Method for the treatment of the complications and pathology of diabetes

The present invention provides a method for the treatment of the complications and pathology of diabetes. The method involves the administration to a diabetic subject of a composition comprising a compound selected from the group consisting of (.beta.-Ala-His).sub.n, (Lys-His).sub.n, a compound of the formula R.sub.1 -X-R.sub.2, pharmaceutically acceptable salts thereof and combinations thereof; and a pharmaceutically acceptable carrier, in which n is 2-5, R.sub.1 is one or two naturally occurring amino acids, optionally alpha-amino acetylated with alkyl or aralkyl of 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms, R.sub.2 is 1 or 2 naturally occurring amino acids, optionally alpha-carboxyl esterified or amidated with alkyl or aralkyl of 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms and X is R.sub.3 -L or D-His (R.sub.4)-R.sub.5 where R.sub.3 is void or .omega.-aminoacyl with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms, R.sub.4 is void or imidazole modification with alkyl-sulphydryl, hydroxyl, halogen and/or amino groups, and R.sub.5 is void or carbonyl (alkyl) amides with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms. Preferably, the compound is camosine.

The present invention relates to a method of treating the complications and 
pathology of diabetes. 
The di-peptide carnosine, was discovered about 90 years ago (Gulewitsch and 
Amiradzibi, 1900) as a heat-stable extract derived from meat; since these 
early origins, considerable data has accumulated on the distribution and 
metabolism of the di-peptide. Carnosine (.beta.-alanyl-L-histidine) and 
its related compounds such as anserine 
(.beta.-alanyl-1-methyl-L-histidine) and homocarnosine 
(.gamma.-amino-butyryl-L-histidine) are present in millimolar 
concentrations in numerous mammalian tissues, including skeletal muscle 
(2-20 mM) and brain (0.3-5 mM). Although no unified hypothesis exists to 
account for physiological function of this group of di-peptides, their 
antioxidant properties, ability to protect DNA from radiation damage, 
ability to chelate divalent cations, and remarkable buffer capacity at 
physiological pH-values has led to the proposal that their primary 
function in vivo is to furnish protection to proteins, lipids and other 
macromolecules. 
In addition to its role as free radical scavenger carnosine has been 
claimed to act as an "immunoregulator" (Nagai, Patent: GB 2143732A) with 
useful properties in the treatment of certain cancers (Nagai, Patent DE 
3424781 A1). Camosine has also been suggested to be useful in the 
treatment of lipid peroxide induced cataracts (Babizhayev, 1989). There is 
also evidence that carnosine can accelerate the process of wound healing. 
Non-enzymatic Glycosylation 
Free-radical damage is not the only process to affect the structure of 
proteins and nucleic acids. Non-enzymatic glycosylation (glycation), the 
Maillard reaction in food chemistry (Maillard, 1912, or browning reaction, 
involves reaction of amino groups with sugar aldehyde or keto groups to 
produce modified amino groups and eventually forming 
advanced-glycosylation-end-products (AGE-products). Although glycation is 
slow in vivo, it is of fundamental importance in ageing and in 
pathological conditions where sugar levels are elevated, e.g. diabetes. 
It is possible to demonstrate glycation of proteins in the test tube. 
Several studies have shown that most proteins and DNA are potential 
targets for non-enzymic glycosylation in which sugars become attached to 
amino groups in the molecule via a Schiff's base. Subsequently a 
rearrangement occurs to give the coloured product (called the Amadori 
product). Further slow and uncharacterised reactions of the Amadori 
products occur. 
Analysis of the preferred glycation sites in proteins shows the epsilon 
amino groups of lysine residues are primary targets, particularly when in 
proximity to histidine residues (Shilton & Walton, 1991). In a search for 
stable peptides with long half-lives in vivo we found that the amino acid 
sequence of carnosine is similar to Lys-His, thus having the potential to 
react with sugars and react as scavenger for aldehyde groups. In addition 
carnosine is virtually non-toxic; well documented toxicity studies have 
indicated that the material can be administered to mammals to a level of 
5-10 g/kg body weight and therefore no toxic side effects are expected 
over long-term treatment. 
So far only one other compound has been shown to slow down glycation by 
reacting with sugars and blocking the Amadori re-arrangement. 
Aminoguanidine can reduce both in vitro and in vivo glucose-derived 
advanced glycation end products. Unfortunately aminoguanidine, a 
nucleophilic hydrazine compound, is nonphysiological and is of unknown 
long-term toxicity. 
Diabetes 
Diabetes is a metabolic disorder caused by an acute or chronic deficiency 
of insulin. It is diagnosed by an increased blood glucose level. The acute 
condition is characterised by a reduced glucose uptake of the 
insulin-dependent tissues. The body counteracts the resulting energy 
deficiency by increasing lipolysis and reducing glycogen synthesis. When 
the diabetic condition is severe, calories are lost from two major 
sources; glucose is lost in the urine, and body protein is also depleted. 
This is because insufficiency of insulin enhances gluconeogenesis from 
amino acids derived from muscle. The acute disorder can be controlled by 
insulin injections but since the control can never be perfect, the 
long-term fate of a diabetic is dependent on complications occurring later 
in life in the eye (cataractogenesis and retinopathy), kidney 
(nephropathy), neurons (neuropathy) and blood vessels (angiopathy and 
artherosclerosis). It is well established that coronary heart disease is 
one of the most common causes of deaths in diabetics and non-diabetics 
alike. 
Analyses of urine protein are usually requested in diabetic patients to 
rule out the presence of serious renal disease (nephropathy). A positive 
urine protein result may be a transient or insignificant laboratory 
finding, or it may be the initial indication of renal injury. The most 
serious proteinuria is associated with the nephrotic syndrome, 
hypertension, and progressive renal failure. In these conditions, the 
glomeruli become increasingly permeable to proteins by mechanisms that are 
poorly understood. The consequences are extremely serious, since they can 
progress rather rapidly to total renal failure and ultimate death. This 
form of proteinuria occurs as a secondary consequence to diseases like 
diabetes, amyloidosis and lupus erythematosus. 
As in other complications of diabetes, consideration of a potential role 
for glycation in the development of retinopathy must be taken into 
account. The retinal capillaries contain endothelial cells which line the 
capillary lumen and form a permeability (blood-retinal) barrier, and 
pericytes (mural cells), which are enveloped by basement membrane produced 
by the two cell types. Intramural pericytes are selectively lost early in 
the course of diabetic retinopathy, leaving a ghost-like pouch surrounding 
basement membrane. Breakdown of the blood-retinal barrier is another 
failure. Aldose reductase inhibitors are under investigation in treatment 
of experimental retinopathy in animals. Their mechanism of action is the 
prevention of the accumulation of sorbitol and resulting osmotic changes. 
However, the link with non-enzymatic glycosylation becomes obvious by the 
fact that Hammes et al (1991) have shown that treatment with 
aminoguanidine inhibits the development of experimental diabetic 
retinopathy. It is very likely that other potential inhibitors of 
glycation like carnosine should also have a positive effect. 
Glycation and Atherosclerosis 
Recent studies have suggested that AGE may have a role in the development 
of atherosclerosis. This is based on the finding that human monocytes have 
AGE specific receptors on their surface and respond when stimulated by 
releasing cytokines. Minor injury to the blood vessel wall may expose 
sub-endothelial AGE and promoting the infiltration of monocytes and 
initiating the development of atherosclerotic lesion. Circulating 
lipoproteins can also undergo glycation which is then taken up by 
endothelial cells at a faster rate than non-glycated lipoprotein. This is 
of importance in diabetes where an increased serum level of glycated 
lipoproteins has been reported. A compound with anti-glycation properties 
like carnosine should therefore have a positive effect on vascular 
diseases. 
The reason for the diabetic complications are not fully understood as a 
continuous release of insulin after subcutaneous injections may not be 
adequate to respond to varying glucose concentrations necessary to avoid 
periodic hyperglycaemic conditions. Therefore, blood sugar levels in 
diabetics can be on average higher than in normal individuals resulting in 
an increased level of glycation. The best example are glycohaemoglobins 
which form non-enzymatically in red blood cells in amounts proportional to 
the cellular glucose levels. The higher percentage of glycated haemoglobin 
and serum albumin is used to monitor the degree of a diabetic's 
hyperglycaemia. 
A controlled dietary intake of compounds which can counteract the long-term 
effects of high glucose levels in blood would be beneficial as an addition 
to a controlled diabetes therapies, such as insulin administration, 
sulfonylurea and biguanide treatment, on the use of amylin blockers. It is 
only the open chain form of reducing sugars like glucose, galactose, 
fructose, ribose and deoxyribose which participate in glycation. By 
scavenging this free aldehyde group and binding it in a non-toxic form we 
believe that it should be possible to decrease the damage caused by high 
sugar levels in vivo and in vitro. The compounds which are proposed for 
the treatment of the complications and pathology of diabetes could be 
peptides with one or more of the following characteristics: 
1) they should be non-toxic even at relatively high doses; 
2) they should be resistant to cleavage by non-specific proteases in the 
intestine and be taken up intact into the blood and organs, but should be 
cleared by kidneys, thereby following a similar tissue distribution to 
glucose in diabetes; 
3) the peptides should react rapidly with reducing sugars compared with 
amino groups on protein surfaces; 
4) the resultant glycated peptides should not be mutagenic, in contrast to 
glycated amino acids, 
5) if the peptide is cleaved by specific proteases in blood and tissue the 
resulting amino acids should be of nutritional value for diabetics, for 
example facilitating gluconeogenesis and counteracting a negative nitrogen 
balance. 
SUMMARY OF THE INVENTION 
The present inventors believe that peptides having similar activity to that 
of canosine may be useful in the treatment of the complications and 
pathology of diabetes. 
Accordingly, in a first aspect the present invention consists in a method 
for the treatment of the complications and pathology of diabetes in a 
diabetic subject comprising administering to the subject a composition 
comprising a compound selected from the group consisting of 
(.beta.-Ala-His).sub.n, (Lys-His).sub.n, a compound of the formula R.sub.1 
-X-R.sub.2, pharmaceutically acceptable salts thereof and combinations 
thereof; and a pharmaceutically acceptable carrier, in which n is 2-5, 
R.sub.1 is one or two naturally occurring amino acids, optionally 
alpha-amino acetylated with alkyl or aralkyl of 1 to 12 carbon atoms, 
preferably 2 to 6 carbon atoms, R.sub.2 is 1 or 2 naturally occurring 
amino acids, optionally alpha-carboxyl esterified or amidated with alkyl 
or aralkyl of 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms and X 
is R.sub.3 -L or D-His (R.sub.4)-R.sub.5 where R.sub.3 is void or 
.omega.-aminoacyl with 1 to 12 carbon atoms, preferably 2 to 6 carbon 
atoms, R.sub.4 is void or imidazole modification with alkyl-sulphydryl, 
hydroxyl, halogen and/or amino groups, and R.sub.5 is void or carbonxyl 
(alkyl) amides with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms. 
In a second aspect the present invention consists in the use of a compound 
selected from the group consisting of (.beta.-Ala-His).sub.n, 
(Lys-His).sub.n, a compound of the formula R.sub.1 -X-R.sub.2, 
pharmaceutically acceptable salts thereof and combinations thereof; in 
which n is 2-5, R.sub.1 is one or two naturally occurring amino acids, 
optionally alpha-amino acetylated with alkyl or aralkyl of 1 to 12 carbon 
atoms, preferably 2 to 6 carbon atoms, R.sub.2 is 1 or 2 naturally 
occurring amino acids, optionally alpha-carboxyl esterified or amidated 
with alkyl or aralkyl of 1 to 12 carbon atoms, preferably 2 to 6 carbon 
atoms and X is R.sub.3 -L or D-His (R.sub.4)-R.sub.5 where R.sub.3 is void 
or .omega.-aminoacyl with 1 to 12 carbon atoms, preferably 2 to 6 carbon 
atoms, R.sub.4 is void or imidazole modification with alkyl-sulphydryl, 
hydroxyl, halogen and/or amino groups, and R.sub.5 is void or carbonxyl 
(alkyl) amides with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms, 
in the preparation of amedicament for the treatment of the complications 
and pathology of diabetes. 
In a preferred embodiment of the present invention R.sub.1 and R.sub.2 are 
L- or D-lysine or L- or D-aspartic acid or L- or D-glutamic acid or 
homologues thereof. In a preferred form of the invention the compound is 
selected from the group consisting of carnosine, anserine, ophidine, 
homocarnosine, homoanserine, D-carnosine, and carcinine, it is presently 
most preferred that the compound is carnosine. 
In a further preferred embodiment of the present invention the composition 
may include other compounds which have a beneficial effect in the 
treatment of the complications and pathology of diabetes, such as 
aminoguanidine. 
Further, as a number of the subjects to be treated may also be on insulin 
sulfonylurea, biguanide or amylin blocker therapy the composition of the 
present invention may be co-administered with the insulin sulponyurea, 
biguanide or amylin blockers therapy. 
Further information regarding sulponylurea acid biguanide therapy can be 
found in Beck-Nielsen "Pharmacology of Diabetes", Eds. C. E. Mogensen and 
E. Standl, 1991, pp 75-92, the disclosure of which is incorporated by 
reference. 
Further information on the use of amylin blocker therapy in diabetes can be 
found in Westermerk et al 1987 DNAS 84, 3881-3885, the disclosure of which 
is incorporated herein by reference. 
One of the major drawbacks with insulin therapy is the continued need for 
injections. The present invention may provide an alternative in the oral 
administration of carnosine with biguanides or sulfonylureas which may be 
more attractive to diabetics. 
The composition of the present invention may be administered in any 
suitable manner such as injection, infusion, ingestion, inhalation, 
iontophoresis or topical application. It is presently preferred, however, 
that the composition is administered orally. 
In yet a further preferred embodiment the active compound is mixed with or 
linked to another molecule which molecule is such that the composition is 
improved in regard to skin penetration, skin application, tissue 
absorption/adsorption, skin sensitisation and/or skin irritation. The 
molecule is preferably selected from the group consisting of sodium lauryl 
sulphate, lauryl ammonium oxide, ozone, decylmethylsulphoxide, lauryl 
ethoxylate, octenol, dimethylsulphoxide, propyleneglycol, nitroglycerine, 
ethanol and combinations thereof. 
It is also possible that the compound may be in the form of a prodrug. 
Further information on prodrug technology can be found in "A Text Book of 
Drug Design and Development", edited by Povl Krogsgaard-Larsen and Hans 
Bundgaard, Chapter 5 "Design and Application of Prodrugs", H. Bundgaard. 
The disclosure of this reference is incorporated herein by 
cross-reference. 
As stated above it is preferred that the composition of the present 
invention is administered orally. As will be understood by those skilled 
in the art numerous modifications can be made to the composition to 
improve the suitability of the composition for oral delivery. Further 
information on oral delivery can be found in "Peptide and Protein Drug 
Delivery" edited by Vincent H. L. Lee, Chapter 16 "Oral Route of Peptide 
and Protein Drug Delivery", V. H. L. Lee et al. The disclosure of this 
reference incorporated herein by cross-reference. 
As stated above the composition may be administered by injection. 
Injectable preparations, for example, sterile injectable aqueous, or 
oleagenous suspensions may be formulated according to methods well known 
to those skilled in the art using suitable dispersion or wetting agents 
and suspending agents. The sterile injectable preparation may also be a 
sterile injectable solution or suspension in a non-toxic parenterally 
acceptable diluent or solvent. Among the acceptable vehicles and solvents 
which may be employed are water, Ringer's solution, and isotonic sodium 
chloride solution. In addition, sterile, fixed oils are conventionally 
employed as a solvent or suspending medium. For this purpose, any bland, 
fixed oil may be employed including synthetic mono- or diglycerides. In 
addition, fatty acids, such as oleic acid find use in the preparation of 
injectables. 
The total daily dose of the composition to be administered will depend on 
the host to be treated and the particular mode of administration. It will 
be understood that the specific dose level for any particular patient will 
depend on variety of factors including the activity of the specific 
compound employed, the age, bodyweight, general health, sex, diet, time of 
administration, route of administration, rate of excretion, and the 
severity of the particular side effects undergoing therapy. It is believed 
that the selection of the required dose level is well within the expertise 
of those skilled in this field. 
It is believed that the dosage for carnosine would be in the range of 20 mg 
to 2 g/kg body weight/day and preferably in the range of 100 mg to 200 
mg/kg bodyweight/day. 
As stated above one of the complications associated with diabetes is 
cataracts. Accordingly, one particularly preferred mode of administration 
of the composition of the present invention is opthalmic administration. 
In this situation the pharmaceutically acceptable carrier will be sterile 
aqueous or non-aqueous solutions, suspensions, emulsions and ointments. 
Examples of suitable pharmaceutically acceptable vehicles for opthalmic 
administration are proplylene glycol, and other pharmaceutically 
acceptable alcohols, sesame or peanut oil and other pharmaceutically 
acceptable oils, petroleum jelly, water soluble opthalmically acceptable 
non-toxic polymers such as methyl cellulose, carboxymethyl cellulose 
salts, hydroxy ethyl cellulose, hydroxy propyl cellulose; acrylates such 
as polyacrylic acid salts, ethylacrylates, polyacrylamides, natural 
products such as gelatine, alginates, pectins, starch derivatives such as 
starch acetate, hydroxy ethyl starch ethers, hydroxy propyl starch as well 
as other synthetic derivatives such as polyvinyl alcohol, polyvinyl 
pyrolidone, polyvinyl methylether, polyethylene oxide, carbopol and 
xantham gum and mixtures of these polymers. Such compositions may also 
contain adjuvants such as buffering, preserving, wetting, emulsifying 
dispersing agents. Suitable preserving agents include antibacterial agents 
such as quartenary ammonium compounds, phenylmercuric salts, benzoyl 
alcohol, phenyl ethanol, and antioxidants such as sodium metabisulphide. 
Suitable buffering agents include borate, acetate, glyconate and phosphate 
buffers. The pharmaceutically opthalmic compositions may also be in the 
form of a solid insert. 
As will be clear from the foregoing discussion the complications and 
pathology of diabetes are treated by reducing or preventing non-enzymatic 
glycosylation. Accordingly, it could be expected that the method of the 
present invention would also be useful in the treatment of other adverse 
complications and pathology of other disease states which are due to 
non-enzymatic glycosylation.

EXAMPLE 1 
Reaction of Carnosine with Sugar 
The rate of reaction between aldehydes and amino groups in glycation is 
dependent solely on temperature and reactant concentration, thus allowing 
the use of non-physiological conditions in in vitro experiments to speed 
up the reaction without affecting the equilibrium. The first step in the 
Maillard reaction, the formation of a Schiff base between aldehyde and 
primary amino group, varies according to amount of linear chain form of 
the sugar. This is followed by an Amadori rearrangement and complicated 
secondary reactions, most of which are poorly understood. 
Incubation of glucose, galactose and dihydroxyactetone (DHA) with carnosine 
produced brown solutions characteristic of glycation as originally 
described by Maillard (1912). The reaction of carnosine with the sugars 
was followed by disappearance of free amino group measured flurometrically 
after HPLC. Glucose, galactose and DHA differed in their reaction with 
carnosine (FIG. 1). Glucose was the least reactive and DHA the most, 
showing at least a fifteen-fold difference. For convenience we chose to 
employ the triose DHA in most subsequent studies. 
EXAMPLE 2 
Prevention of dihydroxyacetone induced modification of bovine serum albumin 
by carnosine 
Physiological concentrations of bovine serum albumin (50 mg/ml in 50 mM 
Na-phosphate buffer pH 7.0) were incubated with or without 250 mM 
dihydroxyacetone in the presence and absence of 250 mM L-carnosine at 
23.degree. C. for 4 weeks. The experiment was performed under sterile 
conditions and the ionic strength was the same in all vials. The results 
are shown in table 1. 
In this long-term experiment dihyroxyacetone had glycated albumin and, as a 
result of an Amadori rearrangement, subsequently induced the formation of 
a solid gel. When carnosine was present the contents of the vial remained 
fluid. 
TABLE 1 
______________________________________ 
Incubation conditions Resultant effects 
______________________________________ 
Albumin + phosphate buffer 
colourless, fluid. 
Albumin + dihydroxyacetone 
brown, firm gel. 
Albumin + dihydroxyacetone 
dark brown, fluid. 
______________________________________ 
The effects of camosine on dihydroxyacetone-induced non-enzymic 
glycosylation of bovine serum albumin. 
EXAMPLE 3 
Comparison of the reaction rate of camosine and different amino acids with 
glucose 
Slow non-enzymic glycosylation of proteins and nucleic acids by glucose can 
be accelerated in vitro by raising the temperature from physiological 
values to 50.degree. C. The main targets of glucose in vivo and in vitro 
are basic amino acids lysine and arginine (either free or following their 
incorporation into proteins). Table 2 shows a comparison of the reaction 
of carnosine and different amino acids with glucose. In order to 
demonstrate the specificity of the Maillard reaction for reducing sugars, 
glucose was substituted by sorbitol (a non-reducing sugar). 500 .mu.l of 
glucose or sorbitol (250 mg/ml in 50 mM Na-phosphate buffer pH 7.0) were 
incubated with different amino acids or carnosine (500 mM) at 50.degree. 
C. for 18 h. The optical densities at 400 nm of the resultant solutions 
were measured (Table 2). Carnosine formed by far the most Maillard 
reaction product, approximately 2-times or 8-times more than the fastest 
reacting amino acid, L-lysine or beta-alanine respectively. A small amount 
of Maillard reaction product is also apparent when carnosine reacts with 
sorbitol, probably due to autoxidation of sorbitol to a reducing sugar. 
TABLE 2 
______________________________________ 
Optical density at 400 mm of Maillard reaction products 
between dipeptides or amino acids with glucose or sorbitol 
Incubation conditions 
OD 400 nm 
______________________________________ 
glucose + PBS 0.175 
glucose + carnosine 
8.455 
sorbitol + PBs 0.000 
sorbitol + carnosine 
0.209 
carnosine + PBS 0.041 
glucose + D, L-alanine 
0.266 
sorbitol + D, L-alanine 
0.008 
glucose + beta-alanine 
1.240 
sorbitol + beta-alanine 
0.010 
glucose + L-arginine 
0.469 
sorbital + L-arginine 
0.010 
glucose + L-lysine 170 
sorbitol + L-lysine 
0.009 
glucose + imidazole 
0.046 
sorbitol imidazole 0.035 
______________________________________ 
EXAMPLE 4 
Reaction of carnosine, related peptides and amino acids with 
dhydroxyacetone 
When the reaction rates of camosine, related peptides and amino acids with 
DHA were compared (Table 3), carnosine reacted faster than lysine, which 
suggested that the dipeptide could compete against other sources of amino 
groups for glycation. However, in this assay lysine had two amino groups 
contributing to its reactivity whereas in proteins only the epsilon amino 
group is usually available. To compare the glycation rate solely of the 
epsilon amino group, N-alpha-carbobenzox-yl-lysine (Z-lysine) which as a 
blocked alpha amino group, was used. When DHA was added to an equimolar 
mixture of carnosine of Z-lysine the dipeptide reacted about ten times 
faster than the blocked amino acid (Table 3). The relative reactivity was 
retained when glucose was employed as the glycating sugar, although the 
experiment took ten days to complete(not shown). Ac-Lys-NHMe, a molecule 
which closely resembles a lysine residue incorporated into proteins, also 
showed a slower reaction with DHA compared to carnosine. The peptide 
Ac-Lys-His-NH.sub.2 resembles the preferential glycation site in proteins 
and showed the same reactivity as does carnosine. The peptide 
beta-alanyl-glycine was virtually unreactive with DHA, confirming the 
requirement for histidine at position two in a peptide for fast glycation 
(Shilton & Walton, 1991). While D-carnosine (beta-alanyl-D-histidine) 
reacted as fast as the naturally-occurring isomer, the higher homologue, 
homocarnosine (gamma-amino-butyryl-L-histidine), reacted slower. This 
indicates that a minor structural change to camosine (the addition of a 
methylene group) reduces its reactivity. It also becomes evident that 
modification of lysine by various groups has a significant effect on the 
reaction rate. Whereas Ac-Lys-NH.sub.2 Me (blocked amino and carboxyl 
group) reacted faster, Z-lysine reacted slower than the free amino acid. 
It was also found that the addition of free imidazole and succinyl 
histidine (alpha-amino group blocked) promoted the reactivity of carnosine 
with DHA as shown by an increase in the rate of disappearance of the 
dipeptide's amino group. This is in agreement with the suggestions that 
imidazole either catalyses the Amadori rearrangement or reacts with an 
intermediate form thereby changing the equilibrium of the reaction towards 
AGE-products (Shilton & Walton, 1991). 
TABLE 3 
______________________________________ 
Compound % reacted 
______________________________________ 
A Beta-Ala-l-His-OH 26 
Beta-Ala-D-His-OH 26 
Ac-Lys-His-NH.sub.2 25 
AC-Lys-NH.sub.2 Me 21 
H-Lys-OH 17 
Gamma-aminobutyryl-His-OH 
15 
Z-Lys-OH 3 
Beta-Ala-Gly-OH 2 
B Beta-Ala-L-His-OH + succinyl-His 
33 
Beta-Ala-L-His-OH + imidazole 
43 
______________________________________ 
Glycation of Peptides and Amino Analogues by DHA. 
A and B: Compounds were reacted with DHA in PBS for 5 hours at 60.degree. 
C. and the loss of free amino group assayed by HPLC. Data are expressed as 
percent of amino groups reacted with DHA (SEM.+-.1% of total peptide or 
amino acid in incubation mixture). B only: Succinyl-His and imidazole were 
added at equimolar concentrations to beta-Ala-L-His-OH and the amino group 
of the latter assayed. 
EXAMPLE 5 
Mutagenic properties of glycated amino acids and glycated carnosine 
Glycated amino acids such as lysine and arginine have been reported to be 
mutagenic (Kim et al., 1991) in an assay system first described by Maron & 
Ames (1983) "Ames Test". Other glycated amino acids like proline and 
cysteine did not exhibit mutagenicity. We have investigated the 
mutagenicity of L-carnosine and the glycated froms of L-carnosine, 
L-lysine and L-alanine (Table 4). All four solutions appear to inhibit the 
indicator strain to some extent, especially at the 250 .mu.l dose. Our 
data confirm earlier results by Kim et al., (1991) that glycated L-lysine 
is mutagenic and may therefore be carcinogenic. The activity is slightly 
enhanced by the rat liver S-9 metabolic activation system. Glycated 
L-alanine showed no mutagenicity in our experiments and only weak 
mutagenicity in the earlier work. Both, free carnosine and glycated 
carnosine are not mutagenic. This would be anticipated should carnosine 
play a significant role in the Maillard reaction in vivo. The reason for 
the difference of the glycated forms of L-carnosine and L-lysine is not 
known. 
TABLE 4 
______________________________________ 
Revertants per Plate with TA 100 
Compound Dose (.mu.l) 
Without S-9 
With S-9 
______________________________________ 
L-carnosine 
250 158 .+-. 11 
149 .+-. 13 
50 154 .+-. 14 
179 .+-. 15 
L-carnosine 
250 142 .+-. 17 
158 .+-. 19 
glycated 50 159 .+-. 7 167 .+-. 10 
L-lysine 250 277 .+-. 21 
244 .+-. 13 
glycated 50 357 .+-. 17 
553 .+-. 19 
L-alanine 250 145 .+-. 6 146 .+-. 9 
glycated 50 160 .+-. 9 181 .+-. 10 
negative control 161 .+-. 6 188 .+-. 10 
+ azide &gt;1000 N/A 
+ 2AF N/A 250 .+-. 33 
+ 2AAF N/A 500 
______________________________________ 
Mutagenic potential of glycated compounds 
Salmonella tyrphimurium TA 100 indicator strain his.sup.- to his.sup.+ 
reversion system. Data represent the mean number of revertants per plate 
and their standard deviation for the test solutions and controls with and 
without metabolic stimulation by rat liver microsomal (S-9) preparation. 
EXAMPLE 6 
Comparison of Camosine with Aminoguanidine as Inhibitors for Non-enzymatic 
glycosylation 
To compare the effect of both carnosine and aminoguanidine on glycation 
bovine serum albumin (BSA) and ovalbumin was incubated with a constant 
amount of DHA and varying concentrations of either anti-glycator at 
60.degree. C. At the start of the reaction and after seven hours aliquots 
were taken and the progress of the reaction analysed by gel filtration on 
a Superose 6 column. Crosslinking or fragmentation of protein became 
clearly visible as a change in retention time compared to the untreated 
protein used as control. Some compounds eluted after theoretical retention 
time for the smallest compound. They tend to interfere with the column 
resin even at high ionic strength and presence of a detergent (Tween 20). 
They are not necessary small compounds but rather highly charged and 
reactive. Table 5 summarises the data. Both compounds seem to react 
differently in this system: Carnosine reduced formation of high molecular 
weight compounds and was slightly more effective at low concentration 
compared to aminoguanidine. In all aminoguanidine samples uncharacterised 
reaction products are formed predominantly at high concentrations 
(described as low molecular weight form "LMW" because the retention time 
is longer then observed for all other compounds). Since the albumin 
monomer peak area is also reduced it is most likely that these are 
reaction products between ovalbumin, aminoguanidine and DHA. All three 
compounds showed no change in retention time or peak area when incubated 
separately under the same conditions for seven hours. LMW were also 
observed when ovalbumin was replaced by bovine serum albumin in the 
incubation mixture (not shown). The LMW forms were never present in the 
camosine samples. A good measure for the effectiveness of an 
TABLE 5 
______________________________________ 
OVALBUMIN 
Percent Area of Chromatogram 
HMW Monomer LMW 
______________________________________ 
t.sub.o hours 
Carnosine samples 
[A] to [D] 0 100 0 
[control] 0 100 0 
Aminoguanidine samples 
[A] to [D] 0 100 0 
[control] 0 100 0 
after 7 hours 
Carnosine samples 
[A] 9 91 0 
[B] 6 94 0 
[C] 3 97 0 
[D] 25 75 0 
[control] 68 32 0 
Aminoguanidine samples 
[A] 0 31 69 
[B] 8 20 72 
[C] 0 41 49 
[D] 38 40 22 
[control] 68 32 0 
______________________________________ 
Legend: ovalbumin was incubated with DHA for 7 hours in the presence of 
various concentrations of either carnosine or aminoguanidine. Reaction 
products were separated on a gelfiltration column (Superose 6) and peaks 
grouped according to their retention times: HMW, high molecular weight 
(15-30 min); albumin monomer (35 min); and late eluting compounds LMW, lo 
molecular weight (&gt;40 min). Carnosine and aminoguanidine concentration 
[control] 0 mM, [A] 600 mM, [B] 300 mn, [C] 100 mM, [D] 50 mM. 
anti-glycator is the amount of unmodified ovalbumin remaining after 7 hours 
of reaction. Here carnosine was more effective at all concentrations 
compared to aminoguanidine. 
EXAMPLE 7 
The Effect of Carnosine on Atherosclerosis 
Coronary heart disease is one of the most common causes of deaths in 
diabetics and non-diabetics alike. Glycation has been implicated in the 
development of atherosclerotic plaques in addition to many diabetic 
complications including diabetic kidney and eye disease. Cholesterol-fed 
rabbits were used to examine the effect of carnosine on atherosclerotic 
plaques formation over a period of 8 weeks. Our studies have shown that 
inhibition of glycation with carnosine can reduce but not prevent plaque 
formation. These results are shown in FIG. 2. 
The two tailed P values for the data were calculated using the Mann-Whitney 
two sample test: thoracic aorta=0.0529; abdominal aorta=0.5368; aortic 
arch=0.6623, all data carnosine (n=6) feeding versus diabetic control 
(n=5). For comparison aminoguanidine gave the following results in a 
similar experiment: thoracic aorta=0.12; abdominal aorta=0.044; aortic 
arch=0.067, all data aminoguanidine (n=11) feeding versus diabetic control 
(n=12). More animals were used in this study giving a statistically better 
result. However, there are clear indications that both inhibitors of 
non-enzymatic glycosylation can reduce plaque formation. 
The body weight of the animals reduced over the 8 week treatment period, 
however there was no difference between the control and carnosine treated 
group. No difference in weight of various organs was observed in control 
versus camosine treatment. 
______________________________________ 
Body Weight in kg 
Week 0 Week 8 
______________________________________ 
Control 3.27 .+-. 0.09 
2.75 .+-. 0.17 
Carnosine 3.33 .+-. 0.09 
2.68 + 0.12 
______________________________________ 
Weight of organs (g) after 8 weeks treatment 
Liver Kidney Heart 
______________________________________ 
Control 135.94 .+-. 5.06 
16.20 .+-. 0.67 
7.92 .+-. 0.53 
Carnosine 
124.40 .+-. 6.36 
17.53 .+-. 0.69 
6.12 .+-. 0.27 
______________________________________ 
EXAMPLE 8 
The Effect of Carnosine on the Formation of Cataracts in Diabetic Rats 
Cataract is an opacification of the ocular lens sufficient to impair 
vision. Diabetes has been associated with cataract for many years and many 
laboratory experiments support the view that diabetes is the cause of one 
form of cataract. Diabetes in animals can be induced by streptozotozin and 
opacity of the lens starts to develop by 20 days after injection but dense 
opacities appear only after about 100 days depending on age at injection. 
In cataract adducts of sugars to proteins including lens proteins have been 
identified and quantified. In most tissues there is little accumulation of 
late Maillard products even in diabetes but proteins in the lens nucleus 
have time not only to accumulate early glycation products but also for 
them to change into yellow Maillard products. The initial attack of a 
sugar leads to a variety of chemical entities and induces structural 
changes to enzymes, membrane proteins and crystallins in the lens and 
therefore several pathways can lead to damage. A compound like carnosine 
with its ability to scavenge the reaction aldehyde group of sugars should 
reduce the onset of cataract. We have tested this in the streptozotocin 
induced diabetic rat model. After 8 week on a carnosine diet the animals 
showed a higher clarity (less opacity) compared to a diabetic control 
group (Mann Whitney two sample test, two tailed p value=0.2092; carnosine 
feeding versus diabetic control) (see FIG. 3). Since this was measured at 
56 days, the half way mark of the experiment, the trend indicates a 
reduction in cataract formation by carnosine feeding. 
Cataract can not only be induced by reducing sugars in animal models. 
Babizhayev (1989) has shown that lipid peroxidation can be one initiatory 
cause of cataract development in animal models. Injection of a suspension 
of liposomes prepared from phospholipids containing lipid peroxidation 
products induces the development of posterior subcapsular cataract. 
According to his finding such modelling of cataract is based solely on 
lipid peroxidation and can be inhibited by antioxidants like carnosine. 
The formation of Maillard reaction products however, is an independent 
pathway and cannot be influenced by antioxidants. 
EXAMPLE 9 
The Effect of Carnosine on Proteinuria, Glycated Haemoglobin and Blood 
Glucose Levels in Diabetic Rats 
At week 8 no significant changes were observed for the following 
parameters: 
______________________________________ 
normal diabetic diabetic + carnosine 
______________________________________ 
Albuminuria 
2.4 .times./.div. 1.3 
2.51 .times./.div. 1.07 
2.51 .times./.div. 1.48 
Percent Glycated Haemoglobin (HbAl.sub.c) 
1.5 .+-. 0.1 
4.83 .+-. 0.23 
4.49 .+-. 0.14 
Blood Glucose (mM mean + SEM) 
10.0 .+-. 1.5 
29.84 .+-. 4.88 
22.63 .+-. 3.00 
______________________________________ 
Changes in proteinuria and retinopathy can only be observed after about 30 
weeks of diabetic condition. The compound aminoguanidine, usually used for 
the prevention of non-enzymatic glycosylation does not reduce the amount 
of glycated haemoglobin in diabetic models even after 30 weeks of feeding. 
This study is presently continuing. 
It will be appreciated by persons skilled in the art that numerous 
variations and/or modifications may be made to the invention as shown in 
the specific embodiments without departing from the spirit or scope of the 
invention as broadly described. The present embodiments are, therefore, to 
be considered in all respects as illustrative and not restrictive. 
Literature: 
Gulewitsch, W., & Amiradzibi, S. (1900) Ber. Dtsch. Chem. Ges. 33, 
1902-1903 
Maillard, L. C. (1912) C.R. Acad. Sci. 154, 66-68 
Shilton, B. H., & Walton, D. J. (1991) J. Biol. Chem. 266, 5587-5592 
Hammes, H. P., Martin, S., Federlin, K., Brownlee (1991) Proc. Natl. Acad. 
Sci USA 88, 11555-11558 
Kim, S. B., Kim, I. S., Yeum, D. M., & Park, Y. H. (1991) Mut. Res. 254, 
65-59 
Maron, D. M. and Ames, B. N. (1983) Mut. Res. 113, 173-215 
Babizhayev, M. A. (1989) Biochimica et Biophysica Acta 1004, 363-371