qphc 1-001 (1) (1)

1Volume 1 Issue 1 001

Quality in Primary Health CareOPUS JOURNALS

Qual Prim Health Care (2017) 11 001

Research ArticleQu

ality in Healthcare

AbstractDiabetes is the seventh leading cause of death based on US death certificates The growing toll of diabetes cost the US nation a record high $245 billion in 2012 a 41 increase from $174 billion in 2007 Global prevalence of diabetes mellitus and its complications throughout the world are interlaced with the specific diverse microvascular and macrovascular pathologies resulted from hyperglycaemia being a leading cause of blindness renal failure nerve damage and diabetes-accelerated atherosclerosis leads to increased risk of myocardial infarction stroke and limb amputation

Several pathophysioloical mechanisms have been considered as emerging targets for the combination therapies of diabetes mellitus and the treatment of diabetic complications increased polyol pathway flux increased advanced glycation end-product (AGE) formation activation of protein kinase C (PKC) isoforms and increased hexosamine pathway flux The listed mechanisms reflect a single hyperglycaemia-induced process of overproduction of superoxide anion radical by the mitochondrial electron-transport chain Besides mediating mitochondrial functions the major biological effects of excessive nonenzymatic glycosylation are leading to increased free radical production and compromised free radical inhibitory and scavenger systems inactivation of enzymes inhibition of regulatory molecule binding crosslinking of glycosylated proteins and trapping of soluble proteins by glycosylated extracellular matrix (both may progress in the absence of glucose) decreased susceptibility to proteolysis abnormalities of nucleic acid function altered macromolecular recognition and endocytosis and increased immunogenicity

This investigation cumulates the data on biological activities of patented carnosine mimetics resistant in formulations to enzymatic hydrolysis with human carnosinases that are acting as a universal form of antioxidant deglycating and transglycating agents that inhibit sugar-mediated protein cross-linking chelate or inactivate a number of transition metal ions (including ferrous and copper ions) possess lipid peroxidase type of activity and protection of antioxidant enzymes from inactivation (such as in a case of superoxide dismutase)

L-Carnosine released systemically from N-acetylcarnosine lubricant eye drops or released from skeletal muscle during exercise may be transported into hypothalamic tuberomammillary nucleus (TMN)-histamine neurons and hydrolyzed The resulting L-histidine may subsequently be converted into histamine acting as metabolic fuel feeding for the hypothalamic histaminergic system the latter represents an important component in the neurocircuitry relevant for diabetes sickness behavior This mechanism could be responsible for the effects of L-carnosine on autonomic neurotransmission and physiological function of pancreas stimulating in vivo regeneration of insulin-producing beta-cells Thus L-carnosine might stimulate insulin secretion and appears to influence hypoglycemic hypotensive and lipolytic activity through regulation of autonomic nerves and with the involvement of the the hypothalamic suprachiasmatic nucleus (SCN)

This study indicates on therapeutic benefits for imidazole-containing antioxidants (nutraceutical non-hydrolized carnosine carcinine D-carnosine ophthalmic prodrug N-acetylcarnosine leucyl-histidylhidrazide and patented formulations thereof) as an essential part of any diabetes management plan

Keywords Peptides Diabetes Complications Nephropathy Retinopathy Neuropathy Atherosclerosis Excessive nonenzymatic glycosylation Increased free radical production Patented carnosine mimetics resistant to enzymatic hydrolysis with human carnosinases Non-hydrolized carnosine Carcinine D-carnosine Ophthalmic prodrug N-acetylcarnosine Leucyl-histidylhidrazide and patented formulations thereof Universal form of antioxidant and transglycating agents Hypothalamic histaminergic system Therapeutic management strategies for Type 2 Diabetes

Advanced Glycation End Products Free Radical Generation by Early Glycation Products as a Mechanism for Long-Term

Complications of Diabetes Mellitus Toxicity Regulation Function and Role in Health Nutrition and Disease

Mark A BabizhayevInnovative Vision Products County of New Castle Delaware USA

Received March 19 2017 Accepted April 26 2017 Published May 03 2017

Corresponding author Dr Mark A Babizhayev Innovative Vision Products Inc Moscow Division Ivanovskaya 20 Suite 74 Moscow 127434 Russian Federation County of New Castle Delaware USA Tel +7(499) 977-2387 E-mail markbabizhayevmailru

Citation Babizhayev MA (2017) Advanced Glycation End Products Free Radical Generation by Early Glycation Products as a Mechanism for Long-Term Complications of Diabetes Mellitus Toxicity Regulation Function and Role in Health Nutrition and Disease Qual Prim Health Care (2017) 11 001



methylglyoxal or 3-deoxyglucosone as well as via depletion of NADPH or glutathione raising intracellular ROS all of which indirectly result in increased formation of AGEs [3-510-12] Several

compounds eg εN-carboxymethyl-lysine pentosidine or methylglyoxal derivatives serve as examples of well-characterized and widely studied AGEs [67]

Several immunoassay-based tests have established higher levels of common AGEs such as εN-carboxymethyl-lysine (CML) or methylglyoxal (MG) in older persons who are otherwise healthy [13] The frequent finding in the aged population of increased ROS [1415] a state known to promote AGEs formation supports the notion of increased endogenous AGEs formation in the elderly

A key characteristic of certain reactive or precursor AGEs is their ability for covalent crosslink formation between proteins which alters their structure and function as in cellular matrix basement membranes and vessel-wall components Other major features of AGEs relate to their interaction with a variety of cell-surface AGE-binding

receptors leading either to their endocytosis and degradation or to cellular activation and pro-oxidant pro-inflammatory events A large body of evidence suggests that AGEs are important pathogenetic mediators of almost all diabetes complications conventionally grouped into micro- or macroangiopathies For instance AGEs are found in retinal vessels of diabetic patients and their levels correlate with those in serum as well as with severity of retinopathy [1617]

Several approaches seeking to reduce AGE interactions either by inhibiting AGE formation blocking AGE action or breaking pre-existing AGE cross-links have been explored The first class of those agents involved the inhibitors of AGE formation which act by inhibiting post-Amadori advanced glycation reactions or by trapping carbonyl intermediates and thus inhibiting both advanced glycation and lipoxidation reactions Aminoguanidine [1819] ALT-946 [1920] 2-3-Diaminophenazine [20] thiamine pyrophosphate [21] benfotiamine [22] and pyridoxamine [23] ORB-9195 [24] constitute known representatives of this group of agents The second class of those agents involved the AGE breakers which ldquobreakrdquo pre-accumulated AGE or existing AGE cross-links leading to the elimination of the smaller peptides through urine PTB (N-phenylthiazolium bromide) [25] and ALT-711 are the best known representatives of this group of agents [2627] Recently it has been shown that antihypertensive drugs such as losartan olmesartan and hydralazine seem to inhibit AGE formation [28-31]

Carnosine (β-alanyl-L-histidine) and related compounds are natural constituents of excitable tissues possessing diverse biological activities [32] The level of carnosine in tissues is controlled by a number of enzymes transforming carnosine into other carnosine related compounds such as carcinine N-acetylcarnosine anserine or ophidine (by decarboxylation acetylation or methylation respectively) or its cleavage into the amino acids histidine and β-alanine Hydrolysis is mainly due to tissue carnosinase (EC 34133)

IntroductionAccording to the American Diabetes Association

diabetes is the seventh leading cause of death in the US There are 103 million people in the US with the disease and an additional estimated 54 million have not yet been diagnosed Many people do not become aware that they have diabetes until they develop one of its life-threatening symptoms such as blindness kidney disease nerve damage and heart disease Diabetes develops due to a diminished production of insulin (in type 1) or resistance to its effects (in type 2 and gestational) (Figure 1) [1] Several predominant well-researched theories have been proposed to explain how hyperglycemia can produce the neural and vascular derangements that are hallmarks of diabetes These theories can be separated into those that emphasize the toxic effects of hyperglycemia and its pathophysiological derivatives (such as oxidants hyperosmolarity or glycation products) on tissues directly and those that ascribe pathophysiological importance to a sustained alteration in cell signaling pathways (such as changes in phospholipids or kinases) induced by the products of glucose metabolism [2] Hyperglycemia is still considered the principal cause of diabetes complications Its deleterious effects are attributable among other things to the formation of sugar-derived substances called advanced glycation end products (AGEs) AGEs form at a constant but slow rate in the normal body starting in early embryonic development and accumulate with time However their formation is markedly accelerated in diabetes because of the increased availability of glucose

AGEs are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins lipids and nucleic acids [3-7] The initial product of this reaction is called a Schiff base which spontaneously rearranges itself into an Amadori product as is the case of the well-known hemoglobin A1c (A1C) These initial reactions are reversible depending on the concentration of the reactants A lowered glucose concentration will unhook the sugars from the amino groups to which they are attached conversely high glucose concentrations will have the opposite effect if persistent

A series of subsequent reactions including successions of dehydrations oxidation-reduction reactions and other arrangements lead to the formation of AGEs AGEs may form by auto-oxidation of glucose or through the glycolytic pathway but also from non-glucose sources including lipid and amino acid oxidation [2-7] In addition neutrophils monocytes and macrophages upon inflammatory stimulation produce myeloperoxidase and activate Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase which can lead to new AGEs by way of amino acid oxidation [3-589]

Binding and activation of cellular AGE Receptor (RAGE) by AGEs or any other ligand can also promote reactive oxygen species (ROS) and AGE formation via the NADPH oxidase and the myeloperoxidase pathways [3-589] Another potential mechanism of AGE formation is the polyol pathway Glucose entering the polyol pathway may form AGEs via reactive intermediates ie glyoxal



Figure 1 Pathophysiology Complications of Diabetes Mellitus and Main symptoms of diabetes Diabetes mellitus is usually not considered a single disease but rather a group of three different disorders that appear to have different causes though they result in similar symptomsThe image comprising the inserted card illustrates effects associated with Type II Diabetes stroke ocular pathology hypertensive heart disease hardening of the kidney hardening of the arteries insulin resistance neuropathy and foot ulcerations



which is widely distributed among different subjects [3334] or serum carnosinase (EC 341320) obtained in brain and blood plasma of primates and humans [3536] Carnosine has been proven to scavenge reactive oxygen species (ROS) as well as alpha-beta unsaturated aldehydes formed from peroxidation of cell membrane fatty acids during oxidative stress [37-39] It can oppose glycation [4041] and it can chelate divalent metal ions The important studies have produced clinical and experimental evidence of beneficial effects of N-acetylcarnosine in treating cataracts of the eyes these and other ophthamological benefits have been proven [42-49] Carcinine (β-alanyl histamine) is an imidazole dipeptide first discovered in the crustacean Carcinus maenas [50] and has subsequently been found in the hearts of several mammalian species [5152] It has been demonstrated that carcinine is metabolically related to histamine histidine and carnosine (β-alanyl-L- histidine) and could be synthesized from histamine and β-alanine [53] 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 [54] Carcinine was shown to act as a natural antioxidant [5556] and to play a role in regulating stress and shock with a 1000-fold less potent hypotensive effect than histamine [5257] suggesting that carcinine might have therapeutic use Overall these low molecular mass antioxidant peptidomimetics add significantly to the defense provided by the enzymes superoxide dismutase catalase and glutathione peroxidases [5556] Combination pharmaceutical products or fixed-dose combinations (FDCrsquos) offer benefits to many drug classes due to the additive nature of therapeutic effect and the reduced level of side-effects associated with their use A widespread acknowledgement and acceptance of these combined therapies as an essential part of any diabetes management plan has now been established

Recently carnosine analogs bearing the histidyl-hydrazide moiety were synthesized and patented in ophthalmic pharmaceutical formulations with N-acetylcarnosine bioactivating prodrug or L-carnosine to moderate the enzymatic hydrolysis of a dipeptide by carnosinase (inhibited by a nonhydrolyzable substrate analog so that this keeps steadier levels of the drug active principle in the aqueous humor) [5859] In this study Leucyl-histidylhydrazide peptidomimetic demonstrated the transglycation activity more pronounced than L-carnosine accounting for the ability of either molecule to reverse pre-existing glycation-induced cross-linking and checking the nonenzymatic glycation cascade in the ophthalmic age-related or diabetic complications pathologies

The present article introduces the experience of launching Combination Products as a new report with the scientific and technology insights that provide detailed strategic guidance for the preparation and successful execution of combination transglycating product launches targeting the diabetic complications and age-related eye diseases The presented therapeutic strategies feature the latest management strategies of diabetes and diabetic complications (including

systemic and ocular complications of diabetes) with transglycating imidazole-containing peptide-based agents to help the establishment effectively and maximize the pipeline productivity of the authorsrsquo Group

Materials and Methods Carcinine (Decarboxy carnosinemiddot2HCl) l-prolylhistamine

and N-acetyl-β-alanylhistamine were synthesized by Exsymol SAM (Monaco Principaute de Monaco) l-Carnosine and N-acetylcarnosine were synthesized by Hamari Chemicals Ltd (Japan) per specifications proposed by Innovative Vision Products Inc Superoxide dismutase from bovine erythrocytes methylglyoxal L-lysine 1133-tetraethoxypropane nitro blue tetrazolium (NBT) lucigenin and other reagents produced by Sigma (USA) were used in the work Malondialdehyde was obtained by acid hydrolysis of 1133-tetraethoxypropane as described in [60] EPR spectra were recorded at room temperature in an E-109E spectrometer (Varian USA) Recording settings were as follows microware power 20 mW microware frequency 915 GHz high frequency modulation amplitude 02 mT Spectrum recording was started 1 min after the mixing of reaction components The reaction mixture (120 microl) was introduced into PTFE 22 gas_permeable capillaries (Zeus Industrial Products USA) The capillaries were placed into a quartz tube for continuous nitrogen or air flow during the measurement EPR spectra were simulated by SimFonia software (Bruker Germany) The EPR signal of the stable synthetic free radical diphenylpicrylhydrazine was used as a standard [61]

Generation of superoxide anion radical (О2-middot) was

detected using two independent methods reduction of nitro blue tetrazolium by the superoxide and О2

-middot induced chemiluminescence of lucigenin The kinetics of accumulation of NBT reduction product formazan was determined by absorption at 560 nm in a Hitachi-557 spectrophotometer (Japan) at 25degC The reaction was initiated by adding 10 mM methylglyoxal or 10 mM MDA to the medium containing 100 microM NBT and 10 mM L-lysine in 100 mM carbonate buffer pH 95 Chemiluminescence was measured by a Lum-5773 chemiluminometer (Russia) in medium containing 20 microM lucigenin 15 mM L-lysine and 15 mM methylglyoxal in 100 mM K Na_phosphate buffer pH 78 Measurements were performed at 37degC under continuous stirring of the reaction medium Statistical treatment of the data was performed using Studentrsquos t-criterion

Molecular modelingLow-energy 3-D conformations of carnosine carcinine

and N-acetylcarnosine were derived using the PM3 method of the MOPAC 60 program (Stewart MOPAC Air Force Academy Boulder CO 80840) The precise energy minima conformations were determined by semi-empirical Quantum mechanics This technique structures a pool of energetically accessible shapes especially suitable for dipeptides comparative to large protein molecules The program is supplemented with ZINDO1 computer software for estimation of chelating properties of dipeptides and related



compounds The conformational geometry optimization was carried out using the revised computer program [6263]

Peroxidation reaction system and reactivity of imidazole-containing compounds to aldehydes

The techniques for phospholipid extraction purification and preparation of liposomes (reverse-phase evaporation technique) have been described previously [6465] Peroxidation of phosphatidylcholine (PC derived from egg yolks) was initiated by adding 25 microM FeSO4 and 200 microM ascorbic acid to the suspension of liposomes (1 mgml) in 01 M Tris-HCl buffer (pH 74) The incubations were performed at 37ordmC The tested compounds either N-acetylcarnosine l-carnosine carcinine or other imidazole-containing compounds were added at 10ndash20 mM concentration to the system of iron-ascorbate-induced liposome PC peroxidation The kinetics of accumulation of lipid peroxidation (LPO) products in the oxidized liposomes were measured by reaction with thiobarbituric acid (TBA) The peroxidation reaction was arrested by adding EDTA to a final concentration of 50 microM or by the addition of 20 ml of ice-cold 025 M HCl containing 15 (wv) three-chloroacetic acid (TCA) TBA (0125 wv) was then added to the mixture and followed by boiling for 15 min The TBA assay was described previously The differential absorbance of the condensation product malonyl dialdehyde (MDA) at 535 and 600 nm was measured spectrophotometrically (ε535=156 x 105 M-1cm-1) The TBA reaction itself was not affected by the components of the radical generators or scavengers used in the study To determine conjugated dienes the lipid residue of the samples was partitioned through chloroform during the extraction procedure This protocol removes any water-soluble secondary oxidation products leaving them in the methanol-aqueous phase Correlation of the extracted lipid concentrations to the measured phosphorus was done by means of characteristic absorption at 206ndash210 nm of the lipid sample (redissolved in 2ndash3 ml of methanolheptane mixture 51 vv) Accumulation of net diene conjugates corresponding to the level of lipid hydroperoxides was assessed from characteristic absorbance of diene conjugates at ~ 230 nm (ηCD=28x104 M-1cm-1) in a Shimadzu UV-260 spectrophotometer (Japan) Absorbance of the secondary LPO products at ~274 nm corresponding to the concentration of conjugated trienes and ketodienes was also measured spectrophotometrically from the lipid spectra [65] An average MW of phospholipid was assumed to be ~ 730 Da Statistical significance was evaluated by the unpaired Studentrsquos t-test and P=001 was taken as the upper limit of significance

Ferroxidase activity of carnosineThe ability of carnosine to decrease the concentration

of free ferrous ions in TrisndashHC1 buffer (100 mM pH 74) was monitored by the 110-o-phenanthroline chelating assay modified from Ref [66] The reaction was started by the addition of 125 microM FeSO4 to the reaction mixture which contained 3ndash20 mM carnosine Sixty minutes after incubation at 37ordm C the reactions were stopped by the

addition 100 microM 110-o-phenanthroline (Serva) and A515 was immediately read The concentration of (Fe2+ndash110-o-phenanthroline) chelating complex was determined using the molar extinction ε515=10 931 M-1 cm-1

Reactivity of carcinine and carnosine to hydroperoxide of linoleic acid

Standard hydroperoxide of linoleic acid (LOOH) and its alcohol form (LOH) were obtained as described by Iacazio et al [67] The reaction conditions of pure 13(S) linoleic acid hydroperoxide with imidazole-containing peptidomimetic compounds (carnosine carcinine) were described earlier [56] The results of experiments demonstrating the lipid peroxidase activity of l-carnosine and carcinine were carefully described [5556]

Electrophoresis assaysOne millimole of 13(S) linoleic acid hydroperoxide were

incubated in phosphate buffer solution (PBS) (01 M pH 74) with a bovine serum albumin (BSA) solution (05 mg ml-1) at 37ordmC with 3 mM of several antioxidants (carcinine (β-alanylhistamine) l-carnosine (β-alanyl-l-histidine) l-prolylhistamine N-acetyl-β-alanylhistamine or vitamin E) In another experiment liposomes made from phospholipids containing unsaturated fatty acids were peroxidized during 2 days by contact with copper [55] In a second step the imidazole-containing antioxidants were introduced in the liposome mixture The representative protein BSA was then added and incubated for 2 days After 2 days incubation a SDS-PAGE electrophoresis (75 polyacrilamide gel containing 01 SDS) was made according to Laemmli 1970 [68] and stained with the normal silver technique [69] The analytical scanner and the appropriate software used to realize figures were purchased from Advanced American Biotechnology

HPLC analysis for detection of lipid hydroperoxide

Following different incubation times a fraction of the solution was processed which contains the fatty acid hydroperoxide BSA and the imidazole peptidomimetic compound After the addition of 100 ml HCl (1N) to the same volume of reaction mixture and a centrifugation (10000 g 10 min) a HPLC analysis was also made The supernatant of each sample was diluted 3 times in methanol 40 μl were used for the following reverse phase HPLC analysis technique

bull Column C18 MachereyndashNagel 46 mm 5 mm 125 cm

bull Elution 5050 acetonitrile-acetic acid 001

bull Controls retention time of 13(S) linoleic acid hydroperoxide = 15 min

bull Retention time of 13(S) linoleic acid alcohol = 128 min

bull (obtained after NaBH4 reduction [67])

bull Spectrophotometer Hewlett Packard HP 1050



Protection of Superoxide Dismutase (SOD) activity with an imidazole-containing peptidomimetic Skin treatment with carcinine during UV irradiation

Porcine ears were heated at 70ordmC for 70 s The epiderm-derm fraction was removed with a mechanical treatment Skin fragments were treated with creams containing 0 05 1 and 2 carcinine during 5 min Skins were then washed with 1 Triton X-100 solution in phosphate buffer to take emulsion off the skin surface After UVA-UVB irradiation (08 Jcm2) skin fragments were cut suspended (30 gl) in phosphate buffer and crushed with ultra turrax (0ordmC for 2 min) Extracts obtained were diluted (13) in Triton X-100 (1) and kept 2 h at 0ordmC The mixtures were then centrifuged at 10000 g for 10 min The SOD-like activity was measured in the supernatant fraction

Measurement of SOD-like activityAnion superoxides produced by the hypoxanthine

xanthine oxidase system react with cytochrome c This reaction induces a ferrous cytochrome formation which absorbs at 550 nm SOD is able to dismute a part of anion superoxides Due to the SOD activity the level of the anion superoxides decreases Thus the cytochrome c is less reduced and the OD values (550 nm) decrease Six hundred microliters of phosphate buffer or of epiderm extract (obtained from 10 g of skinl of phosphate buffer) were added to 400 μl of solution A Solution A contained 45 μM of cytochrome c 540 μM of

hypoxanthine and 1250 units of catalase Xanthine oxidase was solubilized in a phosphate buffer solution (006 unitsml) The reaction was initiated by the addition of 100 μl of xanthine oxidase The kinetics were realized at 25ordmC for 2ndash3 min (550 nm) The rate of cytochrome c reduction (delta ODmin) at 25ordmC was assessed The protective effect of carcinine was obtained with the following formula

Irradiation 0 - Irraditation X x 100Irradiation 0 - No irradiation

ndash No irraditation kinetics obtained from non-irradiated skin fraction It represents the natural SOD-like activity of the skin extract

ndash Irradiation 0 kinetics obtained with irradiated skin fractions treated with the cream containing 0 carcinine It represents the maximum impact of UV irradiation on the SOD-like activity in the extract

ndash Irradiation X kinetics obtained with irradiated skin fractions treated with creams (oilwater) containing 01 05 1 or 2 carcinine It represents the SOD-like activity of the extracts after irradiation and treatment with carcinine

Detection of transglycating activity of imidazole-containing peptides and peptidomimetics

The standard peptide chemistry procedures were employed for the synthesis of carnosine derivatives and the

obtained compounds were purified by liquid chromatography (LC) or HPLC to obtain pure specimens as confirmed by NMR and mass spectroscopy [70]

ESI-MS spectra were acquired with a Mariner (Per-Spective Biosystems) mass spectrometer instrument using a mixture of neurotensin angiotensin and bradykinin at concentration of 1 pmolL as external standard Samples were prepared by dissolving the compound (10-5M) in acetonitrilewater 11 mixture with 1 acetic acid 1H and 13C NMR spectra were recorded with a Bruker Avance DRX 400 spectrometer Chemical shifts (δ) are given in parts per million (ppm) using solvent (CDCl3 or DMSO-d6) as internal standard Reaction courses and product mixtures were routinely monitored by TLC on silica gel (precoated Polygram Sil GUV 254 from Macherey-Nagel) and visualized with UV lamp (254 nm) or iodine vapors Reagents and solvents were of high-purity grade and were purchased from SigmandashAldrich JT Baker and Carlo Erba

13C NMR experiments Glucosendashethylamine (GndashE) was synthesized by incubating 500 mM 13C-glucose and 15N-ethylamine at pH 12 and 37ordmC for 3 h [7071] At the end of the incubation period about 75 of the starting material was converted to glucosendashethylamine in equilibrium with the starting materials NMR experiments were conducted under conditions which stabilized Schiff base enough to be able to observe them by NMR over several hours The reaction mixture (05 mL in a 5 mm NMR tube) included 250 mM Hepes pH 85 10 D2O and 20 mM concentration of carnosine or one histidyl-hydrazide derivative The reaction was performed at room temperature and it was initiated by adding an aliquot of GndashE to produce a final concentration of 20 mM At that time consecutive NMR spectra of 20 min duration were acquired using 580 scans 60ordm pulses and an interpulse delay of 205 s The spectra were analyzed using the information from model compounds and chemical shifts from the literature The area of the GndashE doublet at 9000 ppm was calculated and plotted against time after subtraction of the natural GndashE Schiff base decay measured in a blank experiment Transglycation efficiency of L-carnosine and carnosine derivatives 2ndash7 (Figure 2) was assessed following Szwergold protocol [71] using the Schiff base glucosylndash

Figure 3 Haematoxylin and Eosin (magnification x40) Basal cells vacuolation and lymphocytic infiltration from left concha



ethylamine (GndashE) as a model of the first intermediate in the glycation process of side chain primary amines of proteins 15N labeled ethylamine was used to minimize electric quadrupole moment and obtain a C-1 peak of glucose as a sharp doublet centered at 9000 ppm The kinetics of the transglycation reaction for the control reaction carnosine and compounds 2ndash7 are illustrated in Figure 2 For a better evaluation of the transglycation kinetics of the compounds for each 13C spectrum the integral of the buffer Hepes signals (50ndash55 ppm range) was set as=1 then the integral of the C-1 glucose peak at 9000 ppm was measured and integration values normalized and corrected for the natural decay of the GndashE Schiff base (control curve) were plotted against time The ability of carcinine (decarboxycarnosine) to behave as an ldquoacceptor moleculerdquo for transglycation was determined using a specific experimental design (Figure 3) A model glycosylamine namely Glucosyl-ethylamine was synthesized and the transfer of the glycosyl moiety to decarboxycarnosine (formation of glucosyl-decarboxycarnosine) was monitored by carbon Nuclear Magnetic Resonance (13 C NMR) spectroscopy Glucosyl-ethylamine synthesis this model glycosylamine was obtained by incubating D-glucose and ethylamine (500 mM) for 3 hours at 37degC in alkaline conditions (pH 12) In order to ease the 13C NMR study we have used isotopically enriched [1- 13 C]glucose and [15 N]ethylamine Transglycation reaction experimental conditions were adapted form Szwergold 2005 The glycosylamine (20mM) and decarboxycarnosine (20mM) were reacted at room temperature in an Hepes medium (250 mM) containing 10 of D2O at slightly alkaline pH conditions (pH 85) that enables to conduct the NMR study over a several hours period of time 13 C NMR study NMR spectra were obtained from a Bruker Avance 500MHz Spin-spin coupling between neighboring 13C and 15N atoms enables to obtain a doublet as a characteristic signal for glycosyl-amines (alpha and beta) As in a published study [71] a kinetic study was performed by acquiring consecutive NMR spectra of 20 minutes duration (580 scans) during 240 minutes Reagents including [1- 13C] glucose and [15N] ethylamine were obtained from Sigma-Aldrich-Fluka (SAF Deisenhofen Germany)

Testing of human carnosinase activityIn our issued provided studies [58] human carnosinase

activity was assayed according to a method described by Bando et al [72] modified and adapted to 96 well plates Briefly substrate hydrolysis was carried out in 50 mM Tris-HCl buffer (pH 75) 1 mM carnosine in 100 μl final volume using 025-05 μg of celltissue extract or 10 ng of purified enzyme The reaction was initiated by addition of substrate and stopped after 60 min incubation at 30 degC by adding 50 μl of 1 TCA Liberated histidine was derivatized by adding 50 μl of 5 mgml o-Pthaldialdehyde (OPA) dissolved in 2 M NaOH and 30 min incubation at 30 degC Fluorescence was read using a MicroTek plate reader (Exc 360 nm and Em 460 nm) Reaction blank values were obtained by adding the TCA stop solution 1 min prior to substrate addition Reactions were carried out in triplicate

Results

Mechanism of Superoxide Formation as a Result of Interaction of L-Lysine with Dicarbonyl Glycating Compounds

For the comparative study of the interaction of L-lysine with carbonyl compounds we used the major secondary product of lipid peroxidation (MDA) and its isomer α-ketoaldehyde (α-oxoaldehyde) mdash methylglyoxal Figure 4a shows the results of EPR spectroscopic study of the products of L-lysine reactions with methylglyoxal and MDA The data presented in this figure demonstrate that free radical intermediates are formed under anaerobic conditions in the reaction of L-lysine with methylglyoxal but not with MDA (Figure 4a spectra 1 and 3) The EPR spectrum recorded during the reaction of L-lysine with methylglyoxal has a multicomponent hyperfine structure

Previously in work [73] such EPR spectrum was recorded in reaction mixture containing L-alanine and methylglyoxal In this work using C13- and N15-substituted and deuterated L-alanine derivatives it has been shown that the EPR spectrum is a superposition of signals of methylglyoxal anion radical (MG ˉ) and Schiff base cation radical (dialkylimine) appearing on the interaction of methylglyoxal with the amino acid Based on this we suggest that the EPR spectrum observed in our experiments is also a superposition of signals of MG ˉ and the cation radical of methylglyoxal dialkylimine with lysine

It is important to note that only trace quantities of free radical intermediates were registered under aeration of the reaction mixture (Figure 4 spectrum 2) Substitution of air for nitrogen after incubation of methylglyoxal and L-lysine mixture under aerobic conditions results in a significant (nearly by an order of magnitude) increase in the level of free radicals supposedly dialkylimine and methylglyoxal (Figure 4b) It is significant that under these conditions the content of free radical intermediates increases on addition of superoxide dismutases (SOD) to the reaction mixture (Figure 4b curve 2) The effect of SOD might be due to the fact that this enzyme removes the superoxide radical generated in the tested model system Indeed the data obtained in work [73] indicate that О2ˉ˙ is formed by single_electron oxygen reduction by methylglyoxal semidione in accordance with the reaction

(reaction 1)

Our model system has also demonstrated that О2ˉ˙ is intensively generated on the interaction of L-lysine with methylglyoxal in carbonate buffer pH 95 Superoxide formation was assessed by the accumulation of formazan on NBT reduction The accumulation of formazan under these conditions might not depend on О2ˉ˙ since it is probable that NBT is reduced by other intermediates of L-lysine reaction with methylglyoxal Nevertheless reasoning from the fact that SOD significantly (more than 4 times) inhibited the formation of formazan under the above conditions one can state that the most part of NBT is reduced under the action of О2ˉ˙ (Figure 5a)



Figure 3 13C NMR spectra with characteristic peaks for residual glucose (szlig-Glc α-Glc) and model glycosylamines szlig-glucosyl-ethylamine (szlig-G-E) and α-glucosyl-ethylamine (α-G-E) 13C NMR spectra obtained 4 hours after addition of decarboxycarnosine to the szlig-G-E α-G-E mixture with a characteristic peak for the transglycation product glycosyl-decarboxycarnosine (G-Decarboxy C)



However only insignificant generation of superoxide radical was observed on the interaction of L-lysine with MDA (Figure 5b) The rate of reaction of amino groups with methylglyoxal becomes lower on increasing acidity of the medium [74] It is concluded that the primary step in the reaction involves the formation of a Schiff base

linkage between the lysine side chain and methylglyoxal These findings reaffirm the concept that by the formation of Schiff bases aldehydes can act as electron acceptors in charge transfer interactions with proteins [74] The application of chemiluminescence as a method more sensitive than NBT reduction [75] revealed the formation

Figure 4a EPR spectra of free radical intermediates of the reaction between L-lysine and dicarbonyl compounds The reaction medium contained 160 mM L-lysine and 160 mM methylglyoxal (spectra 1 and 2) or 160 mM MDA (spectrum 3) in KNa_phosphate buffer (02 M pH 78) EPR signals were registered 4 min after mixing the components under aeration (spectrum 2) or under nitrogen (spectra 1 and 3)

Figure 4b Effect of aeration and SOD on the kinetics of accumulation of free radical intermediates recorded by EPR The reaction medium contained 1) 160 mM L-lysine and 160 mM methylglyoxal in 02 M KNa_phosphate buffer pH 78 2) the same as (1) + 400 SOD units



of О2ˉ˙ in the mixture of methylglyoxal with L-lysine at pH 78 (Figure 6) ie under conditions close to physiological SOD under these conditions almost completely inhibits the chemiluminescence of lucigenin which is evidence of the dependence of this process on the presence of superoxide anion radical (Figure 3b curve 2)

The decrease in concentration of free radicals recorded by EPR in aerated reaction medium is probably not associated with inhibition of their formation Indeed with nitrogen purging the content of free radical intermediates reaches its maximum in 8 min after the mixing of reaction components but after the gas medium is replaced by air the level of EPR-revealed free radicals quickly drops (Figure 7 (panel a))

Under these experimental conditions SOD reliably reduced the rate of decline of EPR signal intensity during aeration (Figure 7 (panel a) curve 2) In 2 min after the increase in oxygen concentration in the medium containing L-lysine and methylglyoxal it is impossible to reveal there free radical intermediates (Figure 7 (panel a) curve 1)

Nevertheless the EPR spectrum containing five components of hyperfine structure and a g-factor equal to 20042 were recorded on aeration of the reaction medium in the presence of SOD (Figure 7 (panel b) spectrum 2) According to the literature data the characteristics of the EPR spectrum presented in Figure 7 (panel b) (spectrum 2) correspond to the signal of the cis-form of methylglyoxal

Figure 5a Effect of SOD on kinetics of formazan formation during the reaction of L-lysine with methylglyoxal (a) or MDA (b) The reaction medium contained 1) 100 mM carbonate buffer pH 95 10 mM L-lysine and 10 mM methylglyoxal or MDA 2) the same as (1) + 120 SOD units

Figure 5b Effect of SOD on superoxide_dependent chemiluminescence of lucigenin The reaction medium contained 1) 100 mM KNa_phosphate buffer pH 78 20 microM lucigenin 15 mM L-lysine 15 mM methylglyoxal 2) the same as (1) + 120 SOD units



Figure 6 (A) Kinetics of SOD-like activity in extracts from non-irradiated or irradiated skin previously treated with creams containing 0 or 01 of carcinine The slope obtained with the non-irradiated skin is 01 OD unitsminThe slope obtained with the irradiated skin treated with 0 carcinine is 017 OD unitsmin The slope obtained with the irradiated skin treated with 01 carcinine is 014 OD unitsmin (B) Protection of the SOD activity of isolatedporcine ear dermis-epidermis treated with various concentrations of an imidazole-containing peptidomimetic Average plusmn SEM from 10 independent experiments are given significant differences ( p lt0001) with control(Studentrsquos t-test) Percent of protection is calculated by comparing with the SOD activity of a non-irradiated skin

Figure 7 Effect of oxygen and SOD on the level of free radical derivatives of methylglyoxal and dialklylimine a) Decrease under aeration conditions of the level of MGˉ˙ and dialkylimine cation radical in the absence (1) and presence of SOD (2) Reaction medium composition is the same as in Fig 3a b) EPR spectrum ofSOD containing reaction medium (400 Uml) 8 min after the mixing of lysine and methylglyoxal EPR spectra were recorded under nitrogen purging (1) the same sample 2 min after the beginning of aeration (2) simulation of the spectrum of methylglyoxal anion radical (3) Closed squares on curve 2 (panel (a)) correspond to EPR signals analogous to spectrum 1 (panel (b))open squares correspond to the signal analogous to spectrum 2(panel (b))



anion radical [76] This fact confirms the above assumption that the free radical intermediates of L-lysine reaction with methylglyoxal are MGˉ˙ and the cation radical of dialkylimine Thus molecular oxygen seems to interact directly with the free radical derivatives of methylglyoxal and dialkylimine and the products formed in this reaction are not registered by EPR (Figure 7 (panel a)) However SOD protects the anion radical of methylglyoxal under aerobic conditions which points to the possibility of MGˉ˙ elimination under the effect of superoxide Indeed it has been established that in aqueous media О2ˉ˙ reduces some organic radicals [77] and catalyzes protonation and disproportionation of nitrobenzene anion radical [76] By analogy it can be supposed that superoxide radical interacts with the protonated semidione of methylglyoxal reducing it in accordance with the reaction

(reaction 2)

Chemical and 3-D chemical structures of N-acetylcarnosine l-carnosine and carcinine

Figure 8a shows the formula structure and the energy-minimized 3-D conformation of l-carnosine derived from the chemical structure using space filling model Due to energy differences determined by molecular mechanics PM3 semi-empirical quantum mechanics among different conformations of the natural imidazole-containing

peptidomimetics a dynamic equilibrium of energetically permissible C-linked and N-linked analogs of rotamers exists in aqueous solution The resulting minimized structures indicate that a common characteristic for all the calculated conformations for peptidomimetics is that a claw-like structure of every compound results in proper stabilization and for the possible metal chelating such as when iron (Fe2+)- natural imidazole containing compound complex is obtained (Figure 8b) The data provide the hypothesis supported by 3-D molecular conformational studies that Fe 2+ can be enveloped inside the natural peptidomimetic The claw-like structure of the imidazole-containing molecules and relevant bound activities can lie in the basis of the antioxidant (free-radical scavenging and aldehyde scavenging) properties of the studied imidazole-containing compounds

Effect of L-Carnosine on the decrease of ferrous iron (ferroxidase activity)

L-Carnosine accelerated the decrease of ferrous iron in the ferrous sulfate solution in a concentration-dependent mode of 5ndash20mM l-carnosine pronounced by the 10ndash30 min of incubation (Figure 9a) The kinetic curves presented in Figure 9a demonstrate that there is a dose-dependent increase in the rate of ferrous iron disappearance A strong ferrous iron chelator 33 330 μM EDTA showed a complete decrease of the accessible to 110-o-phenanthroline ferrous ions by the second minute

Figure 8a L-Carnosine energy-minimized structure (ball and stick model)

Figure 8b L-Carnosine- Fe 2+ energy-minimized structure (ball and stick model)



Figure 9a Effect of l-carnosine on the decrease of ferrous iron determined by10-o-phenanthroline assay in the presence of 125 μM ferrous sulfate The data points are the means of two independent determinations and are representative of three independent experiments The standard error of the mean value for each point is le3 of the mean value Details of incubations are presented in Materials and Methods Samples taken at zero time and at the time intervals indicated and were used immediately for measurements (a) ()- Fe 2+ control incubation () ndash Fe 2+ + l-carnosine (5 mM) () ndash Fe 2+ + l-carnosine (10 mM) () ndash Fe 2+ + l-carnosine (20 mM)

Figure 9b Effect of l-carnosine on the decrease of ferrous iron determined by 110-o-phenanthroline assay in the presence of 125 μM ferrous sulfate The data points are the means of two independent determinations and are representative of three independent experiments The standard error of the mean value for each point is le3 of the mean value Details of incubations are presented in Materials and Methods Samples taken at zero time and at the time intervals indicated and were used immediately for measurements(b) ()- Fe 2+ control incubation (in the absence of EDTA) ( ) Fe2+ + EDTA (33 μM) ( )-Fe2+ + EDTA (330μM)



after EDTA addition to the ferrous sulfate solution (Figure 9b curves 56) The rates of decrease of ferrous iron accessible to 110-o-phenanthroline in the presence of l-carnosine are indicative on the autooxidation of ferrous iron (ferroxidase-like activity) of l-carnosine at higher or equal to 5 mM concentrations (Figure 9a curves 2-4) L-Carnosine chelatingferroxidase activity appears weaker than that of EDTA but it is competitive with ferrous iron chelating activity shown by 110-o-phenanthroline Based on the high affinity properties of 110-o-phenanthroline to bind preferably ferrous but not Fe 3+ ions there is a potential preference for Fe 2+ autooxidationchelating by l-carnosine over Fe 3+ that is important for the rationale of presented later experiments The reference curves (56) in the presence of EDTA (3 and 33 μM) and the curves (2-4) of autooxidation of ferrous iron are displayed on Figure 9a Figure 9b The rate of decrease of ferrous iron below the autooxidation curve indicates that l-carnosine worked as a ferroxidase compound at concentrations (5-20 mM) This model system illustrates the competitive binding of ferrous iron ions with the used ferroxidase compound (carnosine) or another peptide based metal ion chelator (carcinine n-acetylcarnosine) so removing them from detector (110-o-phenanthroline) molecule (data not shown)

Antioxidant Activities of L-carnosine N-acetylcarnosine and Carcinine in the Fe2+ascorbate ndashInduced Lipid Peroxidation in Liposomes Scavenging of Free-radical Species of Oxygen and Aldehydes with L-carnosine N-acetylcarnosine (NAC) and Carcinine

The comparative antioxidant activity of NAC and

l-carnosine was assessed in the liposome peroxidation system catalyzed by Fe 2+ + ascorbate (Figure 10) The accumulation kinetics of molecular LPO products such as MDA and liposomal conjugated dienes and trienes are shown in (Figure 10A- Figure C) The results demonstrate that the LPO reactions in the model system of lipid membranes are markedly inhibited by l-carnosine The effective concentrations of l-carnosine are 10 and 20 mM Data on the biological effectiveness of l-carnosine and carcinine as antioxidants preventing PC liposome or linoleic acid peroxidation in physiological concentration ranges of 5ndash25 mM have already been published [566465] The scavenging of lipoperoxide-derived free radicals with l-carnosine and carcinine during the peroxidation of linoleic acid and PC liposomes in the peroxidizing system Fe2+ascorbate was documented (Table 1 Table 2) Figure 10A shows that the level of TBA reactive substances (TBARS) reached at 5-min incubation decreases in the presence of l-carnosine (10 or 20 mM) at 10 min and at later time points (20 mM) which must be due to a loss of existing TBARS or peroxide precursors of MDA and not due to a decreased formation of peroxide compounds The ability of the histidine-containing compound NAC to inhibit the (Fe2++ ascorbate)-induced oxidation of PC liposomes was compared with that of equimolar concentrations of l-carnosine The antioxidant activity of 10 and 20 mM NAC corresponded to 38 and 55 inhibition of LPO for the two concentrations after 60-min incubation NAC exhibited less antioxidant protection than l-carnosine corresponding to 60 and 87 of the equimolar (10 or 20 mM) l-carnosine inhibition percentage Lipid peroxidase activity of NAC was less pronounced than of L-carnosine (Figure 10B) However since N-acetylcarnosine can act as

Figure 10 Accumulation of lipid peroxidation products (TBARS measured as MDA) (A) diene conjugates (B) triene conjugates and ketone and aldehyde products (274 nm absorbing material) (C) in liposomes (1 mgml) incubated for 60 min alone (6 dotted line) and with addition of the peroxidation-inducing system of Fe2+ + ascorbate (1) Antioxidants N-acetylcarnosine (NAC) (10 or 20 mM) (2 3) or l-carnosine (10 or 20 mM) (4 5) were added at the fifth minute of the incubation period to the system containing the peroxidation inducers Samples were taken at zero time and at time intervals indicated in the figures and were used immediately for measurement of TBARS (see lsquolsquoMaterials and methodsrsquorsquo) A similar amount of sample was partitioned through chloroform and used for detection of conjugated dienes and trienes dissolved in 2 ndash 3 ml of methanolndash heptane mixture (5 1 vv)



a time release version metabolized into l-carnosine during its topical and external application to the ocular tissues (but not oral use) the antioxidant activity of NAC in vivo application is significantly increased Once released from NAC in tissues l-carnosine might act against peroxidation during its ophthalmic target pharmaceutical use [78]

Reactivity of l-carnosine and carcinine with lipid hydroperoxide Lipid peroxidase-like activity of imidazole-containing peptidomimetics

The lipid peroxidase-like effect of carnosine and carcinine was preliminary demonstrated [56] The lipid peroxidase-like activity was described as a reduction activity of fatty acid hydroperoxide into the alcohol form that was assayed by TLC analysis The same reducing effect (alcohol formation from hydroperoxides) was found now in a biphasic model system in which the oxidative stress was generated by the 13(S) linoleic acid hydroperoxide (liposoluble) and the target of the oxidation was a sample water soluble protein (bovine serum albumin BSA) The in vitro model system described in Material and methods shows the reaction of linoleic acid hydroperoxide (LOOH) with BSA The reaction products were analyzed by HPLC (Figure 11A- Figure 11C)

Figure 11A Figure 11B show representative chromatograms in quantitative analysis of lipid linoleic acid hydroperoxide and its reduced with NaBH4 alcohol (LOH) product The incubation of BSA with a lipid hydroperoxide would result in the formation of characteristic peaks and indeed numerous polar low-molecular weight degradation products which would not appear when the BSA protein or the peroxide were incubated alone could be detected at 205 nm (Figure 11C) The formation of the reduced product LOH when linoleic hydroperoxide alone was incubated with the imidazole-containing peptidomimetic was also monitored with the HPLC technique The HPLC spectra revealed that carcinine acting as the chemical chaperone would avoid the formation of low-molecular-weight degradation products of BSA and that concomitantly LOH was formed (Figure 11D Figure 11E) It was verified that LOH is harmless for the

protein no breakdown products were observed when BSA was incubated during an extended period of time (12 days) with the reduced form The HPLC analysis substantiates the ability of the naturally occurring imidazole-containing peptidomimetics to reduce (LOOH) into non-toxic alcohols (LOH) The reduction of various lipid hydroperoxides may result from the cleavage of lipid hydroperoxide with a transition metal complex of l-carnosine (carcinine) and supplement with electrons for the reductive reaction LOOH----not LOH [56] The commonly used lipophilic antioxidant vitamin E being only capable of free radical scavenging is therefore ineffective once hydroperoxides are formed

This unique lipoperoxidase activity of imidazole-containing dipeptides as chemical chaperones is correlated with the protection of protein against oxidative cross-linking induced by these toxic lipid peroxides This was demonstrated using SDS-PAGE electrophoresis (Figure 12A) For this purpose the representative protein BSA was incubated in the presence of the chemically well-defined 13(S)-linoleic acid hydroperoxide and in a similar fashion as before the proteinrsquos cross-linking was observed after 2 days of incubation (Figure 12A lane 2) Here again carcinine and l-prolylhistamine (endowed with lipid peroxidase activities and being both strong aldehyde quenchers and chemical chaperones) (lanes 3 and 6) were able to protect the protein while at the same concentrations l-carnosine N-acetyl-β-alanylhistamine or vitamin E were uneffective (lanes 4 5 and 7) Vitamin E cannot act with lipid peroxidase activity and is not an aldehyde quencher in the conditions used

In another experiment the imidazole-containing dipeptides were introduced in the peroxidized liposome mixture The representative protein BSA was then added and incubated for 2 days The protective effect was illustrated by electrophoretic monitoring of the protein molecular weight (Figure 12B) After 2 days of incubation phospholipid peroxides (Figure 12B lane 3) induced protein cross-linking (and to some extent degradation) as indicated by the formation of a multimolecular weight diffuse band around 66 kDa Interestingly carcininersquos (lanes 4 and 5) protective effect was far superior to l-carnosinersquos (lanes 6 and 7) which gave very poor results with this experiment l-Prolylhistamine was the most effective peptidomimetic while N-acetyl-β-alanylhistamine was almost uneffective In these experimental conditions the reference lipophilic antioxidant vitamin E was also completely unable to protect BSA from this kind of cross-linking This test shows that lipid peroxides break down into free radicals and toxic amphiphilic aldehydes resulting in the spread of the oxidative stress from the oily phase (lipid hydroperoxides) to the water phase leading to the oxidation of surrounding proteins (eg collagen BSA SOD etc)

Protection of SOD-like activity with carcinine treatment of the skin after UVA-UVB irradiation

The effectiveness of natural imidazole-containing peptidomimetics to sustain the protein enzyme conformation and activity and in vivo was demonstrated with an ex vivo study performed on a porcine dermis-epidermis fraction

Compound tested at concentration Inhibition of MDA release from oxidative25 mM degradation of linoleic acidl-Carnosine (β-alanyl-l-histidine 59Carcinine (β-alanylhistamine) 47

Detailed experimental procedures are described in Ref 56 Each result represents the mean of 5 experiments Table 1 Percentage of inhibition obtained by comparison with a control experiment with no antioxidant

Compound tested at concentration 10 mM Inhibition of MDA release from oxidative degradation of PC liposomesl-Carnosine (β-alanyl-l-histidine) 53Carcinine (β-alanylhistamine) 42

Detailed experimental procedures are described in Ref 56 Each result representsthe mean of 5 experimentsTable 2 Percentage of inhibition obtained by comparison with a control experiment with no antioxidant



Skin tissues were UV-irradiated (UVA-UVB) and the resulting inactivation of SOD [79] was monitored The oxidative deactivation of SOD in cutaneous cells during a UV irradiation represents both the decrease of a part of the skinrsquos natural antioxidant defenses and the increase of the oxidative stress impact Results obtained with a carcinine treatment are shown in Figure 6A The protective effect of carcinine demonstrated as example on Figure 6A is about 43 ( p lt0001 n =10) The antioxidants were applied as a cream on the tissues prior to irradiation The protective effect was evaluated by measuring the catalytic activity of the SOD after extraction from the cells (Figure 6B) According to the method described in the Materials and methods section a SOD-like activity was measured from the extracts and a pure commercial SOD was used as the reference for quantitation In the ex vivo test the treatment with carcinine containing creamsconfers to the skin a

significant protection against the oxidative stress induced by UVA-UVB irradiation Carcinine in applied creams do not absorb in UVA (320ndash400 nm) or UVB (280ndash320 nm) regions and the action is different from the UV filters The protection of natural skin defenses by a chemical chaperone carcinine such as SOD activity provides the facility of the skin to withstand the oxidative stress such as UV irradiation glycation and aging

Our more recent results (data not shown) also suggest that one of the chemical mechanisms responsible for the aggregated SOD toxicity may be modification by AGEs ie the Maillard reaction Moreover our data also show that at least some of the SOD molecules probably toxic or mutant SOD1 occurring in inclusions in diseases may be modified by the insoluble and deleterious AGEs Therefore formation of the AGE-modified SOD could result in higher toxicity while oxidative stress and protein nitration due to

Figure 11 (A) HPLC spectrum of 13(S) linoleic acid hydroperoxide in a phosphate buffer solution (01 M pH 73) after 15 min of incubation at 37ordmC Absorbance wavelengths used 234 and 205 nm (B) HPLC spectrum of 13(S) hydroxy linoleic acid phosphate buffer solution (01 M pH 73) Monitoring absorbance wavelength used 234 nm (C) HPLC monitoring of protein (BSA) oxidation degradation by linoleic acid hydroperoxide (LOOH) (D) Correlation of the natural imidazole-containing peptidomimetic protective effect with linoleic acid hydroperoxide (LOOH) reduction (E) HPLC spectra recorded at 234 nm wavelength BSA (033 gl) in 01 M phosphate buffer pH=73 was incubated with 15 mM 13(S)-linoleic acid hydroperoxide and 5 mM carcinine during 60 h at 37ordm C



Figure 12 (A) SDS-PAGE of BSA exposed to 13(S)-linoleic acid hydroperoxide 1 BSA control 2 BSA+LOOH 3 BSA+LOOH+carcinine 4 BSA+ LOOH+l-carnosine 5 BSA+LOOH+N-acetyl-β-alanylhistamine 6 BSA+ LOOH+l-prolylhistamine 7 BSA+LOOH+vitamin E Gel silver stain method (B) SDS-PAGE of BSA exposed to peroxidized liposomes after treatment with different imidazole-containing antioxidants 1 BSA control 2 BSA and non-oxidized liposomes 3 BSA and oxidized liposomes 4 BSA oxidized liposomes and 1 equiv (versus ROOH) of carcinine 5 BSA oxidized liposomes and 2 equiv of carcinine 6 BSA oxidized liposomes and 1 equiv of l-carnosine 7 BSA oxidized liposomes and 2 equiv of l-carnosine 8 BSA oxidized liposomes and 1 equiv of N-acetyl-β-alanylhistamine 9 BSA oxidized liposomes and 2 equiv of N-acetyl-β-alanylhistamine 10 BSA oxidized liposomes and 1 equiv of l-prolylhistamine 11 BSA oxidized liposomes and 2 equiv of l-prolylhistamine 12 BSA oxidized liposomes and 1 equiv of vitamin E 13 BSA oxidized liposomes and 2 equiv of vitamin E Gel stained with silver



peroxynitrite may be prevented or reversed with imidazole-containing peptidomimetics in SOD-linked disease in human or mouse by concomitant mechanisms described in this study

Transglycating Activities of Imidazole-containing Peptide-based Compounds

The ability of decarboxycarnosine (carcinine) to behave as an ldquoacceptor moleculerdquo for transglycation was determined using a specific experimental design (Figure 3) A model glycosylamine namely Glucosyl-ethylamine was synthesized and the transfer of the glycosyl moiety to decarboxycarnosine (carcinine) (formation of glucosyl-decarboxycarnosine) or related imidazole-containing peptidomimetics was monitored by carbon Nuclear Magnetic Resonance (13C NMR) spectroscopy (see Materials and Methods 13C NMR experiments section) Reaction between ethylamine and D-glucose leads to the formation of the model glycosylamine glucosyl-ethylamine obtained as a mixture of stereoisomers the beta being predominant in equilibrium with some starting material (szlig-Glc amp α-Glc) Glucosyl-ethylamine is unambiguously identified by the presence of a doublet due to the 13C-15N spin-spin coupling (ie sect3 isotopically enriched starting material was used for the synthesis of the model glycosylamine) The experiment was conducted in slightly alkaline conditions (pH 85) in order to insure optimum stability of the glycosylamine (limitation of spontaneous deglycosylation during NMR analysis) Addition of decarboxycarnosine results in the loss of the characteristic doublet (Figure 3) which is indicative of the cleavage of the covalent bond between ethylamine and the glucosyl moiety Appearance of a new single peak with a chemical shift near to glucosyl-ethylamine doublet is consistent with the formation of the transglycation product glucosyl-decarboxycarnosine (G-Decarboxy C) [718081] More accurately both glycosylamines (szlig-G-E amp α-G-E the major and minor stereoisomers respectively) undergo transglycation in the presence of decarboxycarnosine Another new minor single peak is observed near 87 ppm corresponding to the transglycation product α-glucosyl-decarboxycarnosine

Interestingly subunits of decarboxycarnosine (szlig-alanine imidazole) had very limited or no transglycating properties (data not shown) It can be hypothesized that a particular molecular arrangement participates to the stabilization of glucosyl-decarboxycarnosine A kinetic study was conducted in order to better correlate the doublet peak disappearance (cleavage of glucosyl-ethylamine) and the appearance of the new singlet (glucosyl-decarboxycarnosine formation) It was found that szlig-G-E disappearance kinetics closely follows the szlig-glucosyl-decarboxycarnosine formation kinetics Similar spectral data although moderately well defined were collected for the minor stereoisomer α-G-E and the corresponding transglycation product α-glucosyl-decarboxycarnosine (data not shown) As a whole the presented data support the following experimental findings

A transglycation 13C NMR study with the model glucosyl-ethylamine has shown that decarboxycarnosine (carcinine) is an effective transglycating agent behaving

as an ldquoacceptor moleculerdquo for glucose and releasing a ldquode-glycosylation productrdquo eg the ldquofree aminerdquo

The data presented show that the transglycating efficiency of the tested carnosine imidazole-containing derivatives (Figure 2) is generally lower than that of carnosine with the exception of leucyl-histidylhydrazide (formula 5) which transglycation activity is markedly higher than of carnosine in the tested objective G-E Schiff base decay system logP value and transglycating efficiency of the derivatives show a good correlation (R2 = 038) The hydrazide moiety of leucyl-histidylhydrazide (formula 5) boosts the aldehyde scavenging efficiency of compound [5970] and in combination with a free Nα-amino group concurs in the disruption of the Schiff base adduct GndashE as a model of protein glycation Further structureactivity relationship details the synergistic efficacy of leucyl-histidylhydrazide (formula 5) in therapeutic applications [58] The data are related to sample supporting the IVP invention of the worldwide patented codrug formulation including N-acetylcarnosine (an ophthalmic prodrug of L-carnosine) and a revealed tripeptide peptidomimetic reversing the glycosylation (glucose-derived intermolecular) crosslinks in proteins (Advanced Glycation End Products (AGEs)) and the Schiff bases for the next- generation treatment of ophthalmic complications of Diabetes Mellitus (DM) such as the development of visual impairment or blindness consequent to cataract formation retinopathy or glaucoma [4658] Diabetes affects the (outer) lens middle (vitreous) and inner (retina) areas of the eye

Susceptibility of imidazole-containing peptide based compounds to human carnosinase activity

In mammals two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far and they have been classified as metallopeptidases of the M20 family Human CN1 was identified as a dipeptidase that hydrolyzes Xaa-His dipeptides including those with first residues β-Ala (carnosine) γ-aminobutyric acid (homocarnosine) N-methyl-β-Ala Ala and Gly On the other hand CN2 has a broader specificity than CN1 but it does not hydrolyze homocarnosine and is sensitive to inhibition by bestatin (IC50 7nM) [82] Unlike most other metallopeptidases CN2 requires Mn 2+ for complete activity and Zn 2+ alone cannot activate this enzyme Based on the similarity in primary sequences CN1 and CN2 have been classified as metallopeptidases belonging to the M20 family of clan MH [83] We demonstrate that the synthetic peptides (N-acetylcarnosine L-carnosine leucyl-histidylhydrazide) containing histidine derivatives and pseudodipeptide carcinine are relevant to the activities of the novel genes coding CN1 secreted human carnosinase and the CN2 cytosolic non- specific dipeptidase previously named tissue carnosinase [58] In our issued provided studies [58] the substrate specificity of human carnosinase activity was determined with 18 X-His dipeptides non X-His dipeptides and several His-containing tripeptides at pH 75 Highest enzyme activity was found with carnosine (β-Ala-His) and the other X-His dipeptides served as substrate for this



enzyme including N-Methylcarnosine Ala-His Gly-His and GABA-His (homocarnosine) The non X-His dipeptides β-Ala-Ala Ala-Ala or Ala-Pro as well as tripeptides or tested tripeptide peptidomimetics containing histidine in central or C- terminal position (such as Gly-His-Gly or Gly-Gly-His) or leucyl-histidylhydrazide and other tested histidyl-hydrazide compounds were not degraded indicating that carnosinase is a true X-His dipeptidase

The catalytic efficiencies (kcatKm) of carnosinase activity for carnosine and homocarnosine were 89 mM-1 sec-1 and 13 mM-1 sec-1 respectively When carcinine N-acetylcarnosine or tested histidyl-hydrazide compounds were used no hydrolytic activity was detectable

Results from the studies described in this section provide valuable industrial drug information for optimization of the drugcodrug design and ophthalmic formulation in order to achieve the sustained release of described triple peptide moieties N-acetylcarnosineL-carnosineleucyl-histidylhydrazide during targeted therapy for ocular diseases and diabetic pathology [8485]

DiscussionDiabetic complications such as neuropathy retinopathy

nephropathy and atherosclerosis contribute to the severity of the disease and the mortality of diabetic patients the clinical characteristics of these complications include hyperglycemia hyperlipidemia oxidation stress cytokine imbalance and coagulation predomination [86-89] It was shown that oxidation stress advanced glycation processes inflammation and blood coagulation are strongly associated with diabetes [89-91] and all are involved in the development of diabetic complications Thus it is very important to control these risk factors and biological reactions to delay diabetic deterioration

Possible sources of oxidative stress and damage to proteins in diabetes include free radicals generated by autoxidation reactions of sugars and sugar adducts to protein and by autoxidation of unsaturated lipids in plasma and membrane proteins The oxidative stress may be amplified by a continuing cycle of metabolic stress tissue damage and cell death leading to increased free radical production and compromised free radical inhibitory and scavenger systems which further exacerbate the oxidative stress Structural characterization of the cross-links and other products accumulating in collagen in diabetes is needed to gain a better understanding of the relationship between oxidative stress and the development of complications in diabetes Such studies may lead to therapeutic approaches for limiting the damage from glycation and oxidation reactions and for complementing existing therapy for treatment of the complications of diabetes Free amino groups of proteins react slowly with reducing sugars such as glucose by the glycation or Maillard reaction to form poorly characterized brown fluorescent compounds This process is initiated by the condensation reaction of reducing sugars with free amino groups to form Schiff bases which undergo rearrangement to form the relatively stable Amadori products [9293] The Amadori products subsequently degrade into α-dicarbonyl

compounds deoxyglucosones [94] These compounds are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form cross-links stable end products called advanced Maillard products or advanced glycation end products (AGEs) AGEs are irreversibly formed and found to accumulate with aging atherosclerosis and diabetes mellitus especially associated with long-lived proteins such as collagens [9596] lens crystallines [9798] and nerve proteins [99100] It was suggested that the formation of AGEs not only modifies protein properties but also induces biological damage in vivo [101-105] For example AGEs deposited in the arterial wall could themselves generate free radicals capable of oxidizing vascular wall lipids and accelerate atherogenesis in hyperglycemic diabetic patients [104 105] The molecular structures of some AGEs have been identified as pentosidines [106- 110] pyrrole derivatives [111] pyrazine derivatives [112113] and Nε-carboxymethyllysine [114-118] In the presence of molecular oxygen the formation of these products from sugars is catalyzed by transition metal ions via glycoxidation which oxidizes Amadori products to Nε-carboxymethyllysine [114115] and the autoxidation of glucose which produces superoxide radical anions (О2ˉ˙) H2O2 and α-ketoaldehydes [7119-122] The major pathways of glycation reaction-mediated damage to macromolecules therefore involve both nonoxidative and oxidative processes Their individual contributions to biological damage however are not well understood The formation of α-dicarbonyl compounds seems to be an important step for cross-linking proteins in the glycation or Maillard reaction To elucidate the mechanism for the cross-linking reaction we studied the reaction between a three-carbon α-dicarbonyl compound methylglyoxal and amino acids Our former results showed that this reaction generated yellow fluorescent products as formed in some glycated proteins [59] In addition a few types of free radical species were also produced and their structures were determined by EPR spectroscopy These free radicals are 1) the cross-linked radical cation 2) the methylglyoxal radical anion as the counterion and 3) the superoxide radical anion produced only in the presence of oxygen [73] The generation of the crosslinked radical cations and the methylglyoxal radical anions does not require metal ions or oxygens These results indicate that dicarbonyl compounds cross-link free amino groups of protein by forming Schiff bases which donate electrons directly to dicarbonyl compounds to form the cross-linked radical cations and the methylglyoxal radical anions

Oxygen can accept an electron from the radical anion to generate a superoxide radical anion (О2ˉ˙ ) which can initiate damaging chain reactions Thus it is most likely that oxidative modification of proteins and other biomolecules might be the consequence of local generation of superoxide on the interaction of the residues of L-lysine (and probably other amino acids) with α-ketoaldehydes This phenomenon of non-enzymatic superoxide generation might be an element of autocatalytic intensification of pathophysiological action of carbonyl stress Glycation generation of advanced glycosylation end-products (AGEs)



and formation of protein carbonyl groups play important roles in aging diabetes its secondary complications and neurodegenerative conditions Carnosine has the potential to suppress many of the biochemical changes (eg protein oxidation glycation AGE formation and cross-linking) that accompany aging diabetes and associated pathologies Due to established carnosinersquos molecules antiglycating activity reactivity toward deleterious carbonyls zinc- and copper-chelating ferroxidase type of activities and low toxicity carnosine and related structures could be effective against age-related protein carbonyl stress

This paper comments on the relative efficacy of the potent imidazole-containing therapeutic agents towards diabetic conditions addressing the molecular damages that are presumed to result from the covalent attachment of glucose to amino groups in line with the mindset of the major pharmaceutical companies that seek a single critical molecular target for their drugs in the management of Type 2 diabetes metabolism We have considered that the fragmentation and conformational molecular changes observed in diabetes are dependent upon hydroxyl radicals produced by glucose autoxidation or some closely related process and that imidazole-containing antioxidants dissociate structural damage caused by the exposure of glucose (or glycating ketoaldehyde compound) to protein from the incorporation of monosaccharide into protein We have also provided further support that glycofluorophore formation is dependent upon metal-catalysed oxidative processes associated with ketoaldehyde formation and the considered family of transglycating imidazole-containing compounds exerts aldehyde-scavenging free radical-scavenging and transition metal ions chelating activities (or ferroxidase type of activity relevant for carnosine) Our experimental glycation reaction is an adequate model of tissue damage occurring in diabetes mellitus so these studies indicate a therapeutic role for imidazole-containing antioxidants (non-hydrolized carnosine carcinine D-carnosine ophthalmic prodrug N-acetylcarnosineleucyl-histidylhidrazide and patented formulations thereof) in therapeutic management strategies for Type 2 Diabetes

In this study we suggest that a broad-brush multisite attack should be employed in the treatment of diabetes complications with imidazole-containing compounds based upon the revealed basic biology of the complications of Diabetes-specific Program that encompasses provided basic and clinical research The authors propose that our atented imidazole-containing therapeutic agents in formulations are acting as anti-inflammatory compounds which are also representing a universal form of antioxidant that chelates or inactivates metal ions in this way inhibiting superoxide- mediated biochemical mechanisms for oxygen free radical formation through the inhibition of free-radical propagation chain reactions in addition possess anti (trans)glycating activity with the ability to scavenge dicarbonyls such as methylglyoxal suppress advanced glycation end product formation and reactivity and exert the repairing biological membranes lipid peroxidase type of activity demonstrated in this study It should be noted that the therapeutic agents

also supress or inhibit the principal factors that promote the accumulation of altered proteins and which accompany (or cause) human and animal aging A particular example is the developed non-hydrolized forms of carnosine and carcinine which are naturally found in the brain and muscles of mammals birds fish or crustacea sometimes at surprisingly high concentrations [123124] It has been proposed that carnosine can inhibit generation of many of the protein alterations accompanying aging [125] diabetes and its complications [126]

There is an evidence from the recently published studies that the systemic release of L-carnosine from the ophthalmic prodrug N-acetylcarnosine applied topically to the eyes of patients with sight-threatening eye disorders or L-carnosine leaking out from skeletal muscle during physical exercise affects autonomic neurotransmission improves visual performance organ functions and physiological functions acting through the hypothalamus anatomical nuclei (Figure 13) [127-130] In particular L-carnosine affects the activity of sympathetic and parasympathetic nerves innervating the adrenal glands liver kidney pancreas stomach and white and brown adipose tissues thereby causing changes in blood pressure blood glucose appetite lipolysis and thermogenesis Carnosine-mediated changes in neurotransmission and physiological functions were eliminated by histamine H1 or H3 receptor antagonists (diphenhydramine or thioperamide) and bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN) a master circadian clock Moreover a carnosine-degrading enzyme (carnosinase 2) was shown to be localized to histamine neurons in the hypothalamic tuberomammillary nucleus (TMN) Thus L-carnosine or carcinine released ophthalmically through the systemic absorption from conjunctival sac of the eye upon the topical instillation of lubricant eye drops or from skeletal muscle during exercise may be transported into TMN-histamine neurons and hydrolyzed The resulting L-histidine may subsequently be converted into histamine which could be responsible for the effects of L-carnosine on neurotransmission and physiological function Thus carnosine appears to influence hypoglycemic hypotensive and lipolytic activity through regulation of autonomic nerves and with the involvement of the SCN and histamine These findings are important and discussed herewith in the context of the present and other recent reports including those on carnosine synthetases carnosinases and carnosine systemic absorption and transport [127-130]

Finally we have developed and patented a number of carnosine mimetics with the apparent anti-diabetes and anti-aging activity which possibly derives from their pluripotency although their potential efficacy as targeted pharmaceuticals andor a dietary supplement in the specific formulations in humans has also been claimed [465859129]

ConclusionGlucose and α-dicarbonyl compounds chemically

attach to proteins and nucleic acids without the aid of enzymes Initially chemically reversible Schiff base and



Amadori product adducts form in proportion to glucose concentration Subsequent reactions of the Amadori product slowly give rise to nonequilibrium advanced glycosylation end-products which continue to accumulate indefinitely on longer-lived molecules Excessive formation of both types of nonenzymatic glycosylation product appears to be the common biochemical link between chronic hyperglycemia and a number of pathophysiologic processes potentially involved in the development of long-term diabetic complications

The major biological effects of excessive nonenzymatic glycosylation are leading to increased free radical production

and compromised free radical inhibitory and scavenger systems inactivation of enzymes inhibition of regulatory molecule binding crosslinking of glycosylated proteins and trapping of soluble proteins by glycosylated extracellular matrix (both may progress in the absence of glucose) decreased susceptibility to proteolysis abnormalities of nucleic acid function altered macromolecular recognition and endocytosis and increased immunogenicity This study demonstrates the progress in development of patented carnosine mimetics resistant in formulations to enzymatic hydrolysis with human carnosinases that are acting as a universal form of antioxidant and deglycatingtransglycating

L-carnosine N-acetylcarnosine ophthalmic prodrug

Figure 13 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) Carnosine is not only a radical scavenger but also 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 Since the tuberomammillary nucleus is the exclusive origin of histaminergic neurons several groups of authors have investigated 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 It has been revealed that CN2 is highly expressed in the histaminergic neurons in the tuberomammillary nucleus implying that it may supply histidine to histaminergic neurons for histamine synthesis [128-130] This mechanism could be responsible for the effects of L-carnosine on autonomic neurotransmission and physiological function of pancreas stimulating in vivo regeneration of insulin-producing beta-cells Thus L-carnosine might stimulate insulin secretion and appears to influence hypoglycemic hypotensive and lipolytic activity through regulation of autonomic nerves and with the involvement of the hypothalamic suprachiasmatic nucleus (SCN)



agent that inhibits sugar-mediated protein cross-linking and also chelate or inactivate a number of transition and heavy metal ions (including copper and ferrous ions) Carnosine biological mimetics react with methylglyoxal and they have been described in this study as a glyoxalase mimetics The imidazole-containing carnosine biological mimetics can react with a number of deleterious aldehydic products of lipid peroxidation and thereby suppress their toxicity Carnosine and carcinine can also react with glycated proteins and inhibit advanced glycation end product formation [465859129] Such studies may lead to therapeutic approaches for limiting the damage from glycation and oxidation reactions with developed and patented carnosine mimetics and pharmaceutical and consumer healthcare formulations thereof and for complementing existing therapy for treatment of the complications of diabetes

AcknowledgementThis work was planned organized and supported by

Innovative Vision Products Inc (County of New Castle DE USA) Innovative Vision Products Inc is a Pharmaceutical and Nanotechnology Development Company with a focus on innovative chemical entities drug delivery systems and unique medical devices to target specific biomedical applications Over the last decade IVP has developed a track record in developing these technologies to effectively address the unmet needs of specific diseased populations The biologically significant applications of carnosine mimetics including those in ophthalmology were patented by Dr Babizhayev and the alliance Groups (WO 2004028536 A1 WO 9419325 WO 9512581 WO 2004064866 A1)

DisclosureThe described extensive therapeutic modalities through

the text of the article utilizing the topical ophthalmic formulations of N-acetylcarnosine carcinine lubricant eye drops oral formulations of non-hydrolyzed carnosine andor carcinine and their biomedical uses are the subject of the issued and pending International Patents (WO 2004028536 A1 WO 9419325 WO 9512581 WO 2004064866 A1)

Conflict of InterestDeclaration of interest The author (Dr Mark A

Babizhayev) reports 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 Recently the certain appeals have been made to the President of the USA Japanese Prime Minister Abe HM British Queen Elizabeth II about the Genealogical studies inherent to Dr Mark Babizhayev and the Members of his Family The inheritance has been unequivocally proved with the number of relevant modern and classical approaches Dr Mark Babizhayev is the Senior Great Grandson of Wallis Simpson (Bessie Wallis Warfield-Spencer-Simpson) USA wife of King of UK Edward VIII amp of The King of UK Edward VIII ldquoDavidrdquo (Edward Albert Christian George Andrew Patrick David) Prince of Wales UK Duke amp Duchess of Windsor In other

words Dr Mark Babizhayev has an ldquoUnofficial Hereditary Status of a King of UKrdquo and is the Major relative of the British Royal Family The data are described shortly on The Twitter Albert II Grimaldimarkinmonaco httpstwittercommarkinmonacowith_replies

Recently the certain appeals have been made to the President of the USA Japanese Prime Minister Abe HM British Queen Elizabeth II about the Genealogical studies inherent to Dr Mark Babizhayev and the Members of his Family The inheritance has been unequivocally proved with the number of relevant modern and classical approaches

Dr Mark Babizhayev is the Senior Great Grandson of Wallis Simpson (Bessie Wallis Warfield-Spencer-Simpson) USA wife of King of UK Edward VIII amp of The King of UK Edward VIII ldquoDavidrdquo (Edward Albert Christian George Andrew Patrick David) Prince of Wales UK Duke amp Duchess of Windsor

References1 World Health Organisation Department of

Noncommunicable Disease Surveillance (1999) ldquoDefinition Diagnosis and Classification of Diabetes Mellitus and its Complicationsrdquo

2 Sheetz MJ King GL Molecular understanding of hyperglycemiarsquos adverse effects for diabetic complications JAMA 2002 Nov 27288(20)2579-88 JAMA 2003 Apr 9289(14)1779-80 author reply 1780

3 Vlassara H (2005) Advanced glycation in health and disease role of the modern environment Ann N Y Acad Sci 1043 452-460

4 Vlassara H Palace MR (2002) Diabetes and advanced glycation endproducts J Intern Med 251 87-101

5 Peppa M Vlassara H (2005) Advanced glycation end products and diabetic complications a general overview Hormones (Athens) 4 28-37

6 Fu MX Requena JR Jenkins AJ Lyons TJ Baynes JW et al (1996) The advanced glycation end product Nepsilon-(carboxymethyl)lysine is a product of both lipid peroxidation and glycoxidation reactions J Biol Chem 271 9982-9986

7 Wolff SP Dean RT (1987) Glucose autoxidation and protein modification The potential role of lsquoautoxidative glycosylationrsquo in diabetes Biochem J 245 243-250

8 Anderson MM Requena JR Crowley JR Thorpe SR Heinecke JW (1999) The myeloperoxidase system of human phagocytes generates Nepsilon-(carboxymethyl)lysine on proteins a mechanism for producing advanced glycation end products at sites of inflammation J Clin Invest 104 103-113

9 Vander Jagt DL Hassebrook RK Hunsaker LA Brown WM Royer RE 2001 Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I roles for glutathione in both enzymes and implications for diabetic complications Chem Biol Interact 2001 130-132 549-562

10 Thornalley PJ (2005) Dicarbonyl intermediates in the maillard reaction Ann N Y Acad Sci 1043 111-117

11 Vander Jagt DL Hassebrook RK Hunsaker LA Brown WM Royer RE Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I roles for glutathione in both enzymes and implications for diabetic complications Chem Biol Interact 2001 130-132 549-562

12 Peppa M Raptis SA (2008) Advanced glycation end products and cardiovascular disease Curr Diabetes Rev 4 92-100



13 Uribarri J Cai W Peppa M Goodman S Ferrucci L et al (2007) Circulating glycotoxins and dietary advanced glycation endproducts two links to inflammatory response oxidative stress and aging J Gerontol A Biol Sci Med Sci 62 427-433

14 Stadtman ER (1992) Protein oxidation and aging Science 257 1220-1224

15 Finkel T Holbrook NJ (2000) Oxidants oxidative stress and the biology of ageing Nature 408 239-247

16 Yamagishi S Ueda S Matsui T Nakamura K Okuda S (2008) Role of advanced glycation end products (AGEs) and oxidative stress in diabetic retinopathy Curr Pharm Des 14 962-968

17 Bhatwadekar AD Glenn JV Li G Curtis TM Gardiner TA et al (2008) Advanced glycation of fibronectin impairs vascular repair by endothelial progenitor cells implications for vasodegeneration in diabetic retinopathy Invest Ophthalmol Vis Sci 49 1232-1241

18 Vasan S Foiles PG Founds HW Therapeutic potential of AGE inhibitors and breakers of AGE protein cross-links Expert Opin Investig Drugs 2001 Nov10(11)1977-87

19 Forbes JM Soulis T Thallas V Panagiotopoulos S Long DM et al (2001) Renoprotective effects of a novel inhibitor of advanced glycation Diabetologia 44 108-114

20 Wilkinson-Berka JL1 Kelly DJ Koerner SM Jaworski K Davis B et al (2002) ALT-946 and aminoguanidine inhibitors of advanced glycation improve severe nephropathy in the diabetic transgenic (mREN-2)27 rat Diabetes 51 3283-3289

21 Booth AA Khalifah RG Todd P Hudson BG In vitro kinetic studies of formation of antigenic advanced glycation end products (AGEs) Novel inhibition of post-Amadori glycation pathways J Biol Chem 1997 Feb 28272(9)5430-7

22 Stracke H Hammes HP Werkmann D Mavrakis K Bitsch I et al (2001) Efficacy of benfotiamine versus thiamine on function and glycation products of peripheral nerves in diabetic rats Exp Clin Endocrinol Diabetes 109 330-336

23 Onorato JM Jenkins AJ Thorpe SR Baynes JW Pyridoxamine an inhibitor of advanced glycation reactions also inhibits advanced lipoxidation reactions Mechanism of action of pyridoxamine J Biol Chem 2000 Jul 14275(28)21177-84

24 Nakamura S Makita Z Ishikawa S Yasumura K Fujii W et al (1997) Progression of nephropathy in spontaneous diabetic rats is prevented by OPB-9195 a novel inhibitor of advanced glycation Diabetes 46 895-899

25 Schwedler SB Verbeke P Bakala H Weiss MF Vilar J Depreux P Fourmaintraux E Striker LJ Striker GE N-phenacylthiazolium bromide decreases renal and increases urinary advanced glycation end products excretion without ameliorating diabetic nephropathy in C57BL6 mice Diabetes Obes Metab 2001 Aug3(4)230-9

26 Forbes JM Thallas V Thomas MC Founds HW Burns WC Jerums G Cooper ME The breakdown of preexisting advanced glycation end products is associated with reduced renal fibrosis in experimental diabetes FASEB J 2003 Sep17(12)1762-4 Epub 2003 Jul 18

27 Vaitkevicius PV Lane M Spurgeon H Ingram DK Roth GS Egan JJ Vasan S Wagle DR Ulrich P Brines M Wuerth JP Cerami A Lakatta EG A cross-link breaker has sustained effects on arterial and ventricular properties in older rhesus monkeys Proc Natl Acad Sci U S A 2001 Jan 3098(3)1171-5

28 Sebekovaacute K Schinzel R Muumlnch G Krivosiacutekovaacute Z Dzuacuterik R et al (1999) Advanced glycation end-product levels in subtotally nephrectomized rats beneficial effects of angiotensin II receptor 1 antagonist losartan Miner Electrolyte Metab 25 380-383

29 Miyata T van Ypersele de Strihou C Ueda Y Ichimori K Inagi R Onogi H Ishikawa N Nangaku M Kurokawa K Angiotensin II receptor antagonists and angiotensin-converting enzyme inhibitors lower in vitro the formation of advanced glycation end products biochemical mechanisms J Am Soc Nephrol 2002 Oct13(10)2478-87

30 Parving HH Brenner BM Cooper ME de Zeeuw D Keane WF Mitch WE Remuzzi G Snapinn SM Zhang Z Shahinfar S [Effect of losartan on renal and cardiovascular complications of patients with type 2 diabetes and nephropathy] [Article in Danish] Ugeskr Laeger 2001 Oct 1163(40)5514-9

31 Parving HH Hommel E Jensen BR Hansen HP (2001) Long-term beneficial effect of ACE inhibition on diabetic nephropathy in normotensive type 1 diabetic patients Kidney Int 60 228-234

32 Boldyrev AA Severin SE The histidine-containing dipeptides carnosine and anserine distribution properties and biological significance Adv Enzyme Regul 1990 30 175-194

33 Lenney JF (1976) Specificity and distribution of mammalian carnosinase Biochim Biophys Acta 429 214-219

34 Jackson MC Kucera CM Lenney JF (1991) Purification and properties of human serum carnosinase Clin Chim Acta 196 193-205

35 Lenney JF1 (1990) Separation and characterization of two carnosine-splitting cytosolic dipeptidases from hog kidney (carnosinase and non-specific dipeptidase) Biol Chem Hoppe Seyler 371 433-440

36 Kunze N Kleinkauf H Bauer K Characterization of two carnosine-degrading enzymes from rat brain Partial purification and characterization of a carnosinase and a beta-alanyl-arginine hydrolase Eur J Biochem 1986 Nov 3160(3)605-13

37 Aldini G Facino RM Beretta G Carini M Carnosine and related dipeptides as quenchers of reactive carbonyl species from structural studies to therapeutic perspectives Biofactors 200524(1-4)77-87

38 Aldini G Orioli M Carini M Maffei Facino R (2004) Profiling histidine-containing dipeptides in rat tissues by liquid chromatographyelectrospray ionization tandem mass spectrometry J Mass Spectrom 39 1417-1428

39 Orioli M Aldini G Beretta G Facino RM Carini M LC-ESI-MSMS determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and histidine-containing peptides as early markers of oxidative stress in excitable tissues J Chromatogr B Analyt Technol Biomed Life Sci 2005 Nov 15827(1)109-18

40 Reddy VP Garrett MR Perry G Smith MA Carnosine a versatile antioxidant and antiglycating agent Sci Aging Knowledge Environ 2005 May 42005(18) pe12

41 Rashid I van Reyk DM Davies MJ (2007) Carnosine and its constituents inhibit glycation of low-density lipoproteins that promotes foam cell formation in vitro FEBS Lett 581 1067-1070

42 BabizhayevMADeyevAIYermakovaVNSemile-tovYuADavydovaNG KuryshevaNI Zhukotskii AV-Goldman IM N-Acetylcarnosine a natural histidine-con-taining dipeptide as a potent ophthalmic drug in treat-ment of human cataracts Peptides 2001 22 979-994

43 Babizhayev MA Deyev AI Yermakova VN Semiletov YA Davydova NG et al (2002) Efficacy of N-acetylcarnosine in the treatment of cataracts Drugs R D 3 87-103

44 Babizhayev MA Deyev AI Yermakova VN Remenschikov VV Bours J Revival of the lens transparency with N-acetylcarnosine Current Drug Therapy 2006 1 91-116

45 Babizhayev MA1 Deyev AI Yermakova VN Brikman



IV Bours J (2004) Lipid peroxidation and cataracts N-acetylcarnosine as a therapeutic tool to manage age-related cataracts in human and in canine eyes Drugs R D 5 125-139

46 Babizhayev MA 2004 Method for topical treatment of eye disease and composition and device for said treatment PCT Patent Application International Publication Number WO 2004028536 A1 International Publication Date 8 April 2004

47 Babizhayev MA Ophthalmic pharmacology of N-acetylcarnosine lubricant eye drops Journal of Pharmacology and Toxicology 2006 1(3) 201-233

48 Babizhayev MA Rejuvenation of visual functions in older adult drivers and drivers with cataract during a short-term administration of N-acetylcarnosine lubricant eye drops Rejuvenation Research 2004 7 186- 198

49 Babizhayev MA Yermakova VN Deyev AI Seguin M-C Imidazole-containing peptidomimetic NACA as a potent drug for the medicinal treatment of age-related cataract in humans Journal of Anti-Aging Medicine 2000 3 43-62

50 Arnould JM Frentz R Presence isolation and chemical structure of a substance characteristic of cardiac tissue in Carcinus maenas (L) beta-alanylhistamine Comp Biochem Physiol C 1975 Jan 150(1)59-66

51 Brotman DN Flancbaum L Fitzpatrick JC Fisher H Presence of carcinine (szlig-alanylhistamine) in mammalian tissues FASEB J 1989 3 1028

52 Brotman DN Flancbaum L Kang YH Merrill GF Fisher H Positive inotropic effects of carcinine in the isolated perfused guinea pig heart Crit Care Med 1990 18 317ndash321

53 Flancbaum L Brotman DN Fitzpatrick JC Van Es T Kasziba E et al (1990) Existence of carcinine a histamine-related compound in mammalian tissues Life Sci 47 1587-1593

54 Chen Z Sakurai E Hu W Jin C Kiso Y et al (2004) Pharmacological effects of carcinine on histaminergic neurons in the brain Br J Pharmacol 143 573-580

55 Babizhayev MA Biological activities of the natural imidazole-containing peptidomimetics n-acetylcarnosine carcinine and L-carnosine in ophthalmic and skin care products Life Sci 2006 Apr 1178(20)2343-57 Epub 2006 Jan 4

56 Babizhayev MA Seguin MC Gueyne J Evstigneeva RP Ageyeva EA Zheltukhina GA L-carnosine (beta-alanyl-L-histidine) and carcinine (beta-alanylhistamine) act as natural antioxidants with hydroxyl-radical-scavenging and lipid-peroxidase activities Biochem J 1994 Dec 1304 ( Pt 2)509-16

57 Arnould JM Frentz R (1977) [Carcinine (beta-alanyl-histamine) rapid synthesis and action on vertebrate blood pressure] Arch Int Physiol Biochim 85 339-350

58 Babizhayev MA Meguro K 2004 Combined use of carnosinase inhibitor with L-carnosines and composition WO 2004064866 PCTJP2004000351 Filed 20 January 2003 (20012003)

59 Babizhayev MA Guiotto A Kasus-Jacobi A N-Acetylcarnosine and histidyl-hydrazide are potent agents for multitargeted ophthalmic therapy of senile cataracts and diabetic ocular complications J Drug Target 2009 Jan17(1)36-63

60 Requena JR Fu MX Ahmed MU Jenkins AJ Lyons TJ Baynes JW Thorpe SR Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein Biochem J 1997 Feb 15322 ( Pt 1)317-25

61 Thornalley PJ (1985) Monosaccharide autoxidation in health and disease Environ Health Perspect 64 297-307

62 Stewart JJP 1989 MOPAC FJ Seiler Research Laboratory Air Force Academy Boulder pp 80840

63 Stewart JJ MOPAC Ver6 QCPE Bull1989 910 Revised as Ver 601 by T Hirano University of Tokyo for HITAC and UNIX machines (JCPE Newsletter 1989 110)

64 Babizhayev MA (1989) Antioxidant activity of L-carnosine a natural histidine-containing dipeptide in crystalline lens Biochim Biophys Acta 1004 363-371

65 Babizhayev MA Bozzo Costa E Lipid peroxide and reactive oxygen species generating systems of the crystalline lens Biochimica et Biophysica Acta 1994 1225326ndash337

66 Yoshikawa T Naito Y Tanigawa T Yoneta T Kondo M (1991) The antioxidant properties of a novel zinc-carnosine chelate compound N-(3-aminopropionyl)-L-histidinato zinc Biochim Biophys Acta 1115 15-22

67 Iacazio G Langrand G Baratti J Buono G Triantaphylides CJ Preparative enzymatic synthesis of linoleic acid (13S)-hydroperoxide using soybean lipoxygenase-1 Organic Chemistry 1993 55 1690

68 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680-685

69 Sammons DW Adams LD Nishizawa EE Ultra sensitive silver based color staining of polypeptides in polyacrylamide gels Electrophoresis 1981 2 135

70 Guiotto A Ruzza P Babizhayev MA Calderan A Malondialdehyde scavenging and aldose-derived Schiff basesrsquo transglycation properties of synthetic histidyl-hydrazide carnosine analogs Bioorg Med Chem 2007 Sep 1515(18)6158-63 Epub 2007 Jun 20

71 Szwergold BS (2005) Carnosine and anserine act as effective transglycating agents in decomposition of aldose-derived Schiff bases Biochem Biophys Res Commun 336 36-41

72 Bando K Shimotsuji T Toyoshima H Hayashi C Miyai K Fluorometric assay of human serum carnosinase activity in normal children adults and patients with myopathy Ann Clin Biochem 1984 21 (Pt 6) 510-14

73 Yim HS Kang SO Hah YC Chock PB Yim MB Free radicals generated during the glycation reaction of amino acids by methylglyoxal A model study of protein-cross-linked free radicals J Biol Chem 1995 Nov 24270(47)28228-33

74 McLaughlin JA Pethig R Szent-Gyoumlrgyi A (1980) Spectroscopic studies of the protein-methylglyoxal adduct Proc Natl Acad Sci U S A 77 949-951

75 Tarpey MM Wink DA Grisham MB (2004) Methods for detection of reactive metabolites of oxygen and nitrogen in vitro and in vivo considerations Am J Physiol Regul Integr Comp Physiol 286 R431-444

76 Shumaev KB Gubkina SA Kumskova EM Shepelkova GS Ruuge EK Lankin VZ Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism Biochemistry (Mosc) 2009 Apr74(4)461-6

77 Bisby RH Parker AW Reactions of the alpha-tocopheroxyl radical in micellar solutions studied by nanosecond laser flash photolysisFEBS Lett 1991 Sep 23290(1-2)205-8

78 Babizhayev MA (2008) Ocular drug metabolism of the bioactivating antioxidant N-acetylcarnosine for vision in ophthalmic prodrug and codrug design and delivery Drug Dev Ind Pharm 34 1071-1089

79 Kang JH Protective effects of carnosine and N-acetylcarnosine on salsolinol-mediated CuZn-superoxide dismutase inactivation Bull Korean Chem Soc 2007 208(10) 1881-1884

80 Neglia CI Cohen HJ Garber AR Thorpe SR Baynes JW Characterization of glycated proteins by 13C NMR spectroscopy Identification of specific sites of



protein modification by glucose J Biol Chem 1985 May 10260(9)5406-10

81 Mossine VV Glinsky GV Feather MS The preparation and characterization of some Amadori compounds (1-amino-1-deoxy-D-fructose derivatives) derived from a series of aliphatic omega-amino acids Carbohydr Res 1994 Sep 15262(2)257-70

82 Teufel M Saudek V Ledig JP Bernhardt A Boularand S Carreau A Cairns NJ Carter C Cowley DJ Duverger D Ganzhorn AJ Guenet C Heintzelmann B Laucher V Sauvage C Smirnova T Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase J Biol Chem 2003 Feb 21278(8)6521-31 Epub 2002 Dec 6

83 Unno H Yamashita T Ujita S Okumura N Otani H Okumura A Nagai K Kusunoki M Structural basis for substrate recognition and hydrolysis by mouse carnosinase CN2 J Biol Chem 2008 Oct 3283(40)27289-99 Epub 2008 Jun 12

84 Lee YT Hsu CC Lin MH Liu KS Yin MC Histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation Eur J Pharmacol 2005 Apr 18513(1-2)145-50 Epub 2005 Apr 2

85 Babizhayev MA Current ocular drug delivery challenges for N-acetylcarnosine novel patented routes and modes of delivery design for enhancement of therapeutic activity and drug delivery relationships Recent Pat Drug Deliv Formul 2009 Nov3(3)229-65

86 Amaral ME Oliveira HC Carneiro EM Delghingaro-Augusto V Vieira EC et al (2002) Plasma glucose regulation and insulin secretion in hypertriglyceridemic mice Horm Metab Res 34 21-26

87 Strippoli GF Di Paolo S Cincione R Di Palma AM Teutonico A et al (2003) Clinical and therapeutic aspects of diabetic nephropathy J Nephrol 16 487-499

88 Goldberg RB (2003) Cardiovascular disease in patients who have diabetes Cardiol Clin 21 399-413 vii

89 Locatelli F Canaud B Eckardt K U Stenvinkel P Wanner C Zoccali C Oxidative stress in end-stage renal disease an emerging threat to patient outcome Nephrol Dial Transplant 2003 18 1272ndash1280

90 Yamada T Sato A Nishimori T Mitsuhashi T Terao A Sagai H Komatsu M Aizawa T Hashizume K Importance of hypercoagulability over hyperglycemia for vascular complication in type 2 diabetes Diabetes Res Clin Pract 2000 49 23ndash31

91 Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications Nature 414 813-820

92 REYNOLDS TM (1963) CHEMISTRY OF NONENZYMIC BROWNING I THE REACTION BETWEEN ALDOSES AND AMINES Adv Food Res 12 1-52

93 Reynolds TM (1965) Chemistry of nonenzymic browning II Adv Food Res 14 167-283

94 Kato H Hayase F Shin DB Oimomi M Baba S (1989) 3-Deoxyglucosone an intermediate product of the Maillard reaction Prog Clin Biol Res 304 69-84

95 Monnier VM Vishwanath V Frank KE Elmets CA Dauchot P Kohn RR Relation between complications of type I diabetes mellitus and collagen-linked fluorescence N Engl J Med 1986 Feb 13314(7)403-8

96 Monnier VM Kohn RR Cerami A (1984) Accelerated age-related browning of human collagen in diabetes mellitus Proc Natl Acad Sci U S A 81 583-587

97 Monnier VM Cerami A (1981) Nonenzymatic browning in vivo possible process for aging of long-lived proteins Science 211 491-493

98 Liang JN Hershorin LL Chylack LT Jr (1986) Non-

enzymatic glycosylation in human diabetic lens crystallins Diabetologia 29 225-228

99 Vlassara H Brownlee M Cerami A (1981) Nonenzymatic glycosylation of peripheral nerve protein in diabetes mellitus Proc Natl Acad Sci U S A 78 5190-5192

100 Vlassara H Brownlee M Cerami A (1983) Excessive nonenzymatic glycosylation of peripheral and central nervous system myelin components in diabetic rats Diabetes 32 670-674

101 Hicks M Delbridge L Yue DK Reeve TS (1988) Catalysis of lipid peroxidation by glucose and glycosylated collagen Biochem Biophys Res Commun 151 649-655

102 Bucala R Tracey KJ Cerami A (1991) Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes J Clin Invest 87 432-438

103 Simpson JA Narita S Gieseg S Gebicki S Gebicki JM et al (1992) Long-lived reactive species on free-radical-damaged proteins Biochem J 282 621-624

104 Brownlee M Vlassara H Cerami A (1984) Nonenzymatic glycosylation and the pathogenesis of diabetic complications Ann Intern Med 101 527-537

105 Mullarkey CJ Edelstein D Brownlee M Free radical generation by early glycation products a mechanism for accelerated atherogenesis in diabetes Biochem Biophys Res Commun 1990 Dec 31173(3)932-9

106 Sell DR Monnier VM Structure elucidation of a senescence cross-link from human extracellular matrix Implication of pentoses in the aging process J Biol Chem 1989 Dec 25264(36)21597-602

107 Sell DR Monnier VM (1990) End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen J Clin Invest 85 380-384

108 Grandhee SK Monnier VM (1991) Mechanism of formation of the Maillard protein cross-link pentosidine Glucose fructose and ascorbate as pentosidine precursors J Biol Chem 266 11649-11653

109 Sell DR Nagaraj RH Grandhee SK Odetti P Lapolla A Fogarty J Monnier VM Pentosidine a molecular marker for the cumulative damage to proteins in diabetes aging and uremia Diabetes Metab Rev 1991 Dec7(4)239-51

110 Dyer DG Blackledge JA Thorpe SR Baynes JW Formation of pentosidine during nonenzymatic browning of proteins by glucose Identification of glucose and other carbohydrates as possible precursors of pentosidine in vivo J Biol Chem 1991 Jun 25266(18)11654-60

111 Miyata S Monnier V (1992) Immunohistochemical detection of advanced glycosylation end products in diabetic tissues using monoclonal antibody to pyrraline J Clin Invest 89 1102-1112

112 Namiki M Hayashi T Ohta Y (1977) Novel free radicals formed by the amino-carbonyl reactions of sugars with amino acids amines and proteins Adv Exp Med Biol 86B 471-501

113 Hayashi T Ohta Y Namiki M (1977) Electron spin resonance spectral study on the structure of the novel free radical products formed by the reactions of sugars with amino acids or amines J Agric Food Chem 25 1282-1287

114 Ahmed MU Thorpe SR Baynes JW (1986) Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein J Biol Chem 261 4889-4894

115 Baynes JW (1991) Role of oxidative stress in development of complications in diabetes Diabetes 40 405-412

116 Dunn JA Ahmed MU Murtiashaw MH Richardson JM Walla MD Thorpe SR Baynes JW Reaction of ascorbate



with lysine and protein under autoxidizing conditions formation of N epsilon-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate Biochemistry 1990 Dec 1129(49)10964-70

117 Dunn JA Patrick JS Thorpe SR Baynes JW Oxidation of glycated proteins age-dependent accumulation of N epsilon-(carboxymethyl)lysine in lens proteins Biochemistry 1989 Nov 2828(24)9464-8

118 Dyer DG Dunn JA Thorpe SR Bailie KE Lyons TJ et al (1993) Accumulation of Maillard reaction products in skin collagen in diabetes and aging J Clin Invest 91 2463-2469

119 Thornalley P Wolff S Crabbe J Stern A The autoxidation of glyceraldehyde and other simple monosaccharides under physiological conditions catalysed by buffer ions Biochim Biophys Acta 1984 Feb 14797(2)276-87

120 Jiang ZY Woollard AC Wolff SP Hydrogen peroxide production during experimental protein glycation FEBS Lett 1990 Jul 30268(1)69-71

121 Hunt JV Dean RT Wolff SP Hydroxyl radical production and autoxidative glycosylation Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing Biochem J 1988 Nov 15256(1)205-12

122 Hunt JV Smith CC Wolff SP (1990) Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose Diabetes 39 1420-1424

123 Bonfanti L Peretto P De Marchis S Fasolo A (1999) Carnosine-related dipeptides in the mammalian brain Prog Neurobiol 59 333-353

124 Quinn PJ Boldyrev AA Formazuyk VE (1992) Carnosine its properties functions and potential therapeutic applications Mol Aspects Med 13 379-444

125 Hipkiss AR Would carnosine or a carnivorous diet help suppress aging and associated pathologies Ann N Y Acad Sci 2006 May1067369-74

126 Hipkiss AR Expert Rev Neurother 2009 May9(5)583-5 Comment on Expert Rev Neurother 2009 May9(5)617-30

127 Babizhayev MA Khoroshilova-Maslova IP Kasus-Jacobi A Novel intraocular and systemic absorption drug delivery and efficacy of N-acetylcarnosine lubricant eye drops or carcinine biologics in pharmaceutical usage and therapeutic vision care Fundam Clin Pharmacol 2012 Oct26(5)644-78 doi 101111j1472-8206201100963x Epub 2011 Jul 27

128 Nagai K Tanida M Niijima A Tsuruoka N Kiso Y Horii Y Shen J Okumura N Role of L-carnosine in the control of blood glucose blood pressure thermogenesis and lipolysis by autonomic nerves in rats involvement of the circadian clock and histamine Amino Acids 2012 Jul43(1)97-109 doi 101007s00726-012-1251-9 Epub 2012 Feb 25

129 Babizhayev MA Seguin M-C Coupling product obtained from histamine and an amino acid US Patent Number 5792784 Date of Patent August 11 1998

130 Otani H Okumura A Nagai K Okumura N Colocalization of a carnosine-splitting enzyme tissue carnosinase (CN2)cytosolic non-specific dipeptidase 2 (CNDP2) with histidine decarboxylase in the tuberomammillary nucleus of the hypothalamus Neurosci Lett 2008 Nov 14445(2)166-9 doi 101016jneulet200809008 Epub 2008 Sep 7

Title

Corresponding author

Abstract

Introduction

Materials and Methods

Molecular modeling


Ferroxidase activity of carnosine


Electrophoresis assays


Protection of Superoxide Dismutase (SOD) activity with an imidazole-containing peptidomimetic Skin t

Measurement of SOD-like activity


Testing of human carnosinase activity

Results

Mechanism of Superoxide Formation as a Result of Interaction of L-Lysine with Dicarbonyl Glycating C



Antioxidant Activities of L-carnosine N-acetylcarnosine and Carcinine in the Fe2+ascorbate -Induce

Reactivity of l-carnosine and carcinine with lipid hydroperoxide Lipid peroxidase-like activity of




Discussion

Conclusion

Acknowledgement

Disclosure

Conflict of Interest

Figure 1

Figure 2

Figure 3

Figure 4a

Figure 4b

Figure 5a

Figure 5b

Figure 6

Figure 7

Figure 8a

Figure 8b

Figure 9

Figure 9b

Figure 10

Figure 11

Figure 12

Figure 13

Table 1

Table 2

References



methylglyoxal or 3-deoxyglucosone as well as via depletion of NADPH or glutathione raising intracellular ROS all of which indirectly result in increased formation of AGEs [3-510-12] Several

compounds eg εN-carboxymethyl-lysine pentosidine or methylglyoxal derivatives serve as examples of well-characterized and widely studied AGEs [67]

Several immunoassay-based tests have established higher levels of common AGEs such as εN-carboxymethyl-lysine (CML) or methylglyoxal (MG) in older persons who are otherwise healthy [13] The frequent finding in the aged population of increased ROS [1415] a state known to promote AGEs formation supports the notion of increased endogenous AGEs formation in the elderly

A key characteristic of certain reactive or precursor AGEs is their ability for covalent crosslink formation between proteins which alters their structure and function as in cellular matrix basement membranes and vessel-wall components Other major features of AGEs relate to their interaction with a variety of cell-surface AGE-binding

receptors leading either to their endocytosis and degradation or to cellular activation and pro-oxidant pro-inflammatory events A large body of evidence suggests that AGEs are important pathogenetic mediators of almost all diabetes complications conventionally grouped into micro- or macroangiopathies For instance AGEs are found in retinal vessels of diabetic patients and their levels correlate with those in serum as well as with severity of retinopathy [1617]

Several approaches seeking to reduce AGE interactions either by inhibiting AGE formation blocking AGE action or breaking pre-existing AGE cross-links have been explored The first class of those agents involved the inhibitors of AGE formation which act by inhibiting post-Amadori advanced glycation reactions or by trapping carbonyl intermediates and thus inhibiting both advanced glycation and lipoxidation reactions Aminoguanidine [1819] ALT-946 [1920] 2-3-Diaminophenazine [20] thiamine pyrophosphate [21] benfotiamine [22] and pyridoxamine [23] ORB-9195 [24] constitute known representatives of this group of agents The second class of those agents involved the AGE breakers which ldquobreakrdquo pre-accumulated AGE or existing AGE cross-links leading to the elimination of the smaller peptides through urine PTB (N-phenylthiazolium bromide) [25] and ALT-711 are the best known representatives of this group of agents [2627] Recently it has been shown that antihypertensive drugs such as losartan olmesartan and hydralazine seem to inhibit AGE formation [28-31]

Carnosine (β-alanyl-L-histidine) and related compounds are natural constituents of excitable tissues possessing diverse biological activities [32] The level of carnosine in tissues is controlled by a number of enzymes transforming carnosine into other carnosine related compounds such as carcinine N-acetylcarnosine anserine or ophidine (by decarboxylation acetylation or methylation respectively) or its cleavage into the amino acids histidine and β-alanine Hydrolysis is mainly due to tissue carnosinase (EC 34133)

IntroductionAccording to the American Diabetes Association

diabetes is the seventh leading cause of death in the US There are 103 million people in the US with the disease and an additional estimated 54 million have not yet been diagnosed Many people do not become aware that they have diabetes until they develop one of its life-threatening symptoms such as blindness kidney disease nerve damage and heart disease Diabetes develops due to a diminished production of insulin (in type 1) or resistance to its effects (in type 2 and gestational) (Figure 1) [1] Several predominant well-researched theories have been proposed to explain how hyperglycemia can produce the neural and vascular derangements that are hallmarks of diabetes These theories can be separated into those that emphasize the toxic effects of hyperglycemia and its pathophysiological derivatives (such as oxidants hyperosmolarity or glycation products) on tissues directly and those that ascribe pathophysiological importance to a sustained alteration in cell signaling pathways (such as changes in phospholipids or kinases) induced by the products of glucose metabolism [2] Hyperglycemia is still considered the principal cause of diabetes complications Its deleterious effects are attributable among other things to the formation of sugar-derived substances called advanced glycation end products (AGEs) AGEs form at a constant but slow rate in the normal body starting in early embryonic development and accumulate with time However their formation is markedly accelerated in diabetes because of the increased availability of glucose

AGEs are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins lipids and nucleic acids [3-7] The initial product of this reaction is called a Schiff base which spontaneously rearranges itself into an Amadori product as is the case of the well-known hemoglobin A1c (A1C) These initial reactions are reversible depending on the concentration of the reactants A lowered glucose concentration will unhook the sugars from the amino groups to which they are attached conversely high glucose concentrations will have the opposite effect if persistent

A series of subsequent reactions including successions of dehydrations oxidation-reduction reactions and other arrangements lead to the formation of AGEs AGEs may form by auto-oxidation of glucose or through the glycolytic pathway but also from non-glucose sources including lipid and amino acid oxidation [2-7] In addition neutrophils monocytes and macrophages upon inflammatory stimulation produce myeloperoxidase and activate Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase which can lead to new AGEs by way of amino acid oxidation [3-589]

Binding and activation of cellular AGE Receptor (RAGE) by AGEs or any other ligand can also promote reactive oxygen species (ROS) and AGE formation via the NADPH oxidase and the myeloperoxidase pathways [3-589] Another potential mechanism of AGE formation is the polyol pathway Glucose entering the polyol pathway may form AGEs via reactive intermediates ie glyoxal



























































Results





(reaction 1)


























(reaction 2)












































































































































































































































Title


Abstract

Introduction


Molecular modeling










Results









Discussion

Conclusion

Acknowledgement

Disclosure


Figure 1

Figure 2

Figure 3

Figure 4a

Figure 4b

Figure 5a

Figure 5b

Figure 6

Figure 7

Figure 8a

Figure 8b

Figure 9

Figure 9b

Figure 10

Figure 11

Figure 12

Figure 13

Table 1

Table 2

References



























































Results





(reaction 1)


























(reaction 2)












































































































































































































































Title


Abstract

Introduction


Molecular modeling










Results









Discussion

Conclusion

Acknowledgement

Disclosure


Figure 1

Figure 2

Figure 3

Figure 4a

Figure 4b

Figure 5a

Figure 5b

Figure 6

Figure 7

Figure 8a

Figure 8b

Figure 9

Figure 9b

Figure 10

Figure 11

Figure 12

Figure 13

Table 1

Table 2

References

























(reaction 2)












































































































































































































































Title


Abstract

Introduction


Molecular modeling










Results









Discussion

Conclusion

Acknowledgement

Disclosure


Figure 1

Figure 2

Figure 3

Figure 4a

Figure 4b

Figure 5a

Figure 5b

Figure 6

Figure 7

Figure 8a

Figure 8b

Figure 9

Figure 9b

Figure 10

Figure 11

Figure 12

Figure 13

Table 1

Table 2

References





































































































































































































































Title


Abstract

Introduction


Molecular modeling










Results









Discussion

Conclusion

Acknowledgement

Disclosure


Figure 1

Figure 2

Figure 3

Figure 4a

Figure 4b

Figure 5a

Figure 5b

Figure 6

Figure 7

Figure 8a

Figure 8b

Figure 9

Figure 9b

Figure 10

Figure 11

Figure 12

Figure 13

Table 1

Table 2

References

qphc 1-001 (1) (1)

Health & Medicine