p clinical ntal & light induced damage of

doi: 10.1111/j.1472-8206.2011.00963.x

O R I G I N A L

A R T I C L E

Novel intraocular and systemic absorptiondrug delivery and efficacy ofN-acetylcarnosine lubricant eye dropsor carcinine biologics in pharmaceuticalusage and therapeutic vision care

Mark A. Babizhayeva,b*, Inna P. Khoroshilova-Maslovab,Anne Kasus-Jacobic

aInnovative Vision Products, Inc., 3511 Silverside Road, Suite 105, County of New Castle, DE 19810, USAbMoscow Helmholtz Research Institute for Eye Diseases, 14/19 Sadovaya-Chernogryazskaya, Moscow 103064, RussiacDepartment of Ophthalmology, University of Oklahoma Health Sciences Center, Dean A. McGee Eye Institute,

608 Stanton L. Young Blvd., Oklahoma City, OK 73104, USA

‘‘There is overwhelming evidence that the higher the level

of self-esteem, the more likely one will be to treat others with

respect, kindness, and generosity.’’

Nathaniel Branden

I N T R O D U C T I O N

Eye diseases that occur more often in elderly patients

increase concomitantly as the population ages. The latest

estimates of the World Health Organization (WHO)

Keywords

carcinine and blindness

prevention,

cataract, age-related macu-

lar degeneration and prolif-

erative vitreoretinopathy,

light induced damage of

photoreceptor cells,

N-acetylcarnosine lubricant

eye drops,

vision care and age-related

ophthalmic diseases

Received 30 December 2010;

revised 4 May 2011;

accepted 13 May 2011

*Correspondence and reprints:

[email protected]

A B S T R A C T

The latest estimates of the World Health Organization indicate that there are

161 million visually impaired individuals worldwide, 37 million of whom are blind,

with a yearly increase of 1–2 million. The scientists developed and patented the

lubricant eye drops formulated as 1% N-acetylcarnosine prodrug of L-carnosine

containing a mucoadhesive cellulose-based compound combined with corneal

absorption promoters in an ocular drug delivery system. Carcinine is suitable for

the systemic administration (per oral) for ophthalmic therapeutic indications. The

HPLC analysis was developed to search the pathways of ocular metabolic activities of

1% N-acetylcarnosine and the bioactivation of this drug molecule promoting

transcorneal uptake of L-carnosine in the aqueous humor. A meta-analysis of phase 2

randomized double-blind placebo-controlled clinical trial data was conducted. The

intraocular absorbed L-carnosine demonstrated a number of pharmacological

mechanisms of prevention and reversal of cataracts. Results of systemic absorption

of L-carnosine provide tuberomammillary activation that regulates neuronal func-

tions such as hypothalamic control promoting sensory input in the primary vision

perceptual pathway. The parabulbar, subconjunctival, and intravitreal injection of

carcinine with most of the vehicle removed is not toxic to intraocular structures,

reduces postoperative intraocular inflammation, is a potentially useful tool in the

treatment of proliferative vitreoretinopathy as well as considered as the antiapoptotic

drug for the protection of photoreceptor cells from oxidative light-induced stress. The

discovery of naturally occurring carnosine derivatives introduces N-acetylcarnosine

and carcinine as effective medical treatment for sight-threatening eye disorders.

ª 2011 The Authors Fundamental and Clinical Pharmacology ª 2011 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 1

Fund

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indicate that there are 161 million visually impaired

individuals worldwide, 37 million of whom are blind,

with a yearly increase of 1–2 million – this means

approximately 0.85% of the world population. Another

135 million show visual impairment [1].

Prevalence of blindness increases with age, mainly

after 50, being higher in rural areas and among women,

probably because of their socioeconomical vulnerability,

as well as the obstacles they face to access health services

[2]. Notwithstanding the progress in surgical interven-

tion that has been made in many countries over the last

few decades, cataract remains the leading cause of visual

impairment in all regions of the world, except in the most

developed countries. Other major causes of visual

impairment are, in order of importance, glaucoma,

age-related macular degeneration, diabetic retinopathy,

and trachoma [1]. Blindness or low vision affects

approximately 1 in 28 Americans older than 40 years

[3]. Based on demographics from the 2000 US Census,

an estimated 937 000 (0.78%) Americans older than

40 years were blind (US definition). An additional

2.4 million Americans (1.98%) had low vision. The

leading cause of blindness among white persons was age-

related macular degeneration (54.4% of the cases), while

among black persons, cataract and glaucoma accounted

for more than 60% of blindness.

Cataract was the leading cause of low vision

(Figure 1), responsible for approximately 50% of bilateral

vision worse than 6/12 (20/40) among white, black,

and Hispanic persons. The number of blind persons in

the United States is projected to increase by 70% to

1.6 million by 2020, with a similar rise projected for low

vision [3]. Home health care is the fastest growing

expense in the Medicare program because of the aging

population, the increasing prevalence of chronic disease

and increasing hospital costs [4]. Over 375 000 people

can suffer permanent visual impairment every year as a

result of surgical complications [5]. This means that

surgical complications, and cataract complications in

general, represent a significant obstacle to the success of

any blindness prevention program. This article discusses

the use of anticataract new type of medicated

N-acetylcarnosine lubricant eye drop patented by Inno-

vative Vision Products, Inc. (New Castle, DE, USA) (IVP),

which is important to the successful implementation of

VISION 2020 [6–19].

Proliferative vitreoretinopathy (PVR) is a scar tissue

formation within the eye. This condition has been called

by many names, including massive periretinal prolifer-

ation (MPP) and massive vitreous retraction (MVR), and

was finally dubbed PVR by the Retina Society Terminol-

ogy Committee. ‘Proliferative’ is given because cells

proliferate, and ‘vitreoretinopathy’ is given because the

Figure 1 Cataract. Slit lamp photograph of cataract in human eye.

The elderly patient complained of a slowly progressive, painless loss

of vision.

Figure 2 Proliferative vitreoretinopathy (PVR). In PVR, scar tissue

forms in sheets on the retina, which contract. This marked

contraction pulls the retina toward the center of the eye and

detaches and distorts the retina severely. PVR can occur both

posteriorly (as shown) and anteriorly with folding of the retina

both anteriorly and circumferentially.

2 M.A. Babizhayev et al.

ª 2011 The Authors Fundamental and Clinical Pharmacology ª 2011 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

problems involve the vitreous and retina. PVR (Figure 2)

can be divided into multiple categories based on the

configuration of the retina and the location of the scar

tissue, and this categorization is used by eye care

specialists to describe to one another the severity and

configuration of the retina in PVR. PVR is the most

common complication of a retinal detachment (RD) and

occurs in approximately 8–10% of patients who develop

an RD.

The retina normally lies on a layer of epithelial cells

that, in turn, rest on a bed of blood vessels. Sometimes

the retina becomes partially or completely detached from

its support tissues, which impairs or completely abol-

ishes vision. Successful retinal reattachment can be

achieved surgically in about 90% of cases. However, in

the remaining 10%, the surgery results in contractile

scarring on the surfaces of the retina, preventing

reattachment. This condition is known as PVR, and

blindness or extremely limited vision is an inevitable

consequence. At the time of a retinal detachment and

the formation of a retinal tear, Retinal pigment epithelial

(RPE) cells that are normally under the retina come

through the retinal tear and enter the vitreous cavity.

After the retinal detachment is repaired or not repaired

(if the patient does not seek help), these cells proliferate

on the surface of the retina (and sometimes under the

retina) in sheets, which contract and pull the retina back

off. These sheets can occur in the posterior portion of the

retina, or in any other location of the retina, including in

the far anterior periphery of the retina, causing

redetachment.

The surgery to repair an eye detached from PVR

includes pars plana vitrectomy, membrane peeling where

ophthalmic surgeons use small instruments to peel the

membranes from the surface of the retina, and scleral

buckling. These techniques are combined with fluids

placed in the eye to flatten the retina and reattach it to the

outer wall of the retina followed by laser photocoagula-

tion to connect the retina to the outer layers perma-

nently. Although PVR is a catastrophic complication of

retinal detachment surgery and can cause profound

visual loss, it has gone from being unsuccessfully repaired

in the late 1970s to having a very high success rate in

repairing PVR detachments today.

Current medical therapies are limited to the use of

drugs that act to reduce scar formation by inhibiting

proliferation of the cells that form the scars. However,

these drugs are quickly metabolized within the eye

minimizing their long-term effectiveness. High doses of

these drugs may also be toxic to the retina.

Our PVR researchers have discovered that the forma-

tion and subsequent contractile action of retinal scars is

produced by the movement of retinal pigment epithelial

cells that normally remain stationary. They have there-

fore surmised that if one can prevent movement of scar-

forming cells therapeutically, we can also prevent PVR.

Work has been therefore ongoing within the team to

identify chemical signals called chemoattractants that

stimulate these cells to move. Here, we are presenting the

original experimental data.

In many cases, prodrugs may offer a way to overcome

the poor drug-like properties of a very potent lead and

provide the opportunity to convert a nondevelopable

natural molecule subjected to enzymatic hydrolysis into

a potent patented candidate for clinical use. The

histidine-containing dipeptide L-carnosine (b-alanyl-

L-histidine) and its ophthalmic bioactivating prodrug

N-acetylcarnosine (N-acetyl-b-alanyl-L-histidine) are

part of this group of products [14,20,21]. Carnosine

(b-alanyl-L-histidine) and related compounds are natural

constituents of excitable tissues possessing diverse bio-

logic activities [22,23]. The level of carnosine in tissues is

controlled by a number of enzymes transforming carno-

sine into other carnosine-related compounds, such as

carcinine, N-acetylcarnosine, anserine, or ophidine (by

decarboxylation, acetylation, or methylation, respec-

tively) or its cleavage into the amino acids, histidine

and b-alanine. Hydrolysis is mainly as a result of tissue

carnosinase (EC 3.4.13.3), which is widely distributed

among different tissues [24,25] or serum carnosinase

(EC 3.4.13.20), found in brain and blood plasma of

primates and humans [26,27]. 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 [28]. The concentration of carnosine in trans-

parent crystalline lenses detected was about 25 lM. At

different stages of cataract development, the level of

carnosine fell, reaching about 5 lM [28]. 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 oxida-

tive stress [29–31]. It can oppose glycation [32,33], and

it can chelate divalent metal ions. The important studies

have produced clinical and experimental evidence of

beneficial effects of N-acetylcarnosine in treating cata-

racts of the eyes, and these and other ophthamological

benefits have been proven [6–14]. Research with

N-acetylcarnosine (NAC) demonstrates that it is effective

N-acetylcarnosine lubricant eye drops and carcinine for vision care 3


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 [8].

Carcinine (b-alanyl histamine) is an imidazole dipeptide

first discovered in the crustacean Carcinus maenas [34]

and has subsequently been found in the hearts of several

mammalian species [35,36]. It has been demonstrated

that carcinine is metabolically related to histamine,

histidine, and carnosine (b-alanyl-L-histidine) and could

be synthesized from histamine and b-alanine [37]. In

addition, previous studies have shown that carcinine

contains an imidazole group with flexible ethylene side

chain known to be important for histamine H3 receptor–

ligand interactions [38–41]. From these findings, it

seems that a certain relationship exists between brain

histamine and carcinine and that carcinine might be a

new histamine H3 receptor antagonist. The results of the

recent study provide direct evidence that carcinine, as a

novel histamine H3 receptor antagonist, plays an

important role in histaminergic neurons activation and

might be useful in the treatment of certain diseases, such

as epilepsy, and locomotor or cognitive deficit [42].

However, only few reports have explored this relation-

ship, and little is known about the pharmacological and

physiologic role of carcinine. In two of those reports,

carcinine was shown to act as a natural antioxidant

[21,43] and to play a role in regulating stress and shock

with a 1000-fold less potent hypotensive effect than

histamine [36,44], suggesting that carcinine might have

therapeutic use. Carcinine is officially registered for

cosmetic and cosmeto-pharmaceutical application in

the USA and Europe by Exsymol SAM since 1997.

Overall, these low-molecular-mass antioxidant pepti-

domimetics (Figure 3a–c) add significantly to the defense

provided by the enzymes superoxide dismutase, catalase,

and glutathione peroxidases [21,43].

One of the obscure aspects of the carnosine physiologic

role is the biologic significance of the enzymatic meta-

bolism of carnosine or its derivatives in tissues. We found

that to change the antioxidant status, tissue enzymes

can modify the NAC prodrug molecule and that deacet-

ylation increases in vivo the resistance of lens tissues and

its cells to oxidative stress.

Poor bioavailability (<1%) of drugs from conventional

eye drops is mainly owing to the various precorneal loss

factors, which include rapid tear turnover, systemic drug

absorption through naso-lachrymal duct, transient res-

idence time of the drug solution in the cul-de-sac, and the

relative impermeability of the drugs to corneal epithelial

membrane. 1% N-acetylcarnosine is a universal bioacti-

vating antioxidant for vision in the developed and

patented drug delivery system lubricant eye drop formu-

lations containing mucoadhesive cellulose-based com-

pound combined with corneal absorption promoters. Its

topical administration delivers pure L-carnosine and

allows its increased intraocular absorption into the

aqueous humor surrounding the lens, enabling signifi-

cant improvements in anticataract drug efficacy and the

minimization of side effect from either local or systemic

drug absorption/bioavailability to the eye, and also

creates optimization effects in the number of ocular

degenerative age-dependent disorders [10]. The formu-

lation was also found to be nonirritant and well tolerable.

The developed system can be a viable alternative to

conventional eye drops for the treatment of various

ocular diseases and is suitable for clinical application.

The developed IVP N-acetylcarnosine prodrug and

codrug lubricating eye drop systems (including principal

regulatory registered eye drops design IVP CTM and

Can-CTM lubricating eye drops) have been marketed

under numerous brand labels (Figure 4). N-acetylcarno-

sine prodrug and codrug ophthalmic formulations

applied topically to the eye, and moreover, its controlled

time released ophthalmic ingredient L-carnosine exerts

antiglycation, bioactivating antioxidant properties in the

lens and cornea as a scavenger of lipid peroxides, singlet

oxygen, and OH· radicals and provides the spatial aspects

of intracellular pH regulation [8,11,15]. The marketed

patented famous brand label Can-CTM of N-acetylcarno-

sine eye drops approved by Innovative Vision Products,

Inc. recently achieved the important milestone on over

500 000 bottles sold.

We have recently designed an innovative Halometer

DG tester that overcomes previous deficiencies [7,45–

47]. IVP patented the original Halometer DG concept

and designed the vision diagnostic device for clinical

implementation as predictor of visual functions in

subjects during various daily activities, such as driving

(Figure 5) [7,45–47].

The purpose of this study is to examine the effects of a

most often recommended to patients a short-term 5- to

6-month treatment with 1% N-acetylcarnosine in oph-

thalmic formulation with a lubricant carboxymethylcel-

lulose on improvement in visual impairment and glare

disability in older adult subjects and older patients with

cataract whose realistic occupations frequently involve

driving activities.

We used a randomized design that was ethical since

N-acetylcarnosine is an accepted and proven therapeutic



modality of vision care available on the market of

antiaging medicine since 2002 [16–19].

M A T E R I A L S A N D M E T H O D S

Peptide-based compounds have been manufactured

according to cGMP and GLP certified techniques.

Carcinine (Decarboxy carnosineÆ2HCl) was synthesized

by Exsymol S.A.M. (Monaco, Principaute de Monaco)

(Table I) L-Carnosine and N-acetylcarnosine were

synthesized by Hamari Chemicals Ltd, Osaka, Japan

(Table II) per GLP specifications proposed by IVP.

Experimental design: N-acetylcarnosine lubricant

eye drops studies

Studies of transcorneal uptake and systemic absorption of

carnosine from N-acetylcarnosine ocular drug delivery

formulation

Formulations were used in fifty five Grey Chinchilla

rabbits (male) aged 3–4 months weighing 2–3 kg.

(a)

(c)

(b)

Figure 3 Structures of N-acetylcarnosine (a), carnosine (b), and carcinine (c) shown as chemical structures and energy-minimized structures

(space-filling models). (a) N-acetylcarnosine chemical and energy-minimized structures (space-filling model). (b) Carnosine chemical and

energy-minimized structures (space-filling model). (c) Carcinine chemical and energy-minimized structures (space-filling model). Formula

(N-ethyl-5-imidazolyl)b-aminopropionamide chlorhydrate.



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 lL of formulation A (Can-

CTM) containing 1% N-acetylcarnosine (NAC), and the

control right eyes of the separate rabbits were similarly

instilled with their vehicles (placebo) solutions

(Figure 6a). Formulation A was presented in the final

ophthalmic tubes (per volume of 2.5 mL) and in the

moiety of the plastic bottles. Placebo solution contained

the same ingredients without NAC.

Surgical procedure

Topical anesthesia 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- to

2.0-min intervals). The eye drops of 4% lidocaine

Figure 4 Most popular OTC public and officially registered International brands of N-acetylcarnosine lubricant eye drops worldwide patented

by Dr. Mark A. Babizhayev/IVP, clinically developed in cooperation with alliance Corporate Partners and marketed worldwide through the

number of International Distributors contractual with obligations to IVP. The IVP CTM brand of N-acetylcarnosine eye drops is officially

registered (CE Marked) in all countries of EC, and the appropriate Free Sales Certificate has been granted by the Italian Ministry of Health.

Under the IVP. Global Vision policy, the IVP C. TMBrand of N-acetylcarnosine lubricant eye drops now became officially registered in U.A.E.

(Dubai), Malaysia, and Indonesia. Indonesia comprises 17 508 islands, and with an estimated population of around 237 million people,

it is the world’s fourth most populous country and has the largest Muslim population in the world. Cataract prevalence in adults aged

21 years and older in rural Indonesia is among the highest reported in Southeast Asia. Despite this, there are inadequate resources

available to manage this treatable disease. Allocation of resources to tackle the present burden of cataract with developed and patented to

Babizhayev N-acetylcarnosine lubricant eye drops would likely have large personal, social, and economic benefits.



hydrochloride contain benzaltonium chloride preserva-

tive. When ocular anesthesia 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 transcorn-

eally 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 the

addition of ethanol (0.2 mL), keeping the sample on ice

before extraction.

Extraction of imidazoles from aqueous humor

Extractions of imidazole-containing compounds from the

aqueous humor aliquots were performed according to

Babizhayev et al., [14]. The published data showed that

all the desired imidazole-containing compounds in the

aqueous humor thus obtained could be of good purity and

recovery [14]. Portions of aqueous humor were added to

ethanol as afore mentioned and thoroughly mixed

(20 �C, 15min). Extracts were centrifuged (2000 g,

15 min), and the supernatants were removed. Samples

were frozen in the gradient of temperatures to )70 �C 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 lm directly prior the analysis.

Analytical HPLC for the detection of L-carnosine and

N-acetylcarnosine

Reverse-phase analytical HPLC was performed using a

Breeze chromatography system (Waters corporation,

Milford, MA, USA), detector Waters 2487 Dual kAbsorbance Detector, column (250 · 4.6 mm) Symme-

try 300 C18 5 lm (Waters), loop 20 lL (Figure 6b). The

column was eluted isocratically at 30 �C 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)

(Figure 6b). The quantitative determination of L-carno-

sine and N-acetylcarnosine in the samples was under-

taken using the technique of external standard

according to the area of the peak and linear extra-

polation. 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.

Clinical design: N-acetylcarnosine lubricant eye

drops studies

The examined subjects consisted of 75 older adults with

age-related uncomplicated cataracts in one or both eyes

and 74 adult subjects who did not have cataract in either

eye. Patients in these subsamples suffered from different

degrees of glare problems. Those with cataract ranged in

Figure 5 Principle of the glare test for adults, based on the

measurement of the glare radius (r, mm) a new metric for glare

sensitivity. I0 = Indicatrix of light scatter; u = angle. The technique

utilizes a self-illuminating red or green optotype target and tangen-

tial 2 mm ‘point light source’ seen from a distance of 30 cm. The

patient’s task is to move the optotype closer to the glare source until

it disappears because of the veiling glare from the glare source. A

halometer score is determined as follows. The target is approached

from the source so that the patient becomes unable to distinguish the

target from the source, and then, the target is slowly taken away

until the exact moment when the patient distinguishes the target; at

this time, the incident light angle u between the source and the

target is measured. The target is always fixated with the foveal

vision. The target and the ‘point light source’ are viewed in the

same vertical plane, tangential to the plane of emitted light. In this

case, to measure the angle u of the incident light between the

source and the target, it is necessary only to measure its projection

on this vertical plane, which means to measure the distance

between the source and the target. The measured glare radius is

defined as a target image projection for the vector of light scatter

(indicatrix of light scatter I = I0cos2 u) when the glare source is

activated and the patient is asked to recognize the target during

illumination of the eye with a glare source.



age from 54 to 85 years (mean ± SD, 70 ± 8 years),

with 49% female, 100% white and of non-Hispanic

origin. The no-cataract subjects ranged in age from 54 to

80 years (mean ± SD, 68 ± 8 years); 51% were female,

with 100% being white. Subjects who were cataract-free

had to meet the same inclusion criteria as the subjects

with cataract described previously [15–19,45]. All

subjects with cataract were required to meet the follow-

ing inclusion criteria: (i) cataract in one or both eyes

with best-corrected visual acuity of 20/40 or worse in

one or both eyes as indicated by the medical record;

(ii) no previous cataract surgery in either eye; (iii) a

primary diagnosis of cataract in the medical record; (iv)

living independently in the community; specific items

were needed to be addressed if appropriate: (v) the

driving skills: legally licensed to drive and drove during

the 5 years prior to enrollment. Among participants,

bilateral cataracts were present in 91% of subjects

according to the medical record from the most recent

eye examination (within 1 month of enrollment). In the

right eye, 41% had nuclear sclerotic cataract, 9% had

cortical cataract, 11% had posterior subcapsular cata-

ract, and 39% had a combination of at least two types.

The breakdown was similar in the left eye, with 45%

nuclear sclerotic, 10% cortical, 8% posterior subcapsu-

lar, and 37% combination. Seventy-three percent of

subjects with cataract had no additional ocular

conditions other than refractive error; 7% had early

nonexudative age-related maculopathy, 6% had primary

open-angle glaucoma (POAG) associated with cataract,

5% had diabetic retinopathy, 2% had a combination of

two of these problems, and 7% had another ocular

condition. Subjects who were cataract-free had to meet

the same inclusion criteria as the subjects with cataract,

except that they were required to be free of cataract and

to have a best-corrected visual acuity of 20/25 in each

eye, according to medical record review. No cataract-free

subjects had secondary eye conditions other than

refractive error.

Patients with known or presumed hypersensitivity to

any component of the ophthalmic preparations (active

substances or excipients), and those treated with drugs

that could interfere with this trial, were also excluded

from the study. The subjects were recruited and

examined by ophthalmology practices of Innovative

Vision Products Inc (County of New Castle, DE, USA).

The study protocol was approved by the Corporative

Review Board for Human Use. After the purpose of the

study had been explained, each subject was asked

to sign a document of informed consent before

Table I Characteristics and specification of carcinine (Decarboxy

carnosineÆ2HCl) synthesized by Exsymol S.A.M. (Monaco,

Principaute de Monaco).

Composition of the substance: pure substance

cGMP/GLP characteristics

Purity as a percentage: >99%

Hydrosolubility: soluble in water

Liposolubility: insoluble in mineral and vegetal oils

Fusion point: FP = 195 �CQuantification of heavy metals: <20 ppm

Quantification of chlorides: 25.0–30.5%

Infrared characterization: positive

Physical state at 20 �C under 101.3 kPa: solid

Physical characteristics: white crystalline powder, odorless

Microbiology

Mesophilic aerobic bacteria: <1000/g

Yeasts, molds: <100/g

Escherichia coli (per g): none

Table II Characteristics and specification of (a) L-carnosine and

(b) N-acetylcarnosine synthesized by Hamari Chemicals Ltd.

Test item Requirement

Specification of L-carnosine

Appearance White crystal or crystalline

powder

Odor Odorless

Identification Positive

Optical rotation: ½a� 20D +19.8 to +22.2�

Transmittance NLT 95.0%

Heavy metals NMT 10 ppm

Arsenic NMT 2 ppm

Other amino acids Not detectable

Loss on drying NMT 1.0%

Residue on ignition NMT 0.20%

Assay 98.0 to 101.0%

Test name Specification

Specification of N-acetyl-L-carnosine

Appearance White powder

Identification Positive

Optical rotation ½a� 20D +25.2 to +27.5�

pH 4.5–5.5

Heavy metals NMT 10 ppm

Related substances L-Carnosine: NMT 0.3%

Others: NMT 0.2%

Residual solvent 2-propanol: NMT 500 ppm

Water NMT 5.0%

Residue on ignition NMT 0.10%

Assay NLT 99.5% (HPLC area)

NMT, not more than; NLT, not less than.



enrolling. Demographic data and driving status dur-

ing the prior 5 years were confirmed by interview

(Table IV).

Procedures

After enrollment, subjects were computer-randomized

into two groups assigned according to the double-blind

method: to receive treatment with N-acetylcarnosine 1%

eye drops (Can-CTM) or to a control group who received

placebo eye drops. The blinded testing was carried out by

an independent medical worker who handed out the NAC

vs. placebo eye drops (control group) to the randomized

subject members of the clinical groups. The enrolled

subjects underwent follow-up examinations at baseline

and after 5–6 months of enrollment. Test examiners were

masked to the driving histories of all subjects. Two types

of visual functions were assessed: visual acuity and glare

sensitivity (disability glare). All acuity measurements

were taken while subjects wore the lens correction they

typically used during the performance of everyday

distance activities, including driving. Each eye was

assessed separately. Distance acuity was measured as

described before using the letter chart and its standard

protocol and was expressed as log minimum angle

resolvable [15–19,45,48,49]. For each eye, visual acuity

measurements were grouped into four categories: 20/25

or better, 20/25–20/30, 20/35–20/50, and worse than

20/50. These cut points were chosen because they were

the approximate quartiles of the acuity distribution and

included the practically significant cut point for driving

licensure in many countries (20/40–20/50).

Slit lamp biomicroscopic examination or exampled

photographic registration was performed after pupil

dilation to a minimum of 6 mm with tropicamide.

Disability glare was defined with an optical instrument

and method for measuring susceptibility to glare of a

human vision system as described [15–19,45–47]

and schematically presented in Figure 5. A constant

(a)

(b)

Figure 6 (a) Typical rabbit’s eye used in an ocular pharmacokinetic

study. (b) Configuration of techniques for HPLC analysis of peptides

extracted from the aqueous humor. The BreezeTM 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 preconfigured for different

levels of HPLC operational needs with peptide moieties. Symmetry

column(s) (image below) continues to set the standard for HPLC drug

assays, giving a confidence in the long-term compliance of HPLC

methods. No need to compromise on peak shape, selectivity, column-

to-column and batch-to-batch reproducibility, or other critical

performance characteristics. For laboratory balances weighing to the

nearest 0.0000001 g, for the provided fine measurements of the

mass of extracted peptide 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.



‘point’-like bright glare source is used to create the glare

condition (Figure 5). The examining room was dark (<20

foot-candles) as typical when working with the glare

testers to assure maximum contrast of the projected

target. Tests were performed with the best correction in

place. The indicator of optotypes on the front or back

panels of the instrument indicated the tested optotype to

the patient or clinician, respectively. The diagnostic block

of a device contained source light window (glare source)

and the moving indicator of the optotypes illuminated

with red or green light (Figure 5). The back panel of the

Halometer device facing the clinician was equipped with a

chart/scale and with a moving indicator of the optotype

transition. According to a special embodiment of the

invention, [15–19,45–47] for the clinical testing of glare

sensitivity of a patient, we used an illuminated target with

red or green color, which enables the assessment of the

effect of wavelength on the scattered light.

Treatments with N-acetylcarnosine 1% lubricant eye drops

N-Acetylcarnosine (NAC) eye drops (Can-CTM)

contained a 1% solution of NAC (Babizhayev MA,

Bozzo Costa E. Composizioni farmaceutiche contenenti

N-acetilcarnosina per il trattamento della cataratta.

Italian Patent A61K gruppo 37/00 20122 MI, Priority

Oct. 15, 1993.; Babizhayev MA, Bozzo Costa E. Phar-

maceutical compositions containing N-acetylcarnosine

for the treatment of cataract. Patent PCT/EP 94/03340

SCB 238 PCT, Oct. 10, 1994.; Babizhayev MA. Method

for topical treatment of eye disease and composition and

device for said treatment. PCT Patent Application.

International Publication Number WO 2004/028536

A1. International Publication Date: 8 April 2004) with

a lubricant 0.3% carboxymethylcellulose in the isotonic

ophthalmic formulation in borate buffer with preserva-

tive benzyl alcohol (corneal absorption promoter) and

showed the increased intraocular absorption of the

active principle (L-carnosine) in the aqueous humor

compared with topical administration of a pure 1% NAC

solution:

Deionized water 970 g

Glycerin, 1.0% 13 g

N-acetylcarnosine, 1.0% 10 g

Carboxymethylcellulose, 0.3% 3 g

Benzyl alcohol, 0.3% 3 g

Potassium borate 7.91 ga

Potassium bicarbonate 3.47 ga

aOr what is necessary to bring the solution up to a pH of approximately 6.3–

6.8.

The ophthalmic formulation thus creates a facility to

examine the efficacy of treatment for improvements in

vision during the short-term periods of administration of

N-acetylcarnosine 1% eye drops (5–6 months in this

study). The administration schedule was two drops

instilled twice daily, for patients assigned to NAC and

those assigned to placebo (the same formulation without

N-acetylcarnosine, 1%) alone for 5–6 months. The use of

other topical or nutritional antioxidants was not mea-

sured or evaluated between the two groups. The control

groups and the treated group did not take any prescribed

antioxidant vitamins that might have added to the

antioxidant level. Neither the investigators nor the

patients knew who was receiving NAC.

Carcinine ophthalmic research

Experimental modeling of proliferative vitreoretinopathy

(PVR)

Experimental model of PVR is very important in inves-

tigation of pathogenesis and prophylaxis of this ocular

pathology. Grey Chinchilla male rabbits aged 3–

4 months, each weighing between 2 and 3.0 kg, were

used. All animals were sedated by intramuscular injec-

tions of ketamine-xylazine (10 mg/kg body weight). The

eyes were dilated with one drop of 1% cyclopentolate and

2.5% phenylephrine.

We used the ‘cytokine’-induced model of PVR devel-

oped at Moscow Helmholtz Research Institute for Eye

Diseases [50]. In the in vivo model, rabbit eyes were

injected intravitreously with cytokine complex alone to

induce tractional retinal detachment.

The cytokine complex (each single dose of 600 lg)

included retinal pigment cells (9.1–9.6 · 102 cells in

0.2 mL of medium), endogeneous immunopeptides, and

glycoprotein fibronectin. Formulated in advance hetero-

logic cytokine complex including endogeneous immuno-

peptides with activity of Il-1, TNF-a, MIF, and TGF-b(0.1 mL) was administered with the single intravitreous

injection into both matched rabbit eyes [50]. Separate

control group of rabbits was used for modeling of PVR

with cytokine challenge, placebo (vehicle) administra-

tion [the matched control eyes were injected into the

vitreous with phosphate-buffered saline (PBS)] and the

pure eyes morphological control.

Carcinine (b-alanylhistamine) at a concentration of

10 mM dissolved in PBS was used as a prophylaxis

remedy. This drug was injected into the experimental

eyes using the multiple parabulbar (repeated single daily

injections within consequent 5 days) or single-time

intravitreous administration ways. The therapeutic



injections were provided to the left eyes after the

experimental intravitreous injection of cytokine complex

was completed. The right eyes served as a control. At the

end of the month, the animals were killed, and their eyes

were enucleated.

Parabulbar injections (5) were applied during 5 days

to the left eye started just after the intravitreous injection

of the cytokine complex. In this group (nine rabbits

studied), the rabbit eyes obtained the parabulbar injec-

tions of carcinine after the intravitreal injection of

cytokine. In a separate group of rabbits (six rabbits),

subconjunctival injections were applied instead of para-

bulbar injections according to the described design. In

the separate group (five rabbits), we have applied single-

dot intravitreous injection of carcinine (10 mM, 0.1 mL

in balanced physiologic saline) simultaneously with

cytokine complex to the left eye. The separate group of

rabbit eyes (four rabbits) received postponed parabulbar

injections of carcinine started 3–4 days after the intra-

vitreal challenge with cytokine complex. These post-

poned parabulbar injections of carcinine prolonged

within 5-day follow-up period. The right eyes served as

a control after the intravitreal injection of cytokine in all

cases.

Morphological (light and electron microscopic) inves-

tigation was carried out. Histopathologic examination of

the globes of rabbits was used to evaluate the morpho-

logical change after intravitreous injection of cytokine

complex and carcinine. On day 30 after treatment, all

animals in the experiment were killed by intravenous

injection of an excessive dose of pentothal. Globes were

enucleated immediately and fixed with phosphate-

buffered 10% formaldehyde. The specimens were then

dehydrated with increasing concentrations of ethanol,

cleared with xylene, and embedded in paraffin. Ten-

micron sections were cut and stained with hematoxylin

and eosin. Tissue sections were observed and photo-

graphed by light microscopy. In the control eyes, clinical

and morphological picture of PVR was observed. In the

treated eyes, there was a noticeable inhibition of

inflammation and proliferation in the retina. The mech-

anism of PVR inhibition under the influence of carcinine

was studied.

Carcinine effects against light-induced damage and

photoreceptor cell death in Balb/C mice

Balb/C mice were raised in a 12-h dim light (10 lux)-

dark cycle (Figure 7a). Two-month-old mice were deeply

anesthetized with a single intraperitoneal injection of

xylazine (20 mg/g) and ketamine (40 mg/g). One

microliter of carcinine or PBS (vehicle control) was

injected intravitreally into the eye using a 36-gauge

needle (World Precision Instruments, Sarasota, FL,

USA) through the temporal limbus of the eye. In each

animal, the left eye was injected with carcinine and the

right eye with PBS. Thus, each animal served as its

own control. Mice were returned in dim cyclic light for

48 h before exposure to bright light (4000 lux) for 5 h

to induce oxidative stress in photoreceptor cells, as

described previously [51]. After light damage, mice

were returned in dim cyclic light for 7 days to allow the

retina to clear all dead cells and return to a well-

organized morphology. Mice were killed by CO2 inha-

lation before tissue collection. This method is approved

by the Panel of Euthanasia of the American Veterinary

Medical Association. All procedures were performed

according to the National Institutes of Health Guide

for the Care and Use of Laboratory Animals and

the University of Oklahoma Health Sciences Center

(OUHSC) Guidelines for Animals in Research. Whole

eyes were enucleated after orientation of the superior

half with a permanent dye. Oriented eyes were embed-

ded in paraffin, and sections were cut along the vertical

meridian, through the optic nerve head (ONH). To

quantify the number of remaining photoreceptors,

hematoxylin- and eosin-stained paraffin sections were

prepared from each eye, and the thickness of the outer

nuclear layer (ONL) was measured at 0.24-mm inter-

vals from the ONH to the inferior and superior ora

serrata as described previously [51].

Figure 7 Balb/C mice were raised in a 12-h dim light (10 lux)-dark

cycle.



Study of acute toxicity of carcinine by oral administration

in mice

This experimental series has been conducted to verify

the innocuousness of the carcinine product being tested

in the event of accidental ingestion. Male Swiss mice,

weighing 20 g from the IFFA CREDO (Lyon, France)

Breeding Center, were used in the study. The animals

have been chosen at random, and the batches were

prepared before the beginning of the oral treatment

with carcinine. The mice were kept in cages equipped

with a sterilized sawdust floor. Feeding consisted of

‘SOURIS Entretien’(SA. U.A.R.) food. Water was given

as desired.

Administration of compound being studied (carcinine)

was undertaken under the following conditions. Mice

were deprived of water within 15–20 h. The testing

compound (carcinine) was then administered by gastric

intubation (with an oesophagian probe), one time, to a

group of 10 animals (five males and five females). The

treated animals absorbed 2 g of the tested compound per

kg of body weight (20 mL/kg of a 10% solution), i.e., the

maximal quantity permitted for gnawing animals (JO.CE:

L 383 A/111). In the event of death, the compound to be

studied was administered to another group of 10 mice

(five males and five females). They absorbed 1 g/kg of

body weight (10 mL/kg of a 10% solution). If the LD 0

value was <1 g/kg, the LD 50 was calculated. If the LD 0

was more than 1 g/kg, the compound was said to be

nontoxic.

Statistical analyses

Statistical analysis was performed by Student’s t test;

P = 0.05 was taken as the upper limit of significance. To

assess associations, correlation and linear regression

analyses were used.

R E S U L T S

Intraocular transcorneal and systemic absorption

of L-carnosine from N-acetylcarnosine ocular drug

delivery system (lubricant eye drops Can-CTM):

suitable molecular therapeutic interventions and

visual sensory signaling responses

Histidyl compounds of aqueous humor have been

examined by reverse-phase analytical HPLC (see Exper-

imental design: N-acetylcarnosine lubricant eye drops

studies) as used in separation of imidazole-containing

amino acids and dipeptides described earlier [52]. Amino

acids can be detected by the absorbance of carboxylate

(approximately 200 nm) and peptides by the absorbance

of carboxylate and the peptide bond (200–220 nm).

Chromatograms of solutions of L-carnosine and its

putative N-acetyl derivative (Figure 8a) show that these

compounds are well separated. The elution order of the

compounds was compared with a predicted order based

on their relative hydrophobicities as outlined by Rekker

[53]. The chromatographic system is suitable to monitor

the behavior of other histidine-containing derivatives of

carnosine [14]. The calibrating chromatograms show

the predicted elution order and the average elution times

for each standard of L-carnosine and NAC in mixtures

(Figure 8a) and the presence of majority of NAC in the

lubricant eye drops Can-CTM employed in pharmaco-

kinetic experiments (Figure 8b). Peaks were unequivo-

cally identified by comparison of their retention times to

those of the authentic standard compounds or of

Figure 8 (a) HPLC of a mixture of L-carnosine and N-acetylcarnosine (NAC). Peak fractions were examined for specific chemical reactivity.

The integrated calibrating concentrations for the standard peaks were (a) 140 lg/mL, 3.216 min for carnosine; 82.5 lg/mL, 6.017 min for

NAC. The retention times of compounds in mixtures did not vary significantly from those run singly. Peak at 3.00 min is the solvent front.

(b) HPLC analysis of the imidazole moieties in the eye drops formulation. Can-CTM after appropriate dissolution of initial solutions of eye drops

by at least 100-fold for HPLC chromatography analysis purposes (see, Experimental design: N-acetylcarnosine lubricant eye drops studies

section). The high peak of N-acetylcarnosine (NAC) is detectable with a retention time of 6.078 min. The presence of L-carnosine in the

mixture with retention time of 3.232 min is negligible. The small peak at 3.00 min is the solvent front. (c) 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 lg/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 with a predicted order based up their relative hydrophobicities, and the

chromatographic system was shown to be suitable to monitor the behavior 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 evidence for the

identification of L-carnosine and N-acetylcarnosine [14].



putative acetylated compound run singly (Figure 8a).

Tests for specific chemical reactivity [54] provided

additional evidence for the identification of L-carnosine

and N-acetylcarnosine.

As a result of its relative hydrophobicity compared with

L-carnosine, NAC might cross the cornea of the treated eye

gradually and maintain longer the concentration of the

active principle (L-carnosine) reaching the aqueous

(c)

(a) (b)



humor. In the present section of the study, we considered

whether NAC acts in the ophthalmic formulation with

lubricants and preservatives when topically administered

to the eye as a time release carrier (prodrug) of L-carnosine.

The HPLC pattern of an extract of the aqueous humor

obtained 30 min after instillation to the rabbit eye of

ophthalmic formulation containing 1% NAC, lubricants

carboxymethylcellulose, glycerin, and preservative benzyl

alcohol in the borate buffer confirms that the peak

characteristic of L-carnosine has a concentration and a

retention time (3.225 min) clearly distinct from N-acet-

ylcarnosine (6.0 min) and basically different from the

dead time of the column (3.0 min) (Figure 8c). This

identified peak of L-carnosine quantified and integrated

by the data processor showed that virtually all N-acetyl-

carnosine after the overall extraction efficiency is con-

verted into the L-carnosine compound with a retention

time of 3.225 min (Figure 8c). The data on the L-carno-

sine-related product were blanked with the control

placebo data applied to the paired eyes of the animals.

The mean ratio of L-carnosine (C)/(NAC) relevant to the

L-carnosine release in the aqueous humor 30 min after

instillation of formulation A (Can-CTM) with 1%

N-acetylcarnosine into the rabbit eye corresponded to

C/NAC = 6.64 ± 0.06 (n = 8, where n = number of the

examined treated rabbit eyes; only right eyes were

treated). In the control placebo formulation-treated eyes,

the same indices could not be quantified at statistically

significant rate. Concentrations of imidazole products in

the aqueous humor corresponded to those of intact rabbit

eyes and refer to baseline values of L-carnosine

0.19 ± 0.10 lg/mL related products variously detected

in extracts from normal animals.

Our data demonstrate that topical administration of

pure L-carnosine (1% solution) to the rabbit eye (instil-

lation and subconjunctival injection) does not lead to

accumulation of this natural compound in the aqueous

humor over 30 min in concentration exceeding that in

the placebo-treated matched eyes, and its effective

concentration is exhausted more rapidly [52,55]. In

another aspect, the data presented in Results sections

demonstrate the prospects of applications of an ophthal-

mic composition comprising NAC or its pharmacologi-

cally acceptable salt in combination with a cellulose

compound or its pharmaceutically acceptable salt, which

are effective to treat the eye complex of symptoms. This

complex of symptoms may have an oxidative component

in their genesis, such as senile cataract, glaucoma,

inflammation, or diabetic ocular complications. The

topical administration of N-acetylcarnosine in the devel-

oped and patented lubricant eye drop formulation

delivers pure L-carnosine and allows its increased

intraocular absorption into the aqueous humor

surrounding the lens, thus enabling significant improve-

ments in anticataract efficacy [10]. This formulation also

optimizes beneficial effects in a number of ocular

degenerative age-dependent disorders [15–19].

The important pharmacokinetic observation activities

of the tested ophthalmic formulation Can-CTM including

1% N-acetylcarnosine and lubricant carboxymethyl-

cellulose relate to the accumulation of L-carnosine in the

aqueous humor of the contralateral rabbit eyes after

30 min of instillation of the medication to the tested rabbit

eyes. The detected measure of L-carnosine in the aqueous

humor of the contralateral untreated eyes corresponded to

1.45 ± 0.08 lg/mL (n = 9). The data indicate that

intraocular route for the administered medication in-

cludes (at least partially) a systemic drug absorption in the

preferred intraocular site of L-carnosine released from 1%

N-acetylcarnosine ophthalmic prodrug enhanced with the

addition into the ophthalmic formulation of a cellulose

derivative, i.e., carboxymethylcellulose (bioadhesive and

absorption enhancer) that is also used as a mucoadhesive

carrier for the patented ocular drug, owing to its ability to

coat the cornea and remain on the eye for a longer time.

This route of systemic absorption of biologically active

compound avoids the first-pass effect normally observed

after oral presentation of a compound, and the pharma-

cological sequelae resemble those seen after an intrave-

nous administration. This pharmacokinetic phenomenon

has been given in clinical situations with topical admin-

istration of antiglaucoma medicines to the eye, because

the systemic effects of drugs such as timolol can be quite

pronounced [56].

The systemically absorbed L-carnosine released from

the 1% N-acetylcarnosine ophthalmic prodrug Can-CTM

as lubricant eye drops, which is topically administered to

the eyes, not only acts as a radical scavenger but also

represents a possible neurotransmitter-like molecule that

regulates neuronal functions such as hypothalamic

control of the autonomic nervous system. CN2 (CNDP2)

is a cytosolic enzyme that can hydrolyze carnosine to

yield L-histidine and beta-alanine. CN2-immunoreactivity

was highly concentrated in neuronal cells in the dorsal

part of the tuberomammillary nucleus of the posterior

hypothalamus [57]. As the tuberomammillary nucleus is

the exclusive origin of histaminergic neurons, further

investigations were focused to find whether CN2 is

present in the histaminergic neurons. It was found that

CN2-immunoreactivity was colocalized with that of



histidine decarboxylase, which is the key enzyme for

histamine biosynthesis specifically expressed in the

histaminergic neurons of the tuberomammillary nucleus

[57]. These results clearly indicate that CN2 is highly

expressed in the histaminergic neurons in the tubero-

mammillary nucleus, implying that systemically

absorbed L-carnosine released from the Can-CTM ophthal-

mic formulation topically administered to the eyes serves

as a metabolic source to supply histidine and activates

histaminergic neurons for histamine synthesis (Figure 9).

Axons of the tuberomammillary nucleus project primar-

ily to the cerebral cortex, thalamus, basal ganglia, basal

forebrain, and hypothalamus. Tuberomammillary acti-

vation usually results in a rapid and significant increase

in the amplitude of baseline activity and visual responses

in lateral geniculate nucleus (LGN) neurons [58].

Tuberomammillary activation causes a small phase lag

in the visual response that is similar at all spatial frequen-

cies, consistent with the induced shift from burst to tonic

firing mode. These results indicate a significant histamin-

ergic effect of L-carnosine on LGN thalamocortical cells,

with no clear effect on thalamic inhibitory neurons.

The histaminergic system appears to strengthen

central transmission of afferent information, intensifying

but not transforming the retinally derived signals. The

increase in visual response developed rapidly within the

first visual stimulus cycle and persisted for the duration

of tuberomammillary activation. Promoting sensory

input in addition to the direct anticataract activities of

N-acetylcarnosine prodrug supported through the num-

ber biochemical mechanisms [19,58] may be one way in

which the histaminergic system plays a role in enhance-

ment of visual functions and responses in elderly patients

with cataracts and no-cataract adult subjects who

usually note the brightness, relaxation, decrease in

disability glare, and clarification of vision in their treated

eyes already after the short-term administration of 1%

N-acetylcarnosine lubricant eye drops (1 week – 5- to

6-month periods of treatment) (this study) and during

the more prolong evaluation period and follow-up

(9 months) described previously [15–19]. Tubero-

mammillary histaminergic neuron activity could be

inhibited by a GABAergic pathway, originating in the

ventrolateral preoptic area, which was activated during

sleep [59].

Inhibition of experimental proliferative

vitreoretinopathy by carcinine in a rabbit model

Proliferative vitreoretinopathy was modeled that was a

complicated ocular pathologic process. Morphologically,

there is an extensive intraretinal proliferation accompa-

nied with the formation of epiretinal membrane in

pathogenesis of PVR.

Contractile properties of epiretinal membrane lead to

the retractive retinal detachment. Initially, we used the

cytokine-induced model in 24 rabbits. During modeling,

the natural complex of cytokines (NCC) that included

endogeneous immunopeptides with activity of IL-1,

TNF-a, MIF, and TQF was injected into the vitreous

body of both contralateral eyes of the animals in a

standard amount of 0.1 mL (single dose of 600 lg). The

main evaluation of the pathologic process was carried

out with clinico-morphological study. The results below

are presented after 1 month of follow-up. Natural

imidazole-containing pseudodipeptide carcinine is ubiq-

uitous in mammalian tissues and presents rather stabil-

ity in the area of inflammation to the action of

proteolysis with specific and nonspecific peptidases

[43,60].

Figure 9 Neurons of tuberomammillary nucleus of hypothalamus

as a target of a systemically absorbed L-carnosine (see, formula) in

the activation (arousal) of vision responses. Possible mechanism of

brightness, relaxation, and clarification effects on vision of adults

and elderly patients after topical administration of carnosine to the

eyes in the form of 1% N-acetylcarnosine ophthalmic prodrug

(lubricant eye drops including carboxymethylcellulose bioadhesive

and absorption enhancer).



In this experimental series, carcinine is indicated for

administration as a medicine for ophthalmic treatment

in the injection formulation (subconjunctival, ocular

parabulbar, and intravitreous injections). Recent study

demonstrated the reactivity of carcinine with ROS and

aldehyde products [43]. Carcinine noticeably limits

4-hydroxynonenal (4-HNE)-induced cross-linking of col-

lagen. Carcinine is utilized for ophthalmic protection

based on its antioxidant including hydroxyl-radical-

scavenging and lipid peroxidase activities patented by

Babizhayev et Exsymol [43,47]. Carcinine is working as

a water-soluble universal antioxidant both in the lipid

phase of cellular and biologic membranes and in an

aqueous environment [43,47].

Peculiarities and staging of PVR morphogenesis in the

cytokine-induced model

Proliferative processes occurred in 100% of the control

eyes challenged with cytokine complex. Epiretinal and

subretinal localization of PVR was detected among the

cytokine-challenged control eyes. The spreading of the

process varied (Figure 10). The most frequent was the

paraneural localization of the PVR. Proliferative changes

were characterized by two types of changes.

The first one is the formation of the limited prolifer-

ative hearth in which the fiber elements are combined

with proliferating fibroblast-like cells. Originating from

proliferation, the shaft-braces are protracted inside the

vitreous body. The second mode of changes is the

creation of flat limited formations of the fiber structure

like epiretinal membrane, which are spreaded above

limitans interna of the retina with the single fibroblast

cells as well as with glyal elements. The same structures

form the veil-like thickenings of the internal boundary

retinal membrane. Such formations have rather ex-

tended character. The formed areas of epiretinal and

subretinal proliferation led to the folds of the retina and

its detachment. Among proliferations that contained the

spindle-shaped cells, the accumulations of macrophages

and lymphocytes did occur. In several cases, the massive

fibrotic protractions appeared extended from the funnel

of the nervus opticus toward the lens. The mode of

proliferative processes exhibited hearth-like character

near the accumulations of macrophages at the internal

boundary membrane intermitted with the zones of the

intact retina. The cases of more extended epiretinal

membrane occurred from ora serrata to the disk of the

nervus opticus.

Such extension is regarded with multiple strains of the

internal boundary membrane in which glyal and fibro-

blastic elements are actively proliferated with formation

of the fiber structures of the epiretinal membrane. Often,

combination of epiretinal proliferation with the subreti-

nal proliferation in the control group of animal eyes took

place in such modeling.

Clinical and Morphological data provided with the

cytokine-induced modeling of PVR revealed:

• The role of macrophages as the trigger cells reaction

involved in the pathogenesis of PVR, especially initial

stage of PVR.

• Inhibitory action of carcinine on the PVR progres-

sion, its especial role at the initial stage of PVR, based on

the property of carcinine to block the functional activity

of macrophages in disease.

Morphological stages in the development of PVR:

initial, advanced, and terminal

At the initial stage of PVR, three morphological compo-

nents were noticed.

• Accumulation of macrophages: There was an

accumulation of macrophages attracted from blood

vessels by cytokines into the vitreous of the eye

(Figure 10). Macrophages represent the cells, acting as

regulators and a polyfunctional complex. Macrophages

synthesize different enzymes, peroxide compounds, bio-

logic active substances, and growth factors, promoting to

proliferation of fibroblasts and fibril material. Macrophag-

es are capable for adhesion depending on function

forming conglomerates.

• The damage of the inner limiting membrane: The

conglomerates of active macrophages showed cytotoxic

effect to the environment, especially to the retina. The

result of such process was the damage of the inner

limiting membrane (ILM) and reactive proliferation of

glyal cells (see, Figure 10c).

• Formation of epiretinal membrane: Active prolifera-

tion of glyal and fibroblastic cells around the place of ILM

damage formed an epiretinal membrane. It was possible

to distinguish not only cells but also the collagenous

fibrils and accumulation of the extracellular matrix

fibronectin–like protein component (see, Figure 10d).

Morphology of the advanced PVR stage is character-

ized by the following features: Spread of the epiretinal

membranes with contractile properties leads to the

appearance of the retinal folds (Figure 10e).

Morphology of the terminal stage of PVR is charac-

terized by spreading of the epiretinal and vitreoretinal

proliferation with retractive retinal detachment

(Figure 10f). In the group of rabbit eyes treated with

postponed parabulbar injections started 3–4 days after



the intravitreal ocular challenge with cytokine complex

of the rabbit eyes within 5-day follow-up period, less

pronounced inhibitory effect was observed to PVR

morphogenesis than in the case of parabulbar or

subconjunctival injections started immediately after the

intravitreal injection of cytokine (1 parabulbar injection

per day prolonged for 5 days).

In all control cytokine-challenged rabbit eyes, the

inflammatory reaction was noted, which was charac-

terized by the small lymphocyte infiltration inside the

ciliary body and the appearance of inflammatory cells

inside the vitreous cavity, sedimented preferably onto

the collagen fibers of the carcass elements and on the

internal boundary membrane of the retina. Among the

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 10 (a) Accumulation of macrophages, attracted from blood vessels by cytokines into the vitreous. Model rabbits of proliferative

retinopathy were generated as described in Materials and Methods. Cytokine complex was injected intravitreally into the experimental eyes,

while phosphate-buffered saline was injected into the matched eyes as a control. (a–e) The morphology of the eyes injected with cytokine

complex is presented. (b) Formation of macrophagal conglomerates at the internal limiting membrane of the retina. (c) Damage of the inner

limiting membrane and reactive proliferation of glyal cells. (d) Collagen-like fibrils and accumulation of extracellular integrin matrix–

fibronectin-like material. (e) Retinal folds because of the contraction of epiretinal membranes. (f) Spread of epiretinal and vitreoretinal

proliferation with retractive retinal detachment. (g) Significant inhibition of proliferative vitreoretinopathy with carcinine in the cytokine-

treated eyes. Carcinine (10 mM) was applied in parabulbar injections to the rabbit eyes, 1 injection per day, 5 days. 1-month follow-up.

Parabulbar injections were started on the day after the ocular challenge with cytokine. (h) Incomplete inhibition of inflammatory and

proliferative process after the single intravitreal injection of carcinine concurrently with intravitreal ocular challenge of the rabbit eye

with the cytokine complex.



cells of inflammatory infiltration, lymphocytes and

macrophages have been presented. The latter formed

conglomerates. In the cytoplasm of macrophages, the

grainy inclusions were detected during the accumula-

tion of these cells near the hemorrhage areas. The

significant inflammatory reaction occurred in the area

of the nervus opticus cup around its central vessels. A

combination of atrophic and proliferative processes was

also detected in the control cytokine complex-chal-

lenged eyes. Atrophic changes in the retina were noted

in the majority of cases and were significantly expressed

and localized predominantly in one half of the ocular

fundus.

In the atrophic areas, the retina was significantly

thinned because of the complete reduction in granular

layers. External and internal granular layers were fused

into one, the thickness of which was reached 1–2 rows of

nuclei. Topographically, such changes were localized

predominantly near the nervus optical disk or were

located at the peripheral retina. In the area of the retinal

atrophic changes, the sites of the retinal glyal substitu-

tion occurred.

PVR treatment with carcinine

Morphological investigations have shown the significant

inhibition of PVR in the group of eyes where carcinine

was applied in parabulbar injections (eight rabbit eyes)

started just after the intravitreal ocular challenge of the

rabbit eyes with the cytokine complex, and the parabul-

bar injections with carcinine were continued for 5 days

(five injections during 5 days and one injection daily).

The experimental clinical and morphological investiga-

tions revealed that the retina remained unchanged in all

such carcinine-treated cytokine-challenged animal eyes

compared with the cytokine-treated control. Inflamma-

tory effect was absent too. Carcinine showed the

remarkable preventive action and inhibition of PVR in

this treated group of eyes (see, Figure 10g).

Subconjunctival injections of carcinine were similarly

effective; however, more anti-inflammatory effect of

carcinine appeared after parabulbar injections than after

the single intravitreal administration of carcinine

compound. In the separate group of five rabbit eyes

treated with the single carcinine injection directly into the

vitreous cavity, the retina was conserved; however, there

was possible to find small zones of epiretinal membrane

formation. The inhibition of inflammatory and prolifera-

tive processes was incomplete in such cases (Figure 10h).

Thus, under the treatment with parabulbar injections

and intravitreal injection of carcinine, the distinct

inhibition of PVR was observed in the eyes challenged

with cytokine complex. The inhibitory effect of carcinine

on the PVR overall was more pronounced when it was

administrated at the early stages of pathologic process,

when the link with the inflammatory reaction mediated

by macrophages was a leading cell reaction component

of PVR. In general, upon the treatment with carcinine,

inflammatory processes were less exhibited and charac-

terized only by single lymphocytes, monocyte–macro-

phages adhered onto the internal boundary retinal

membrane (membrana limitans interna), sometimes in

the form of conglomerates. Atrophic changes were

absent at the big extension of the retina and occurred

in the single eyes (one eye) in the form of the single

hearth. The rest of the animals treated with carcinine

showed the intact retina with the intact internal

boundary membrane. Proliferative processes in the form

of epi- and subretinal membrane were completely absent

in the carcinine-treated group. The formation of very

thin one or two rows of the cell elements from epiretinal

membrane has occurred in one eye. In the contralateral

cytokine-challenged eye without treatment with carci-

nine, the massive cellular epiretinal membrane was

formed with 3-fold higher thickness than in the eyes

treated with carcinine. The experiments demonstrate the

clinico-morphological evidence for the inhibitory action

of the universal bioactivated antioxidant carcinine to

proliferative disorders. Based on the established biologic

activities of carcinine, the therapeutic effects on PVR

were because of the block of peroxide compounds and

reactions induced by macrophages in the vitreous cavity.

Apparently, macrophages lost their activity including

chemotaxis-induced cellular signaling and mitogenic

activities in most cases accompanied with proinflamma-

tory oxygen free radical species–mediated pathways and

a potent nitric oxide response [61].

Carcinine protection of photoreceptor cells from

light-induced damage

In photoreceptor cells, oxidative stress is induced by

exposure to bright light causing photoreceptor cell

damage and apoptosis [62]. Current thinking suggests

that ROS formed during oxidative stress can directly

attack polyunsaturated fatty acids (PUFAs) and initiate a

chain reaction that amplifies lipid peroxidation (LPO) in

cellular membranes. This causes a nonenzymatic PUFA

degradation into a variety of oxidized products, including

short- and medium-chain (hydroxy) aldehydes. 4-HNE,

one of the most abundant and the most toxic products of

LPO, is a mediator of the apoptotic response induced by



oxidative stress [63–66]. 4-HNE reacts with histidine,

cysteine, or lysine residues of proteins forming Michael

adducts [67]. This reaction leads to a variety of cyto-

pathologic effects, such as inhibition of enzyme activity;

inhibition of protein function; inhibition of protein, RNA,

and DNA synthesis; cell cycle arrest; and apoptosis

[66,68,69].

To test the protective effect of carcinine against light-

induced damage in photoreceptors, we performed intra-

vitreal injections of carcinine in wild type Balb/C mice,

followed by exposure to bright light, and quantified the

remaining photoreceptor cells (Figure 11). The ONL

thickness of mice raised in dim cyclic light is 50 lm as

previously published [70]. As shown in Figure 11, our

conditions of light exposure–induced apoptosis of 50–

60% of photoreceptor cells in the PBS-injected eye.

Carcinine significantly protected the other eye from light-

induced photoreceptor apoptosis, presumably through its

universal antioxidant and scavenging effect on 4-HNE

and other LPO products.

Oral administration and acute toxicity test of

carcinine in mice

The behavior, physiologic state, and death of the animals

were studied every day for 14 days following the

treatment of mice (females-5, males-5) with carcinine

chlorohydrate (posology 2 g/kg body mass) (Table III). It

was shown by the data that carcinine represents a

nontoxic product upon oral administration (LD0 > 2

g/kg).

Phase 2 randomized, double-masked, placebo-

controlled clinical trial of N-acetylcarnosine

lubricant eye drops

Sample characteristics

Table IV lists the demographic and ergonomic occupa-

tional characteristics of the cataract (n = 75) and no-

cataract groups (n = 74). Those with cataract were

similarly older on average with the no-cataract group of

subjects.

Both groups were split evenly between males and

females and had similar racial composition, with the

totality white population.

Table V lists the visual function for both groups

enrolled in the study and the distribution of visual

acuity and disability glare scores for subjects with

cataract and those without. As would be expected by

the case definition for cataract group membership, those

in the cataract group had impairments in visual function

as compared with the no-cataract group. This was true

for both the ‘worse’ and ‘better’ eyes. In addition, visual

acuity in the range of 20/35–20/50 and disability glare

readings in the range of glare radius more than 12 mm

was associated with driving difficulties (such as crash

involvement) (Table IV). Although not statistically sig-

nificant, there was also a possible relationship between

visual acuity worse than 20/50 and crashing during

driving experience [71]. Disability glare (glare radius

measured in millimeters) was correlated for statistical

significance with visual acuity at red and green targets at

baseline and 5- to 6-month examination interval in the

total samples of older subjects with cataract and no-

cataract older adult subjects (Table VI).

Older subjects enrolled in the study were divided into

two groups: treated with NAC and control group

(Tables VII–IX). Table VII lists the analogous and adjusted

analyses for the worse eye, which generated results in

the eyes with cataract upon treatment with NAC

prodrug ophthalmic formulation qualitatively similar to

those for the better eye. None of the baseline differences

between the different groups was significant. The two

groups were similar in smoking history, sunlight expo-

sure, and alcohol use. There was not any substantial

difference in the use of sunglasses, where the patients

lived or occupational hazard exposure between the two

groups.

Ophthalmic examinations indicated that the method-

ological variances of measurements were approximately

equal. Correlations of glare sensitivity at red vs. green

targets were significant (Table VI). Intraoperator corre-

lation coefficients obtained as repeated measurements for

each combination of operator, eye (right or left), and

glare radius (at red and green targets) were statistically

significant and presented earlier [6,7,45]. Overall,

the reproducibility for the one operator was good.

Tables VI–IX summarize the effects of study treatment

on VA and glare sensitivity over 9 months in older

subjects with cataract and no-cataract. In the control

placebo-treated group, comparison with baseline values

showed some variability of data in gradual worsening of

glare sensitivity at red and green targets and minimal VA

changes over 5–6 months (Table VIII). Glare sensitivity

indicated mostly changes in lens clarity and confirmed

the tiny changes in the optical media of the eye at the

short-term follow-up examinations when slit lamp–

associated image analysis, data, and densitometric

readings did not differ significantly with baseline

[6,7,12,13,45].

In the NAC-treated group, 5- to 6-month follow-up

generally showed an improvement in VA (according to



the distribution score of distance acuities in worse and

better eyes), and a significant improvement in glare

sensitivity at red and green targets was documented in

worse and better eyes using a critical cut point halometer

score (Tables VII and IX). VA was mostly improved in

older subjects with cataract in worse and better eyes, and

an improvement in glare sensitivity was found in both

older subjects with cataract and no-cataract older adult

subjects in worth and better eyes after 5–6 months of

treatment with NAC. The exampled images of cataract

reversal in older subjects are presented on the slit lamp

images just for note (see, Figure 12a–c).

The NAC-treated eyes had statistically significant

difference in VA and glare sensitivity compared with

the control group (P < 0.001) at 5- to 6-month time

point of treatment, as supported by the overall t-test

results of the ratio of the follow-up data to the baseline

values (Table IX). The previously published data illustrate

examinations over 24 months of the eyes treated with

NAC to show that the effect of treatment is sustainable

over more prolonged periods [6,7]. In the eyes of older

subjects with different localization and grade of cataract

and in no-cataract older adult subjects, short-term and

prolonged treatment with NAC did not seem to result in

(a)

(b)

OS

Dim cyclic light

Dim cyclic light

Inferior Superior60

50

40

30

ON

L th

ickn

ess

(µm

)

20

10

02.0 1.0 0 1.0 2.0

Bright light/carcinine

Bright light/carcinine

Bright light/PBS

Bright light/PBS

Distance from ONH (mm)

ISONL

OPL

INL

IPLGCL

Figure 11 Carcinine protects against light-induced oxidative stress and photoreceptor cell death in Balb/C mice. Mice were raised in dim

cyclic light for 8 weeks. Each mouse was injected in one eye with 1 lL phosphate-buffered saline (PBS) and in the other eye with 1 lL of 2 M

carcinine diluted in PBS. Mice were returned in dim cyclic light for 48 h before light damage was initiated. Light damage was then induced in

mice by 5 h exposure to 4000 lux of diffuse, cool, white fluorescent light. After light exposure, mice are returned in dim cyclic light for 7 days

to allow the retina to clear all dead cells and return to a well-organized morphology. (a) Representative histologic sections through the

inferior retina near the optic nerve head (ONH) are shown. The outer nuclear layer (ONL) thickness is indicated by arrows. (b) The thickness

of the ONL was measured at 0.24-mm intervals from the ONH to the inferior and superior ora serrata. Graph shows results from six mice, and

error bars indicate SEM. OS, photoreceptor outer segment; IS, photoreceptor inner segment; OPL, outer plexiform layer; INL, inner nuclear

layer; IPL, inner plexiform layer; GCL, ganglion cell layer.



a worsening of the visual outcome in this study and

elsewhere [15–19]. Topical short- or long-term admin-

istration of 1% NAC to the eye was well tolerated, with

no ocular or systemic adverse effects, no hyperemia of

conjunctival vessels, and no signs of allergy or other

toxic manifestations being reported. No clinically signif-

icant changes from baseline and no statistically signif-

icant differences between the treatment and control

groups were observed regarding ocular comfort and

ocular signs and symptoms (lack of burning and

stinging, blurred vision, ocular dryness, superficial

punctate keratitis, foreign body sensation, itching,

ocular discharge, ocular pain, tearing, ocular inflam-

mation, and photophobia). All patients completed the

study without any problems related to their allocated

treatment.

Table III Recapitulative table. Study of mortality and behavior

during the time following the intubation (D0) of carcinine within

14 days (D1–D14).

Days

Mortality

BehaviorMales Females

D0 0 0 N

D1 0 0 N

D2 0 0 N

D3 0 0 N

D4 0 0 N

D5 0 0 N

D6 0 0 N

D7 0 0 N

D8 0 0 N

D9 0 0 N

D10 0 0 N

D11 0 0 N

D12 0 0 N

D13 0 0 N

D14 0 0 N

Groups 0 0 N

N, normal.

Table IV Demographic and ergonomic occupational characteristics

of cataract and no cataract adult subjects enrolled in the study.

Cataract No Cataract

n % n %

Total 75 74

Age groups

54–59 years 16 21 24 32

60–69 years 25 34 37 50

70–85 years 34 45 13 18

Sex

Female 37 49 38 51

Male 38 51 36 49

Race White 75 100 White 74 100

Driving exposurea

Total 42 56 44 60

<150 km/week 25 59 19 43

‡150 km/week 17 41 25 57

Data are presented as numbers and percentages.aDriving subjects were classified into two categories according to whether

they drove more or less than the median number of km (150 km) driven per

week based on the distribution of all subjects. Although this was a self-report

measure, prior studies indicate that older adults can provide valid estimates

of driving exposure [12].

Table V Distribution of visual acuity and disability glare in the

cataract and no-cataract groups of the adult subjects enrolled in the

study at baseline examination.

Adult subjects

Cataract group

No cataract

group

n % n %

Total 75 74

Worse eye

Visual acuity

20/25 or better 7 9 53 71

20/25 to 20/30 16 21 12 16

20/35 to 20/50 47 63 5 7

Worse than 20/50 5 7 4 6

Disability glare readings (glare radius)

At red target

<12 mm 12 16 32 43

‡12 mm 63 84 42 57

At green target

<12 mm 10 13 34 46

‡12 mm 65 87 40 54

Better eye

Visual acuity

20/25 or better 14 19 56 76

20/25 to 20/30 33 44 11 15

20/35 to 20/50 23 31 4 5

Worse than 20/50 5 6 3 4


At red target

<12 mm 31 41 42 57

‡12 mm 44 59 32 43

At green target

<12 mm 21 28 35 47

‡12 mm 54 72 39 53

Normal measures of glare sensitivity of young subjects (20–30 years) with

best correction without cataracts are 3 ± 2 mm (mean ± SD) of at least four

measurements at red and green targets in the daytime [47].



We find that repurchase announcements during 5

recent years of follow give the credible figure in excess of

52 000 patients compliant using their Can-CTM eye

drops for published therapeutic indications (including

treatment of cataracts) and excellent safety on the daily

basis. We examine the factors influencing repurchase

behavior and find that repurchases in the latest quarters

are associated with a number of variables, including the

efficacy of the product and its persistent safety to control

the signs of glare sensitivity, cataract amelioration, and

quality of vision during the daily life activities.

The patented N-acetylcarnosine lubricant all-in-one

eye drops formula Can-CTM can provide beneficial results

for cataracts and recommendations for Rational Pre-

scribing with the following ophthalmic disorders and

visual disturbances in patients:

• Presbyopia [10].

• Open-angle primary glaucoma (in combination with

beta-blockers or prostaglandin derivatives, for example,

lumigan is instilled over night and Can-CTM is instilled as

adjunct therapy twice daily by 10 AM and 2 PM).

• Corneal disorders.

• Computer vision syndrome.

• Driving and night vision disability glare. Perceived

driving disability at night.

• Eyestrain.

• Ocular inflammation.

• Blurred vision.

• Dry eye syndrome.

• Retinal diseases.

• Vitreous opacities and lesions.

• Ocular complications of diabetes mellitus and other

systemic diseases.

• Contact lens difficulties, particularly with soft contact

lenses. (Not only do the lubricants in the Can-CTM

N-acetylcarnosine eye drop help to make wearing

contact lenses more comfortable, but N-acetylcarnosine

is also believed to reduce the build up of lactic acid in the

eye cornea, thus enabling the soft contact lens to be left

safely in the eye for longer).

Of the more than 51 408 patients who were enrolled

in the postmarket evaluation, the several classic various

types of cataracts under following classification (Table X)

are prescribed and delivered for the medical treatment of

eye diseases with the Can-CTM N-acetylcarnosine eye

drop therapeutic platform. The persons with best-

corrected vision of no <20/70 or better in the worse

eye are recommended for eye drop correction.

Nuclear cataracts included the following clinical

symptoms/history:

• Age.

• Occupation hazards.

• Cigarette smoking increases the risk of nuclear sclerosis.

Symptoms/history

• Gradual progressive loss of vision.

• Second sight – development of myopia owing to

increased lenticular refractive index.

• Monocular diplopia.

• Decreased color discrimination especially blue.

Clinical features

• Central yellow to brown discoloration of the lens.

• Myopic shift.

• Bilateral.

• Decreased penetration of cobalt blue slit beam through

lens.

Posterior subcapsular cataracts were characterized

with the following clinical features:

Epidemiology/risk factors

• Younger patients than with nuclear or cortical cata-

racts.

• Diabetes mellitus.

• Primary open-angle glaucoma.

• Radiation.

• Corticosteroids.

• Uveitis and retinitis pigmentosa.

• Smoking.

Symptoms/history

• Progressive loss of vision, sometimes rapid.

• Disability glare, halos.


Table VI Linear correlation coefficients (R) between the character-

istics of older subjects with cataract and no-cataract older adult

subjects measured by visual acuities (VA) and glare radius (GR at

red and green targets) at baseline and at 5- to 6-month follow-up

ophthalmic examinations.

Parameter

Baseline study 5–6 months

VA

GR red

target

GR green

target VA

GR red

target

GR green

target

Older subjects with cataracta

VA X )0.61* )0.64* X )0.41 )0.58

GR red target X +0.81* X +0.84*

GR green target X X

Older adult no-cataract subjectsb

VA X )0.63* )0.68* X )0.44 )0.45

GR red target X +0.83* X +0.80*

GR green target X X

*P < 0.01.aNumber of eyes examined 75.bNumber of eyes examined 74.



Clinical features

• Axial opacity of the posterior cortical material that

initially has an iridescent sheen and appears in the

posterior cortex that is followed by granular and plaque-

like opacities.

• Can be confused with posterior polar cataract and

mittendorf dot.

• Can see with direct but best viewed with red reflex

through slit lamp.

Cortical Cataracts are characterized with

Epidemiology/risk factors

• Younger patients than with nuclear cataracts.

• Diabetes mellitus.

• Sunlight.

• Trauma.

• Smoking.

Symptoms/history

• Progressive loss of vision, sometimes rapid.

• Disability glare, halos.


Clinical features

• Opacity of the cortical fibers from posterior to central.

• Sometimes wedge shaped, forming cortical spokes.

• Can progress to intumescent or hypermature cataract.

• Usually medial and inferior from UV exposure.

Among 51 408 patients treated with N-acetylcarno-

sine eye drops (Can-CTM), the data on combined

N-acetylcarnosine eye drops therapeutics in the

treatment of patients with cataract and POAG were

comprehensively studied, and all aspects and variations

of the combined procedure were assessed. As the disease

progressed, a person with glaucoma noticed his or her

Table VII Visual function in the better and worse eyes after

5–6 months of treatment with N-acetylcarnosine 1% eye drops

(Can-CTM) vs. baseline examination.

Adult subjects

Cataract group No cataract

group

n % n %

Total

Baseline examination

42 39

Worse eye

Visual acuity

20/25 or better 4 9 26 67

20/25–20/30 10 24 6 15

20/35–20/50 25 60 4 10

Worse than 20/50 3 7 3 8


At red target

<12 mm 6 14 17 44

‡12 mm 36 86 22 56

At green target

<12 mm 4 9 16 41

‡12 mm 38 91 23 59

Better eye

Visual acuity

20/25 or better 6 14 28 71

20/25–20/30 18 43 5 13

20/35–20/50 15 36 3 8

Worse than 20/50 3 7 3 8


At red target

<12 mm 14 33 21 54

‡12 mm 28 67 18 46

At green target

<12 mm 13 31 12 31

‡12 mm 29 69 27 69

After 5–6 months of treatment with N-acetylcarnosine 1% eye drops

Worse eye

Visual acuity

20/25 or better 8 19 27 69

20/25–20/30 15 36 7 18

20/35–20/50 17 40 2 5

Worse than 20/50 2 5 3 8


At red target

<12 mm 11 26 23 59

‡12 mm 31 74 12 31

At green target

<12 mm 13 31 22 56

‡12 mm 29 69 17 44

Better eye

Visual acuity

20/25 or better 12 28 30 76

20/25–20/30 25 60 6 15

Table VII (Continued).

Adult subjects

Cataract group

No cataract

group

n % n %

20/35–20/50 4 10 1 3

Worse than 20/50 1 2 2 5


At red target

<12 mm 18 43 30 77

‡12 mm 24 57 9 23

At green target

<12 mm 19 45 17 44

‡12 mm 23 55 22 56



side vision gradually failing. That is, objects in front may

still be seen clearly, but objects to the side could be

missed. As glaucoma remains untreated, individuals

missed objects to the side and out of the corner of their

eye. Without treatment, individuals with glaucoma

slowly lost their peripheral (side) vision. Over time,

straight-ahead vision may decrease until no vision

remains. Applied N-acetylcarnosine eye drops therapy

has improved the success rate and reduced the compli-

cation rate previously associated with extracapsular

cataract extraction combined with trabeculectomy. A

Table VIII Visual function in the better and worse eyes after 5–

6 months of treatment with placebo (control group) vs. baseline

examination.

Adult subjects

Cataract group

No cataract

group

n % n %

Total

Baseline examination

33 35

Worse eye

Visual acuity

20/25 or better 3 9 27 77

20/25–20/30 6 18 6 17

20/35–20/50 22 67 1 3

Worse than 20/50 2 6 1 3


At red target

<12 mm 6 18 15 43

‡12 mm 27 82 20 57

At green target

<12 mm 6 18 18 51

‡12 mm 27 82 17 49

Better eye

Visual acuity

20/25 or better 8 24 28 80

20/25–20/30 15 46 6 17

20/35–20/50 8 24 1 3

Worse than 20/50 2 6 0 0


At red target

<12 mm 17 52 21 60

‡12 mm 16 48 14 40

At green target

<12 mm 8 24 23 66

‡12 mm 25 76 12 34

After 5–6 months of treatment with placebo

Worse eye

Visual acuity

20/25 or better 2 6 22 63

20/25–20/30 4 12 11 31

20/35–20/50 24 73 1 3

Worse than 20/50 3 9 1 3


At red target

<12 mm 4 12 13 37

‡12 mm 29 88 22 63

At green target

<12 mm 4 12 15 43

‡12 mm 29 88 20 57

Better eye

Visual acuity

20/25 or better 6 18 25 71

20/25–20/30 17 52 7 20

Table VIII (Continued).

Adult subjects

Cataract group

No cataract

group

n % n %

20/35–20/50 8 24 3 9

Worse than 20/50 2 6 0 0


At red target

<12 mm 15 45 18 51

‡12 mm 18 55 17 49

At green target

<12 mm 6 18 19 54

‡12 mm 27 82 16 46

Table IX Mean ± SD of changes (improvement) in visual functions.

Treatment group Visual acuity Glare radius

5- to 6-month follow-up of older subjects with cataract

Control group 0.79 ± 0.03 (n = 33) 1.58 ± 0.06 (n = 33)

NAC-treated group 1.65 ± 0.05*,** (n = 42) 0.38 ± 0.05* (n = 42)

5- to 6-month follow-up of older adult no-cataract subjects

Control group 0.91 ± 0.03 (n = 35) 1.37 ± 0.05 (n = 35)

NAC-treated group 1.21 ± 0.05* (n = 39) 0.49 ± 0.05* (n = 39)

The measure of visual acuity readings after 5–6 months of treatment was

divided by the clinical baseline measure of visual acuity for each eye

individually to get ratios, and then, the average of those ratios through each

clinical group of eyes was calculated. Similarly with glare, the calculation of

the ratio of glare sensitivity at red and green target after 5–6 months of

treatment to the baseline reading of glare sensitivity for each eye was made,

and then, the ratios were averaged through the whole groups of eyes.

NAC, N-acetylcarnosine (Can-CTM).

*P < 0.001 compared with control group who received placebo eye drops.

**P < 0.001, where an improvement in visual acuity is statistically signif-

icantly better in the group of older subjects with cataract than an

improvement in visual acuity in the group of older adult no-cataract subjects.



mean reduction in intraocular pressure (IOP) of 3–

7 mmHg was achieved. Can-CTM was recommended to

combine with another antiglaucoma medication, such as

beta-blocker treatment. Progressive loss of optic nerve

axons and retinal ganglion cells (RGCs) results in

characteristic optic nerve atrophy and visual field defects

in glaucoma patients. In many patients with glaucoma,

IOP is higher than the statistically normal limits; our

extensive evidence supports that elevated IOP-initiated

factors are important for the initiation and progression of

neuronal damage in these patients. Current therapeutic

management of glaucoma therefore aims to halt disease

progression by reducing elevated IOP. However,

although IOP lowering treatment can provide neuropro-

tection and retard the disease progression in many

glaucoma patients, it is not always sufficient to fully

prevent disease progression. This is a reason that our

recent efforts have been focused on the rational pre-

scription of alternative N-acetylcarnosine eye drops

treatment strategies for neuroprotection. In addition,

IOP is not elevated in all of the observed eyes exhibiting

characteristics of glaucomatous neurodegeneration.

(c)

(b)

(a)

1 2

Figure 12 (a) The pictures show the treatment of human cataract in the older subject with the patented by Innovative Vision Products, Inc

formula of 1% N-acetylcarnosine lubricant eye drops for the period of 5 months. The left image shows the appearance of cataract of a

76-years-old woman with opacified cortical layers, and the right image shows that this opacity has partially disappeared after the cited

period after treatment with N-acetylcarnosine is completed. The lens has become clearer. (b) Treatment of posterior subcapsular cataract

in the older subject for the period of 9 months with 1% N-acetylcarnosine lubricant eye drops. Left image – before treatment; right image –

after 9 months interval of treatment. The opalescence lens opacities areas are reversed, and the lens becomes clearer (look like dark greenish

zones) at the right image. (c) Treatment of cortical cataract in the upper segment of the pupil image of the older woman for the period

of 9 months. Upper image: the lens before treatment. Lower image- after 9 months of treatment with the 1% N-acetylcarnosine lubricant

eye drops. The appearance of rose reflex at the lower image demonstrates that the lens becomes clearer.

Table X Types of cataract treated with 1% N-acetylcarnosine

lubricant eye drops (Can-CTM) in population in excess of 51 408

patients.

Type Age of onset

Main clinical

symptoms (complaints)

Nuclear 60–72 Myopic shift

Blurred vision

Loss of blue/yellow

color perception

Posterior

subcapsular

40–63 Disability glare

Diminished reading

Monocular diplopia

Cortical 40–65 Disability glare

Monocular diplopia



Additional supportive evidence for oxidative stress dur-

ing glaucomatous neurodegeneration came from our

studies in glaucoma patients aided with N-acetylcarno-

sine eye drops (Can-CTM).

The N-acetylcarnosine lubricant eye drops have been

successfully used for medically oriented home health

care usually helping seniors recover or exercise with aid

in recovery from visual impairment or illness including

cataracts (Figure 13). At IVP, we are focused on provid-

ing patients with the selection and recommendation of

specialty home eye health care products and services for

vision and tailored to meet the specific needs for eye

health recovery and maintenance.

D I S C U S S I O N

The overarching aim of eye-care and vision-related

rehabilitation programs is to improve the quality of life

of visually impaired individuals, but resources for these

programs are limited [72]. Vision impairment has a

significant impact on length and quality of life [73,74].

Difficulty with daily activities as a result of impaired

vision is commonly reported by older adults and often

does not correlate well with clinical measures of vision or

ability to perform specific tasks [75–77]. Previous

research has shown that vision impairment is associated

with an increased risk of falls, hip fractures, depression,

social isolation, greater need for community services,

and greater risk of admission to nursing homes [73,78].

The age- and sex-standardized 7-year cumulative mor-

tality rate was 26% among persons with any visual

impairment and 16% in persons without visual impair-

ment. After adjusting for factors found significantly

associated with mortality, including age, male sex, low

self-rated health, low socioeconomic status, systemic

medical conditions, and negative health risk behaviors,

the presence at baseline of any visual impairment was

independently associated with increased mortality risk

[risk ratio (RR), 1.7; 95% confidence interval, 1.2–2.3].

The presence of age-related cataract, either nuclear (RR,

1.5), cortical (RR, 1.3), or posterior subcapsular cataract

(RR, 1.5), was also significantly associated with

increased mortality risk. These associations remained

statistically significant when visual impairment and each

type of cataract were included simultaneously in the

multivariate Cox model [79].

Nursing homes have changed dramatically over the

past several decades. These changes have been driven by

government regulations and consumer pressures.

Today’s nursing homes are highly regulated, high-

quality institutions for the care and treatment of older

adults who have severe physical health and/or mental

disabilities. Almost half of all individuals who live in

nursing homes are 85 years or older. Relatively few

residents are younger than 65 years. Most are women

(72%), many of whom are without a spouse (60%

widowed) and with only a small group of family

members and friends for support. Some type of disability

or impairment with activities of daily living (ADLs) is the

most common reason that older individuals live in

nursing homes. In the absence of any preventive

measures that can be applied in a public health setting,

the only recourse is surgery. This work intends to

demonstrate that it is possible in a developing country

setting to establish self-sustaining, high-quality home

eye care that is affordable and accessible to poor people

for the community global vision strategy.

The cost-effectiveness and safety of the NAC lubricant

eye drops developed and applied for medical cataract

therapy has been demonstrated [17,18]. In addition to

the restoration of sight, there is an enhancement of the

quality of life in the developing world using available

N-acetylcarnosine ophthalmic therapeutic technology

[16–19].

Use of NAC to treat cataract and aging lens can lead to

diminishing of light scattering units in the lens which

raise glare effect, probably by prevention of the oxidative

modification of crystallins and utilization of lipid perox-

ides [8,9]. After 5–6 months, the worse and better eyes

of older adult subjects and older subjects with cataract

treated with 1% NAC generally showed an improvement

Figure 13 The Innovative Vision Products, Inc. patented Vision

products’ initiatives honor and recognize the exceptional efforts of

homecare, hospice, and private duty providers across the globe

actively engaged in strategic initiatives to improve quality of vision

care and organizational performance.



in visual functions as compared with baseline. The

results of glare testing show a reduction in light

scattering in the worse and better eyes of older drivers.

Glare sensitivity improvement was accompanied by

improvement in VA more pronounced in the eyes with

cataract during treatment with NAC. The data of visual

functions (VA and glare sensitivity) in older adult

subjects and older subjects with cataract treated with

1% NAC showed significant improvement as compared,

in contrast to the control group that showed generally

no improvement in visual functions, with no difference

from baseline in VA and glare sensitivity readings. In

most of the patients treated, study treatment was well

tolerated, and no ocular or systemic adverse events were

reported.

NAC releases carnosine for intraocular uptake and

systemic absorption, which acts as a vision sensory input

signaling molecule and locally in the lens as universal

antioxidant both in the lipid phase of the cellular lens

membranes and in their aqueous environment and

protects the crystalline lens from oxidative stress-induced

damage [8,9,17]. The treatment with NAC is improving

eyesight in older adult cataract-free patients as

L-carnosine promotes vision sensory input through the

activation of the histamine-containing neurons in the

tuberomammillary nucleus of the hypothalamus.

Besides, the intraocular released carnosine promoted

by specific ophthalmic formulation including muco-

adhesive compound carboxymethylcellulose and corneal

absorption promoters reacts with oxygen radicals, LPO

products, and carbohydrate oxidation products accumu-

lated in the crystalline human lens with aging [8,9].

When cataract was accompanied with POAG, 1% NAC

prodrug was prescribed 15 min prior the topical appli-

cation of beta-blocker, specifically used to decrease the

IOP.

The entire body of supporting research on N-acetyl-

carnosine and its bioactivated principle carnosine reveals

its promise as a water-soluble universal antioxidant that

works at several levels to defend against the oxidative

stresses to the lens, glycosylation problems, and protect

the lens proteins and the membrane lipids from oxidative

damages thus preventing and reversing age-related

cataracts in human eyes. The presented results can be

explained in part by the adduction of the various LPO

products directly by carnosine following de-acetylation of

N-acetylcarnosine. The ability of L-carnosine to inhibit

LPO reactions as well as to diminish the content of LPO

products makes N-acetylcarnosine applied with lubricant

carboxymethylcellulose a prominent tool in the therapy

especially, of the posterior subcapsular and cortical

cataracts, whose mechanism can be related with the

toxic effects of LPO products [80].

The most prevalent age-related ocular diseases are

macular degeneration, open-angle glaucoma, cataract,

and diabetic retinopathy [81,82]. There are several

clinical types of PVR: proliferative diabetic retinopathy,

PVR following rhegmatogenous retinal detachment,

and posttraumatic PVR. PVR occurs after rhegmato-

genous retinal detachment, perforating injury to the

posterior segment of the eye, and retinal detachment

surgery [83]. It is the leading cause of failure in retinal

detachment surgery and is characterized by the

formation of proliferating fibrocellular membrane on

both sides of retinal surfaces and in the vitreous

cavity. Further contraction of the fibrocellular mem-

brane causes tractional retinal detachment (TRD). RPE

cells, fibroblasts, glial cells, macrophages, and myo-

fibroblast-like cells are found in the proliferating

membrane [84].

They are surrounded by various amounts of extracel-

lular matrix (ECM), including collagen, fibronectin,

vitronectin, and laminin [85,86]. PVR is the leading

cause of failure after retinal detachment surgery.

With the advancement of vitreoretinal surgery, the

prognosis of PVR has been improved. Long-term success,

however, often is prevented by the recurrence of

proliferative contractile membranes [87]. Repeated sur-

gery often exacerbates the inflammatory reaction within

the vitreous cavity. Pharmacologically, various drugs,

including antimetabolites, colchicine, and steroids, have

been tested as potential therapeutic agents for the

treatment of PVR [88]. However, many of the cited

inhibitors of cellular proliferation have side effects that

are toxic to the eye. Thus, further understanding the

pathogenesis of PVR and searching for more appropriate

and effective therapeutic agents are critical.

This pilot study suggests that parabulbar, subconjunc-

tival, or intravitreal injection of carcinine with most of

the vehicle removed is not toxic to intraocular struc-

tures, reduces postoperative intraocular inflammation,

and may be a potentially useful additional tool in the

treatment of PVR as well as can be considered as the

antiapoptotic drug for the protection of photoreceptor

cells from oxidative light-induced stress.

Preliminary publications about photoprotector and

antioxidant activities of carcinine have been issued

[16,21,43,89–92]. Carcinine is resistant to hydrolysis

with natural enzymatic systems. Our study shows the

reactivity of carcinine with ROS and aldehyde products



[43,89,91]. Carcinine noticeably limits 4-HNE-induced

cross-linking of collagen. For comparison, vitamin E

(lypophilic antioxidant) is not active against this type of

toxic agent. Aminoguanidine is also not efficient in

protection from the aldehyde-induced cross-linking of

collagen. Carcinine is acting as the universal antioxidant

and transition metal and heavy metal ions chelating

agent. It exerts the revival of the enzymatic superoxide

dismutase (SOD) activity in tissues following the expo-

sure with UV or glycation reactions [43,89,91]. Carci-

nine acts as an efficient water-soluble scavenger of

oxygen-derived free radicals and is also able to inhibit

metal-catalyzed free radical oxidation of lipids

[16,21,43,89–92].

The critical role of mitochondria in programmed cell

death leads to the design of mitochondriotropic agents as

a strategy in regulating apoptosis. Recently, phospho-

lipid peroxidation products gained a reputation as key

regulatory molecules and participants in oxidative

signaling pathways. Oxidation of two anionic phospho-

lipids – cardiolipin (CL) in mitochondria and phospha-

tidylserine (PS) in extramitochondrial compartments – is

important signaling event, particularly during the exe-

cution of programmed cell death and clearance of

apoptotic cells. Quantitative analysis of CL and PS

oxidation products is central to understanding their

molecular mechanisms of action [93,94]. Furthermore,

specific characteristics of CL in mitochondria – –its

asymmetric transmembrane distribution and mecha-

nisms of collapse, the regulation of its synthesis, remod-

eling, and fatty acid composition – are given significant

consideration [94,95]. Cytochrome c (cyt c) acts as a CL-

specific peroxidase very early in apoptosis. At this stage,

the hostile events are still secluded within the mitochon-

dria and do not reach the cytosolic targets. CL oxidation

process is required for the release of proapoptotic factors

into the cytosol. Manipulation of cyt c interactions with

CL, inhibition of peroxidase activity, and prevention of

CL peroxidation are prime targets for the discovery of

antiapoptotic drugs acting before the ‘point-of-no-return’

in the fulfillment of the cell death program.

During apoptosis, a mitochondria-specific phospho-

lipid, CL, interacts with cytochrome c (cyt c) to form a

peroxidase complex that catalyzes CL oxidation; this

process plays a pivotal role in the mitochondrial stage of

the execution of the cell death program. Several works

were focused on redox mechanisms and essential struc-

tural features of cyt c’s conversion into a CL-specific

peroxidase that represent an interesting and may be still

unique example of a functionally significant ligand

change in hemoproteins [93,94]. Recently, it was

demonstrated that peroxidase cyt c/CL complexes can

utilize free fatty acid hydroperoxides (FFA-OOH) at

exceptionally high rates that are approximately three

orders of magnitude higher than for H2O2 [93].

Accordingly, the new concepts in drug discovery based

on the design of mitochondria-targeted inhibitors of cyt

c/CL peroxidase and CL peroxidation with antiapoptotic

effects have appeared. Therefore, mitochondria-targeted

disruptors and inhibitors of cyt c/CL peroxidase com-

plexes and suppression of CL peroxidation represent new

strategies in antiapoptotic drug discovery [96].

We have originally discovered that both carnosine and

carcinine (10–25 mM) are capable of inhibiting the

catalysis of linoleic acid and phosphatidylcholine

liposomal peroxidation by the O2)•-dependent iron-

ascorbate- and lipid-peroxyl-radical-generating linoleic

acid 13-monohydroperoxide (LOOH)-activated hemo-

globin systems, as measured by thiobarbituric-acid-

reactive substance. Carcinine and carnosine are good

scavengers of OH· radicals, as detected by iron-depen-

dent radical damage to the sugar deoxyribose. This

suggests that carnosine and carcinine are able to

scavenge free radicals or donate hydrogen ions. The

iodometric, conjugated diene and t.l.c. assessments of

lipid hydroperoxides (13-monohydroperoxide linoleic

acid and phosphatidylcholine hydroperoxide) showed

their efficient reduction and deactivation by carnosine

and carcinine (10–25 mM) in the liberated and bound-

to-artificial-bilayer states. This suggests that the perox-

idase activity exceeded that susceptible to direct reduc-

tion with glutathione peroxidase. Imidazole, solutions of

beta-alanine, or their mixtures with peptide moieties did

not show antioxidant potential [21]. Because of the

combination of weak metal chelating (abolished by

EDTA), OH• and lipid-peroxyl-radicals-scavenging, and

reducing activities to liberated fatty acid and phospho-

lipid hydroperoxides, carnosine and carcinine appear to

be physiologic antioxidants able to efficiently protect the

lipid phase of biologic membranes and aqueous environ-

ments and act as the antiapoptotic natural drug com-

pounds.

The unwanted proliferation of intraocular tissue, such

as vascular retinal cells in eyes with ischemic retino-

pathies, subretinal neovascular tissue in eyes with

exudative age-related macular degeneration, and retinal

pigment epithelium cells in the case of PVR, is one of the

important problems still mostly unsolved in clinical

ophthalmology. Based on clinically oriented experimen-

tal observations and pathogenic considerations provided



in this study, we have originally suggested that a single

intravitreal injection or several parabulbar injections of

carcinine can locally suppress intraocular inflammation

and proliferation of cells, especially during the modeling

of PVR. Further provided clinical studies (data not

shown) have correspondingly revealed that a single

intraocular injection of carcinine may be a therapeutic

option as adjunctive treatment of exudative age-related

macular degeneration, diabetic cystoid macular edema,

and proliferative diabetic retinopathy.

Although the retina has a remarkable capacity for

morphological and biochemical adaptation to different

environmental luminances, prolonged light exposure

can cause photoreceptor cell damage by nonthermal

mechanisms. It is now clear that light damage involves a

complex series of events that depend on light intensity

[96–98], duration [99,100], and wavelength [101] and

on the distribution of absorbing chromophores in the

retina [102,103]. As it was suggested that light induces

oxidative reactions that result in damage to the photo-

receptor cells, [104] evidence that LPO occurs in the

retina in response to light has gradually accumulated.

An increase in the level of peroxides in rod outer segment

(ROS) extracts [105] and specific loss of docosahexaenoic

acid (22 : 6) from ROS membranes [106] have been

measured after exposure to an intense light. The

hypothesis that oxygen free radicals mediate LPO [107]

has been confirmed in vivo by several investigators

[106,108–110].

Oxygen radicals are generated through a series of

reactions (including the Haber-Weiss and Fenton reac-

tions) [111] that produce the hydroxyl radical (•OH),

which then initiates LPO. The vertebrate retina is very

vulnerable to free radical assault because it contains

high levels of polyunsaturated fatty acids, which are

choice substrates for peroxidation [112]; it consumes

much oxygen compared with other tissues [113], and it

acts as a light processor, which initiates the formation of

free radicals [114].

In this study, the authors have determined through

histologic measurements that carcinine is also effective

in reducing retinal light damage in mice. Unfortunately,

these studies provide no insight on the functional

survival of photoreceptor cells, and the question arises

whether the anatomic rescue of photoreceptors is

accompanied by preservation of the retinal function. In

this study, we have made use of the model of retinal

photodegeneration we had developed. The further clin-

ical analysis of the protective effects of oral carcinine

against retinal degeneration revealed that the observed

carcinine protection of photoreceptor cells from oxidative

stress is morphometric and functional.

Carcinine is indicated for administration as a phar-

maceutical drug in the injection formulation (subcon-

junctival, ocular parabulbar, and intravitreous

injections).

Carcinine is suitable for the systemic administration

(per oss) for ophthalmic and CNS pharmaceutical indi-

cations. This is supported by the fact that carcinine is

only slightly affected by the enzymatic hydrolysis with

natural peptidases [89–91]. The lack of the oral acute

toxicity of carcinine introduces the experimental proof

for this regular oral therapeutic application modality.

The oral therapeutic administration of carcinine and its

bioavailability in patented by IVP oral formulations

thereof have been supported in the matched studies

[115].

In the previous studies, the authors have determined

through histologic measurements that carcinine, a

decarboxylated carnosine natural derivative, is also

effective in reducing retinal light damage in mice

[10,89–91,116]. The further clinical analysis of the

protective effects of lubricant carcinine eye drops against

retinal degeneration revealed that the observed carcinine

protection of photoreceptor cells from oxidative stress is

morphometric and functional. This is supported by the

fact that carcinine is only slightly affected by the

enzymatic hydrolysis with natural peptidases [10,89–

91,115,116]. The lack of the acute toxicity of carcinine

introduces the experimental proof for this regular ocular

topical therapeutic application modality. The therapeutic

administration of carcinine with lubricant eye drops and

its bioavailability in patented by IVP eye drop lubricant

formulations thereof have been supported in the

matched studies [10,89–91,116]. In the previous stud-

ies, we have also presented a growing evidence that

demonstrates the ability of N-acetylcarnosine (lubricant

eye drops) or natural pseudodipeptide carcinine

(b-alanylhistamine) to act as pharmacological chaper-

ones [117–119]; these beta-alanine-containing chaper-

ones can decrease oxidative stress and ameliorate

oxidative and excessive glycation stress-related eye

disease phenotypes, suggesting that the field of chaper-

one therapy might clinically benefit novel treatments for

age-related cataracts, age-related macular degeneration

(AMD), and ocular complications of diabetes (OCD).

Molecular chaperones induced by L-carnosine or carci-

nine antiapoptotic ophthalmic therapeutics facilitate and

regulate protein conformational change within cells.

This encompasses many fundamental cellular processes:



including the correct folding of nascent chains; protein

transport and translocation; and signal transduction and

protein quality control. Imidazole-containing peptidom-

imetics acting as pharmacological chaperones are

important in several forms of human disease, including

age-related cataracts and neurodegeneration. Within the

retina, the highly specialized photoreceptor cell presents

a fascinating paradigm to investigate the specialization of

molecular chaperone function and reveals unique chap-

erone requirements including the transglycating activity

of carcinine essential to photoreceptor function [117–

119]. Exploiting natural peptide diversity: imidazole-

containing pharmacological chaperone molecules was a

one driving factor that led IVP pharmacologists in

cooperation with alliance groups to develop scientifically

proven grooming ophthalmic drug products for oral,

intravitreal administration, or topical lubricant eye drops

drug formulations with active ingredients characterized

by interaction to other proteins and mediating their

folding, transport, and interaction with other molecules,

LPO products and membranes. Our research group has

revealed that toxic aldehydes, such as 4-HNE, are

produced in photoreceptor cells by LPO, induced during

exposure to bright light. If not detoxified, they disrupt

photoreceptor functions and induce apoptosis. The

naturally occurring dipeptide-based compounds either

L-carnosine or carcinine have been shown to quench

4-HNE, forming specific adducts [16]. Specifically, we

investigated whether carcinine, a decarboxylated

derivative of L-carnosine offering the advantage of being

more resistant to enzymatic hydrolysis with endogenous

peptidases, had the same ability to quench 4-HNE,

therefore protecting against its toxic effects. Our exper-

iments were conducted in cultured cells and in mice. The

photoreceptor cell line 661W was preincubated with 0,

2, and 20 mM carcinine for 2, 4, 6, and 24 h. After

removing and washing extracellular carcinine, cells were

treated with increasing concentrations of 4-HNE over-

night, and cell death was quantified using an LDH assay.

Balb/C mice were injected intravitreally with carcinine in

one eye and PBS in the other eye. Two days after

injection, mice were exposed to bright light (4000 lux for

5 h) to induce oxidative stress and endogenous produc-

tion of 4-HNE. Protection of photoreceptors by carcinine

was quantified by histology. We found that carcinine

protects 661W cells against 4-HNE-induced apoptosis in

a time- and dose-dependent manner. This suggests that

carcinine can be transported inside the cells and quench

4-HNE [16]. The earlier established by our group lipid

peroxidase and transglycating free-radical-scavenging

activities of carcinine propose the capacity of carcinine

to act as the antiapoptotic drug and mitochondria-

targeted disruptor and inhibitor of cyt c/CL peroxidase

complexes and suppressor of CL peroxidation [16,117–

119]. Carcinine does not protect when preincubated

<6 h, suggesting an additional mechanism of induction

of protective genes, or chaperone proteins, an effect

reported recently with another Zn carnosine derivative.

Carcinine significantly protected photoreceptors against

light-induced apoptosis, suggesting that this compound

is sufficiently resistant to degradation and can be used in

vivo representing new strategies in antiapoptotic

ophthalmic drug discovery. The biologic properties of

carcinine offer a potential treatment strategy for a wide

range of eye diseases linked to oxidative damage. For

example, we have shown that the photoreceptor-specific

enzyme RDH12 offers endogenous protection against

4-HNE. In absence of a functional RDH12 gene, an early

onset congenital vision loss develops. We will further

explore possibilities of replacing the disrupted activity of

RDH12 with a similar functioning molecule such as

carcinine [16,117–119].

C O N C L U S I O N

There is a growing trend worldwide to evaluate cataract

disease and disability prevention on the basis of costs

incurred and benefits accrued. Public health interven-

tions to prevent blindness are particularly revealing in

this respect, as cost savings and return on investment

accrue, because of the avoided rehabilitative costs, on

the one hand, and the gains in productivity, on the

other. Accurate data on the total economic and social

costs of blindness and severe visual impairment are not

available. Moreover, the cost estimates vary widely from

country to country, based on the prevalence and causes

of blindness, the age of those affected, the employment

situation in the country concerned, the wage structure,

and the existence and coverage of social, rehabilitative,

and educational services. However, in every country,

such costs have been shown to be a considerable strain

on the national economy. In 1990, the aggregated cost

of blindness to the federal budget in the United States

was estimated to be approximately US$4.1 billion. A

minimal federal budgetary cost of a person-year of

blindness (vision <6/60 in the better eye) for a working-

age adult was estimated to be US$11 896.

More importantly, it has been estimated that in the

United States, if all the avoidable blindness in persons

under 20 and working-age adults were prevented, a



potential saving of US$1.0 billion per year would accrue

to the federal budget. In a study from India in 1989, a

conservative estimate of the aggregated costs of blind-

ness to the national economy, including a minimal

subsistence allowance for the blind, amounted to

approximately US$4.6 billion per year. The economic

burden of blindness and visual impairment is only part of

the story. The person who is blind and his/her family

face important social constraints. Both directly and

indirectly, visual impairment interferes with various

activities of daily living. Blind children have to face

developmental challenges. In adult life, employment

opportunities for the visually disabled are extremely

limited, and their participation in a host of leisure

activities is seriously hampered. In addition, in many

societies, visual impairment results in a loss of status and

self-esteem. These physical and, especially, the psycho-

social implications of visual impairment and blindness

cannot be accurately quantified in monetary terms.

However, they do erode the quality of life of both the

affected individuals and their families. Our study in the

UK indicated that visually disabled individuals were

poorer on average and had lower educational levels,

lower employment, and less social life than sighted

individuals.

These findings probably hold true in all societies. Also,

in African settings, it has been reported that blind persons

die earlier compared with the sighted population. The

Nepal blindness survey showed that life expectancy of

blind individuals was 3 years. Cataracts are the most

common cause of avoidable blindness. Worldwide, there

are an estimated 16 million persons who are blind as a

result of cataracts. The majority of these individuals live in

the rural areas of economically developing countries. The

burden on the country is significant.

In this study, we present the evidence that N-acetyl-

carnosine lubricant eye drops globally patented by IVP

have now reached clinical trials and showed encourag-

ing results as anticataract drug or medical device (CE-

marked product) that warrant further investigation. The

discovery of naturally occurring carnosine derivatives:

N-acetylcarnosine and carcinine as effective medical

treatment for sight-threatening eye disorders, such as

cataract, PVR, macular degenerations, POAG, and

ocular complications of diabetes is likely to make global

impact on eye health. The current aims of this paper are

to introduce the updated data on the drug therapy of

cataract with N-acetylcarnosine lubricant eye drops, on

the carcinine treatment of experimental PVR, to cover

the carcinine protection of photoreceptor cells from the

light-induced damages, and to provide updates of the

latest development.

A C K N O W L E D G E M E N T S

This work was planned, organized, and supported by

IVP. Innovative Vision Products, Inc. is a holder of the

worldwide patent (including PCT International Publica-

tion Number WO 2004/028536 A1) for the application

of N-acetylcarnosine for the Treatment of ophthalmic

disorders, including cataracts as well as (PCT Interna-

tional Publication Number WO 2004/064866 PCT/

JP2004/000351) protecting the described in the article

therapeutic applications. The biologically significant

applications of carnosine mimetics including those in

ophthalmology were patented by Dr. Babizhayev and

the alliance Groups (WO 2004/028536 A1; WO 94/

19325; WO 95/12581; WO 2004/064866 A1). Inno-

vative 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 biomedical

applications. Over the last decade, IVP has developed a

track record in developing these technologies to effec-

tively address the unmet needs of specific diseased

populations.

C O N F L I C T O F I N T E R E S T

Declaration 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.

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