WO2001004632A1

WO2001004632A1 - Oligosaccharide metabolites of glycosaminoglycans and their use in the diagnosis and treatment of complications of diabetes

Info

Publication number: WO2001004632A1
Application number: PCT/EP2000/006474
Authority: WO
Inventors: Ivo Volpato; Bernard Bizzini; Giovanni Scapagnini; Lorenzo Volpato; Maurizio Magara; Flavio Veneroni
Original assignee: Bidifarm S.R.L.
Priority date: 1999-07-08
Filing date: 2000-07-07
Publication date: 2001-01-18
Also published as: AU6270600A; ITMI991503A0; ITMI991503A1; EP1198709A1

Abstract

Oligosaccharide metabolites of glycosaminoglycans which may be found in biological liquids are useful in the diagnosis and treatment of diabetic complications, since their concentration is proportional to the severity of the course of the pathology.

Description

OLIGOSACCHARIDE METABOLITES OF GLYCOSAMINOGLYCANS AND

THEIR USE IN THE DIAGNOSIS AND TREATMENT OF COMPLICATIONS OF

DIABETES

Field of the invention The present invention concerns oligosaccharide metabolites of glycosaminoglycans and their use in the diagnosis and treatment of diabetes, as well as the methods for detecting and titrating them. State of the art Pathologies with a chronic course are generally characterised by the appearance and development of physiological dismetabolites originating from alterations at both cellular and humoral level.

The extent of the variation of the humoral parameters may be influenced by the general condition of the subject, so it does not always correctly reflect the state of evolution of the disease and may provide an incorrect indication of the extent of the damage produced and of the consequent risk of onset of related pathological complications.

A correct assessment of the cellular damage would be desirable as it would be able to provide useful indication on the severity of the damage produced, on the speed of progression of the disease, on its regression following pharmacological treatment and on the potential onset of related pathologies.

However, to date it has not always been possible to make a correct diagnosis of the cellular damage with methods that did not imply invasive intervention on the patient. Actually this type of investigation is generally carried out with cytometric methods which involve collecting a sample of tissue. Diabetes is one of those chronic diseases for which the control of the humoral and cellular parameters is particularly interesting in order to understand the methods of the course of the pathology, which presents periods of latency of different length, varying from person to person. Contrary to what was initially believed, the humoral alterations in diabetes may be an expression, not only of a malfunction of the Islands of Langerhans, but also of a simultaneous endocrine disorder such as hyperpituitarism, hyperadrenocorticism, hyperthyroidism, etc. The aim of treating a diabetic patient is to maintain the blood glucose levels as close as possible to normal, also in order to limit the onset and progress of complications. The most frequent risks of complications in diabetes are microvascular disorders, retinopathy, nephropathy and neuropathy. On average these tend to appear about 10-15 years after the onset of the disease, but only the renal complications and disorders of the retina seem to be directly related to the severity of the hyperglycaemia, while this is not the case for atherogenic and neurological disorders. It must be stressed that some complications do not seem to regress significantly even when the glucose levels are reduced for 1-3 years. The form of monitoring most commonly used is to determine the glucose in order to highlight states of hyper- or hypoglycaemia. An important test concerns the measurement of glycated proteins, but, due to the relatively short half-life of such proteins, this type of tests allows only a short-term control of the glycaemic state of the patient, and consequently a short-term assessment of the treatment that would be most effective in keeping the patient in the desired normo-glycaemic state and of the progression of the complications.

Hence it may be deduced that the current methods of diagnosis of forecast and onset of diabetic pathologies present severe gaps and limitations. The genesis and chronic development of many pathological disorders imply alterations of the extra-cellular matrix. Among its specific components, the basement membrane contains the proteoglycan of heparansulphate (also known as proteoheparansulphate) and traces of other proteoglycans. Proteoglycans are structures formed by a central proteinaceous filament to which polysaccharides called glycosaminoglycans (hereinafter referred to as GAGs), composed of repetitive disaccharide units containing a derivative of an amino sugar, glucosamine or galactosamine are connected by acidic bonds. At least one of the disaccharide sugars possesses a carboxylate or sulphate group with negative charge. The presence of proteoheparansulphate, with its strong negative charge, can endow the basement membrane with the ability to block the passage of proteins from the blood to the urine; through its links with other structures of the membrane, it is also able to influence the processes involved in the inflammatory, atherosclerotic or thrombogenic pathogenesis, etc.

It has been stressed that the decrease of the concentration of proteoheparansulphate can cause alterations of the basement membrane structure that are typical of diabetic pathology (Rorbach D.H. et al., "Alterations of the basement membrane in diabetes, in extracellular matrix", S.Hawkes e J.L.Wang ed., 1982, Academic Press, N.Y., pages 407-411 ). On the long term, the diabetic basement membrane appears more thickened and porous than normal, particularly in the areas through which large volumes of fluids pass, such as in the renal glomeruli and the blood capillaries. In particular, thickening has been attributed to low levels of proteoheparansulphate (Rorbach D.H. et al. supra).

In mellitus diabetes there is an increase of the values of β-N- acetylglucosaminidase (NAG) and of β-D-glucuronidase, two enzymes which participate in the degradation of mucopoiysaccharides and glycoproteins. (Whiting P.H, et al., Clinica Chimica Acta, 1979, 92, 459-463; Reglero A. et al., Clinica Chimica Acta, 1980, 103, 156-158) describe the seric activity of these two enzymes with relation to the severity of diabetic complications, such as microangiopathy, and an increase of the activities in proportion to the severity of the diabetes itself.

Pitkanen E. et al, Diabetologica, 1980, 18(4), 275-278, Gosiewska A. et al., Pd. Tyg. Lek., 1992, 47(1-2), 28-30, and Maisant A. et al., Padiatr. Padiol., 1993, 28(3), 77-80 have assumed that the urinary levels of NAG may reflect the renal damage in the diabetic patient and that its seric levels may, in some way, give some indications as to the development and prognosis of microangiopathy. Besides being a correlating parameter between diabetes and some of its complications, the determination of the NAG would seem to be independent of the blood glucose levels, which would indicate a probable direct relationship with tissue damage underlying the complications of the disease. However, up till now these remarks have not led to any practical diagnostic system since the enzymes concerned, and NAG in particular, have not proved to have an unmistakable linear relationship with the diabetic pathology and its course. In fact, as it is a protein, it is metabolised at renal level, and the part that survives this process has its concentration decreased by its role in the catabolism of gycosaminoglycans which, being present also in the urine, continue to stimulate its intervention. Moreover the determination of NAG would be related to a humoral parameter, while it is much more desirable to have a method that can detect a cellular parameter so as better to assess the damage caused by the pathology at these levels.

Summary of the invention Surprisingly, it has now been found that oligosaccharide metabolites of the GAGs originating from the cellular membrane may be detected in the biological liquids in concentrations proportional to the possible onset of the diabetes or of its pathological complications. They may also be a therapeutic tool for the treatment of complications of diabetes. Brief description of the figures

Figure 1 illustrates the electrophoretic bands of oligosaccharide metabolites of

GAGs extracted from the urine of healthy subjects, before and after treatment with chondroitinase A/C compared with standard GAGs (Sigma).

Figure 2 illustrates the electrophoretic bands of oligosaccharide metabolites of GAGs extracted from the urine of diabetic subjects, before and after treatment with chondroitinase A/C compared with standard GAGs (Sigma).

Figure 3 illustrates the electrophoretic run obtained from the urine of a diabetic subject.

Figure 4 illustrates the trend of the reading of the optic density in the affinity/specificity antigen/antibody test.

Figures 5 and 6 illustrate the calibration lines of the affinity test for lectin from

Triticum vulgaris.

Description of the invention

The present invention therefore refers to the use of oligosaccharide metabolites of GAGs as markers in biological fluids for assessing the state, severity, risk of onset of complications of diabetes, and for combating onset and progression thereof, and to the methods for revealing and titrating them.

The term oligosaccharide metabolites of GAGs refers, more specifically, to heparansulphate, chondroitinsulphates A and C, dermatansulphate and their metabolic fragments. Particularly preferred for the purposes of this invention is the use of heparansulphate and of its metabolic fragments.

The quantity of metabolites of the invention present in the biological liquids depends on the extent of the cellular damage caused by diabetes, but it is independent of the pharmacological control of hyperglycaemia. For this reason the determination of their quantity may be used in the control of the risk of onset of complications associated with diabetes, in monitoring the seriousness or regression of the pathology, in predicting the onset in subjects at risk. Since the concentration of metabolites of the invention is a value that does not depend on the variations of the humoral biochemistry, that is on the concentration of glucose in the subject's biological fluids, it may be considered as a real reference parameter of the state, severity, risk of onset, possibility of the genesis of complications, monitoring of the treatment of the diabetic disease. The metabolites of the present invention may be detected in the biological liquids, preferably in serum and in urine, and even more preferably in urine. Said oligosaccharide metabolites are extracted from the biological liquids with specific methods, and characterised both by electrophoresis and by a specific reaction in response to a nitrosation process.

The methods developed for determining these metabolites are of a ponderal, immonoenzymatic type, for example, competitive ELISA, immunoenzymatic-like, immunochromatographic-colorimetric, colorimetric and chromatographic, for example HPLC as described in the Journal of Chromatography, 212 (1981 ), 65- 73. Preferably the metabolites to which the present invention refers are detected with a competitive method, for example by means of the biotin/avidin system, by colorometric determination of glucuronic acid and of glucosamin; or by means of an immunoenzymatic-like method using lectins, for example from Triticum vulgaris. The use of lectins to detect metabolites of glycosaminoglycans is particularly important for the purpose of this invention. Lectins have been used for some time in a chromatographic method for purifying sugars. Surprisingly, it has now been found that the use of lectins may also be applied to the recognition of glycosaminoglycans in immunoenzymatic-like systems.

A further object of the present invention is therefore a kit for detecting glycosaminoglycans based on lectin adhering to a microplate or, alternatively, bonded to a detecting enzyme, for example peroxidase. The lectins particularly preferred for the purpose of the present invention are those derived from Triticum vulgaris.

Preferably, the kit based on lectins of the present invention works according to the so-called sandwich technique whereby the lectin or a suitable antibody is anchored to a solid support, made to react with the molecule that is to be detected. The latter is in turn reacted with, respectively, a suitable antibody or lectin, both marked with an enzyme, which then interacts with a chromogen in the presence of substratum for the final detection. Such a kind of technique is simpler to carry out and supplies results more rapidly.

The oligosaccharide metabolites of the present invention are also able to combat the onset and progression of complications of diabetes. In fact, metabolites taken, for example, from the urine of healthy subjects can compete in vivo for the enzymes produced in excess during the course of the diabetic pathology, for example β-N-acetyl-glucosaminidase and β-D-glucosidase. In other words, the administration to a diabetic subject of the metabolites of the invention constitutes an alternative substratum which prevents the above- mentioned enzymes from attacking the proteoglycans in the cell wall and producing the well known diabetic complications, that is, for example, microangiopathy, nephropathy, vasculopathy and angiopathy. The oligosaccharide metabolites of GAGs are therefore useful as therapeutic agents in nephropathy, retinopathy, vasculopathy, angiopathy and microangiopathy of diabetic origin.

As has been said above, the metabolites of the present invention may be obtained by purifying the urine of healthy subjects using techniques well known to the expert in this field, for example by chromatographic purification procedures. The oligosaccharide metabolites to which of the present invention may be administered both orally and parenterally. In the case of oral administration, the dose to be administered varies from 10 to 50 mg thrice a day. In the case of the parenteral administration, the dose varies from 5 to 50 mg once or twice a day. Suitable pharmaceutical forms useful for the oral administration of the oligosaccharide metabolites of the present invention are, for example, tablets, capsules, sugar-coated pills, granulates, solutions and suspensions even of an extemporary nature, syrups. Suitable pharmaceutical forms for the parenteral administration of the oligosaccharide metabolites of the present invention are, for example, solutions for both intramuscular and intravenous injection, and also sublingual tablets, creams and ointments. The extraction, characterisation and quantitative detection of the oligosaccharide metabolites object of the present invention will now be illustrated by the following examples.

Unless otherwise specified, in the following examples the buffer solution PBS (phosphate buffer) was used 0.1 M and with pH 7.4. EXAMPLE 1 Isolating the oligosaccharide metabolites of GAGs from human urine Extraction of glycosaminoglycans (GAGs) from urine

Urine (10 I) of healthy human volunteers or subjects with confirmed diabetic pathogenesis was added with 0.1 M NaOH (40 g), stirred for 15 minutes and left to sediment for 4 hours, then filtered and the clear supernatant liquor was recovered. Triton X100 (300 ml) was added to the supernatant liquor and the pH brought to 6 with concentrated HCI. DEAE-Sephadex A-25 (5 g) was then added and the whole was kept under stirring for 20 minutes, then filtered. The recovered resin was loaded in a column, washed with 0.3M NaCI/Triton X100 3% (200 ml) and eluted with 1 M NaOH (20 ml), then 0.1 M (80 ml). Acetic acid (1.2 g) and absolute ethanol (400 ml) were added to the eluate and the mixture was kept for one night at 20°C. The formed precipitate was recovered by centrifugation at 1500xg for 10 minutes, washed 3 times with absolute ethanol and then dried at

60°C.

The following Table 1 shows the weight results of the recovery of glycosaminoglycans from human urine.

Table 1

From this table it may be clearly seen that the quantity of GAGs present in the urine of subjects with confirmed diabetes is significantly greater than in that of the healthy volunteers. Treatment with chondroitinase A/C

This phase is useful for eliminating chondroitins A/C from the urine, then checking the behaviour of HS in the disease.

The product obtained in phase a) was dissolved in a buffer solution 0.1 M Tris , pH=8, 0.1 M NaCl to give a concentration of 20 mg/ml. The enzyme SIGMA C- 3667 was then added so as to have 0.1μg/ml. The whole was then left to react for 24 hours, stirring slowly, protected from the light and at room temperature, then placed in a dialysis tube with cut 1000 against water. Triton X100 (as required to 3%), HCI diluted at pH=6, DEAE-Sephadex 1-25 (50 mg/ml) were added to the dialysed product, and the whole was kept under stirring for 20 minutes. The resin was recovered and transferred to a column, washed with 0.3M NaCl / Triton X100 at 3% (20 ml/g of resin) and eluted with the smallest possible volume of 1 M NaOH. The eluate was brought to pH 5 using acetic acid, added with absolute ethanol, and kept in the freezer for one day. The precipitate was recovered by centrifugations at 1500xg for 10 minutes, washed 3 times with absolute ethanol and dried at 60°C.

The following Table 2 shows the weight results of the recovery of glycosaminoglycans free from chondroitin A/C from human urine.

Table 2

From this table it may be clearly seen that the quantity of GAGs free from chondroitin (prevalently HS) present in the urine of subjects with confirmed diabetes is significantly greater than in that of the healthy volunteers.

EXAMPLE 2 Characterisation of the oligoheterosaccharide metabolites of proteoheparansulphate

Electrophoretic method

Electrophoretic characterisation was performed following the teachings of

Cappelletti R. et al., Anal. Biochem., 1979, 93, 37, and Cappelletti R. et al., It. J. Biochem., 1982, 31 (3), 229, using samples obtained as described in example 1.

Figure 1 illustrates the Sigma standard electrophoretic bands (a), metabolites of a healthy subject before treatment with chondroitinase A/C (b), and metabolites of a healthy subject after treatment with chondroitinase A/C (c).

Figure 2 illustrates the Sigma standard electrophoretic bands (a), metabolites of a diabetic subject before treatment with chondroitinase A/C (b), and metabolites of a diabetic subject after treatment with chondroitinase A/C (c).

Nitrosation reaction method

Nitrosation is a characteristic reaction of the HS by which the stain that identifies it disappears from the electrophoretic run. This reaction was carried out using the method described by Cappelletti R. et al. , Anal. Biochem., 1980, 105, 430. Figure 3 illustrates the Sigma standard electrophoretic run (a), metabolites of a healthy subject before nitrosation treatment (b) and metabolites of a diabetic subject after nitrosation treatment (c). The immunological characterisation was carried out by the production of antibodies. To achieve this, two immunogens of GAGs were prepared, one with BSA (bovine sero-albumin) as a working immonogen, injected into the animal (rabbit) to produce the antibody; the second with OVA (ovalbumin), as a control immunogen for checking the specificity of the antibody. The synthesis of these immunogens is described below. EXAMPLE 3

Preparation of the conjugate GAGs-BSA

Hydrazide of BSA

BSA (45 mg) was dissolved in a physiological solution (20 ml), a 0.5M solution of dihydrazide of adipic acid was added (175 mg in 2 ml), at pH 5, and the volume was brought to 24 ml with physiological solution. An aqueous solution of carbodiimide was added (192 mg in 10 ml, 0.1 M), and it was left to react for 6 hours at room temperature, keeping the pH under 5 by adding HCI. The whole was then dialysed against an acetate buffer 10mM, pH=9. Periodic oxidation of the HS GAGs (400 mg) were dissolved in 4 ml of a fresh solution of 0.02M sodium metaperiodate (4.28 g/l) in 0.1 M acetate buffer at pH=4. The mixture was stirred for 30 minutes at room temperature, then dialysed for one night at room temperature against 1mM acetate buffer, pH=4.4, after adding ethylene glycol (0.4 ml). Conjugation

The solution obtained in point b) was added to the one of point a) and the whole was stirred for 2 hours at room temperature. NaBH₄ was added (100 mg) and the reaction allowed to continue for 4 hours at 4°C. The reaction mixture was dialysed against physiological solution, then concentrated from 25 to 5 ml by dialysis against PEG 20, and eluted with a column of sepharose. In this way about 560 mg of conjugate HS-BSA were obtained (90% yield). EXAMPLE 4

Preparation of the conjugate GAGs-OVA

Proceeding as described in example 3, the conjugate in title was obtained with substantially quantitative yield. EXAMPLE 5

Production of antibodies

Rabbits weighing about 1.5 kg were used. At time 0 (start of treatment) the animals were treated intradermically in 20 sites of the abdomen, with 0.1 ml/site of a suspension of PBS (1 ml) containing the immunogen GAGs-BSA (2 mg) obtained as described in example 3, and a complete Freund's adjuvant (1 ml, Sigma), in order to have the sensitisation reaction. After 21 days, the animals were treated intramuscularly with 1 ml of a suspension of PBS (1 ml) containing the immunogen GAGs-BSA obtained as described in example 3) and incomplete Freund's adjuvant (1 ml, Sigma), in order to have the priming reaction. After 8 days a blood sample was taken from the marginal vein of the ear for checking with the affinity/specificity method described in example 6. Subsequently, 20 days later, a repeated cycle of treatment was carried out, injecting intramuscularly the same suspension used for the priming reaction, and samples were taken in order to ascertain that specific antibodies had been produced. EXAMPLE 6

Affinity/specificity antigen/antibodv test a) Preparation of the microplate

Solutions were prepared with 40 μg/l of the two immunogens GAGs-BSA and

GAGs-OVA prepared as described, respectively, in examples 3 and 4, in PBS. Using a 96-well microplate for ELISA testing, 100 μl of a solution containing GAGs-BSA were deposited in each well. In parallel, in a second microplate, the same operation was carried out with a solution of GAGs-OVA. The plates were placed in a thermostat at 37°C for 3 hours for the adhesion of the immunogen. Subsequently, 150 μl of a 2% casein solution (p/v) were added to each well for saturation of the non reacted sites. The plates were again placed in a thermostat at 37°C for 1 hour, then the wells were washed 3 times with 300 ml of a solution of PBS containing 0.2% of Tween 20 and perfectly dried by shaking the plate. b) Preparation of the sample

The rabbit blood was taken from the marginal vein as described in the example and centrifuged at 3,000 rpm for 10 minutes. The resulting serum was diluted with PBS buffer solution in serial dilutions (1/20, 1/40, 1/80, 1/160, 1/320, 1/640, 1/1280). c) Preparation of the conjugate for measurement

The quantity of immunoglobulins (antibodies) which bind with the immunogen adhering to the plate was measured by means of a rabbit anti-lgG antibody (Sigma) conjugated with the peroxidase enzyme (HRP) and diluted 1 :1000 with PBS. d) Performing the test

In the wells of the microplate previously prepared, 100 ml of the different dilutions of the sample were added (one in each well). In the first well, 100 μl of PBS were added as blank. The microplates were placed in a thermostat at 37°C for 1 hour.

They were then washed 3 times and dried in the same way as in phase a). 100 μl of diluted anti-lgG-HRP conjugate were added to each well and the plates were put back in the thermostat at 37°C for 1 hour. They were washed again 2 times and dried in the same way as in phase a) and 100 μl of substratum for the enzyme (OPD - Sigma) were added to each well; the sample was again incubated at 37°C and finally 50 μl of stopper (Sigma) were added.

The optical density was read at 492 nm within 30 minutes, resetting against the blank. The results are expressed by the diagram in Figure 4.

The antibody formed in the rabbit was confirmed to be directed against the GAGs, since an overlapping reading was obtained for the two immunogens (HS-BSA and

HS-OVA).

The following examples concern various diagnostic methods for detecting and dosing the markers of the invention.

EXAMPLE 7 Competitive immunoenzvmatic method on a sample containing chondroitins

GAGs and their metabolic fragments compete with biotinilated GAGs added in standard concentrations against the anti-GAGs antibody adhering to a solid phase (microplate). The concentration of biotinilated GAGs which binds with the antibody is inversely proportional to the concentration of GAGs and of its metabolic fragments in the sample. Detection is performed by titrating the biotin radical by a specific reaction with avidin-peroxidase. a) Preparation of the immunoglobulin IgG anti-GAGs and metabolites

From the rabbit antibody serum (10 ml) obtained as described in example 5, the proteins were precipitated by adding 50% ammonium sulphate; it was then centrifuged at 3,000 rpm for 15 minutes and the supernatant liquor was eliminated. The precipitate was taken up with PBS buffer solution (2 ml) and subjected to dialysis in a 10 kD tube, for 48 hours at 2-8°C. The protein yield was about 7/100 ml.

The IgG were purified from the dialysed substance by low-pressure chromatography on a Tryacril column. On the purified IgG, proteinaceous titration was carried out using the Lowry micromethod (Sigma kit code 690.A). The yield in

IgG was 1.8-2 g/ml of serum.

The purified IgG were collected and brought to the final concentration of 12.9 mg/ml by adding PBS, then diluted 1 :100 with PBS for the subsequent preparation of the microplates. b) Preparation of the conjugate HS-biotin (biotinisation of HS) or of GAGs-biotin Conjugation was carried out on a 50% mixture of HS and metabolites extracted anhd purified from normal subjects (HSN) and from diabetic subjects at different stages of the disease, more or less affected by complications (HSP), in order to have competition with the largest possible panel of metabolites.

Two initial solutions of HSN and HSP were prepared (10 mg/ml in water). 230 μl of each were placed in a bath of ice. A separate solution of 0.1 M NalO₄ was prepared in 0.1 M acetate buffer, pH 5.5, and this too was cooled in a bath of ice. 3.52 ml of this solution of NalO₄ were then added to the solutions of HSN and HSP and the reaction was allowed to proceed for 20 minutes in a bath of ice. 150 μl of 1 M ethylenglycol were then added and allowed to react for 30 minutes at room temperature. The whole was placed in a Centricon 3,000 and centrifuged at 1 ,000 x g for 30 minutes (the operation was repeated 5 times). The volume was brought to 4 ml and 20.8 mg of biotin-hydrazide were added. The reaction was allowed to continue for 2 hours at room temperature, then centrifugation in the Centricon was repeated and the final mixture was brought to a volume of 4.6 ml with PBS (final concentration: 1 mg/ml). c) Preparation of the microplates

In 96-well microplates for ELISA testing, 100 μl of the IgG-anti-GAGs solution obtained as in point a) above were placed in each well. The plates were placed in a thermostat at 37°C for 3 hours, then the wells were emptied, washed 3 times with 300 μl of PBS and 0.2% Tween 20, and dried completely by shaking them. Subsequently, 150 μl of a 2% casein solution (p/v) in PBS buffer were added to each well and left to incubate at 37°C for 4 hours. After that the wells were emptied, washed and dried as described above. d) Test calibration curve

PBS buffer solutions were prepared, containing, in a volumetric ratio 1 :1 , a fixed concentration of 100 μg/ml biotinilate GAGs and scalar concentrations of standard GAGs starting from a maximum concentration of 100 μg/ml (1^st point) and diluting in scale 1 :2 for the subsequent points. 100 μl of the various solutions were placed in each well, except one to which only the buffer was added (blank). The microplates were incubated at 37°C for 1 hour, then the wells were emptied, washed and dried as described above. 100 μl of a 1 :500 dilution of avidin peroxidase were added to each well (Sigma, code A7419), and the microplates were put back to incubate in a thermostat at 37°C for 30 minutes, after which the wells were emptied, washed and dried as described above. 100 μl of substratum for the enzyme were added (OPD Sigma), prepared according to the supplier's instructions. The plates were again put in the thermostat at 37°C for 30 minutes and 50 μl of stopper (Sigma) were added. The optical density was read at 490 nm, resetting against the blank. e) Determination of the concentration in the sample not treated with chondroitinase of urinal and seric polysaccharide metabolites. As regards the serum, it was obtained from venous blood taken in the absence of anticoagulant and centrifuged at 3,000 rpm for 15 minutes. The volume of serum used was 100 μl. The same volume was used for the urine, after filtration, if it did not appear perfectly clear. The indicated sample volume was added in a volumetric ratio of 1 :1 to the solution of biotinilated GAGs (amounting to 100 mg/ml). 100 μl of this mixture were placed in a well in the microplate sensitized with anti-GAGs antibody, then left to incubate for 1 hour at 37°C. The measurement was carried out in the same way as described for calibration in point d), reading at 490 nm against the blank. The optic density value (D.O.) extrapolated on the line of calibration and multiplied by the dilution factor supplies the concentration of GAGs and of its oligosaccharide fragments in the sample.

Table 3 shows the values obtained from the urine of diabetic subjects compared with that of healthy subjects.

Table 3

The concentration of GAGs in the urine of sick subjects is dramatically higher than that in the urine of healthy subjects.

EXAMPLE 8

Competitive immunoenzvmatic method on a sample of urine treated with chondroitinase A/C

As regards the microplate, the conjugate, the method of operation and the preparation of the calibration curve, they are the same as those described in example 7. a) Preparation of the sample

10^"3 U of chondroitinase AC (Sigma C 3667) were added to 1 ml urine and the reaction was allowed to proceed under stirring at room temperature for 24 hours, protected from the light. At the end of that period the whole was transferred into a 1 ,000 D dialysis tube against water and left to dialyse for 24 hours with slight stirring. The dialysed product (100 μl) was then mixed with the biotinilated HS solution in a ratio of 1 :1 v/v. 100 μl of this mixture were then placed in a well in the microplate sensitised with anti-GAGs antibody. The detection and reading of the HS and of its metabolites in the sample were carried out with the same method described in example 7. The results are shown below in Table 4. Table 4

The concentration of HS and of its metabolites, following treatment with chondroitinase A/C, in the urine of sick subjects is dramatically higher than that in the urine of healthy subjects.

The increased concentraton of urinary GAGs in diabetic subjects in comparison with healthy ones is found both before and after treatment with chondroitinase A/C, so, on application level, both parameters are equally significant. EXAMPLE 9

Affinity method for lectin from Triticum vulgaris

1. Method with the antibody adhering to the plate and detection with lectin-

HRP. a-1 ) Preparation of the microplate

IgG anti-GAGs were adhered to a 96-well microplate for ELISA testing, using the same technique described in the example 7,c). b-1 ) Lectin-HRP conjugate

Lectin-Peroxidase Labeled Triticum vulgaris was used (Sigma L.3892) diluted before use 1 mg in 500 ml of PBS buffer. c-1 ) Principle of the method

Triticum vulgaris lectin has affinity for N-acetyl-β-D-glucosamine residues and for

N-acetyl-β-D-glucosamine oligomers. The latter are components of HS and of its structural analogs. HS, the analogs and their metabolic fragments in the sample of biological fluid bind with the specific antibody adhering to the wall of the microplate. They are detected by adding lectin conjugate of Triticum vulgaris- peroxidase and subsequent spectrophotometric determination of the reaction that develops between the enzyme and the specific substratum

(orthophenyldiamine=OPD). Performing the test

On a microplate sensitized with IgG anti-GAGs antibodies, 100 μl of different solutions were deposited according to the following pattern: B C₂ s. C₂ s₂ C₃ s₃ C₃ s₄ c₄ s₅ c₄ c₁ •

C • where: B = blank (urine without GAGs)

S,, S₂, S₃, S₄ e S₅ = standards, composed of a mixture of GAGs or of HS from normal and diabetic subjects in a PBS buffer solution with the following scalar concentrations in μg/ml (respectively 200, 50, 12.5, 3.125, 0.781 ). C^ C₂, C₃, and C₄ = samples (urine).

The plates are placed to incubate in a thermostat at 37°C for 1 hour. They were then washed 3 times consecutively by adding 300 μl of PBS buffer solution containing 0.2% Tween in each well, and the wells were then dried perfectly by shaking. 100 μl of lectin-HRP conjugate, prediluted as described above in point b- 1 ), were added to each well. The plate was then put back in the thermostat at 37°C for 1 hour and 3 washes were carried out as described above, after which each well was added with 100 μl of chromogen obtained by dissolving, at the time of use, one 2 mg tablet of OPD (Sigma) in 5 ml of PBS buffer solution and adding 10 μl of H₂O₂ at 15%. The plate was put back in the thermostat at 37°C for 30 minutes, then 50 μl of 2N H₂SO₄ were added to each well.

The optical density was read at 492 nm within 30 seconds, resetting against the blank. e-1 ) Calibration line and determination of the sample

Plotting the concentrations of the standard points and the respective optical densities (D.O.) on the logarithmic scale, we obtain the test calibration line (illustrated in Figure 5), obtained on standard samples with or without treatment with chondroitinase.

The optical densities read for the samples prepared according to the methods described are marked on the calibration line, allowing extrapolation of the content in metabolic derivatives of the sample expressed in μg/ml.

The following Table 5 shows the total values of GAGs and HS and the respective metabolic fragments from the same samples from normal and pathological subjects, obtained with the sandwich method and detection with lectin from Triticum vulgaris conjugated with peroxidase (HRP). For the titration of HS and its fragments, the biological samples were treated beforehand with chondroitinase. Table 5

Method with lectin of Triticum vulgaris adhering to the microplate and detection with antibody conjugated with peroxidase.

HS, GAGs and their metabolites in the sample bind by affinity with the lectin of

Triticum vulgaris adhering to the solid phase (microplate). They are detected by adding conjugate between the specific IgG antibody directed against them and the peroxidase enzyme, and subsequent spectrophotometric determination of the colouring developed by the reaction between the blocked enzyme and the specific substratum. a-2) Preparation of the microplate A solution of lectin of Triticum vulgaris (Sigma) in buffer 0.1 M CO3²7HCO3\ pH 9.5 was deposited in the wells of a microplate in doses of 100 μl/well, and the plate was incubated at 37°C for 2 hours. After the washing procedure already described above, 150 μl of a 2% casein solution in PBS buffer were added to each well. The microplate was left for one night at 4°C, then washed 3 times following the procedures described above. b-2) Antibody-enzyme conjugate (Ab-HRP)

Conjugation was carried out between IgG obtained by purification of the anti- GAGs antiserum and the HRP enzyme. The purification of the antiserum IgG was carried out as described in the example 7, a), while the conjugate was prepared as follows. A solution of HRP enzyme (5.1 mg) in 0.1 M NalO₄ (1.275 ml) in 0.1 M acetate buffer, pH 5.5, was left to react for 20 minutes while stirring at room temperature, protected from the light. 1 M (76.5 μl) ethylene glycol was then added and the reaction allowed to continue for further 5 minutes. The resulting enzyme solution was chromatographed on a Sephadex column (eluent: 0.1 M CO3²7HCO3^_ buffer, pH 9.5). IgG (10.2 mg) was added to the fractions containing activated HRP and they were left to react for 2 hours while stirring at room temperature, protected from the light. 510 μl of NaBH₄ (4 mg/ml of water) were added and the reaction allowed to continue for a further 2 hours while stirring at room temperature, protected from the light. The mixture was dialysed in a 100kD tube against PBS buffer. To perform the test, the resulting solution was diluted in 0.1 M PBS , pH 7.4 in a ratio of 1 :900. c-2) Performing the test Carried out in the same way as described above in point d-1 ), but using IgG-HRP as the detecting conjugate instead of lectin-HRP, and in this case the lectin was adhering to the microplate. d-2) Calibration line and determination of the sample

Figure 6 shows an example of a calibration line applying the same system as in point e-1 ) above. Table 6 shows the values of the contents of the biological liquids obtained operating on the same samples and with the same procedures. Table 6

EXAMPLE 10

Determination by means of the chromatographic-colorimetric method on a microcolumn

The GAGs metabolites and their fragments are blocked on a chromatographic column which may be composed of a matrix such as, for example, IgG anti-GAGs or anti-HS adhering to an inert support, or lectin of Triticum vulgaris immobilised on Sepharose 6MB (Sigma L.6257). The eluent may be 0.1 N HCI or 0.5-1.5M N- acetyl-β-D-glucosamine. The concentration of GAGs or HS and their metabolic fragments is determined by detection of the content of uronic acids (glucuronic) or of glusosamine, carried out with spectrophotometric methods.

1. Immobilisation of the anti-GAGs antibodies on glass beads

IgG anti-GAGs (200 mg) were bonded to Sigma G4643 beads (1 g equal to

7.7-10^"5 moles of NH₂). 2. Determination of uronic acid

Three test tubes containing 0.0125M sodium tetraborate (1.2 ml) in H₂SO₄ at 96%, and 0.2 ml of a sample, or 0.2 ml of standard or 0.2 ml of blank (PBS) were placed in a bath of ice and stirred gently, then for 5 minutes in bain-marie ar 100°C and then cooled. m-Hydroxy-diphenyl 0.15% in NaOH at 0.5% (40 μl) was added and after 5 minutes the reading of the optical density was taken at 520 nm resetting against the blank. 3. Preparation of the kit and performing the test

Chromatographic columns (0 0.6x10 cm) were loaded with glass beads with the immobilised anti-GAGs antibody, suspended again in PBS buffer solution. The chromatographic bed was washed with the same buffer solution, eluted with 30 ml of sample urine and washed again with the same buffer. It was then eluted with a solution of 0.1 M N-acetyl-β-D-glucosamine (5 ml) and the eluate was subjected to spectrophotometric determination of the concentrations of uronic acids in the urinary GAGs and their metabolites. The sample concentrations were defined from the ratio of the titre determined with reference to the respective standards.

The following Table 7 shows the values of urinary GAGs and their fragments isolated from the sample.

Table 7

The size of the Δ% values is a clear indication of how the concentration of GAGs in the urine is in proportion to the severity of the diabetic pathology and to the onset of complications.

EXAMPLE 11

Immunochromatographic method on a nylon membrane

The fragments of sulphate GAGs present in the sample are bonded to the specific antibody adhering to the membrane. They are detected by adding lectin of

Triticum vulgaris bonded to HRP. The intensity of the colouring compared with the predosed standards gives a quality-quantity indication of the sample analytes concentration. 1. Preparation of the membrane

A solution of IgG anti-HS (30 mg/ml in PBS) was deposited on a membrane in a dose of 3 μl. The membrane was dried and then immersed in 3% casein in PBS. After 15 minutes it was drained on paper and placed in a thermostat at 37°C for 30 minutes, then placed on the dedicated support.

2. Preparation of the conjugate

The lectin-HRP conjugate of marked Triticum vulgaris (Sigma L.3882) (1000 μl) was diluted with 10 ml of PBS 3% casein, and the mixture was stirred where protected from the light. 3. Performing the test

Each well was added with PBS 3% casein (blank), the fresh urine to be analysed

(C and the standards (S„ S₂, S₃), following this pattern:

B. S_{1 f} S₂, S₃,Cι,C| wherein B, S and C are as defined above. The wells were subjected to a cycle of 3 washes with 300 μl of PBS buffer solution containing 0.2% Tween 20, then 5 μl of lectin-HRP of marked Triticum vulgaris (Sigma L.3882), diluted in PBS, were added. After 10 minutes the wells were washed again as above and 10 μl of tetramethylbenziline (TMB) in a buffer solution (chromogen substratum) were added. The results were visually assessed, comparing the intensity of colouring of the wells containing the sample with the positive control wells.

Claims

1 1. Use of oligosaccharide metabolites of glycosaminoglycans as markers in biological fluids for assessing the state, severity, risk of onset of complications of diabetes, and for combating the onset and progression thereof.

1 2. Use of the metabolites according to claim 1 selected from the group consisting of heparansulphate, chondroitinsulphates A and C, dermatansulphate and their metabolic fragments.

1 3. Use of the metabolites according to claim 1 wherein the metabolite is heparansulphate and its metabolic fragments.

1 4. Use of the metabolites according to claim 1 wherein the metabolites are detected from serum and urine.

1 5. Diagnostic method for the determination of oligosaccharide metabolites of glycosaminoglycans from biological fluids making use of a competitive immunoenzymatic technique (ELISA).

1 6. Diagnostic method according to claim 5 wherein the competitive technique is based on the biotin/avidin detection system.

1 7. Use of the method according to claim 5 for assessing the state, severity, risk of

2 onset, possibility of the genesis of complications, monitoring of the treatment of

3 the diabetic disease. l 8. Diagnostic kit based on the method according to claim 5.

1 9. Diagnostic method for the determination of oligosaccharide metabolites of

2 glycosaminoglycans from biological fluids which consists of the colorimetric

3 determination of glucuronic acid and of glucosamine.

1 0.Use of the method according to claim 9 for assessing the state, severity, risk of

3 the diabetic disease. l 11. Diagnostic kit based on the method according to claim 9.

1 72. Diagnostic method for the determination of oligosaccharide metabolites of

2 glycosaminoglycans from biological fluids which makes use of a sandwich type

3 immunoenzymatic-like method by means of chromatographic/colorimetric

4 detection with lectins or their conjugates. l 13. Method according to claim 12 wherein the lectins come from Triticum vulgaris.

1 14. Method according to claim 12 wherein the lectins are conjugated with

2 peroxidase.

1 15. Use of the method according to claim 12 for assessing the state, severity, risk

2 of onset, possibility of the genesis of complications, monitoring of the treatment

3 of the diabetic disease. l 16. Method according to claim 12 applied to urine. l 17. Diagnostic kit based on the method according to claim 12.

1 18. Use of lectins for the chromatographic/colorimetric detection of

2 oligosaccharide metabolites of glycosaminoglycans from biological fluids.

1 19. Use of lectins according to claim 18 for detecting oligosaccharide metabolites

2 of glycosaminoglycans from urine.

1 20. Use of lectins according to claim 18 for assessing the state, severity, risk of

3 the diabetic disease. l 21. Use of lectins of Triticum vulgaris according to claim 18. l 22. Use of lectins in conjugate form according to claim 19. l 23. Use of lectins in conjugate form with peroxidase according to claim 22.

1 24. Diagnostic method for the determination of oligosaccharide metabolites of

2 glycosaminoglycans from biological fluids which makes use of a HPLC

3 technique.

1 25. Use of the method according to claim 24 for assessing the state, severity, risk

3 of the diabetic disease. l 26. Diagnostic kit based on the method according to claim 24.

1 27. Use of metabolites according to claim 1 for the therapy of microangiopathy,

2 nephropathy, retinopathy vasculopathy, angiopathy of diabetic origin.

1 28. Use according to claim 27 wherein the metabolites are extracted from the

2 urine of healthy subjects.

1 29. Pharmaceutical composition containing a therapeutically effective quantity of

2 metabolites according to claim 1 together with pharmaceutically acceptable excipients. 30. Pharmaceutical composition according to claim 29 suitable for oral administration and containing from 10 to 50 mg of the metabolites of claim 1. 31. Pharmaceutical composition according to claim 29 suitable for parenteral administration and containing from 5 to 50 mg of the metabolites of claim 1.