AU660047B2 - Method for the treatment of the complications and pathology of diabetes - Google Patents

Description

OP: DATE 05/04/93 AOJP DATE 10/06/93 APPLN. ID 25532/92 I 11111 IIII llll lI i IIIlllll III III PCT NUMBER PCT/AU92/0048o I I0iii 111111111 1 11111111111111 11111 1 AU9225532 INTERNATIONAL APPLICATION PUBLISiHItU UNUtK It t I A tIv- I LUUrtKI UlN i ntATY (PCT) (51) International Patent Classification 5 International Publication Number: WO 93/04690 A61K 37/02 A (43) International Publication Date: 18 March 1993 (18.03.93) (21) International Application Number: PCT/AU92/00480 (74) Agent: F.B.RICE COMPANY; P.O. Box 117, (28A Montague Street), Balmain, NSW 2041 (AU).

(22) International Filing Date: 9 September 1992 (09.09.92) (81) Designated States: AU, CA, JP, US, European patent (AT, Priority data: BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, PK 8279 9 September 1991 (09.09.91) AU NL, SE).

(71) Applicants (for all designated States except US): PEPTIDE Published TECHNOLOGY LIMITED [AU/AU]; 4-10 Inman With international search report.

Road, Dee Why, NSW 2099 KING'S COLLEGE LONDON [GB/GB]; The Strand, London WC2R 2LS

(GB).

(72) Inventors; and Inventors/Applicants (for US only) MICHAELIS, JOrgen [DE/AU]; 10 Honiton Avenue East, Carlingford, NSW 2118 HIPKISS, Alan, Roger [GB/GB]; 83 Gables Close, Lee, London SE12 OUF PANAGIOTOP- OULOS, Sianna [AU/AU]; 2 Ryall Court, Doncaster, VIC 3108 (AU).

660-0 4 7 (54)Title: METHOD FOR THE TREATMENT OF THE COMPLICATIONS AND PATHOLOGY OF DIABETES (57) Abstract 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 (P- Ala-His)n, (Lys-His)n, a compound of the formula RI-X-R 2 pharmaceutically acceptable salts thereof and combinations thereof; and a pharmaceutically acceptable carrier, in which n is 2-5, R, 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 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 3 -L or D-His (R 4

)-R

5 where R 3 is void or o-aminoacyl with I to 12 carbon atoms, preferably 2 to 6 carbon atoms, R 4 is void or imidazole modification with alkyl-sulphydryl, hydroxyl, halogen and/or amino groups, and R 5 is void or carbonyl (alkyl) amides with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms. Preferably, the compound is carnosine.

WO 93/04690 PCT/AU92/00480 Method for the Treatment of the Complications and Pathology of Diabetes The present invention relates to a method of treating the complications and pathology of diabetes.

The di-peptide carnosine, was discovered about 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 (p--alanyl-L-histidine) and its related compounds such as anserine (p-alanyl-l-methyl-L-histidine) and homocarnosine 7 -amino-butyryl-L-histidine) are present in millimolar concentrations in numerous mammalian tissues, including skeletal muscle (2-20mM) and brain (0.3-5mM). 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 Al). Carnosine 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 WO 93/04690 PCF/AU92/00480 2 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 glycatiLn end products. Unfortunately aminoguanidine, a nucleophilic hydrazine compound, is nonphysiological and is of unknown long-term toxicity.

WO 93/04690 PC/AU92/00480 3 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 insulindependent 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" WO 93/04690 PCT/AU92/00480 4 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 WO 93/04690 PCT/AU92/00480 5 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; WO 93/04690 PCT/AU92/00480 6 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 paptides 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 p-Ala-His)n, (Lys-His)n, a compound of the formula R 1

-X-R

2 pharmaceutically acceptable salts thereof and combinations thereof; and a pharmaceutically acceptable carrier, in which n is

R

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 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 3 -L or D-His (R 4

)-R

5 where R 3 is void or w -aminoacyl with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms, R 4 is void or imidazole modification with alkyl-'sulphydryl, hydroxyl, halogen and/or amino groups, and R 5 is void or carbonxyl (alkyl) amides with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms.

WO 93/04690 PCF/AU92/00480 7 In a second aspect the present invention consists in the use of a compound selected from the group consisting of P-Ala-His)n, (Lys-His)n, a compound of the formula R 1

-X-R

2 pharmaceutically acceptable salts thereof and combinations thereof; in which n is 2-5, R 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 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 3 -L or D-His (R 4

)-R

5 where R 3 is void or w -aminoacyl with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms, R 4 is void or imidazole modification with alkyl-sulphydryl, hydroxyl, halogen and/or amino groups, and R 5 is void or carbonxyl (alkyl) amides with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms, in the preparation of a medicament for the treatment of the complications and pathology of diabetes.

In a preferred embodiment of the present invention

R

1 and R 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 WO 93/04690 PCT/AU92/00480 8 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 Pov1 Krogsgaard-Larsen and Hans WO 93/04690 PCT/AU92/00480 9 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 "Paptide 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 o 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, WO 93/04690 PMTAU92/00480 10 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 20mg to 2g/kg body weight/day and preferably in the range of 100mg to 200mg/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, yolyvinyl methylether, polyethylene oxide, carbopol and xantham gum and mixtures 3 of these polymers. Such compositions may also contain adjuvants such as buffering, preserving, wetting, emulsifying dispersing agents. Suitable preserving agents include antibacterial agen', such as quartenary ammonium compounds, phenylmercuric salts, benzoyl alcohol, phenyl ethanol, and antioxidants such as sodium metabisulphide.

WO 93/04690 PCT/AU92/00480 11 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.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples and figures in which:- Figure 1 shows the reaction rate of L-carnosine with sugars. L-carnosine (60mM) was reacted with sugars (180mM) in 50mM na-phosphate buffer pH7 for five hours at 600 and the loss of the free amino group of carnosine assayed by HPLC. SEM 1% of total carnosine in incubation mixture; Figure 2 shows the effect of carnosine on atherosclerosis m cholesterol, d cholesterol plus carnosine); and Figure 3 shows the effect of carnosine on formation of cateracts in diabetic rats control, a diabetic, o diabetic plus carnosine).

Detailed Description of the Invention Methods: Reaction of Peptides and Amino Acid Derivativas with Sugars Unless otherwise stated, reactions were carried out in phosphate buffered saline, PBS, (140mM-NaCl/ phosphate, pH 7.4) in sealed microcentrifugation vials in a C waterbath. The reaction mixture contained peptide and 500 mM sugar. At specified time points samples WO 93/04690 PCT/AU92/00480 12 were taken, diluted 1:20 with water, and stored at -20 0

C

prior to analysis by HPLC.

Detection of Amino Groups For the detection of free amino groups on peptides the Waters AUTO. OPA TM was used (Waters AUTO.TAGTM operation manual). In brief, peptides were reacted with o-phthalaldehyde and the fluorescent derivative separated by HPLC on a Radical-PAKTM C18 column using a 10% (v/v) to 90% methanol gradient over 15 min as solvent. A Waters 470 fluorescence detector set at excitation 340nm/emission 440 nm was used.

Gelfiltration Chromatography of Glycated Proteins HPLC Specifications: COLUMN Superose 6, Pharmacia SAMPLE; 100p of Protein Sample (Approximately 100pg) ELUENT: 10mM phosphate buffer pH 7.38, 140 mM NaCI, 2mM KC1, 0.02% NaN 3 0.05% Tween FLOR RATE: DETECTION: 280nm.

Calibration was performed using Pharmacia high molecular weight (HMW) and low molecular weight (LMW) calibration kit.

CALIBRATOR COMPONENTS MOLECULAR RETENTION HMW Blue Dextran Thyroglobulin Ferritin Catalase Aldolase Blue Dextran Albumin Ovalbumin Chymotrypsin A Ribonuclease A

WEIGHT

)2,000,000 669,000 440,000 232,000 158,000 Co-elute )2,000,000 67,000 43,000 25,000 13,700 Co-elute

TIMES

15.49 25.84 29.73 32.58 32..58 15.65 33.74 35.57 39.23 39.23 n=6 n=6 n=6 n=6 n=6 n=6

LMW

WO 93/04690 PCT/AU92/00480 13 Analysis of mutagenic potential of qlycated compounds, "Ames Test" Preparation of glycated compounds was performed according to Kim et al. (1991). In brief, D-glucose (lM) and each of the following: L-carnosine, L-lysine, L-alanine (all 1M) were dissolved in distilled water, the pH adjusted to 7 and the mixtures heated at 100 0 C for min. The solutions (50pl and 250pl) were evaluated against strain TA 100 of Salmonells typhimurium using the plate incorporation method (Maron Ames, 1983) with or without metabolic activation by a standard rat liver microsomal preparation. 2-AF and 2-AAF were used as positive controls for the experiments with metabolic stimulation, otherwise sodium azide was included as strain specific positive control.

The Effect of Carnosine on Atherosclerosis Male New Zealand white cross rabbits fed a high cholesterol diet were randomised to control or carnosine treatment 2% and plasma was assayed for cholesterol and triglycerides and carnosine. All'animals received 100g of food pellets/day and free access to water.

At the end of the 8 week treatment period, the rabbits were anaesthetised with pentobarbitone (325mg/kg) and the total aorta removed. The arch, thoracic and abdominal regions were isolated by cutting the aorta circumferentially 1.0cm proximal to the first pair of intercostal arteries and 0.3cm above the coeliac artery.

The adventitia was carefully dissected away, and the artery longitudinally cut to expose the intimal surface.

The aorta was fixed in 10% buffered formalin for 48 hours. The lipid plaques in these vessels were then stained using Sudan IV and mounted with an aqueous mounting medium (Kaiser's Glycerol Jelly). The lesioned areas were directly traced from the mounted sections using an image analyser (Eye Com 850 image processor).

WO, 93/04690 PCT/AU92/00480 14 Computer-aided planimetry was used to macroscopically determine the percentage of the affected area.

Representative segments of atherosclerotic plaque were removed for confirmation by light microscopy. Results are expressed as mean SEM.

Formation of Cataracts in Diabetic Rats Male Sprague-Dawley rats weighing 200-250 g and 6 weeks of age were randomised to the following three treatment groups: control, diabetic, and carnosine-treated diabetic rats. Diabetes was induced with streptozotozen STZ(60mg/kg body weight in citrate buffer, pH 4.5, and all animals with plasma glucose levels )20mM after 1 week were included in the study.

Diabetic rats were randomised to receive either no therapy or 2% carnosine in drinking water.

Results 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, WO 93/04690 PCI'/AU92/00480 15 galactose and DHA differed in their reaction with carnosine (Fig. 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 dihvdroxyacetone induced modification of bovine serum albumin by carnosine Physiological concentrations of bovine serum albumin (50mg/ml in 50mM Na-phosphate buffer pH 7.0) were incubated with or without 250mMr dihydroxyacetone in the presence and absence of 250mM L-carnosine at 23 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 carnosine on dihydroxyacetone-induced non-enzymic glycosylation of bovine serum albumin.

Example 3 Comparison of the reaction rate of carnosine 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 0 C. The main targets of glucose in vivo and in vitro are basic amino acids lysine and arginine (either free or WO 93/04690 PCT/AU92/00480 16 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). 5001 of glucose or sorbitol (250mg/ml in 50mM Na-phosphate buffer pH 7.0) were incubated with different amino acids or carnosine (500mM) at 50 0 C for 18 h. The optical densities at 400nm of the resultant solutions were measured (Table 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.

WO 93/04690 PCT/AU92/00480 17 Table 2 Optical density at 400mm of Maillard reaction products between dipeptides or amino acids with glucose or sorbitol Incubation conditions OD 400nm glucose PBS 0.175 glucose carnosine 8.455 sorbitol PBs 0.000 sorbitol carnosine 0.209 carnosine BS 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 4.170 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 carnosine, 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-carbobenzoxyl-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 WO 93/04690 PCT/AU92/00480 18 times faster than the blocked amino acid (Table 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 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-Lhistidine), reacted slower. This indicates that a minor structural change to carnosine (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 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).

WO 93/04690 WO 9304690PCT/AU92/00480 19 Table 3 Compound reacted A Beta-Ala-i-His-OH1 26 Beta-Ala-D-His -OH 26 Ac-Lys-His-NH 2 A C-Lys -NH 2 Me 21 H-Lys -OH 17 Gaxma-aminobutyryl-His-OH 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 hours at 60 0Cand the loss of free amino group assayed 23P by HPLC. Data are expressed as percent of amino groups reacted with DHA (SEN 1% of total peptide or amino acid in incubation mixture). B only: Succiivyl-His and imidazole were added at equimolar concentrations to beta-2Aia-L-His-OH and the ami-uo group of the latter assayed.

ExampDl Mutqcwenic properties of clycated amino acids and glycated carnosi-ne Glycated amnino acids such as lysine and arginine have been reported to be mutagenic (Kim et al., 1991) in an assay system first described by IMaron Ames (1983) "Ames Test". Other glycated amino acids like proline and WO 93/0465d PC/AU92/00480 20 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 All four solutions appear to inhibit the indicator strain to some 'extent, especially at the 250 yi 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.

WO 93/04690 PCT/AU92/00480 21 Table 4 Revertants per Plate with TA 100 Compound Dose 1p Without S-9 With S-9 L-carnosine L-carnosine glycated L-lysine glycated L-alanine glycated 250 250 50 250 50 250 50 158 11 154 14 142 17 159 7 277 21 357 17 145 6 160 9 149 13 179 158 19 167 244 13 553 19 146 9 181 negative control azide 2AF 2AAF 161 6 )1000

N/A

N/A

188

N/A

250 33 500 Mutagenic potential of glycated compounds Salmonella typhimurium TA 100 indicator strain his- to his+ 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 preparation.

WO 93/04690 PCT/AU92/00480 22 Example 6 Comparison of Carnosine with Aminoquanidine 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 0

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 (Tteen 20). They are not necessary small compounds but r-aher highly charged and reactive. Table 5 summarises Lne 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 lonaer 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 carnosine samples. A good measure for the effectiveness of an WO 93/04690 PCT/AU92/00480 23 TABLE

OVALBUMIN

Percent Area of Chromatogram HMW Monomer LMW to hours Carnosine samples to 0 100 0 [control] 0 100 0 Aminoguanidine samples to 0 100 0 [control] 0 100 0 after 7 hours Carnosine samples 9 91 0 6 94 0 3 9' 0 25 75 0 [control] 68 32 0 Aminoguanidine samples 0 31 69 8 20 72 0 41 49 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, low molecular weight 40 min).

Carnosine and aminoguanidine concentration [control] OmM, 600mM, 300mn, 100mM, WO 93/04690 PCT/AU92/00480 24 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-diabet:cs 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 week3. Our studies have shown that inhibition of glycation with carnosine can reduce but not prevent plaque formation. These results are shown in Figure 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 feeding versus diabetic control 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=ll) feeding versus diabetic control More animals were used in this study giving a statistically better result. However, there are clear indications that both inhibitors of non-enzymatic glycosylati6a 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 carnosine treatment.

WO 93/04690 PCT/AU92/00480 25 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 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 Th, Effect of Carnosine op 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 iizuced 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 adduccs of sugars to proteins including lens proteins have been identified and quantified. In most tissues there is little accumulation of late Maillard nroducts 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 WO 93/04690 PCT/AU92/00480 26 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 Figure 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 11 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.

WO 93/04690 PCT/AU92/00480 27 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 2.4x/-1.3 Albuminuria diabetic 2.51x/41.07 diabetic carnosine 2.51x/.1.48 Percent Glycated Haemoglobin (HbAlc) normal diabetic diabetic carnosine 0.1 4.83 0.23 4.49 0.14 Blood Glucose (mM mean SEM) normal diabetic diabetic carnosine 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 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.

WO 93/04690 WO 9304690PCr/AU92/00480 28 Literature: Gulewitsch, Axniradzibi, S. (1900) Ber. Dtsch. Chem.

Ges. 33, 1902-1903 Maillard, L.C. (1912) C.R. Acad. Sci. 154, 66-68 Shilton, Walton, D.J. (1991) J. Biol. Chem. 266, 5587 -55DJ2 Hamnmes, Martin, Federlin, Brownlee (1991) Proc. Natl. Acad. Sci USA 88, 11555-11558 Kim, Kim, Yeum, 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

Claims (11)

1. 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 p-Ala-His)n, (Lys-His)n, a compound of the formula R1-X-R2, pharmaceutically acceptable salts thereof and combinations thereof; and a pharmaceutically acceptable carrier, in which n is 2-5, R 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 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 3 -L or D-His (R 4 )-R 5 where R 3 is void or w -aminoacyl with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms, R 4 is void or imidazole modification with alkyl-sulphydryl, hydroxyl, halogen and/or amino groups, and R 5 is void or carbonxyl (alkyl) amides with 1 to 12 carbon atoms, preferably 2 to 6 carbon atoms.

2. A method as claimed in claim 1 in which the compound is selected from the group consisting of carnosine, anserine, ophidine, homocarnosine, homoanserine, D-carnosine and carcinine.

3. A method as claimed in claim 2 in which the compound is carnosine.

4. A method as claimed in claim 1 in which R 1 and R 2 are L- or D-lysine or L- or D-aspartic acid or L- or D-glutamic acid or homologues thereof.

A method as claimed in any one of claims 1 to 4 in which the composition further comprises aminoguanidine.

6. A method as claimed in any one of claims 1 to 5 in which the composition is co-administered with insulin sulfonylureas, biguanides and/or amylin blockers. WO 93/04690 PCT/AU92/00480 30

7. A method as claimed in any one of claims 1 to 6 in which the composition is administered by injection, infusion, ingestion, inhalation, opthalmically, iontophoresis or topical application.

8. A method as claimed in any one of claims 1 to 7 in which the composition is administered orally or opthalmically.

9. A method as claimed in any one of claims 1 to 7 in which the 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.

A method as claimed in claim 9 in which the molecule iL selected from the group consisting of sodium lauryl sulphate, lauryl ammonium oxide, ozone, decylmethylsulphoxide, lauryl ethoxylate, octenol, dimethylsulphoxide, propyleneglycol, nitroglycerine, ethanol and combinations thereof.

11. A method as claimed in any one of claims 1 to 10 in which the compound is in the form of a prodrug. consisting of p-Ala-His)n, (Lys-His)n, a compoun of the formula R -X-R2, pharmaceutically acce ale salts thereof and combinations thereof; i which n is R 1 is one or two naturally occurring ino acids, optionally alpha-amino acetylate with alkyl or aralkyl of 1 to 12 carbon atoms, prefe rly 2 to 6 carbon atoms, R 2 is 1 or 2 naturally oc ring amino acids, optionally alpha-carboxyl es ified or amidated with alkyl or aralkyl of 1 12 carbon atoms, preferably 2 to 6 carbon atoms an is R 3 -L or D-His (R 4 )-R 5 where R 3 is voi or w -aminoacyl with 1 to 12 carbon atoms, referably 2 to 6 carbon atoms, R is void or imidazole eati-n with alkyl-sulphydryl, hydroxyl, halogen