al. [57] was aimed at testing the potential of the lens capsule and the fibres to af-fect the mitotic activity of the lens epithelial cells in human donor eyes. Pairs of human lenses used were subjected to two different experimental conditions. In the first, only a small anterior capsulorhexis was made. In the other, fibres were separated from the epithelium by gentle hydrodissec-tion, the nucleus was rotated and, either left in the capsular bag or removed by hydroexpression. The specimens were cultured for 48 hr in MEM with 2% serum. Mitotic activity was assessed by immunohistochemistry using the bromode-

oxyuridine (BrdU) incorporation technique. Fellow lenses of the same donors were cultured intact as controls. Anterior capsulorhexis showed a small but significant (P < 0.02) in-crease in the number of BrdU positive nuclei in the equato-rial region but not in the wound area. Lenses where fibres were separated from the epithelium showed a large increase in BrdU incorporation in the germinative region, as com-pared to the intact control lenses (P < 0.01). BrdU incorpora-tion was highest when fibres were removed. Lens capsule integrity and the presence of contacts between fibres and epithelial cells have an impact in the control of the mitotic activity of the lens epithelium [57]. Removal of lens fibres significantly elevated the proliferative activity of the remain-ing LECs. Suppression by newly formed differentiated lens fibres in the in vivo capsular bag may be responsible for the return to control levels of mitotic activity of LECs in the posterior capsule opacification (PCO) specimens [58]. The dividing lens epithelium of 8-week-old CF1 mice consists of a monocellular layer of about 31,000 cells and does not in-clude the postmitotic cells of the meridional rows and an-other postmitotic zone of seven cell positions' width immedi-ately anterior to the rows. The latter two populations contain approximately 3,600 and 9,000 cells, respectively, for a total of 44,000 cells in the entire lens epithelium [59]. The divid-ing cell population consists largely of a slowly cycling stem cell group, dividing once about every 17-20 days, and con-sisting of some 5,000 cells. A subpopulation may exist which undergoes two rapid consecutive divisions before be-coming postmitotic, but this is rather small to make a signifi-cant contribution to lens fiber production. Four days are re-quired to transit the postmitotic zone, and an additional 43 or so are needed to transit the meridional rows and differentiate into anucleate lens fibers [59]. The germinative LEC undergoes a controlled amount of mitosis thoughout life that contributes to the continuous growth of the lens, and the equatorial LEC undergoes termi-nal differentiation into lens fiber cells [53, 60-64, 19-29]. Lens epithelial cells explanted together with their capsule into serum-free medium underwent cell division. The extent of cell division depended on the age of the donor rat. After explantation, lens epithelia from newborn and 5 day rats showed decreased mitotic activity from day 0 to day 3 whereas epithelia from 15 day rats demonstrated a marked increase in mitotic activity which peaked at day 3 [60]. The morphology, cell density and mitotic index of human lens epithelial cells were determined according to the region of the opacity as follows: (1) nuclear; (2) posterior subcapsular; (3) cortical; (4) nuclear and posterior subcapsular; (5) corti-cal and posterior subcapsular. Epithelial abnormalities, con-sisting of enlarged, polymorphous cells and intercellular clefts, were found in cataractous lenses involving posterior subcapsular and/or cortical opacities. In contrast to the nor-mal pattern of cell density, the cell density of cataractous epithelia in these categories decreased from the central to the peripheral epithelium [64]. Aberrant proliferation and posterior migration of the LEC are changes that occur in LEC during cataractogenesis and secondary cataract (also known as posterior capsule opacifi-cation [PCO]). These phenomena were previously referred to as fibrous metaplasia or pseudometaplastic changes [65]. These LEC have genotypic and phenotypic changes which


are thought to be a wound healing response of LEC in an attempt to repopulate the lens capsule [66-69]. Lens epithe-lial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. EMT of primary lens epithelial cells in vitrodepends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury invivo or to exposure to exogenous TGF-beta in organ culture [66]. Shirai et al. have utilized a new animal model of ante-rior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factor beta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model [67]. Two days post-burn of the ocular surface, lens epithelium underwent epithelial-mesenchymal transition (EMT) as evidenced by the upregu-lation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type lens cells. Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract, Smad3 signaling is involved [67]. In the other published work to Medvedovic et al. in 2006, it has been supported that after extracapsular lens re-moval by anterior capsulotomy in the mouse, the lens can be regenerated [68]. As the capsular bag is filled with fibers, epithelial to mesenchymal transition (EMT), an event which is common after cataract surgery as well, takes place during early stages. It has been revealed from the analysis of the gene clustering data that initially there is a response to in-jury, extensive matrix remodeling, and severe downregula-tion of genes encoding lens structural proteins. The patterns returned gradually to normal three weeks after surgery. New genes were identified from the clustering results that might be potential regulators of EMT and lens differentiation [68]. Fibroblast growth factors, in combination with other growth factors, are key regulators of lens fiber cell differen-tiation [69]. While members of the transforming growth fac-tor (TGFbeta) superfamily have been implicated to play a role in lens fiber differentiation, inappropriate TGFbeta sig-naling in the anterior lens epithelial cells results in an EMT that bears morphological and molecular resemblance to forms of human cataract, including anterior subcapsular (ASC) and posterior capsule opacification (PCO; also known as secondary cataract or after-cataract), which occurs after cataract surgery. Numerous in vitro and in vivo studies indi-cate that this TGFbeta-induced EMT is part of a wound heal-ing response in lens epithelial cells and is characterized by induced expression of numerous extracellular matrix proteins (laminin, collagens I, III, tenascin, fibronectin, proteogly-cans), intermediate filaments (desmin, alpha-smooth muscle actin) and various integrins (alpha2, alpha5, alpha7B), as well as the loss of epithelial genes [Pax6, Cx43, CP49, al-pha-crystallin, E-cadherin, zonula occludens-1 protein (ZO-1)] [69]. Recent studies implicate other factors [such as fi-broblast growth factor (FGFs), hepatocyte growth factor, integrins], present in the lens and ocular environment, in the pathogenesis of ASC and PCO. For example, FGF signaling can augment many of the effects of TGFbeta, and integrin

signaling, possibly via ILK (Integrin Linked Kinase), ap-pears to mediate some of the morphological features of EMT initiated by TGFbeta [69]. Lenticular EMT is accompanied with the migration of the LEC onto the posterior lens capsule and with the production of aberrant extracellular matrix pro-teins, which result in subcapsular plaque opacities [70]. These molecular rearrangements are seen in advanced senile cataracts, in inherited, congenital, diabetic, traumatic, and ultraviolet radiation (UV)-induced cataracts and during PCO regardless of cause or species [71-76]. Domination of prolif-erative changes in traumatic and complicated cataracts, in contrast to senile cataracts, results from the availability of different mitogenic factors in injured and inflammed eyes and may be due to the younger age of patients in this group [72]. The Emory mouse cataract, an interesting model to study age-related cataracts, is a late-appearing lens opacity which may serve as an animal model for some human senile cataracts. It is inherited as an autosomal dominant trait and has a typical course of development. Lens opacities may become readily apparent as early as 6-8 months in mice hav-ing a familial history of early cataractogenesis [73]. UV-B radiation (290-320 nm) was very effective at inducing poste-rior cortical cataracts in mice similar to those seen in the human senile lens. UV-A radiation, in contrast, was at most weakly cataractogenic. The posterior opacities induced by UV-B began to appear 5-6 months after daily exposure to 8 X 10-6 W cm-2 for 12 hr per day or 10-4 W cm-2 for 1 hr per day. Progression was more rapid following the more intense exposure rate [75]. Results of physiological studies suggest that UV photons interact with proteins of the epithelial cell membranes, in particular tryptophan residues, transport AT-Pases and cytoskeletal proteins. One hypothesis is that dam-age to ion pumps and channels accumulates over the years as repair processes incompletely restore membrane function. Peroxidative damage is likely in view of the formation of UV-induced lipid peroxides in the lens epithelial membranes [76].

5. TELOMERES AND TELOMERASE ACTIVITY IN NORMAL AND CATARACTOUS CANINE LECs

Telomeres are specialized structures present at the ends of eukaryotic chromosomes, consisting of tandem arrays of highly conserved hexameric (TTAGGG)n repeats in verte-brates [77-80]. Telomeres are the components of chromo-some ends that provide stability and allow the complete rep-lication of the ends [79]. Telomeres have been revealed to be implicated in stabilizing linear chromosomes from exonu-cleolytic degradation and chromosome-to-chromosome fu-sions. Telomeres are actively involved in preventing other forms of aberrant recombination and the attachment of chromosomes to the nuclear matrix. Telomeres act as a “mi-totic clock” in determining the maximum replicative capacity of human somatic cells [77-80]. The DNA of telomeres--the terminal DNA-protein complexes of chromosomes--differs notably from other DNA sequences in both structure and function. The published studies have highlighted its remark-able mode of synthesis by the ribonucleoprotein reverse tran-scriptase, telomerase, as well as its ability to form unusual structures in vitro. Multiple studies of telomerase suggested that telomeres are maintained by an elegant but relatively simple and highly conserved mechanism of telomerase-


medicated replication [81]. As such, different from normal DNA replication, telomere replication is mainly executed by a ribonucleoprotein enzyme complex telomerase that con-tains a catalytic protein subunit and a polymerase template RNA [81-83]. Telomere synthesis by telomerase has been shown to be essential for telomere maintenance and long-term viability (Fig. 8) [77]. Maintaining a balance between telomere short-ening and lengthening is essential for cell viability [78]. In human cells telomere length is not maintained and telom-erase is not active in some tissues. In tumors, however, te-lomerase is active and may be required for the growth of cancer cells. Evidence from various organisms suggests that several factors influence telomere length regulation, such as telomere binding proteins, telomere capping proteins, telom-erase, and DNA replication enzymes [79]. In vivo analysis of telomere elongation kinetics shows that telomerase does not act on every telomere in each cell cycle but that it exhibits an increasing preference for telomeres as their lengths decline [80]. In the presence of the cognate template RNA, telom-erase adds telomeric sequences onto chromosome ends, pro-viding a mechanism to balance the loss of repeats from chromosome ends during cell division [77-80]. Telomerase has a role in healing DNA strand breaks and its activity is increased by exposure to ultraviolet- (UV) and X-irradiation in some cell types, the effects that can be attributable with an impact for cataractogenesis and the oxidative damage to the lens tissues and LECs [84-86]. It has been documented the induction of telomerase activity following exposure to DNA-damaging agents like UV radiation in Chinese hamster cells in vitro [84]. The published data suggest that telomerase ac-tivation is involved at an early stage of human skin carcino-genesis and that activation may precede the acquisition of UV-associated p53 mutations in the skin. Telomerase activ-ity was also found in plucked hair follicles and enzymatically separated epidermis, which may be associated with the pres-ence of stem cells in the skin [85]. Hande et al. have studied the expression of telomerase, telomere status and chromo-some rearrangements in mouse splenocytes following differ-ent doses (0.5, 1.0, 2.0 or 3.0 Gy) of X-irradiation in vivo up to 224 days post-exposure. A dose-dependent increase in telomerase activity up to 2 Gy X-ray exposure was observed immediately after irradiation [86]. In the absence of telomere synthesis, telomeres shorten with each cell division [87]. Correct telomere length setting is crucial for long-term sur-vival. The telomere length reserve must be sufficient to avoid premature cellular senescence and the acceleration of age-related disease [88]. Pendergrass WR et al. have investigated whether the av-erage relative telomere length of LECs from brown Norway rats decreases with the age of the donor animal, and whether chronic caloric restriction (CR) of the rats delays the te-lomere shortening [89]. The previous reviewed studies have demonstrated that clonal proliferative potential of rodent LECs as well as the in vivo rate of DNA synthesis decreases with age and that this decrease is slowed by chronic lifelong caloric restriction (CR) (reviewed in Ref. [89]). In order to determine if telomeric shortening might be involved in this loss of proliferative potential, Pendergrass WR et al. have examined relative telomeric lengths in young, old ad lib fed (AL), and old calorically restricted (CR) brown Norway rats

[89]. The average telomere lengths of interphase nuclei in the old AL rat LECs were found to be 21% shorter than paired young AL controls (P < 0.01 by Wilcoxian signed rank test). The calorically restricted old rats had less te-lomere erosion (12%) than the old AL group (P < 0.05). It is not clear whether such moderate telomeric erosion can limit cell division in rodent LECs; the telomeric shortening corre-lated well with the studies demonstrating reduced clonal, replicative potential, and reduced rates of in vivo DNA repli-cation in LECs from old rodents and a delay in this attenua-tion in animals on chronic CR [89]. Colitz et al. have identified telomerase activity in canine lens epithelium (LEC) and demonstrated that higher levels of activity were found in cataractous LEC (reviewed in refer-ence [32]). Although LECs have detectable telomerase activ-ity, normal LECs, especially those in the central region of the lens, are not highly proliferative [90]. Cell proliferation in the lens epithelium is controlled by multiple factors in-cluding, but not necessarily limited to, the retinoblastoma protein (pRB) family [90]. It has been generally proposed that telomeres may serve as important monitors of oxidative damage over a cell’s lifespan [91-94]. It has been revealed that oxidative damage accelerates telomere shortening [91]. Accordingly, it has been considered that telomeres in addi-tion to their well-recognized role in "counting" cell divisions are also, through their GGG sequence, important monitors of oxidative damage over the life span of a cell [91]. Te-lomeres, having the repeated sequences that cap chromo-some ends, undergo shortening with each cell division, are sensitive to oxidative stress, and serve as markers of a cell's replicative history. Telomere length shortening has been re-ported to relate to oxidative stress with aging process and aging-associated diseases, but the telomeric changes were not always identical, especially in change of telomere length distribution and subtelomeric methylation [92]. Telomeres are sensors for oxidative damage in the genome. Telomeres shorten during in vivo aging as well; however, there are sig-nificant differences between individuals. Cumulatively, the published data suggest that telomere shortening in vivo could reflect the cumulative amount of oxidative damage to the organism. It might be useful as a biomarker of aging [93]. Based on the preliminary published data, Colitz et al.have postulated that telomerase in LECs does not play a role in proliferation but perhaps has a significance in protection of telomeres from oxidative stress or represents a marker of exaggerated oxidative stress in canine LECs during catarac-togenesis [17]. Telomerase is responsible for maintaining telomere length in LECs, preventing chromosomal degrada-tion and recombination, and repairing DNA strand breaks. These activities are believed to be important in preventing LECs senescence. Indeed, it is established that the epithe-lium of the crystalline lens is comprised of a population of cells with diverse mitotic potential including the germinative epithelium which contains cells with the potential for unlim-ited replicative capacity, equatorial cells which terminally differentiate into lens fibers, and the central epithelium which are considered to be quiescent and nonreplicative un-der normal circumstances. Canine lens fiber cells, corneal epithelium and endothelium and nonpigmented ciliary epi-thelium were telomerase negative [17]. Since telomerase activity is associated with an immortal phenotype, the presence


Fig. (8). The clinical and experimental findings advancing our understanding on telomere attrition and telomerase regulation in normal LECs and those underwent to cataractogenesis. (A) Primary canine lens epithelial cell cultures. An anterior capsulorhexis specimen from a dog (8-year-old male dog of Terrier breed) with naturally developing mature cataracts was obtained prior to routine phacoemulsification cataract extraction [25]. A small cut was made in the anterior capsule of the lens, the free edge was grasped with forceps, and the capsule with at-tached lens epithelium was placed in a 30-mm tissue culture dish immediately after the operation. Two milliliters of Eagle’s minimal essential medium (MEM) was supplemented with 20% fetal calf serum and 50 �g/ml gentamicin. The culture was maintained at 37°C in a water-saturated air atmosphere containing 5% CO2. (B) In the separate experimental series, telomeres have been labelled using FISH, they are the yellow dots at the ends of chromosomes. (C) Three-dimensional molecular structure of a telomere (G-quadruplex). Fe 2+ /Fenton oxidant-mediated nicking is preferentially localized to a (TTAGGG)n mammalian telomere insert inducing site-specific oxidative damage to telomere DNA structures. (D) The protein portion of telomerase uses the RNA portion of telomerase as a template for synthesizing the telomeres. The localization of TERT in normal LEC corresponded with the LEC’s regional functions. There was more cytoplasmic TERT in the central re-gion that corresponds with the need for inhibited apoptosis and for proliferative capabilities; there was more nuclear TERT in the germinal and equatorial regions corresponding with the need for proliferative capabilities. In addition, cataractous LEC demonstrated increased TERT protein and increased TERT and TR mRNA expression than normal LEC corresponding with their increased proliferative potential [53]. The obtained results at Innovative Vision Products, Inc. (IVP) suggest that the dog telomere biology is similar to that in humans and may represent an alternative model system for studying telomere biology and telomere lengths and telomerase-targeted therapies [16].

A.

B.

C.

D.


Fig. (9A). Configuration of techniques for HLPC analysis of peptides extracted from the aqueous humor of the rabbit eye treated with 1% N-acetylcarnosine lubricant eye drops containing cellulose compound in the patented formulation (Can-C®). The Breeze™ HPLC System delivers technology and performance in an affordable, compact, and user-friendly system platform. Complete with software, pump, detector and injector, the Breeze comes pre-configured for different levels of HPLC operational needs with peptide moieties. Symmetry column(s) (image in the centre) continues to set the standard for HPLC drug assays, giving a confidence in the long-term compliance of applied HPLC methods. No need to compromise on peak shape, selectivity, column-to-column and batch-to-batch reproducibility, or other critical perform-ance characteristics. For lab balances weighing to the nearest 0.0000001 g, for the provided fine measurements of the mass of extracted pep-tide residues from the aqueous humor it can be sure to use a METTLER TOLEDO balance that matches the specific weighing needs during precise works with peptides. Formulations and animals fifty five Grey Chinchilla rabbits (male) aged 3–4 months weighing 2–3 kg were used. Animal experiments conformed to the guidelines of the ARVO Resolution on the Use of Animals in Research. Thirty minutes prior to the ocular incision right eyes of rabbits were instilled with 80 �l of formulation A (Can-C TM) containing 1% N-acetylcarnosine (NAC) and the control right eyes of the separate rabbits were similarly instilled with their vehicles (placebo) solutions. Placebo solution contained the same ingredients without NAC.

Topical anaesthesia of the rabbit eyes was performed after 25 min of instillation of the formula ophthalmic solutions with instillations of 4% lidocaine hydrochloride solution eye drops (three times with 1 drop at 1.5–2.0 min intervals). The eye drops of 4% lidocaine hydrochloride contain benzaltonium chloride preservative. When ocular anaesthesia was achieved, the lids were extended and fixed with the lid-holder and the ocular bulb was fixed by tweezers in the area of the inferior rectus muscle. A stab incision was performed transcorneally 1.0–2.0 mm from the limbus in the temporal superior quadrant. Aqueous humor (0.1–0.2 ml) was aspirated from the anterior chamber of a rabbit eye with 25-gauge needle connected to an insulin syringe and immediately introduced into an Eppendorf tube with addition of ethanol (0.2 ml), keeping the sample on ice before extraction.

Extractions of imidazole-containing compounds from the aqueous humor aliquots were performed according to Babizhayev et al., 1996 [160]. The published data showed that all the desired imidazole-containing compounds in the aqueous humor thus obtained could be of good purity and recovery [157]. Portions of aqueous humor were added to ethanol as above and thoroughly mixed (20ºC, 15min). Extracts were centri-fuged (2000 x g, 15 min) and the supernatants removed. Samples were frozen in the gradient of temperatures to -70ºC and lyophilized using the apparatus JOAN (France). The lyophilized residue was dissolved in 1 ml of 0.1 M Na2 HPO4 (pH 2.1 adjusted with 85% phosphoric acid) and filtrated through the membrane filter with the dimensions of pores 0.22 �m directly prior the analysis.

Reverse phase analytical HPLC was performed using a Breeze chromatography system (USA), detector Waters 2487 Dual � Absorbance Detector, column (250x 4.6 mm) Symmetry 300 C18 5 �m (Waters), loop 20 �l (Fig. 9B). The column was eluted isocratically at 30ºC with the cited phosphate buffer 0.1 M Na2 HPO4 (pH 2.1) over 25 min at a flow rate of 1.0 ml/min. Eluates were monitored for absorbance at 210 nm. The standards of L-carnosine and N-acetylcarnosine were prepared by weighing of the dry material using the analytical balance Mettler Toledo (accuracy 0.00004) and were further dissolved in the phosphate buffer 0.1 M Na2 HPO4 (pH 2.1). The quantitative determination of l-carnosine and N-acetylcarnosine in the samples was undertaken using the technique of external standard according to the area of the peak and linear extrapolation. The standards of eye drops were prepared by dissolution of initial solutions of eye drops by 100 fold using the phosphate buffer 0.1 M Na2 HPO4 (pH 2.1). Statistical significance was evaluated by the unpaired Student’s t-test and P=0.05 was taken as the upper limit of significance.


Fig. (9B). HPLC of extract of aqueous humor aspirated 30 min after the instillation of ophthalmic formulation with 1% NAC and lubricants into the rabbit eye. The integrated concentration of the carnosine related product (3.392 �g/ml, 3.225 min) is attributed to accumulation of carnosine in the ophthalmic formulation-treated eye. Chromatograms of solutions of L-carnosine and its putative N-acetyl derivative show that these compounds are well separated. The elution order of the compounds was compared to a predicted order based upon their relative hydrophobicities and the chromatographic system was shown to be suitable to monitor the behaviour of other histidine containing derivatives of L-carnosine. The calibrating chromatograms showed the predicted elution order and the average elution times for each standard of L-carnosine and N-acetylcarnosine in mixtures. Peaks were unequivocally identified by comparison of their retention times to those of the authentic standard compounds or of putative acetylated compound run singly. Tests for specific chemical reactivity provided additional evi-dence for the identification of L-carnosine and N-acetylcarnosine [157, 160].


Fig. (9C). Can-C eye drops Composition: Glycerin USP 1% w/v, Carboxy Methyl Cellulose USP 0.3% w/v. Excipients: Benzyl Alcohol (as preservative) USP 0.3% w/v, N-acetyl Carnosine 1% w/v, Potassium Bicarbonate USP q.s., Boric Acid NF q.s.

of telomerase activity in the canine lens epithelial cells may function to prevent conversion to senescence. Consistent with in vitro findings in cell cultures, there were no senes-cent cells present on the lens capsule when the lens was ini-tially dissected for culture, but an increasing number of cells were senescent with each passage, correlating well with the loss of telomerase activity. Telomerase activity is likely im-portant in the germinative epithelium to maintain its prolif-erative potential and prevent cell senescence [17]. The demonstrated activity of telomerase in the canine LECs represents a hallmark of the exaggerated oxidative stress in cataractogenesis of the canine species. Telomerase activity and telomere maintenance are important prerequisites for cellular immortality mainly due to its telomere stabilising and proliferation promoting function. However, in the literature, there have been described also telomere-independent functions of telomerase in tumourigenesis [95]. To understand the relationship between telomere length, telomerase expression, and sensitivity to genotoxic or oxida-tive stresses, Rubio et al. have used human fibroblasts with defined telomere lengths and followed their ability to prolif-erate after damage by genotoxic or oxidant agents [96]. Ru-bio et al. have shown that telomerase can confer resistance to such agents, but the resistance depends on the presence of short, presumably near-dysfunctional, telomeres and the abil-ity of telomerase to elongate the short telomeres. Although long telomeres did not protect cells from the effects of ioniz-ing radiation, bleomycin, or etoposide, they sensitized cells

to hydrogen peroxide. Thus, the cellular response to geno-toxic and oxidative stress depends on the combination of the nature of the stress, presence of telomerase, and length of the shortest telomeres. Rubio et al. have concluded from their studies that telomerase influences the stress response indi-rectly by altering telomere length, mainly by elongating the shortest telomeres. Thus, telomere length not only deter-mines replicative senescence, but also influences the senes-cence response to genotoxic agents [96]. Guanine residues, which are enriched in the telomeric repeat sequence, are par-ticularly vulnerable to oxidative damage [97]. Oxidized gua-nines may disrupt the telomere structure, without altering length, which may be sufficient to trigger a senescent re-sponse [98]. From the published data, it is essential to imply that telomeric DNA could protect coding DNA from oxida-tive damage and might also link oxidative damage and iron load to telomere shortening and aging [97]. Telomere lengths may be associated with cataractogene-sis. The human telomerase reverse transcriptase (hTERT) introduced into human lens epithelial cells (HLECs) prevents replicative senescence through telomere synthesis. Among the human, bovine, and rabbit lenses examined, only the cen-tral epithelium from the 6-month rabbit lens displayed te-lomerase activity. In both transparent and cataractous human lenses, hTERT activity and expression were not detected. However, the template RNA was present in both types of human lenses. The telomeres in transparent lenses were ap-proximately 1 kb longer than those in cataractous human lenses [99]. During UV-induced DNA damage and repair in

N-acetylcarnosine

OH

OH

OOH

O

O ONa

O

OOH

O O

O ONa

O

......

Sodium Carboxymethylcellulose Benzyl Alcohol(corneal absorption promoter and preservative)


human lens epithelial cells, telomerase activity was upregu-lated and the expression of stress-related proteins levels was increased. Upregulated telomerase activity may play both a protective and a proliferative role in human lens epithelial cells. Increased stress-related proteins level is critic in UV-induced DNA damage and repair in human lens epithelial [100]. Colitz et al. [32] have monitored the levels of the stress proteins gadd45 and p16 and the stress and prolifera-tion-related protein, proliferating cell nuclear antigen (PCNA) in normal canine LECs. In the experimental series of acute studies, lenses were exposed to tert-butyl-hydroperoxide (TBHP) for 0–120 min. In recovery studies, lenses were exposed to TBHP for 1 hr, then allowed to re-cover for up to 18 hr. In acute studies, telomerase activity was increased, p16 initially decreased then normalized, PCNA levels did not change significantly even in the over-night recovery groups, and gadd45 was decreased in some TBHP exposed groups. In recovery studies, telomerase activ-ity was increased in all groups, gadd45 decreased then be-came elevated, and p16 levels were decreased at later recov-ery times. PCNA levels remained constant during the stud-ies, indicating that there was no change in proliferation [32]. The provided studies showed that elevated telomerase activ-ity did not correlate with increased proliferation in lens epithelial cells; instead of this, increased telomerase activity was associated with increased levels of the stress-related protein gadd45 only in the later recovery times [32]. These experimental findings generally support the hypothesis that telomerase plays a protective rather than a proliferative role in lens epithelial cells and the measurable activity of telom-erase in LECs of canines is a biomarker of increased oxida-tive stress and cataract. Based on the established data supporting the role of oxi-dative stress in the regulation of telomerase, it might be con-cluded that the telomerase-induced telomere length manipu-lations may have a therapeutic utility for tissue engineering and for dissecting the molecular mechanisms underlying age-related diseases [101] and the treatment of cataracts in ca-nines. In the next section of the article we describe the specific mechanisms of oxidative stress which make the greater im-pact on canine LECs telomere length and provoking senes-cence of LECs and cataractogenesis in dogs which will re-main a bone of contention and thus an object of therapeutic strategy and research of cataracts in canines.

6. SPECIFIC FEATURES AND FACTORS OF OXI-DATIVE STRESS (IRON-INDUCED OXIDATIVE DAMAGE) DURING CATARACT FORMATION IN CANINES THREATENING THE APOPTOTIC CELL DEATH, TELOMERE DEGRADATION AND TE-LOMERASE ACTIVATION IN CANINE LECs

Ferrous ions (Fe 2+) are implicated in cataractogenesis of humans and animals, due to their ability to catalyze the for-mation of free radical oxygen species and the capacity to be involved in the damage of biomolecules (Fig. 8). Aged cata-ractous lenses have generally higher levels of iron [102] and an increased capacity for free-radical formation [103]. The published studies have shown that lenticular levels of Fe and Cu are elevated in age-related cataract. The work to Garner

et al. [102] has verified if these metals are present in a state that is permissive for redox reactions that may lead to the formation of free radicals. The aim of the work to Garner etal. was to determine the distribution of ferritin and the redox-availability of Fe and Cu in healthy and cataractous lenses . Lens ferritin distribution was assessed by ELISA and immu-nohistochemistry. A modified ELISA revealed ferritin in an 'insoluble' lens protein fraction. Ferritin levels were not sig-nificantly different in the cortex vs nucleus of healthy lenses. In contrast, ferritin levels in the cataractous lens nuclei ap-peared to be 70% lower compared to the cortex. In normal lenses, ferritin staining was most intense in the epithelium, with diffuse staining observed throughout the cortex and nucleus [102]. The study to Garner et al. [102] has provided an evidence that a chelatable pool of potentially redox-active Fe is present at increased concentrations in human catarac-tous lenses. In contrast, it seems that lenticular Cu may not be readily available for participation in redox reactions. The post-translational modification of crystallins by hydroxyl radicals/Fenton systems seems to dominate their in vivo oxi-dation, and it could explain the known features of such nu-clear cataractogenesis [103]. It is experimentally and clinically observed that excess iron is normally safely stored in ferritin, a ubiquitous protein with a highly conserved structure (reviewed in Reference [104]). Ferritin is the intracellular protein responsible for the sequestration, storage and release of iron. Ferritin can accu-mulate up to 4500 iron atoms as a ferrihydrite mineral in a protein shell and releases these iron atoms when there is an increase in the cell's need for bioavailable iron. Ferritin rep-resents a heteromultimeric complex consisting of 24 subunits of two types: light (L)- chain (19 kDa) and heavy (H)-chain (21 kDa). The L and H chains are architectured in a tissue-specific ratio in a way that each subunit has a complemen-tary role in storing iron. The H chain has ferroxidase activity towards ferrous ions and facilitates iron oxidation and uptake into ferritin. The ferritin subunits perform different functions in the mineralization process of iron. The ferritin protein shell can exist as various combinations of these two subunit types, giving rise to heteropolymers or isoferritins. Isofer-ritins are functionally distinct and characteristic populations of isoferritins are found depending on the type of cell, the proliferation status of the cell and the presence of disease. The synthesis of ferritin is regulated both transcriptionally and translationally [105]. It has been revealed that the L chain translocates ferric ions into the core of ferritin for a long-term deposition and storage in tissue and cellular structures [106]. In vitro studies have shown that ferritin iron incorporation is mediated by a ferroxidase activity associated with ferritin H subunits (H-Ft) and a nucleation center associated with ferritin L subunits (L-Ft). The results of the study to Picard et al. provide the invivo direct demonstration of the capacity of H-Ft to sequester cell iron and to regulate the levels of the labile iron pool [106]. Differences in total ferritin concentration, the structure of the chains, and their ratio can significantly alter the ability of ferritin to control redox-active, free iron, and to protect cells from iron-catalyzed oxidative damage [107]. Ferritin inhibits oxidant-mediated cytolysis in direct relation to its intracellular concentration. Apoferritin, when added to cul-tured endothelial cells, is taken up in a dose-responsive man-


ner and appears as cytoplasmic granules by immunofluores-cence; in a similar dose-responsive manner, added apofer-ritin protects endothelial cells from oxidant-mediated cytoly-sis [107]. Ferritin is present throughout the entire noncataractous and cataractous canine lenses [102, 108]. There are signifi-cant differences in the characteristics of ferritin H-chain and its distribution in canine cataractous lenses compared with noncataractous lenses. The higher content of H-chain in assembled ferritin allows this molecule to sequester more iron. In addition, the accumulation of H-chain in deeper fiber layers of the canine lens may be part of a defense mechanism by which the cataractous lens limits iron-catalyzed oxidative damage to the lens tissue and cellular structures [108]. The concentration of assembled ferritin is comparable in noncata-ractous and cataractous lenses of similarly aged dogs. The ferritin L-chain detected in both lens types was modified and was approximately 11 kDa larger (30 kDa) than standard L-chain (19 kDa) purified from canine liver [108]. The H-chain identified in cataractous fiber cells (29 kDa) differed from the 21-kDa canine H-chain and from the 12-kDa modified H-chain present in fiber cells of noncataractous lenses. His-tologic analysis revealed that the H-chain was distributed differently throughout cataractous canine lenses compared with noncataractous lenses. There is a difference in subunit makeup of assembled ferritin between the two canine lens types. Ferritin from cataractous canine lenses contained more H-chain and bound 11-fold more iron than ferritin from non-cataractous lenses [108]. Thus, ferritin chains are signifi-cantly altered in lens fiber cells, in comparison to those of lens epithelial cells and other tissues [108]. Particularly, fer-ritin-heavy (H) and -light (L) chains in lens fiber cells are modified in comparison to those in LECs. Chain modifica-tions identified in human and canine fiber cells include trun-cation (H chains; 10-12 kDa), increased size (canine L chains; 30 kDa), acidification, and polymorphism of both chains, which increases with age [108]. Goralaska et al. have revealed that canine LECs differentially regulate concentra-tion of both ferritin chains in the cytosol [109]. The overex-pressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin [109]. Post-transfectional expression of ferritin H- and L-chains in ca-nine LEC appears to be regulated differentially [110]. Over-expression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage [110]. The outcome of the published works to Goralaska et al. [104, 108] has been that both ferritin H and L chains were modified in lens fiber cells from normal and cataractous canine lenses (Fig. 10). These modifications were not age-related, and most likely could occur during the differentiation of epithelial cells to fiber cells, since only normal-sized chains have been found in LECs of canine lenses. Moreover, there was a specific and distinct distribu-tion of these modified chains throughout the lens fiber mass. The striking differences between normal and cataractous lenses fiber cells were the appearance of normal-sized fer-ritin H chains and the relatively even distribution of iron

binding capacity throughout the fiber mass of the canine cataractous lenses. These differences may reflect a specific response of the canine lens to increased oxidative stress dur-ing cataractogenesis in canines [104,108]. A better under-standing of the distribution of ferritin and ferritin chains in different lens layers of canines provide essential information on the possible involvement of ferritin in the cataractogene-sis in canines and possibly, a compensatory or temporary prevention of lens opacification caused by iron-induced oxi-dative damage (Figs. 8, 9 and 10). In contrast to the ascorbic acid-induced increase in trans-lation of ferritin, H2O2 substantially decreased de novo fer-ritin synthesis [111]. High levels of H2O2 in aqueous humor of the eye could decrease the concentration of ferritin within the lens. Since ferritin sequesters iron and has been shown to decrease oxidative damage by limiting the availability of iron to catalyse free radical reactions, H2O2-induced reduc-tion in ferritin concentration in the lens could have deleteri-ous effects [111]. The ability of ascorbic acid to increase ferritin concentration in lens epithelial cells could provide an additional protective mechanism for the antioxidant effects of ascorbic acid. In view of the antioxidant role of ascorbic acid and the glutathione redox cycle in the lens, Sasaki et al.have studied the relationship of the cycle to reduction of the oxidized product of ascorbic acid, dehydroascorbic acid (DHA), in lens epithelium [112]. Treatment of lens cells with 1 mM DHA for 0.5 to 3 hours in the absence of glucose (glucose is required for the reduction of GSSG through the glutathione redox cycle) produced from 60% to complete oxidation of reduced glutathione (GSH) (controls contained negligible oxidized glutathione (GSSG)) and distinct mor-phologic changes (cell contraction and blebbing), as shown by scanning electron microscopy [112]. The exposure of intact rabbit lenses and cultured dog lens epithelial cells to DHA under conditions in which the glutathione redox cycle was compromised resulted in the disappearance of GSH in the tissues and the appearance of GSSG. The reduction of DHA was shown to be linked to the glutathione redox cycle by a nonenzymatic interaction between GSH and DHA. Re-duction of DHA in the lens is important because of the po-tential toxicity of this oxidant and/or its degradation products [112]. In the published study to Harned et al. of LECs, the ef-fects of ceruloplasmin and transferrin on intracellular distri-bution and efflux of iron were determined [113]. Both ceru-loplasmin and transferrin increased iron efflux from these cells and their effects were additive. Ceruloplasmin had sig-nificant effects on extracellular iron distribution in cases of iron ions overload. Surprisingly, both transferrin and ceru-loplasmin had significant effects on intracellular iron distri-bution. Under physiological conditions, ceruloplasmin in-creased iron incorporation into the storage protein, ferritin. Under conditions of iron overload, it decreased iron incorpo-ration into ferritin, which is consistent with increased efflux of iron. Measurements of an intracellular chelatable iron pool indicated that both transferrin and ceruloplasmin increased the size of this pool at 24 h, but these increases had different downstream effects. Finally, LECs made and secreted trans-ferrin and ceruloplasmin [113]. These results indicate an impo-rtant role for these proteins in iron metabolism in the lens.


(A) (B) Fig. (10) (A). L-Carnosine- Fe 2+ energy-minimized structure (space filling model). (B). Effect of L-carnosine on the decrease of ferrous iron deter-mined by 1,10-o-phenanthroline assay in the presence of 12.5 �M ferrous sulfate. The data points are the means of two independent determi-nations and are representative of three independent experiments. The standard error of the mean value for each point is 3% of the mean value.

Ferroxidase activity of carnosine

The ability of carnosine to decrease the concentration of free ferrous ions in Tris–HC1 buffer (100 mM, pH 7.4) was monitored by the 1,10-o-phenanthroline chelating assay modified from Ref. [137]. The reaction was started by the addition of 12.5 �M FeSO4 to the reaction mixture which contained 3–20 mM carnosine. Sixty minutes after incubation at 37º C, the reactions were stopped by the addition 100 �M 1,10-o-phenanthroline (Serva), and A515 was immediately read. The concentration of (Fe2+–1,10-o-phenanthroline) chelating complex was deter-mined using the molar extinction �515=10 931 M-1 cm-1. Samples taken at zero time and at the time intervals indicated and were used immedi-ately for measurements.

(a) : (�)- Fe 2+, control incubation; (�) – Fe 2+ + L-carnosine (5 mM);

(�) – Fe 2+ + L-carnosine (10 mM); (�) – Fe 2+ + L-carnosine (20 mM).

Molecular formula of L-Carnosine : C9H14N4O3; Molecular mass : 226.23, CAS number : 305-84-0.


Ferritin scheme (stores iron) Ferritin-Protein shell

C D

Fig. (10) C. Iron is bound and transported in the tissue naturally via transferrin and stored in ferritin molecules. Once iron is absorbed, there is no physiologic mechanism for excretion of excess iron from the body or the target tissue structures, such as LECs.�Thus an important natural mechanism for overcoming iron pro-oxidant toxicity and solubility is ferritin, a ubiquitous iron storage, detoxification and biomineralizing protein found in the canine lens structures. In mammals, ferritins are composed of 24 subunits of two types, H and L. These subunits have complementary roles in iron oxidation and mineralization: the H-chain subunit has ferroxidase activity associated with a di-iron binding cen-ter that is responsible for the rapid oxidation of Fe2+ to Fe3+:

2 Fe 2+ + O2 + 4 H2O ~ 2 FeOOH(core) + H2O2 + 4 H+

4 Fe 2+ + O2 + 6 H2O ~ 4 FeOOH(core) + 8 H+

2 Fe 2+ + H2O2 + 2 H2O ~ 2 FeOOH(core) + 4 H+ ,whereas the L-chain subunit appears to provide efficient sites for iron nucleation and mineralization. The distribution and composition of ferritins in canines is lens organ- dependent, emphasizing the importance of the L-chain in mineral core nucleation and the H- chain in rapid iron oxidation and detoxification.

The observed reduction of Fe uptake by the subcultured and cataract cell lines probably has reflected a decrease in transferrin receptor expression and in the activity of an alter-native pathway for Fe transferrin uptake occurring over time [114]. There was occurred a distinct relationship between the amount of Fe-transferrin added and the amount of Fe cap-tured up, which was linear for the primary cultures but sig-nificantly reduced for the secondary, tertiary and cataract cultures (252 +/- 21, 169 +/- 14, 153 +/- 14 and 96 +/- 2 ng Fe/mg protein, respectively). Transferrin receptor expression in lens cell cultures was reduced 10-fold within 2 days of addition of serum to cells grown in low-Fe, serum-free me-dium for 1 week. The reduced Fe uptake may result from long-term exposure of LECs to relatively high Fe concentra-tion in the media [114]. Transferrin, being the plasma iron transport protein, supports cell proliferation and differentia-tion and has bacteriostatic, antioxidant and anti-inflammatory activity, transferrin has been found in relatively high concen-trations in the intraocular fluids (reviewed in ref. [115]). Transferrin content of aqueous and vitreous humors and whole lenses was determined by ELISA. Transferrin was secreted into the bathing medium from lens epithelial cell cultures, but not from either the pigmented or non-pigmented epithelial cells of the ciliary body cultures [115]. Cyclo-heximide inhibited secretion of transferrin from the LECs. Lenses from inflamed eyes contained higher levels of trans-ferrin than their contralateral controls [115]. To summarize and assess the state of iron ions reactions in the canine lens and its surrounding intraocular fluids, it is important to consider that the synthesis of the iron storage protein, ferritin in LECs is regulated by iron levels and redox

state conditions. Proper iron storage is important to protect against damaging iron-catalysed free radical-induced oxida-tion reactions. In the study to McGahan et al., the concentra-tion of ferritin was measured in cultured canine LECs [116]. In vitro conditions, the baseline ferritin concentration ranged from 76-163 ng (mg protein) -1; thus LECs cultured in low-iron media had significantly lower ferritin levels than cells cultured in iron-supplemented media. Alternatively, addition of a large excess of iron as hemin resulted in an eight-fold increase in ferritin concentration in LECs. The iron chelator, Desferal, significantly decreased ferritin concentration in LECs. The reducing agent dithiothreitol decreased the he-min-induced increase in ferritin levels, but not baseline lev-els in LECs [116]. In contrast, ascorbic acid induced a large increase in ferritin content. The induction of ferritin synthe-sis can protect against oxidative damage. Regulation of fer-ritin levels in the lens may represent a mechanism by which the lens epithelium is protected from oxidative damage to the biomolecules. In vivo conditions, LECs are normally ex-posed to much lower iron concentrations than the cultured LECs. However, in pathological cataractogenesis circum-stances, the iron content and redox state of the aqueous hu-mor is dramatically altered and may affect the steady state levels of ferritin within the lens and the course of metal-catalyzed oxidation reactions of biological molecules in LECs. Preferential cleavage sites have been documented for Fe2+/H2O2-mediated oxidations of DNA biomolecules moie-ties [97]. The cytotoxicity of hydrogen peroxide to DNA is mediated by highly reactive oxidants generated by the reac-


tion of reduced transition metal ions with H2O2 via Fenton reaction [117], (Fig. 8). Fe2+ + H2O2 + H+

� Fe2+ + �OH + H2O (1) DNA is an important target in H2O2-mediated cell killing

through transition metal ions associated reactions [118,119]. The published studies have identified that the direct DNA oxidant and damaging species is a derivative of hydrogen peroxide whose formation is dependent on cell metabolism. The generation of this oxidant depends on the availability of both reducing equivalents and an iron species, which to-gether mediate a Fenton reaction in which ferrous iron re-duces hydrogen peroxide to a reactive radical [118]. To minimize the toxicity of oxygen radicals, the cell utilizes scavengers of these radicals and DNA repair enzymes. On the basis of observations with the model system, it has been proposed that the cell may decrease such toxicity by dimin-ishing available NAD(P)H and by utilizing oxygen itself to scavenge active free radicals into superoxide, which is then destroyed by superoxide dismutase [119]. The oxidizing spe-cies involved in DNA damage are probably not merely freely diffusible hydroxyl radicals (�OH), but rather a localized �OH and/or related iron-oxo species [120-123]. Fe2+ + H2O2 � FeO2+ + H2O (2)

The ferryl ion is considered to be kinetically equivalent to the hydroxyl radical species in regards to the oxidizing activity towards biomolecules in many respects [121]. The oxidizing species of iron which are assigned as ferryl, FeO2+, or Fe(IV) = O was generated effectively in the presence of ADP even at low Fe2+ concentrations. In general, as the Fe 2+

concentration was increased, the ferryl species predominated over the hydroxyl radical except for the case of Fe(II)- dieth-ylenetriaminepentaacetic acid (DETAPAC), which generated only hydroxyl radicals as the oxidizing species [121]. Using the Marcus theory and the published data in the literature, it is shown that in most cases the reaction of the transition metal ion complexes with H2O2 is unlikely to occur via an outer-sphere electron-transfer mechanism [122]. It is sug-gested that the first step in this process is the formation of a transient complex LmM-H2O2n+, which may decompose to an �OH radical or a higher oxidation state of the metal, LmM(n + 2)+, or it may yield an organic free radical in the presence of organic substrates. Thus, the question whether free �OH radicals are being formed or not via the Fenton reaction depends on the relative rates of the decomposition reactions of the metal-peroxide complex and that of its reac-tion with organic substrates [122]. Fenton-type reaction and reactivity has been implicated in the oxidative processes in-volved in the pathology of many disease and degenerative states of organisms (reviewed in reference [123]). The oxi-dizing properties of the localized �OH radical generation and/or related iron-oxo species are governed by the chelation state of the iron to biomolecules upon which these species are formed [97]. Preferential cleavage sites have been deter-mined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mM H2O2, preferential cleavages occurred at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a se-quence found in a majority of telomere repeats. Telomeric DNA can protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and cell aging [84]. DNA can chelate Fe2+ in sev-

eral ways, and DNA damage in vivo and in vitro exhibit pe-culiar dose responses to H2O2 concentration [118,124]. The following forming oxidant species appear to damage DNA at lower H2O2 concentrations [97]: �OH + H2O2 � HO�

2 + H2O (3) The binding of metal ions to preferred sequences within DNA and the relationship of such binding of transition met-als ions to DNA damage by the Fenton reaction have been the subject of published studies [97,125-128]; it has been proposed accordingly that transition metal ions in the pres-ence of H2O2 might cause sequence-specific damage to DNA [97,129,132]. Iron promotes DNA damage by catalyzing hydroxyl radical formation. Enright et al. have examined the effect of chromatin structure on DNA susceptibility to oxi-dant damage [126]. Oxygen radicals generated by H2O2,ascorbate and iron-ADP (1:2 ratio of Fe 2+:ADP) extensively and randomly fragmented protein-free DNA, with double-strand breaks demonstrable even at 1 microM iron ions. In contrast, polynucleosomes from chicken erythrocytes were converted to nucleosome-sized fragments by iron-ADP even up to 250 microM iron. Cleavage occurred only in bare areas where DNA is unassociated with histone [126]. Protection of DNA by histone was dependent on nucleosome assembly and did not simply reflect presence of scavenging protein. In contrast to this specific cleavage of internucleosomal linker DNA by iron-ADP, iron-EDTA cleaved polynucleosomes indiscriminately at all sites. Oxygen radicals generated by iron-ADP indiscriminately cleaved naked DNA but cleaved chromatin preferentially at internucleosomal bare linker sites, perhaps because of nonrandom iron binding by DNA. The published results suggest that the DNA-damaging ef-fects of iron may be nonrandom, site-directed and modified by histone protein [126]. Reactive oxygen species (ROS) induce several modalities of DNA damage, such as single strand breaks, double strand breaks, modified bases, abasic sites, DNA-protein cross-links. These damages are of bio-logic significance, because many ROS-induced base modifi-cations are mutagenic (reviewed in ref. [128]). Prevention of the site-specific free radical damage can be accomplished by using selective chelators for iron and copper, by displacing these redox-active metals with other redox-inactive metals such as zinc, by introducing high concentrations of hydroxyl radicals scavengers and spin trapping agents, and by apply-ing protective enzymes that remove superoxide or hydrogen peroxide. Histidine is a specific amino acid reagent that can intervene in free radical reactions in variety of modes [129]. The results of DNA ferrous ions mediated DNA damage might be interpreted in terms of a 'site-specific' Fenton mechanism according to which the binding of the transition metal ions to the biological target is a prerequisite for the production of damage. The bound metal ion is reduced either by O(2), ascorbate or other reductants and is subsequently reoxidized by H2O2 yielding OH· radicals. Due to the fact that autooxidation of ascorbic acid requires the presence of molecular oxygen and is accompanied by generation of hy-drogen peroxide, the participation of superoxide radicals in the process has been claimed [130]. The cyclic redox reac-tion of the metal generates OH· radicals which react with vital macromolecules with a high probability of causing 'multi-hit' damage. This 'site-specific' formation of OH· radi-cals, which takes place near the target molecules, accounts


both for the high damaging efficiency and for the failure of OH· scavengers to protect against it [130]. Decreases of mi-tochondrial membrane potential and intracellular glutathione level were found to be critical events in the H2O2-mediated apoptosis. Additional experiments revealed that H2O2 ex-erted its apoptotic action through the formation of hydroxyl radicals via the Fenton rather than the Haber-Weiss reaction. Moreover, intracellular redox-active iron, but not copper, participated in the H2O2-mediated apoptosis [131]. Several laboratories have proposed that, whereas iron associated with DNA has been repeatedly implicated in cytotoxic DNA damage sequence preference for damage has been reported only with copper/ascorbate copper/NADH, with Fe 2+ bound to chelators or larger molecules, or with bleomycin (reviewed in Ref. [97]). In contrast, other laboratories have revealed that nucleoside damage following exposure of DNA to Fe 2+ and H2O2 was not equally distributed among the nucleosides [132]. Damage by iron-mediated Fenton reactions under aerobic or anaerobic conditions to deoxycytidine, deoxy-cytidine-5'-monophosphate, d-CpC, d-CpCpC, and dCMP residues in DNA resulted in at least 26 distinguishable prod-ucts [132]. In the recent study the degradative products formed from the exposure of derivatives of thymidine to iron-mediated Fenton reactions were identified by chroma-tographic resolution and analysis by UV absorption spectros-copy, radio-labeling, and positive and negative mode fast atom bombardment mass spectrometry. Fe(2+)/H(2)O(2) and Fe(3+)/H(2)O(2) were utilized to generate oxy-radicals [133]. Substrates included thymidine, 3'- and 5'-dTMP, TpT, oligo(dT), and oligo(dT)·poly(dA) and the results have been compared to those reported for ionizing radiation. It has been revealed from the comparisons of the reaction products that the damaging radical species and the product distribution are perturbed by interaction of the iron atom with the various phosphomono- and diester species [133]. The consensus se-quences, which appear to reflect specific modes of iron bind-ing by DNA, might be involved in regulatory processes of the cell [97], such as LECs. Certain DNA sequences are known to be unusually sensi-tive to nicking via the Fe 2+-mediated Fenton reaction. Most notable are a purine nucleotide followed by three or more G residues, RGGG, and purine nucleotides flanking a TG com-bination, RTGR [134]. The af�nity of Fe 2+ for binding at AGGG sequences in LECs may have a bearing on the role of oxidative DNA damage in telomere shortening and cellular senescence, since mammalian telomeres consist of TTAGGG repeats (Fig. 8). While telomeres are normally protected by telomeric binding proteins that stabilize the capped T-loop structure [135], it is possible that localization of endogenous ferrous ions and their specific binding to biomolecules in LECs during the stochastic open state might enhance te-lomere shortening under conditions of oxidative stress during cataractogenesis in canines. Signi�cantly, hydrogen perox-ide-induced senescence of LECs and oxygen free radical –induced cataractogenesis in canines can be prevented natu-rally by ferritin accumulated in LECs (see information pre-sented above) and during the treatment of canine cataracts with imidazole-containing compounds in the moieties of lubricant eye drops (1% N-acetylcarnosine ophthalmic pro-drug of L-carnosine, 1% carcinine ophthalmic solutions ad-mixed with carboxymethylcellulose) endowed with ferroxi-

dase or the iron-speci�c chelating activities and the capaci-ties to prevent the attrition of telomere structures and cellular senescence [136,137].

7. THE CURING ANTI-CATARACT EFFECTS OF PATENTED OPHTHALMIC COMPOSITION OF 1% N-ACETYLCARNOSINE OPHTHALMIC PRODRUG ADMIXED WITH CARBOXYMETHYLCELLULOSE DURING CANINE CATARACTOGENESIS THROUGH PROTECTIONS FROM TELOMERE ATTRITION IN LECs

Carnosine (�-alanyl-L-histidine) and related imidazole-containing compounds are natural constituents of excitable tissues possessing diverse biological activities [138,139, 140]. Carnosinase and its substrate carnosine have been linked to neuropathophysiological processes [141]. The level of carnosine in tissues is controlled by a number of enzymes transforming carnosine into other carnosine related com-pounds, such as carcinine, N-acetylcarnosine, anserine or ophidine (by decarboxylation, acetylation or methylation, respectively) or its cleavage into the amino acids, histidine and �-alanine. Hydrolysis is mainly due to tissue carnosinase (EC 3.4.13.3) which is widely distributed among different tissues [142,143] or serum carnosinase (EC 3.4.13.20), found in brain and blood plasma of primates and humans [144,145, 146]. Both carnosine and N-acetylcarnosine compounds are found primarily in the heart and skeletal muscles and in the brain. We have found appreciable levels of L-carnosine in transparent human lenses which are markedly depleted in mature cataracts [147]. The concentration of carnosine in transparent crystalline lenses detected was about 25 M. At different stages of cataract development, the level of carnosine fell, reaching about 5 M [147]. Carnosine has been proven to scavenge reactive oxygen species (ROS) as well as alpha-beta unsaturated aldehydes formed from peroxidation of cell membrane fatty acids during oxidative stress [148-150]. It can oppose glycation [151,152] and it can chelate divalent metal ions . The important studies have produced clinical and experimental evidence of beneficial effects of N-acetylcarnosine in treating cataracts of the eyes, these and other ophthamological benefits have been proven [153-160]. Research with N-acetylcarnosine (NAC) demonstrates that it is effective not only in preventing cataracts but also in treating them. NAC has been shown to improve vision by partially reversing the development of the cataract, thus increasing the transmissivity of the lens to light [155]. One of the obscure aspects of the carnosine physio-logical role is the biological significance of the enzymatic metabolism of carnosine or its derivatives in tissues. We found that, in order to change the antioxidant status, tissue enzymes can modify the NAC prodrug molecule and that deacetylation increases in vivo the resistance of lens tissues and its cells to oxidative stress. 1% N-acetylcarnosine (Fig. 9A-C) is a universal bioactivating antioxidant for vision in the developed and patented drug delivery system lubricant eye drop formulations containing mucoadhesive cellulose-based compound combined with corneal absorption promot-ers. Its topical administration delivers pure L-carnosine and allows its increased intraocular absorption into the aqueous humor surrounding the lens, enabling significant improve-ments in anti-cataract drug efficacy and the minimization of


side-effect from either local or systemic drug absorp-tion/bioavailability to the eye, and also creates optimization effects in the number of ocular degenerative age-dependent disorders [136]. The standard ocular eye drop formulation was also found to be non-irritant and well tolerable. The de-veloped system can be a viable alternative to conventional eye drops for the treatment of various ocular diseases and is suitable for clinical application in animals. N-acetylcarnosine prodrug and codrug ophthalmic formulations applied topi-cally to the eye and moreover, its controlled time released ophthalmic ingredient L-carnosine exerts anti-glycation, bio-activating antioxidant properties in the lens and cornea as a scavenger of lipid peroxides, singlet oxygen and OH· radi-cals and provides the spatial aspects of intracellular pH regu-lation [155, 157, 159-163]. The provided studies with N-acetylcarnosine ophthalmic prodrug confirmed the in-creased intraocular uptake of L-carnosine active ingredient in the aqueous humor (Fig. 9B) [9, 160, 163,164]. In the recently published works, we have proposed that the existence of shortened telomeres in the LECs is acting as the molecular clock that triggers cellular senescence pheno-type during aging and cataract progression and is the result of oxidative attack to the lens and the lens cellular membrane structures in the lack of efficacy of the antioxidant protection in the lens towards the damaging oxidant promoters (Fig. 8)[15,16]. Telomere length in the crystalline lens cells is a re-flection of aging, cataractogenesis, and lifespan in biogeron-tological studies. The oxidative stress form and intensity might determine the lens senescence rate and cataract type, making efforts in the cataract prevention challenge more complex. The analyzed challenge in the published work is that the reduction in telomere shortening rate and damages in telomeric DNA make an important contribution to the anti-cataract and life-extension effect of carnosine administered systemically in the formulations stabilizing a dipeptide from the enzymatic hydrolysis with carnosinase, or topically ad-ministered to the eye with carnosine ophthalmic prodrug N-acetylcarnosine and lubricant formulations thereof includ-ing corneal absorption promoters (Fig. 9C) [15]. Prevention of cellular senescence with ophthalmic prodrug N-acetylcarnosine may be a novel therapeutic target in a management of cataract [16]. The cumulative results from the literature demonstrated that carnosine at physiological concentration might remarka-bly reduce the rate of telomere shortening in the continu-ously surviving lens cells subjected to oxidative stress in-duced by phospholipid hydroperoxides, toxic aldehyde mo-lecular products of LPO and oxidative glycosylation, cata-lytic transition metal ions and free radical oxygen species in the lack of efficient antioxidant lens protection [15,16,136, 137, 165-169]. Carnosine and related dipeptides have been shown to prevent peroxidation of model membrane systems (Figs. 10 and 11) leading to the suggestion that they repre-sent water-soluble counterparts to lipid-soluble antioxidants such as alpha-tocopherol in protecting cell membranes from oxidative damage [9]. Other roles ascribed to these dipep-tides in ophthalmics include actions as neurotransmitters, modulation of enzymic activities, chelation of heavy metals and transition metal ions, the inhibition of nonenzymic gly-cosylation of proteins (transglycating activities) [136, 137, 161, 162, 170]. We proposed a deglycation system involving

removal, by transglycation of sugar or aldehyde moieties from the Schiff bases by ophthalmic aldehyde scavenger L-carnosine derived from its ocular bioactivating sustained release prodrug 1% N-acetylcarnosine (NAC) lubricant eye-drops containing a mucoadhesive cellulose compound com-bined with corneal absorption promoters in drug delivery system [161] (Fig. 9). Carnosine attenuates the development of senile features when used as a supplement to a standard diet of senescence accelerated mice (SAM) devoid of serum carnosinase activity [171]. Its effect is apparent on physical and behavioral parameters and on average life span. A strik-ing anti-senescence effect of carnosine was demonstrated by McFarland and Holliday [166-168]. They showed that hu-man diploid fibroblasts grown in 20 mM carnosine had an extended lifespan, both in population doublings (PDs) and chronological time. The dipeptide L-carnosine had beneficial effects on cultured human fibroblasts. Physiological concen-trations in standard media prolong their in vitro lifespan and strongly reduce the normal features of senescence. Late pas-sage cells in normal medium were rejuvenated when trans-ferred to medium containing carnosine, and became senes-cent when carnosine was removed [166]. Neither D-carnosine, (beta-alanyl-D-histidine), homocarnosine, anser-ine, nor beta-alanine had the same effects as carnosine on human fibroblasts. In the recent work [169], the authors stud-ied the effect of carnosine on a human fetal lung fibroblast strain (HPF), which was either kept in a continuously prolif-erating or proliferation-inhibited state. The results indicate that carnosine can reduce telomere shortening rate possibly by protecting telomere from damage. Cells continuously grown in 20 mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. The authors have suggested that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine [169].

In this article we propose the effects of carnosine on te-lomeres of the canine LECs during aging and cataract forma-tion based on the established clinical efficacy of L-carnosine ophthalmic prodrug N-acetylcarnosine to partially reverse and prevent cataract formation and progression in canines in clinics (Figs. 9-12) [16, 22,155,156]. Due to the incomplete replication of linear chromosomes by DNA polymerase, te-lomeric DNA shortens with repeated cell divisions until the telomeres reach a critical length, at which point the cells enter senescence. Telomere length in LECs in canines is an indicator of biological aging, trends to cataractogenesis and dysfunction of telomeres in LECs is linked to age-related pathologies like cataract disease in canines and a predictor of mortality. Telomere length has been shown to be positively associated with nutritional status in animal studies. Various nutrients, such as a dipeptide L-carnosine derived from the ophthalmic prodrug – N-acetylcarnosine lubricant eye drops, influence telomere length in LECs of canines potentially through mechanisms that reflect their role in cellular func-tions including inflammation, oxidative stress, DNA integ-rity, DNA methylation and activity of telomerase, the en-zyme that adds the telomeric repeats to the ends of the newly synthesized DNA [15,16, 22, 155, 166]. Loss of functional


Fig. (11a). Accumulation of lipid peroxidation products (TBARS, measured as MDA) (A), diene conjugates (B), triene conjugates and ketone and aldehyde products (274 nm absorbing material) (C)) in liposomes (1 mg/ml) incubated for 60 min alone (6, dotted line) and with addition of the peroxidation-inducing system of Fe 2+ + ascorbate (1). The experimental technique is presented in details in Refs. [9,175].

The comparative antioxidant activity of N-acetylcarnosine (NAC) and L-carnosine was assessed in the liposome peroxidation system cata-lyzed by Fe 2+ + ascorbate. The accumulation kinetics of molecular LPO products such as MDA and liposomal conjugated dienes and trienes are shown in Figures (A,B,C). The results demonstrate that the LPO reactions in the model system of lipid membranes are markedly inhibited by L-carnosine. The effective concentrations of L-carnosine are 10 and 20 mM. Figure A shows that the level of TBA reactive substances (TBARS) reached at 5-min incubation decreases in the presence of L-carnosine (10 or 20 mM) at 10 min and at later time points (20 mM), which must be due to a loss of existing TBARS or peroxide precursors of MDA and not due to a decreased formation of peroxide compounds. The ability of the histidine-containing compound NAC to inhibit the (Fe 2+ + ascorbate)-induced oxidation of PC liposomes was compared with that of equimolar concentrations of L-carnosine. The antioxidant activity of 10 and 20 mM NAC corresponded to 38% and 55% inhibi-tion of LPO for the two concentrations after 60-min incubation. NAC exhibited less antioxidant protection than L-carnosine, corresponding to 60% and 87% of the equimolar (10 or 20 mM) L-carnosine inhibition percentage. However, since NAC can act as a time release version me-tabolized into L-carnosine during its topical and external application to the tissues (but not oral use), the antioxidant activity of NAC in vivoapplication is significantly increased. Once released from NAC in tissues, L-carnosine might act against peroxidation during its target phar-maceutical administration to the animal eyes in the fields of veterinary ophthalmology.

Fig. (11b). N-acetylcarnosine chemical formula C11H16N4O4 and energy-minimized structure (space filling model). Molecular mass: 268.27 g/mol, CAS number : 56353-15-2.


(Fig. 11) contd….


(Fig. 11) contd….

Figs. (11 c-f). The accumulation of lipid peroxides: TBA-reactive substances (c), conjugated diene (d); triene conjugates, ketone and aldehyde products (274 nm absorbing material) (e) in the liposomes (0.5 mg/ml) incubated alone (control) or in the lens-containing medium B at room temperature for 3 h. Sam-ples were taken at zero time and at varying time intervals as indicated in the figures. The above fixed aliquots of samples (50-500 l) were directly used for the measurement of TBA-reactive substances. Also, a similar amount of incubated sample was partitioned through chloroform as described in the lipid extraction procedure and after dissolution in 2-3 ml of methanol-heptane mixture (5:1,v/v) was used for detection of conjugated diene and triene conjugate/ketones (274 nm absorbing material). (1) Transparent rabbit lenses (IDLC 0-8%), Mean ± S.E.M., n=5; (2) transparent human lenses (IDLC 0-10%), n=4, initial cataract (IDLC 10-40%), n=3; (3) human mature cataractous lenses (IDLC 64-100%), n=5; (4) six normal mice lenses (IDLC 0-10%), n=3; (5) (control) liposomes, n=5. Experimental details are given in the text.

(f): Effect of various oxygen radical scavengers on lipid peroxide formation in liposomes added to the incubation medium of the normal rabbit lens (IDLC 0-8%). In a total vol of 3.0 ml the incubation mixture of the lens containing medium B, 0.5 mg/ml liposome suspension and the appropriate concentrations of scavenger as indicated. Mean values for the MDA concentrations are given for a representative experiment, with the error bars indicating the standard deviation obtained for the group of 3-5 lenses. telomere length below a critical threshold in LECs of canines during the effect of UV and chronic oxidative stress or meta-bolic failure, can activate programs leading to lens epithelial cell senescence or death. Telomere length represents a bal-ance between the loss of terminal telomeric repeats, which occurs during cell division with incomplete DNA replication, and the addition of telomeric repeats by the unique RNA-dependent DNA polymerase telomerase. Although most so-matic cells do not express telomerase, telomerase is induced in LECs of canines at critical stages of cataractogenesis ini-tiation and exposure to oxidative stress through the involve-ment of catalytically active prooxidant transition metal (iron) ions. Telomerase expression thus may prolong the replicative

capacity of LECs in canines and thereby enhance their func-tion in responses to oxidative stress of various origin. It might be possible to regulate physiologically the telomerase activity with N-acetylcarnosine ophthalmic prodrug respon-sing to ferrous ions prooxidant catalytic challenge and to manipulate with this drug effect on transcriptionally altered telomerase expression on telomere length and, consequently, on LECs and senescence function in the homeostatic way by inactivating catalytically active transition metal ferrous or cupric ions through the established ferroxidase activity and chelating properties of carnosine established in our provided and matched studies (Fig. 10) [15, 16, 22,137,155,156,173]. It is proposed that N-acetylcarnosine ophthalmic prodrug of


carnosine in the moiety of lubricant eye drops and related peptides (such as 1% carcinine lubricant eye drops in formu-lations with cellulose compound) might be explored as po-tential therapeutic agents for cataract pathologies in canines that involve the protection of LEC telomeres length modifi-cation and telomerase expression mediated by pro-oxidant stimuli of transition metal ions, production of peroxyl radi-cals generated in the lipid peroxidation (LPO) [15,16, 22, 137, 155, 156,173]. The present investigation indicates that carnosine derived from N-acetylcarnosine ophthalmic pro-drug can preserve telomere structures in the lens epithelial cells, mitochondrial function, cell viability, and ATP levels in canine lens cells during oxidative stress. Although the precise mechanism responsible for protection by the carnosine against oxidative stress remains to be determined, the ability of non-hydrolized carnosine, to protect against peroxide toxicity indicates that this type of biological activ-ity is likely to be mediated through scavenging effect of carnosine towards toxic aldehydes, phospholipid hydroper-oxide compounds (lipid peroxidation products), transition metal ions inducing shortening of telomeres in the lens epithelial cells. Carnosine promotes the protection of normal cells from acquiring phenotypic characteristics of cellular senescence switching canine lens into the process of ageing and cataractogenesis. The staggering statistics highlights the problem for vet-erinary ophthalmologists seeking to investigate the prevalence of cataract in the canine population. Provision of a geographic population-based survey of the pet animal population is exceptionally difficult, since census data for animals do not exist [15, 16, 22, 155, 156, 174]. Although several patterns of cataractogenesis in dogs are associated with aging, direct evidence of lens epithelial cellular senes-cence and the mechanism of senescence associated with chronic oxidative stress and accompanying ocular and sys-temic abnormalities or diseases during aging and cataracto-genesis in dogs is lacking. This study provides the canine lens-specific role of the LECs in cataract formation in dogs and highlights the role of telomere length and telomerase activity in the LECs of canines in the physiology and therapeutic treatment of cataractogenesis in dogs with N-acetylcarnosine lubricant eye drops. These findings establish that telomere erosion as causing senescence in LECs and telomerase upregulation might serve as the therapeutic target for carnosine released in the aqueous humor and the lens from the topically applied to the eyes of ophthalmic prodrug N-acetylcarnosine lubricant eye drops combined with carboxymethylcellulose and corneal absorption promoters in the therapeutic management of cataract in canines [15, 16, 155, 156]. This work demonstrates that transition metal ions, such as ferrous ions catalytic oxidants might induce prema-ture senescence in LECs of canines, telomere shortening with increased telomerase activity as adaptive response to UV light, oxidative and metabolic stresses. In pathological circumstances, the iron content and redox state of the aque-ous humor are dramatically altered and may affect the steady state levels of ferritin within the lens endowed with intrinsic ferroxidase activity as the natural detoxifying mechanism towards initiation of peroxide attack to the LECs and distor-tion of telomeres and DNA by ferrous ions. Prevention of cellular senescence and cataractogenesis in canines with oph-

thalmic prodrug N-acetylcarnosine may be a novel therapeu-tic target in a management of cataract in animals, basic pre-ventive health care of animals in veterinary ophthalmology. The anti-cataract activity of N-acetylcarnosine ophthalmic prodrug of L-carnosine acting as a universal ophthalmic an-tioxidant and transglycating agent is enhanced due to the fact that hydrolysis of carnosine in the aqueous humor, possibly by carnosinase, is not exhibited as dogs are devoid of serum carnosinase activity [176]. The physiological function of this enzyme seems to be the hydrolysis of homocarnosine in the brain and the splitting of carnosine and anserine in the blood stream. Six higher primates were found to have serum carnosinase. Twelve nonprimate mammals including canines were tested; all were lacking the serum enzyme except for the Golden hamster, which had very high concentrations of a carnosinase having somewhat different properties than the higher primate enzyme [176]. Therefore, we have achieved the evidence and consider the significant impact of 1% N-acetylcarnosine lubricant eye drops towards prevention and dissolution of ripe cataracts in canines [15,16, 22,156,175] (Fig. 12).

8. CONCLUSIONS

The dissolution of ripe cataracts in canines is based on the proposed findings on stabilizing properties of carnosine and related substances (carcinine) on biological membranes based on the ability of the imidazole-containing dipeptides to interact with lipid peroxidation products and active oxygen species and to prevent membrane damage and delute the associated with membrane fragements protein aggregates responsible for the increased light scatter in the lens forming cataracts [15, 16, 22, 155, 156, 175]. The advent of therapeu-tic treatment of cataracts in canines with N-acetylcarnosine lubricant eye drops through targeting the loss of functional telomere length below a critical threshold and “flirting” with an indirect effect with telomerase expression in LECs of canines during the effect of UV and chronic oxidative stress, has substantially improved the success rate of cataract cure challenges in home veterinary care, whereas the develop-ment of artificial lens implantation might alternatively and invasively improve postoperative visual acuity.

Throughout this manuscript, the authors have centered their discussions on cataracts in canines. The adoption of the whole concept on the telomeres length protection with N-acetylcarnosine lubricant eye drops prodrug to humans has been recently undertaken [9, 15, 16, 153-164]. The erosion and shortening of telomeres in human lens epithelial cells in the lack of telomerase activity has been recognized as a pri-mary cause of premature lens senescence phenotype that trigger human cataractogenesis. Carnosine, released oph-thalmically from N-acetylcarnosine prodrug lubricant eye drops, at physiological concentration might remarkably re-duce the rate of telomere shortening in the lens cells sub-jected to oxidative stress in the lack of efficient antioxidant lens protection. The data of visual functions (visual acuity, glare sensitivity) in older adult subjects and older subjects with cataract treated with 1% N-acetylcarnosine lubricant eye drops showed significant improvement as compared, by contrast with the control group which showed generally no improvement in visual functions, with no difference from baseline in visual acuity and glare sensitivity readings.


Fig. (12). Therapeutic treatment with 1% N-acetylcarnosine lubricant eye drops (patented formulation by Dr. Mark A. Babizhayev/Innovative Vision Products, Inc. [15, 16, 22,137,155,156]). Of age-related cataract in canine eye (A(1-3)).Pictures A (1-3) showing the reduction of cataract in canine eye. Top picture: this shows the canine cataract before treatment with Can-CTM

including 1% n-acetylcarnosine eye drops. A(2) picture: this shows 2 weeks of the treatment of the canine cataract with topical administration of Can-CTM eye drops including 1% N-acetylcarnosine. A(3): Results of the treatment after 1 month (bottom image). Already one begins to see the break-up of the impaired proteins - an effect that for obvious reasons has been described as "melting snow". The eyes of animals in each group were examined at regular intervals using a Zeiss SL-10 slit lamp. The appearance and progression of opacity was different in ani-mals so the staging varied. Posterior subcapsular opacity (PSC) and cortical opacities occurred during the early stages of cataract formation. Treatment of the 6-month rabbit eye with traumatic cataract for 2 weeks (B(1-3)).Estimation of opacity of the lens by quantitative morphometric analysis; The figures in the upper right segment of the images demonstrate that the area of the lens opacity is significantly diminished during the treatment with 1% N-acetylcarnosine lubricant eye drops.

A (1-3) B (1-3)


Prevention of cellular senescence with ophthalmic prodrug N-acetylcarnosine may be a novel therapeutic target in a management of cataract, basic preventive human health care and in arresting of after-cataract following extracapsular cataract extraction [9,15, 16,153-164].

CONFLICT OF INTEREST

The authors report the interest in the Intellectual Property of the described modalities protected with the patents. The authors bear primary responsibility for accuracy of made statements and employment of the described products and for the content and writing of the paper.

ACKNOWLEDGEMENTS

This work was planned, organized, and supported by Innovative Vision Products, Inc. (County of New Castle, DE, USA). Innovative Vision Products, Inc. is a holder of the worldwide patent (including PCT International Publication Number WO 2004/028536 A1) for the application of N-acetylcarnosine for the treatment of ocular disorders, in-cluding cataracts in canines and other animals as well as (PCT International Publication Number WO 2004/064866 PCT/JP2004/000351) protecting the described in the article therapeutic applications. Innovative Vision Products Inc. is a Pharmaceutical and Nanotechnology Development Company with a focus on innovative chemical entities, drug delivery systems, and unique medical devices to target specific bio-medical applications. Over the last decade IVP has devel-oped a track record in developing these technologies to ef-fectively address the unmet needs of specific diseased spe-cies.

PATIENT CONSENT

Declared none.

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Received: December 07, 2012 Revised: January 18, 2013 Accepted: January 28, 2013

DISCLAIMER: The above article has been published in Epub (ahead of print) on the basis of the materials provided by the author. The Edito-rial Department reserves the right to make minor modifications for further improvement of the manuscript.

PMID: 24093642

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