Qphc 1-001 (1) (1)

1Volume 1: Issue 1: 001
Quality in Primary Health Care
OPUS JOURNALS
Qual Prim Health Care (2017)
1:1 001
Research Article
Qua
lity in Healthc
are
Abstract
Diabetes is the seventh leading cause of death based on U.S. 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 Babizhayev*
Innovative 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: markbabizhayev@mail.ru
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) 1:1 001

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-
5,10-12]. Several
compounds, e.g., ε
N-carboxymethyl-lysine, pentosidine,
or methylglyoxal derivatives, serve as examples of well-
characterized and widely studied AGEs [6,7].
Several immunoassay-based tests have established
higher levels of common AGEs, such as ε
N-carboxymethyl-
lysine (CML) or methylglyoxal (MG) in older persons who
are otherwise healthy [13]. The frequent finding in the aged
population of increased ROS [14,15], 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
[16,17].
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 ofAGE
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 [18,19], ALT-946 [19,20],
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 “break” 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 [26,27]. 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 3.4.13.3)
Introduction
According to the American Diabetes Association,
diabetes is the seventh leading cause of death in the US.
There are 10.3 million people in the US with the disease,
and an additional estimated 5.4 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
importancetoasustainedalterationincellsignalingpathways
(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 [3-5,8,9].
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-
5,8,9]. Another potential mechanism of AGE formation is
the polyol pathway. Glucose entering the polyol pathway
may form AGEs via reactive intermediates, i.e. 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 symptoms.
The 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 [33,34]
or serum carnosinase (EC 3.4.13.20), obtained in brain and
blood plasma of primates and humans [35,36]. 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 [40,41] 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 [51,52] . 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 [55,56] and to play
a role in regulating stress and shock with a 1000-fold less
potent hypotensive effect than histamine [52,57] , 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
[55,56]. Combination pharmaceutical products, or fixed-
dose combinations (FDC’s), 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) [58,59]. 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 authors’ Group.
Materials and Methods
Carcinine(Decarboxycarnosine·2HCl),l-prolylhistamine
and N-acetyl-β-alanylhistamine were synthesized by
Exsymol S.A.M. (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,
1,1,3,3-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 1,1,3,3-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 9.15 GHz, high frequency modulation amplitude
0.2 mT. Spectrum recording was started 1 min after the
mixing of reaction components. The reaction mixture (120
µl) 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
-
·) was
detected using two independent methods: reduction
of nitro blue tetrazolium by the superoxide and О2
-·
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 25°C. The reaction was
initiated by adding 10 mM methylglyoxal or 10 mM MDA
to the medium containing 100 µM NBT and 10 mM L-lysine
in 100 mM carbonate buffer, pH 9.5. Chemiluminescence
was measured by a Lum-5773 chemiluminometer (Russia)
in medium containing 20 µM lucigenin, 15 mM L-lysine,
and 15 mM methylglyoxal in 100 mM K, Na_phosphate
buffer, pH 7.8. Measurements were performed at 37°C
under continuous stirring of the reaction medium. Statistical
treatment of the data was performed using Student’s
t-criterion.
Molecular modeling
Low-energy 3-D conformations of carnosine, carcinine
and N-acetylcarnosine were derived using the PM3
method
of the MOPAC 6.0 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 ZINDO/1 computer software for
estimation of chelating properties of dipeptides and related

compounds. The conformational geometry optimization was
carried out using the revised computer program [62,63].
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 [64,65].
Peroxidation of phosphatidylcholine (PC, derived from
egg yolks) was initiated by adding 2.5 µM FeSO4
and 200
µM ascorbic acid to the suspension of liposomes (1 mg/
ml) in 0.1 M Tris-HCl buffer (pH 7.4). The incubations
were performed at 37ºC. The tested compounds, either
N-acetylcarnosine , l-carnosine, carcinine or other
imidazole-containing compounds were added at 10–20
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 µM or by the addition of 2.0
ml of ice-cold 0.25 M HCl containing 15% (w/v) three-
chloroacetic acid (TCA). TBA (0.125% w/v) 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
=1.56 x 105
M-1
cm-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 206–210 nm of the lipid sample (redissolved in
2–3 ml of methanol/heptane mixture 5:1, v/v).Accumulation
of net diene conjugates corresponding to the level of lipid
hydroperoxides was assessed from characteristic absorbance
of diene conjugates at ~ 230 nm (ηCD=2.8x104
M-1
cm-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 Student’s t-test, and P=0.01 was taken as the upper
limit of significance.
Ferroxidase activity of carnosine
The ability of carnosine to decrease the concentration
of free ferrous ions in Tris–HC1 buffer (100 mM, pH 7.4)
was monitored by the 1,10-o-phenanthroline chelating
assay modified from Ref. [66]. The reaction was started
by the addition of 12.5 µM FeSO4 to the reaction mixture
which contained 3–20 mM carnosine. Sixty minutes after
incubation at 37º C, the reactions were stopped by the
addition 100 µM 1,10-o-phenanthroline (Serva), and A515
was immediately read. The concentration of (Fe2+
–1,10-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 [55,56] .
Electrophoresis assays
One millimole of 13(S) linoleic acid hydroperoxide were
incubated in phosphate buffer solution (PBS) (0.1 M, pH
7.4) with a bovine serum albumin (BSA) solution (0.5 mg
ml-1
) at 37ºC 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 (7.5% polyacrilamide gel
containing 0.1% 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.
• Column C18 Macherey–Nagel, 4.6 mm, 5 mm, 12.5
cm.
• Elution: 50/50 acetonitrile-acetic acid 0.01%.
• Controls: retention time of 13(S) linoleic acid
hydroperoxide = 15 min
• Retention time of 13(S) linoleic acid alcohol = 12.8
min
• (obtained after NaBH4
reduction [67]).
• 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 70ºC for 70 s. The epiderm-
derm fraction was removed with a mechanical treatment.
Skin fragments were treated with creams containing 0%,
0.5%, 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 (0.8 J/cm2
), skin fragments were cut, suspended
(30 g/l) in phosphate buffer and crushed with ultra turrax
(0ºC for 2 min). Extracts obtained were diluted (1/3) in
Triton X-100 (1%) and kept 2 h at 0ºC. The mixtures were
then centrifuged at 10,000 g for 10 min. The SOD-like
activity was measured in the supernatant fraction.
Measurement of SOD-like activity
Anion 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 skin/l 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 (0.06
units/ml). The reaction was initiated by the addition of 100
μl of xanthine oxidase. The kinetics were realized at 25ºC
for 2–3 min (550 nm). The rate of cytochrome c reduction
(delta OD/min) at 25ºC was assessed. The protective effect
of carcinine was obtained with the following formula.
Irradiation 0% - Irraditation X%
x100
Irradiation 0% - No irradiation
– No irraditation: kinetics obtained from non-irradiated
skin fraction. It represents the natural SOD-like
activity of the skin extract.
– 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.
– Irradiation X%: kinetics obtained with irradiated skin
fractions treated with creams (oil/water) containing
0.1%, 0.5%, 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 pmol/L as external standard. Samples
were prepared by dissolving the compound (10-5
M) in
acetonitrile/water 1:1 mixture with 1% acetic acid. 1
H and
13
C 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 G/UV 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 Sigma–Aldrich, J.T. Baker, and Carlo Erba.
13
C NMR experiments Glucose–ethylamine (G–E)
was synthesized by incubating 500 mM 13
C-glucose and
15
N-ethylamine at pH 12 and 37ºC for 3 h [70,71]. At the end
of the incubation period, about 75% of the starting material
was converted to glucose–ethylamine 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 (0.5 mL in a 5 mm NMR tube) included 250
mM Hepes, pH 8.5, 10% D2
O, 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 G–E to produce a final concentration
of 20 mM. At that time consecutive NMR spectra of 20 min
duration were acquired using 580 scans, 60º pulses, and an
interpulse delay of 2.05 s. The spectra were analyzed using
the information from model compounds and chemical shifts
from the literature. The area of the G–E doublet at 90.00
ppm was calculated and plotted against time after subtraction
of the natural G–E Schiff base decay measured in a blank
experiment. Transglycation efficiency of L-carnosine and
carnosine derivatives 2–7 (Figure 2) was assessed following
Szwergold protocol [71] , using the Schiff base glucosyl–
Figure 3: Haematoxylin and Eosin (magnification x40) :
Basal cells vacuolation and lymphocytic infiltration from
left concha

ethylamine (G–E) as a model of the first intermediate in the
glycation process of side chain primary amines of proteins.
15
N labeled ethylamine was used to minimize electric
quadrupole moment and obtain a C-1 peak of glucose as
a sharp doublet centered at 90.00 ppm. The kinetics of the
transglycation reaction for the control reaction, carnosine,
and compounds 2–7 are illustrated in Figure 2. For a better
evaluation of the transglycation kinetics of the compounds,
for each 13
C spectrum the integral of the buffer Hepes
signals (50–55 ppm range) was set as=1, then the integral
of the C-1 glucose peak at 90.00 ppm was measured and
integration values, normalized and corrected for the natural
decay of the G–E Schiff base (control curve), were plotted
against time. The ability of carcinine (decarboxycarnosine)
to behave as an “acceptor molecule” 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-
ethylaminesynthesis:thismodelglycosylaminewasobtained
by incubating D-glucose and ethylamine (500 mM) for 3
hours at 37°C in alkaline conditions (pH 12). In order to ease
the 13
C 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 D2
O, at slightly
alkaline pH conditions (pH 8.5) 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 13
C and
15
N 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- 13
C]
glucose and [15
N] ethylamine, were obtained from Sigma-
Aldrich-Fluka (SAF, Deisenhofen, Germany).
Testing of human carnosinase activity
In 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 7.5), 1 mM carnosine in 100 μl final volume
using 0.25-0.5 μg of cell/tissue extract or 10 ng of purified
enzyme. The reaction was initiated by addition of substrate
and stopped after 60 min incubation at 30 °C by adding 50 μl
of 1% TCA. Liberated histidine was derivatized by adding
50 μl of 5 mg/ml o-Pthaldialdehyde (OPA) dissolved in 2 M
NaOH and 30 min incubation at 30 °C. 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
ResultofInteractionofL-LysinewithDicarbonyl
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) — 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 О2
ˉ˙ is formed by single_electron oxygen
reduction by methylglyoxal semidione in accordance with
the reaction:
(reaction 1)
Our model system has also demonstrated that О2
ˉ˙ is
intensively generated on the interaction of L-lysine with
methylglyoxal in carbonate buffer, pH 9.5. Superoxide
formation was assessed by the accumulation of formazan on
NBT reduction. The accumulation of formazan under these
conditions might not depend on О2
ˉ˙, 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 О2
ˉ˙ (Figure 5a).

Figure 3: 13
C NMR spectra with characteristic peaks for residual glucose (ß-Glc, α-Glc) and model glycosylamines ß-glucosyl-
ethylamine (ß-G-E) and α-glucosyl-ethylamine (α-G-E). 13
C NMR spectra obtained 4 hours after addition of decarboxycarnosine
to the ß-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 K,Na_phosphate buffer (0.2 M, pH 7.8). 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 0.2 M K,Na_phosphate buffer, pH 7.8; 2) the
same as (1) + 400 SOD units.

of О2
ˉ˙ in the mixture of methylglyoxal with L-lysine at pH
7.8 (Figure 6), i.e. 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
2.0042 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 9.5, 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 K,Na_phosphate buffer, pH 7.8, 20 µM 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 0.1% of carcinine. The slope obtained with the non-irradiated skin is 0.1 OD units/min.
The slope obtained with the irradiated skin treated with 0% carcinine is 0.17 OD units/min. The slope obtained with the
irradiated skin treated with 0.1% carcinine is 0.14 OD units/min. (B) Protection of the SOD activity of isolated
porcine ear dermis-epidermis treated with various concentrations of an imidazole-containing peptidomimetic. Average ±
SEM from 10 independent experiments are given. *: significant differences ( p <0.001) with control
(Student’s 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 of
SOD containing reaction medium (400 U/ml) 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 О2
ˉ˙ 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 5–20mM l-carnosine pronounced by the 10–30 min of
incubation (Figure 9a). The kinetic curves presented in Figure
9ademonstratethatthereisadose-dependentincreaseintherate
of ferrous iron disappearance.Astrong ferrous iron chelator 33,
330 μM EDTA showed a complete decrease of the accessible
to 1,10-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 by,10-o-phenanthroline assay in the presence
of 12.5 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 ≤3% 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; (▲) – Fe 2+
+ l-carnosine (5 mM); (●) – Fe 2+
+ l-carnosine (10 mM); (■) – Fe 2+
+ l-carnosine
(20 mM).
Figure 9b: Effect of l-carnosine on the decrease of ferrous iron determined by 1,10-o-phenanthroline assay in the presence
of 12.5 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 ≤3% 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 5,6). The rates of decrease of ferrous iron accessible
to 1,10-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 chelating/
ferroxidase activity appears weaker than that of EDTA but it
is competitive with ferrous iron chelating activity shown by
1,10-o-phenanthroline. Based on the high affinity properties
of 1,10-o-phenanthroline to bind preferably ferrous but not Fe
3+
ions, there is a potential preference for Fe 2+
autooxidation/
chelating by l-carnosine over Fe 3+
that is important for the
rationale of presented later experiments. The reference curves
(5,6) 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 (1,10-o-phenanthroline)
molecule (data not shown).
Antioxidant Activities of L-carnosine,
N-acetylcarnosine and Carcinine in the
Fe2+
/ascorbate –Induced 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
MDAand 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 5–25
mM have already been published [56,64,65]. 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.Theabilityofthehistidine-containingcompound
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 mg/ml) 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 ‘‘Materials and methods’’). A similar amount of sample was
partitioned through chloroform and used for detection of conjugated dienes and trienes dissolved in 2 – 3 ml of methanol–
heptane mixture (5 :1 v/v).

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-
---¬ 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 protein’s 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, carcinine’s (lanes 4 and 5)
protective effect was far superior to l-carnosine’s (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 (e.g. 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 oxidative
25 mM degradation of linoleic acid
l-Carnosine (β-alanyl-l-histidine 59
Carcinine (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 liposomes
l-Carnosine (β-alanyl-l-histidine) 53
Carcinine (β-alanylhistamine) 42
Detailed experimental procedures are described in Ref. 56.
Each result represents
the mean of 5 experiments.
Table 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
skin’s 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 <0.001, 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 creams,confers to the skin a
significant protection against the oxidative stress induced
by UVA-UVB irradiation. Carcinine in applied creams do
not absorb in UVA (320–400 nm) or UVB (280–320 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 ;
i.e., 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 (0.1 M; pH 7.3) after 15 min
of incubation at 37ºC. Absorbance wavelengths used: 234 and 205 nm. (B) HPLC spectrum of 13(S) hydroxy linoleic acid
phosphate buffer solution (0.1 M; pH 7.3). 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 (0.33 g/l) in 0.1 M phosphate buffer, pH=7.3 was incubated with 1.5 mM 13(S)-linoleic acid hydroperoxide
and 5 mM carcinine during 60 h at 37º 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 “acceptor molecule” 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
MagneticResonance(13
CNMR)spectroscopy(see,Materials
and Methods : 13
C 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 (ß-Glc & α-Glc).
Glucosyl-ethylamine is unambiguously identified by the
presence of a doublet due to the 13
C-15
N spin-spin coupling
(i.e §3: isotopically enriched starting material was used for
the synthesis of the model glycosylamine). The experiment
was conducted in slightly alkaline conditions (pH 8.5),
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) [71,80,81]
. More accurately, both glycosylamines (ß-G-E & 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 (ß-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.Akinetic 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 ß-G-E disappearance kinetics closely
follows the ß-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 13
C NMR study with the model
glucosyl-ethylamine has shown that decarboxycarnosine
(carcinine) is an effective transglycating agent, behaving
as an “acceptor molecule” for glucose, and releasing a “de-
glycosylation product”, e.g. the “free amine”.
Thedatapresentedshowthatthetransglycatingefficiency
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
= 0.38). The hydrazide moiety of
leucyl-histidylhydrazide (formula 5) boosts the aldehyde
scavenging efficiency of compound [59,70] , and in
combination with a free Nα
-amino group, concurs in the
disruption of the Schiff base adduct G–E as a model of
protein glycation. Further structure/activity 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
[46,58] . Diabetes affects the (outer) lens, middle (vitreous),
and inner (retina) areas of the eye.
Susceptibilityofimidazole-containingpeptide
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 7.5. 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 (kcat/Km) of carnosinase
activity for carnosine and homocarnosine were 8.9 mM-1
sec-1
and 1.3 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 drug/codrug design and ophthalmic formulation in
order to achieve the sustained release of described triple
peptide moieties N-acetylcarnosine/L-carnosine/leucyl-
histidylhydrazide during targeted therapy for ocular diseases
and diabetic pathology [84,85].
Discussion
Diabetic 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 [92,93]. 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 [95,96], lens
crystallines [97,98] , and nerve proteins [99,100]. 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 [112,113], 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 [114,115], and the
autoxidation of glucose, which produces superoxide radical
anions (О2
ˉ˙), H2
O2
, and α-ketoaldehydes [7,119-122]. 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 (О2
ˉ˙ ) , 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 (e.g., protein
oxidation, glycation, AGE formation, and cross-linking) that
accompany aging ,diabetes and associated pathologies. Due
to established carnosine’s 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-acetylcarnosine,leucyl-
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 [123,124]. 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-
agingactivitywhichpossiblyderivesfromtheirpluripotency,
although their potential efficacy as targeted pharmaceuticals
and/or a dietary supplement in the specific formulations in
humans has also been claimed [46,58,59,129].
Conclusion
Glucose 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 deglycating/transglycating
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
[46,58,59,129]. 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.
Acknowledgement
This 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 2004/028536
A1; WO 94/19325; WO 95/12581; WO 2004/064866 A1).
Disclosure
The 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 and/
or carcinine and their biomedical uses are the subject of the
issued and pending International Patents : (WO 2004/028536
A1; WO 94/19325; WO 95/12581; WO 2004/064866 A1).
Conflict of Interest
Declaration 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 & of The King of UK Edward VIII “David”
(Edward Albert Christian George Andrew Patrick David)
Prince of Wales UK, Duke & Duchess of Windsor . In other
words, Dr. Mark Babizhayev has an “Unofficial Hereditary
Status of a King of UK” and is the Major relative of the
British Royal Family. The data are described shortly on The
Twitter: Albert II Grimaldi@markinmonaco https://twitter.
com/markinmonaco/with_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 & of The King of
UK Edward VIII “David” (Edward Albert Christian George
Andrew Patrick David) Prince of Wales UK, Duke &
Duchess of Windsor.
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2003 Apr 9;289(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
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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:
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protein modification. The potential role of ‘autoxidative
glycosylation’ in diabetes. Biochem J 245: 243-250.
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Heinecke JW (1999) The myeloperoxidase system of human
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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.

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