For those following the carnosine thread....
from
http://www.peptech.com.au/ncs-carn.htm
Carnosine - Novel strategies to prevent the formation of Advanced Glycation
End Products using Carnosine and Carnosine Stabilizers
Background
Carnosine, the dipeptide beta-alanalyl-L-histidine, is a naturally occurring
molecule, found at levels as high as 20 mM in skeletal muscle and 5 mM in
the brain. These high levels appear to be due to resistance of carnosine to
proteolytic cleavage and low activity of specific carnosinases.
The biological role of carnosine remains unclear, but homeostatic or
protective functions have been proposed. Carnosine is believed to decrease
oxygen free radical-mediated damage to cellular macromolecules either by
chelating divalent cations, or by scavenging hydroxyl radicals through its
imidazole moiety.
Peptech scientists and their collaborators have discovered additional
activities of carnosine that suggest it may have considerable therapeutic
potential in reducing or preventing some age-related degenerative
conditions.
Carnosine inhibits reactions related to ageing in the skin
Maintenance of the skin requires continual proliferation of fibroblasts and
production of new collagen. Modelling of this process in cell culture
systems shows that human fibroblasts have limited proliferative capacity.
Cells taken from aged individuals have lower capacity for further new growth
than do cells taken from the young. Peptech scientists and their
collaborators have shown that in such model systems, carnosine is able to
prolong the lifespan potential of skin fibroblasts, and to revitalise cells
which are already well advanced towards senescence.
These results suggest the potential use of carnosine in improving wound
healing, particularly in the elderly.
An unexpected property for carnosine - inhibition of the formation of
advanced glycosylation end product proteins
Non-enzymatic glycosylation (glycation), the Maillard reaction in food
chemistry, involves reaction of amino groups with sugar aldehyde or keto
groups to produce reactive chemical entities. These provoke cross-linking,
with eventual formation of advanced glycosylation end products (AGE).
Although glycation is slow in vivo, it is of fundamental importance in
ageing and in pathological conditions where sugar levels are elevated, eg in
diabetes, and can result in abnormalities of connective tissue, for example
cross-linking of collagen.
Analysis of the preferred glycation sites in proteins shows that the epsilon
amino groups of lysine residues are primary targets, particularly when in
proximity to histidine residues. This preferred sequence closely resembles
that of carnosine.
In in vitro tests, Peptech scientists have shown that carnosine can readily
react with sugars such as glucose, galactose and dihydroxy acetone (DHA) to
produce brown solutions characteristic of glycation as originally described
by Maillard. The reactive rate of carnosine with sugars was determined by
disappearance of free amino groups (measured fluorimetrically after HPLC).
Of the three sugars tested, DHA was the most reactive.
Carnosine reacted with DHA faster than did lysine, suggesting that the
dipeptide could compete against other sources of amino groups for glycation.
Variants of the dipeptide were tested in glycation reactions and it was
found that minor structural changes to carnosine (eg the addition of a
methylene group) reduced its reactivity. In addition, carnosine strongly
inhibited glycation of the dipeptide Ac-Lys-His-NH2 by DHA. Since this
latter sequence resembles the preferred glycation site in proteins, it
indicates that carnosine may be able to block glycation of proteins.
Further investigation showed that carnosine was able to inhibit the
cross-linking of bovine serum albumin resulting from glycation of the
protein by DHA. All in vitro results were observed at relatively high levels
of carnosine (60-250 mM), of the same order as the concentration of sugars
(0.2-2 M) or protein amino groups present in the reaction mixtures. This is
consistent with a mechanism for carnosine action whereby it acts as a
competitive acceptor in the glycation reaction. Thus the concentration of
carnosine required to inhibit protein damage in vivo should be dependent on
the levels of reactive sugars being generated.
Properties of glycated carnosine
Glycated amino acids such as lysine and arginine have been reported to be
mutagenic in the Ames test. Other glycated amino acids such as proline and
cysteine do not exhibit mutagenicity. Peptech scientists investigated the
mutagenicity of L-carnosine, and the glycated forms of L-carnosine, L-lysine
and L-alanine, by Ames testing. As expected from published work,
mutagenicity was recorded for L-lysine but not for L-alanine. Neither
L-carnosine nor glycated carnosine were mutagenic.
Therapeutic significance of glycation inhibition
It is believed that AGE product formation is of particular significance in
diabetes, where levels of blood glucose are periodically elevated. AGE's are
strongly implicated in development of degenerative changes including
cataract formation and atherosclerosis.
Carnosine, administered to animals in their drinking water, has been
demonstrated by Peptech scientists to reduce the occurrence of
AGE-associated degenerative changes. Taken over eight weeks, carnosine
reduced changes associated with cataract formation in the eyes of rats in
which diabetes was induced by treatment with streptozotocin. Further,
carnosine reduced the extent of artherosclerotic plaque formation over eight
weeks in rabbits fed a high cholesterol diet. Carnosine showed activity
equivalent to that of the known glycation inhibitor, aminoguanidine. These
experiments are of a preliminary nature only, and more extended periods of
treatment will be needed to fully assess carnosine's efficacy in reducing
the occurrence of these degenerative conditions.
Strategies to modify carnosine metabolism
Another approach undertaken by Peptech scientists has been to modify the
metabolism of carnosine in our effort to increase its half-life in serum and
thus extend its activity. A screening program has isolated several promising
lead compounds which significantly improve the stability of carnosine in
vitro.
Further testing will be required to determine the efficacy of these
compounds in vivo.
Conclusions
Peptech scientists and their associates have demonstrated that the naturally
occurring dipeptide carnosine is able to directly block protein damage
resulting from AGE formation. In its effects, carnosine is equipotent with
aminoguanidine, the only well-documented glycation inhibitor. However,
carnosine intervenes at an earlier step in the glycation process than
aminoguanidine and effectively diverts the reaction to production of
non-damaging and rapidly cleared products. Carnosine is a natural product
and, unlike aminoguanidine, has extremely low toxicity. Despite its peptide
nature, it is fully effective following oral administration.
In future work, extended animal trials to demonstrate efficacy for carnosine
in appropriate models of diabetes-associated degenerative changes are
required. Carnosine, and closely related molecules with similar properties,
represent an exciting new entry to the treatment of age-related degenerative
responses, particularly in groups of patients, such as diabetics, where
these degenerative processes may be accelerated. Peptech is using its
findings on carnosine as a starting point for design of improved molecules
to act as antiglycating agents in vivo.
Further information
For further information on Carnosine, please contact:
Dr Deborah Rathjen
Manager, Business Development
Peptech Limited
35-41 Waterloo Road
North Ryde, Sydney, NSW 2113, Australia
Tel: +61 2 9870 8788
Tel: +61 2 9870 8786
e-mail: drathjen@peptech.com
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