WO2009121959A1 - Use of glycosaminoglycan for restoring glycocalyx - Google Patents

Abstract

The invention is directed to the use of glycosaminoglycans for restoring glycocalyx functions or preventing glycocalyx damage. The effectiveness of glycosaminoglycans is directed in all pathologies where glycocalyx damage or this function may be etiologically cause of many pathologies such as: vascular disease, septic states, atherosclerosis syndrome, inflammation states and disease related to ischemia, peritoneal impairment, myocardial infarction, cerebrum- vascular events, alteration of enzymatic glycosylation in diabetes, kidney injury, intestinal disease as ulcerous colitis conditions, pneumoconiosis involving alteration of metabolic activity of pulmonary endothelium.

Description

Use of glycosaminoglycan for restoring glycocalyx

BACKGROUND OF THE INVENTION

The glycocalyx is a 0.5 micron thick gel layer, lining the inner wall of healthy blood vessels, it is the first line of defence against many diseases.

The glycocalyx is at today a complex and poorly understood structure. Recent studies have shown that hyperglycaemia can alter the glycocalyx structure, and parallel findings have shown that the apparent increase in permeability demonstrated in hyperglycaemia may be due to an increase in the permeability of the vessel to water, but not an increase in protein permeability.

The problem of the restoring the integrity in the functions of glycocalyx or the prevention of glycocalyx damage is a problem involving more than one mechanism in the permeability of the vessel endothelium.

In view thereof, there remains a pressing need to uncover aetiological factors and mechanisms responsible for these degenerative alterations, since targeting such processes will provide further avenues to prevent and ameliorate vascular damage and the accompanying pathological increase in vascular protein permeability in subjects.

In QJM. 101(7), 513, (2008) Noble et al. describe that in patients with diabetes and metabolic syndrome the first step in atherosclerosis syndrome is the glycocalyx dysfunction. In Am. J.Physiol. Heart. Circ. Physiol. 286(5), H 1672 (2004) the authors say that glycocalyx is involved in inflammation states and ischemia -reperfusion.

Flessner in Perit. Dial. Int. 28(1), 6, 2008 reports that endothelial glycocalyx is involved in transcapillary transport from the plasma to the tissue interstitium and for that the glycocalyx is important in peritoneal dialysis, as the capillary wall represents the initial resistance to solute transfer from the plasma through the tissue to the dialysate.

The mechanism of the putative increased permeability in respect of diabetes is not well understood. Perrin RM et al. in Cell Biochem. Biophys. 49(2), 65 (2007) report that recent studies have shown that hyperglycaemia can alter the glycocalyx structure and the apparent increase in permeability may be due to an increase in the permeability of the vessel to the water.

The enzymatic activity changes can give a defect in the carbohydrate content of some glycoproteins that may participate in the endothelial cell dysfunctions in diabetic microangiopathy. Nieuwdorp et al. report in Diabetes 55: 1127-322006 that patients with diabetes mellitus manifest both microvascular complications, comprising neuropathy, nephropathy and retinopathy, as well as macrovascular complications, mainly myocardial infarction and cerebrovascular events.

Correlation studies have suggested that in diabetes the vessel walls appear to be more vulnerable towards the traditional risk factors for vascular damage compared to non-diabetics. Hyperglycaemia is suspected to play a central role in impairment of the vascular function (Nieuwdorp et al. 2006. Diabetes 55: 480-6; Nieuwdorp et al. 2005. Curr Opin Lipidol 16: 507- 11).

Oturai P. S. in Clinical and Exper. Pharmacol Physiol. 26, 411(1999) disclose the treatment with LMW and UFH on vascular dysfunction in diabetic rats. This article concludes that heparin treatment does not affect diabetes -induced vascular dysfunction as expressed by increased transcapillary escape rate of albumin (TER_aι_b) and after treatment with LMWH or UFH for a period of 12 weeks the TER_aι_b did not change.

Palazzini and Gambaro in WO 01/93850 disclose the use of Sulodexide for the treatment of diabetic nephropathy to reduce the protein level in blood, and they give a safe range of Sulodexide concentration to use in human but they do not recognize the cause of the damage and the solution to the damage. Moreover this document focalizes the damage only to diabetic disease.

Palazzini and Rubbi in EP 0 950 413 disclose the use of Sulodexide for the treatment of diabetic retinopathy giving the effectiveness of Sulodexide with a decrease of the capillary permeability in diabetic patients. The authors related the efficacy of Sulodexide only in diabetic patients.

In the present invention it has been unexpectedly found that glycosaminoglycans (GAGs) and in particular sulodexide, have the property to restore the glycocalyx integrity.

SUMMARY OF THE INVENTION

The present invention regards the use of glycosaminoglycans in restoring of endothelial glycocalyx functions and thereby countering the pathologies related with this function including: vascular disease, septic states, atherosclerosis syndrome, inflammation states and disease related to ischemia, peritoneal impairment, myocardial infarction, cerebrum-vascular events, alteration of enzymatic glycosilation in diabetes, kidney injury, intestinal disease as ulcerous colitis conditions, pneumoconiosis involving alteration of metabolic activity of pulmonary endothelium. The inventors have surprisingly found that Sulodexide is active in the restoring of glycocalyx functions or in the preventing its damage. Therefore Sulodexide may be useful in all the pathologies where glycocalyx is involved. The invention can be applied in prophylactic and/or therapeutic interventions aimed at protecting and/or reconstituting glycocalyx integrity using glycosaminoglycans, can prevent or ameliorate glycocalyx perturbations that underlie pathologically increased vascular permeability.

Accordingly, aspects of the invention relate to:

- a glycosaminoglycan or a mixture of two or more glycosaminoglycans for restoring glycocalyx integrity in a subject having increased vascular protein permeability, or preventing glycocalyx damage;

- a pharmaceutical composition comprising a glycosaminoglycan or a mixture of two or more glycosaminoglycans for restoring glycocalyx integrity; or preventing glycocalyx damage; use of a glycosaminoglycan or a mixture of more than one glycosaminoglycan for the manufacture of a medicament or in the preparation of a medicine for restoring glycocalyx integrity and/or functions or preventing glycocalyx damage;

- a method for restoring glycocalyx integrity , comprising administering to said subject a therapeutically or prophylactically effective amount of a glycosaminoglycan or a mixture of two or more glycosaminoglycans.

The term "glycosaminoglycan" (GAG) as used herein refers to a group of polysaccharides, each having a repeating unit of disaccharide containing an amino sugar (e.g., N-acetyl-hexosamine) and a hexose (e.g., galactose) or a hexuronic acid. The disaccharide units may be optionally modified by alkylation, acylation, sulphonation (O- or N-sulphated), sulphonylation, phosphorylation, phosphonylation or the like. The degree of such modification may vary, and may be on a hydroxyl group or an amino group. Typically, C6 hydroxyl and C2 amine may be sulphated. Illustrative examples include hyaluronic acid, chondroitin, chondroitin 6-sulphate, chondroitin 4-sulphate, dermatan sulphate, heparin, heparan sulphate, keratan sulphate, as well as polymers containing N-acetyl monosaccharides (such as N-acetyl neuraminic acid, N- acetyl glucosamine, N-acetyl galactosamine, and N-acetyl muramic acid), and the like. Glycosaminoglycans may be extracted and purified from natural sources and optionally derivatised. Alternatively, they may be synthetically produced or synthesised by modified microorganisms such as bacteria. The term "glycosaminoglycans" as used herein also encompasses pharmaceutically acceptable salts, solvates, hydrates, and clathrates of glycosaminoglycans. The above aspects preferably employ sulodexide, a natural mixture of GAGs constituted by from about 60% to about 90% (w/w) heparin, more particularly the fast-moving or low molecular weight heparin fraction (iduronylglycosaminoglycan sulphate or IGGS), and between about 10% to about 40% (w/w) dermatan sulphate. Preferably, sulodexide comprises about 80% (w/w) heparin and about 20% (w/w) dermatan sulphate. The fast-moving heparin component (IGSS) may typically have a low to medium molecular weight of about 7 kD.

Compared to slow-moving or unfractionated heparin, IGGS comprises the same dimeric component but with lower amounts of iduronic acid-2-O-sulphate and a different amount of glucosamine N-acetylated-glucuronic acid dimer. Sulodexide as used herein also encompasses pharmaceutically acceptable salts, solvates, hydrates, and clathrates of sulodexide.

Sulodexide can be extracted from mammalian intestinal mucosa (Radhakrishnamurthy et al. 1978. Atherosclerosis 31: 217-229; the preparation of sulodexide is also described in US 3,936,351, herein incorporated by reference in its entirety). Sulodexide is marketed in Europe under the trademark VESSEL DUE F®. The inventors thus realised that restoration of glycocalyx integrity using glycosaminoglycans can reduce vascular protein permeability and thereby be prophylactically or therapeutically beneficial particularly in subjects having pathologically increased vascular protein permeability.

Accordingly, further aspects of the invention relate to:

- a glycosaminoglycan or a mixture of two or more glycosaminoglycans for reducing vascular protein permeability;

- a pharmaceutical composition comprising a glycosaminoglycan or a mixture of two or more glycosaminoglycans for reducing vascular protein permeability; use of a glycosaminoglycan or a mixture of two or more glycosaminoglycans for the manufacture of a medicament for reducing vascular protein permeability; - a method for reducing vascular protein permeability in a subject, comprising administering to said subject a therapeutically or prophylactically effective amount of a glycosaminoglycan or a mixture of two or more glycosaminoglycan.

- A pharmaceutical composition for restoring or preventing glycocalyx damage comprising a therapeutically effective amount of glycosaminoglycan or a mixture of more than one glycosaminoglycans with pharmaceutically acceptable excipients.

These aspects preferably employ sulodexide as the glycosaminoglycan, or as one of the at least two glycosaminoglycans in the composition.

The term "vascular protein permeability" refers to the propensity for the passage of proteins out of the blood vessel lumen across the blood vessel wall. The term is used herein in a general sense encompassing protein passage through any blood vessel walls including without limitation capillaries as well as the kidney's glomerulus's filtration barrier.

Advantageously, the above aspects may be used for reducing vascular protein permeability in a subject having pathologically increased vascular protein permeability, such as in preferred examples, in a subject having proteinuria (i.e., excreting an abnormal amount of serum protein in urine) or albuminuria (i.e., excreting an abnormal amount of plasma albumin in urine), including microalbuminuria and macroalbuminuria, diabetic neuropathy, nephropathy or retinopathy, atherosclerosis, thrombosis, insulin resistance, etc.

In embodiments, the subject having pathologically increased vascular protein permeability, particularly where such manifests as proteinuria or albuminuria, may suffer from a condition or disorder characterised by hyperglycaemia. As used in this specification, such conditions or disorders may include particularly but without limitation hyperglycaemia, prediabetes (diagnosed, e.g., as impaired fasting glucose and/or impaired glucose tolerance), insulin resistance, diabetes type 1, diabetes type 2, metabolic syndrome, and the like. Accordingly, further aspects of the invention relate to:

- a glycosaminoglycan or a mixture of two or more glycosaminoglycans for restoring glycocalyx integrity in a subject having a condition or disorder characterised by hyperglycaemia;

- a pharmaceutical composition comprising a glycosaminoglycan or a mixture of two or more glycosaminoglycans for restoring glycocalyx integrity in a subject having a condition or disorder characterised by hyperglycaemia;

- use of a glycosaminoglycan or a mixture of two or more glycosaminoglycans for the manufacture of a medicament or in the preparation of a medicine for restoring glycocalyx integrity in a subject with hyperglycaemia or a patient having a condition or disorder characterised by hyperglycaemia;

- the glycosaminoglycan or the mixture of two or more glycosaminoglycans for restoring glycocalyx function and/or integrity in a subject having a condition or disorder characterized by hyperglycaemia, wherein the subject has prediabetes, insulin resistance, diabetes type 1, diabetes type 2 or metabolic syndrome. - a method for restoring glycocalyx integrity in a subject having a condition or disorder characterised by hyperglycaemia, comprising administering to said subject a therapeutically or prophylactically effective amount of a glycosaminoglycan or a mixture of two or more glycosaminoglycans. Hereby, the symptoms and complications associated with pathologically increased vascular protein permeability in disorder characterised by hyperglycaemia can be ameliorated.

The above aspects preferably employ sulodexide as the glycosaminoglycan, or as one of the at least two glycosaminoglycans in the composition. The present invention shows that hyperglycaemic conditions increase albumin permeability through a cultured monolayer of HUVEC cells, which can be restored by GAGs such as Sulodexide.

Endothelial permeability for albumin after incubation with Sulodexide decreased to values lower than baseline albumin leakage. Increased coloration of the HUVEC cells with a GAG specific glucosamine staining after incubation with sulodexide supported the restoration by showing increased incorporation of GAG on the endothelial surface. According to these results, amelioration of the vascular function through restoration of glycocalyx damage caused by hyperglycaemia is a promising new use of the GAGs in the prevention and treatment of vascular protein permeability especially in hyperglycaemic disorders.

These and further aspects and embodiments are described in the following sections and in the claims.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows the permeability of HUVEC cells grown under normo- and hyperglycaemia.

High glucose (black bar) increases the endothelial albumin permeability by 22% compared to normal glucose (white bar) (p<0.01). Y-axis: % albumin through monolayer.

Figure 2 shows increase of albumin permeability of HUVEC cells in the presence of glycocalyx inhibitors 4-methylumbelliferone and sodium chlorate compared to baseline conditions. Y-axis: % change of albumin through monolayer in presence of inhibitors.

Figure 3 shows that the lowest concentration sulodexide of 0,06 μg/ml sulodexide has a very beneficial effect on the restoration of hyperglycaemic-induced increase in permeability of HUVEC layer. Y-axis: % change in albumin permeability compared to normoglycaemic control. Figure 4 shows restoration of glycocalyx barrier in hyperglycaemic cells following sulodexide incubation of HUVEC cells. Y-axis: % change from normoglycaemic control; white bars: normoglycaemia; striped bars: hyperglycaemia. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

All documents cited in the present specification are hereby incorporated by reference in their entirety.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

The term "sample" generally refers to material, in non-purified or purified form, obtained from a biological source. A biological sample may be typically removed from its biological source, such as from a subject of interest, by appropriate methods for collecting, drawing, biopsy or resection, etc. of bodily fluids, tissues, cells or the like. Particularly useful samples in the present invention include whole blood, plasma, serum and urine derived from subjects. A biological sample may be further processed to prepare suitable derivatives thereof, such as, without limitation, cell or tissue lysates, homogenates, supernatants, fractions, etc. A sample may be subdivided to isolate or enrich for parts thereof (such as for example parts that are expected to contain analytes of interest) to be used in the diagnostic methods of the invention. Hence, a sample can be applied to the methods of the invention directly or can be processed, extracted or purified to varying degrees before being used.

As used herein, "glycocalyx" generally refers to a polysaccha ride-rich extracellular matrix on the luminal surface of vascular endothelial cells. Glycocalyx is primarily comprised of proteoglycans, glycosaminoglycans, glycolipids and glycoproteins (e.g., selectins, adhesion molecules, etc.) which associate in vivo with water and numerous molecules including inter alia plasma proteins, lipids and enzymes from the circulating blood.

The present aspects generally provide for restoration of glycocalyx integrity in subjects having particular symptoms (e.g., increased vascular protein permeability) and conditions (e.g., hyperglycaemia) which, as realised by the inventors, lead to glycocalyx degeneration.

Such glycocalyx degeneration may be characterised, e.g., by decreased glycocalyx volume or dimension, increased glycocalyx permeability, increased shedding of glycocalyx (i.e., resulting in reduced thickness of the glycocalyx layer), increased activity of glycocalyx-degrading enzyme(s) and/or decreased activity of glycocalyx-synthesising enzyme(s).

Glycocalyx perturbation may be diagnosed in samples removed from subjects, such as e.g. blood, plasma or serum samples, comprising detecting in said samples the presence and/or concentrations of inter alia glycocalyx-derived molecules such as, e.g., oligo- or polysaccharides, glycosaminoglycans, hyaluronan, heparan sulphate or proteoglycans; enzymes that catalyse glycocalyx anabolism or catabolism, such as, e.g., hyaluronidase; and/or endogenous or exogenous (e.g., infused) substances that can become incorporated or otherwise associate with glycocalyx, and can thus provide information about (systemic) glycocalyx volume or dimension and/or its molecular accessibility. By means of example but without limitation, profiles of endogenous lectin-like proteins that normally associate with glycocalyx, as determined in samples e.g. in plasma or urine samples, can provide suitable information about glycocalyx volume or dimension and/or its molecular accessibility. By means of example and not limitation, alterations in indicators or markers of glycocalyx homeostasis such as stated above can be compared to control subject or subjects to assess whether a significant difference occurs that may be indicative of a pathological phenotype. The presence and/or concentration of the above or other glycocalyx markers may be, sequentially or simultaneously, detected by assay technologies known in the art, such as immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), colorimetric and fluorimetric enzyme activity assays, etc.

Consequently, treatments with glycosaminoglycans as taught herein can counter one or more of such degenerative processes, thereby improving glycocalyx structure and function, i.e., restoring glycocalyx integrity.

Conditions of pathologically increased vascular protein permeability as contemplated herein can manifest particularly but without limitation as proteinuria and albuminuria. Proteinuria and albuminuria can be routinely diagnosed using inter alia spot urine dipstick tests performed on random urine samples, or by measuring the respective protein amounts excreted in urine over a 24-hour period (24-hour excretion test).

In the spot urine dipstick test, proteinuria may be concluded when the concentration of protein exceeds 8 mg/dL, more preferably exceeds 30 mg/dL In the 24-hour excretion test, proteinuria may be concluded when the total amount of protein > 150 mg/day, more preferably > 300 mg/day. In the spot urine dipstick test, albuminuria may be concluded when the concentration of albumin exceeds 3 mg/dL. In the 24-hour excretion test, microalbuminuria may be concluded when the total amount or value of albumin is between 30 and 300 mg/day, and macroalbuminuria may be concluded at > 300 mg/day. One aspect of the invention therefore relates to the use of pharmaceutical composition for restoring glycocalyx integrity and/or function or preventing glycocalyx damage comprising a therapeutically effective amount of glycosaminoglycan or a mixture of more than one glycosaminoglycans with pharmaceutical acceptable excipients to lower proteinuria value and/or albuminuria including microalbuminuria or macroalbuminuria values. Another aspect relates to the glycosaminoglycan or the mixture of two or more glycosaminoglycans for restoring glycocalyx integrity and/or function in a subject having increased vascular protein permeability, wherein the subject has proteinuria; albuminuria, including microalbuminuria or macroalbuminuria; diabetic nephropathy; diabetic neuropathy; or diabetic retinopathy. As used herein, disorders characterised by hyperglycaemia encompass all disorders in which disturbance of physiologically normal homeostasis of circulating glucose belongs to, i.e., is comprised amongst, symptoms of the said disorders, and/or belongs to aetiological factors of the said disorders.

The diagnosis of a hyperglycaemic disorder in a subject, i.e., the differentiation between physiologically normal vs. abnormal glucose, may be done by a number of methods well-known in clinical practice, including but not limited to measurement of fasting glucose level; and oral or intravenous glucose tolerance test.

A typical fasting blood glucose test measures the concentration of glucose in blood, serum or plasma of a subject after a fasting period of usually at least 10-12 hours. The normal range of whole blood glucose concentrations (normoglycaemia) in this test is between 60 mg/dL (3.0 mmol/L) and 110 mg/dL (5.6 mmol/L).

A typical oral glucose tolerance test (OGTT) is carried out as follows: after an overnight fasting period (e.g., 10 to 12 hours), a subject drinks a solution containing a known amount of glucose; blood is drawn before the subject drinks the glucose solution, and blood is drawn again every 30 to 60 minutes after the glucose solution is consumed for up to 3 hours. The normal ranges of whole blood glucose concentrations in a 75-gram oral glucose tolerance test (normoglycaemia) are: between 60 mg/dL (3.0 mmol/L) and 110 mg/dL (5.6 mmol/L) after fasting; less than 200 mg/dL (10.1 mmol/L) at 1 hour after consumption of the glucose solution; less than 140 mg/dL (7.1 mmol/L) at 2 hours after consumption of the glucose solution.

Hyperglycaemia may be concluded when glucose concentrations in at least one and preferably both above tests, or analogous tests commonly employed in the art, fall above the normoglycaemic ranges, e.g., as indicated above, at least on one occasion and preferably on two or more occasions.

Further, for example, in the above fasting blood glucose test, a diagnosis of impaired fasting glucose (IFG) may be made when the whole blood glucose concentration of a subject is above 110 mg/dL (5.6 mmol/L) but less than 126 mg/dL (6.4 mmol/L). Diabetes mellitus may be diagnosed when the whole blood glucose concentration of a subject is above 126 mg/dL (6.4 mmol/L). In the above OGTT test, impaired glucose tolerance (IGT) may be diagnosed when the whole blood glucose concentration of a subject at 2 hours after consumption of the glucose solution is higher than 140 mg/dL (7.1 mmol/L) but less than 200 mg/dL (10.1 mmol/L). Diabetes mellitus may be diagnosed when the whole blood glucose concentration of a subject at 2 hours after consumption of the glucose solution is higher than 200 mg/dL (10.1 mmol/L). When at least one of IFG and IGT, and preferably both IFG and IGT, are diagnosed, the condition may be referred to as "prediabetes".

Hence, as used herein conditions or disorders "characterised by hyperglycaemia" or "hyperglycaemic disorders" particularly encompass hyperglycaemia, prediabetes, insulin resistance, diabetes type 1, diabetes type 2, metabolic syndrome, and the like. Exemplary manifestation of diabetes which may particularly profit from the present treatments include inter alia diabetic angiopathies such as diabetic neuropathy, nephropathy and retinopathy.

The aspects of the invention relate to treatments using glycosaminoglycans and particularly sulodexide that restore glycocalyx integrity. Thereby vascular protein permeability can be advantageously reduced, with particularly useful applications in subjects manifesting pathologically increased vascular permeability, such as due to a disease associated with hyperglycaemia.

The terms "subject" or "patient" refer preferably to animals, more preferably warm-blooded animals, yet more preferably vertebrates, and even more preferably mammals specifically including humans and non-human mammals, that have been the object of treatment, observation or experiment. The term "mammal" includes any animal classified as such, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs, cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes. Particularly preferred are human subjects, including both genders and all age categories thereof.

The present treatments are particularly to be given to subjects in need thereof, which phrase includes subjects that would benefit from treatment of a given condition, such as pathologically increased vascular protein permeability or a hyperglycaemic disorder. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in whom said condition is to be prevented.

The terms "treat" or "treatment" encompass both the therapeutic treatment of an already developed disorder, such as the therapy of an already developed glycocalyx perturbation, pathologically increased vascular protein permeability or hyperglycaemic disorder, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent the chances of contraction and progression of glycocalyx perturbation, pathologically increased vascular protein permeability or a hyperglycaemic disorder. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

The term "prophylactically effective amount" refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or disorder being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses.

In particular, prophylactically or therapeutically effective amounts as contemplated herein can produce an observable and significant effect on glycocalyx structure and/or function, i.e., on glycocalyx integrity, and more preferably can prevent an increase of or achieve a measurable and significant reduction of vascular protein permeability, such as prevent an observable increase or achieve a measurable and significant reduction in the extent of proteinuria or albuminuria or even achieve return of the corresponding values to normal physiological range.

Glycosaminoglycans useful for prophylactic and/or therapeutic repair of glycocalyx in the above- discussed subjects and disease states include by preference sulodexide as described elsewhere in this application.

For purposes of treatments as described herein the glycosaminoglycans such as sulodexide to be administered may be advantageously formulated and administered as pharmaceutical formulations.

Such pharmaceutical compositions typically comprise one or more glycosaminoglycans, preferably sulodexide, or a pharmaceutically acceptable form thereof such as an addition salt, solvate, hydrate or clathrate thereof as the active ingredient, and one or more pharmaceutically acceptable carrier/excipient.

The term "pharmaceutically acceptable salts" means pharmaceutically acceptable acid or base addition salts. The pharmaceutically acceptable acid or base addition salts are meant to comprise therapeutically active non-toxic acid and non-toxic base addition salt forms which the present GAG are able to form. The GAG which have basic properties can be converted in their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g., hydrochloric or hydrobromic acid, sulphuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e., butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. The GAG which have acidic properties may be converted in their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g., the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g., the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the GAG, its use in the therapeutic compositions may be contemplated. Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) use, liposomes or surfactant- containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.

Pharmaceutical compositions of the invention may be formulated for essentially any route of administration, such as without limitation, oral or pulmonary administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous, intramuscular, intraperitoneal or intrasternal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intra-tracheal instillation, and the like. In this way, the therapeutic effects attainable by the methods and compositions of the invention can be, for example, systemic, local, tissue-specific, etc., depending of the specific needs of a given application of the invention. Preferably, pharmaceutical compositions of the invention may be formulated for oral administration or parenteral administration.

Sulodexide can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.

An aqueous aerosol is made, for example, by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically-acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. For example, the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) and distearoyl phosphatidyl ethanolamine (DSPE). Typically, a surfactant:active substance molar ratio in an aqueous formulation will be from about 10:1 to about 1:10, more typically from about 5:1 to about 1:5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest. Aerosols generally are prepared from isotonic solutions.

When rectally administered in the form of suppositories, these formulations may be prepared by mixing the active substances according to the invention with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.

Transdermal patches have the added advantage of providing controlled delivery of Sulodexide to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the invention.

For example, for oral administration, pharmaceutical compositions may be formulated in the form of pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, caplets, granules, hard and soft gelatine capsules, troches, dragees, aqueous, alcoholic or oily solutions, syrups, dispersions, emulsions or suspensions, patches and the like, including sustained release formulations and gastroresistant formulations known in the art. Because of their ease of administration, tablets and capsules are preferred. See, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th edition, 2005, LV Allen, NG Popovich and HC Ansel, Baltimore, Md: Lippincott Williams & Wilkins; Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publ. Co., Easton, Pa, for guidance on pharmaceutical formulations.

The compounds of the present invention may also be administered by controlled release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in US 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, the disclosures of which are each incorporated herein by express reference thereto.

These pharmaceutical compositions can be used to provide slow or control led-release of one or more of the active ingredients therein using, for example, hydropropyl methyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable control led-release formulations known to those of ordinary skill in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, caplets, and the like, that are adapted for controlled-release are encompassed by the present invention.

All controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations may include: 1) extended activity of the drug; 2) reduced dosage frequency; and 3) increased patient compliance.

Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

The controlled-release of an active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled-release component" in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like, or a combination thereof, that facilitates the controlled-release of the active ingredient. In an example, without limitation, preparation of oral dosage forms may be is suitably accomplished by uniformly and intimately blending together a suitable amount of the active substance in the form of a powder, optionally also including finely divided one or more solid carrier, and formulating the blend in a pill, tablet or a capsule. Exemplary but non-limiting solid carriers include calcium phosphate, magnesium stearate, talc, sugars (such as, e.g., glucose, mannose, lactose or sucrose), sugar alcohols (such as, e.g., mannitol), dextrin, starch, gelatine, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. Compressed tablets containing the pharmaceutical composition can be prepared by uniformly and intimately mixing the active ingredient with a solid carrier such as described above to provide a mixture having the necessary compression properties, and then compacting the mixture in a suitable machine to the shape and size desired. Moulded tablets maybe made by moulding in a suitable machine, a mixture of powdered active substance moistened with an inert liquid diluent. Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc. By means of example, each tablet, cachet, caplet, or capsule may contain from about 10 mg to about 1000 mg of GAG preferably sulodexide, more preferably from about 25 mg to about 1000 mg of GAG preferably sulodexine, more preferably from about 25 mg to about 250 mg GAG preferably sulodexide.

Gelatine capsule unit dosage form for oral administration may be formulated using conventional methods well known in the art (Ebert 1977. Pharm Tech 1: 44-50). Soft elastic gelatine capsules have a soft, globular gelatine shell somewhat thicker than that of hard gelatine capsules, wherein a gelatine is plasticized by the addition of plasticizing agent, e.g., glycerine, sorbitol, or a similar polyol. The hardness of the capsule shell may be changed by varying the type of gelatine used and the amounts of plasticizer and water. The soft gelatine shells may contain a preservative, such as methyl- and propylparabens and sorbic acid, to prevent the growth of fungi. The active ingredient may be dissolved or suspended in a liquid vehicle or carrier, such as vegetable or mineral oils, glycols, such as polyethylene glycol and propylene glycol, triglycerides, surfactants, such as polysorbates, or a combination thereof.

For example, for parenteral administration, pharmaceutical compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc. Suitable solvents are, without limitation, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. The active substances and pharmaceutically acceptable salts thereof of the invention can also be lyophilised and the lyophilisates obtained used, for example, for the production of injection or infusion preparations. For example, one illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI). Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil- in-water emulsion. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.

Where aqueous formulations are preferred, such may comprise one or more surfactants. For example, the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) and distearoyl phosphatidyl ethanolamine (DSPE). Typically, a surfactant: active substance molar ratio in an aqueous formulation will be from about 10:1 to about 1:10, more typically from about 5:1 to about 1:5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.

One skilled in this art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulations techniques and pharmaceutically-acceptable excipients and carrier solutions are well-known to those skilled in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

The present active substances may be used alone or in combination with any therapies known in the art to restore glycocalyx integrity, reduce vascular protein permeability or treat hyperglycaemic conditions ("combination therapy"). Combination therapies as contemplated herein may comprise the administration of at least one active substance of the present invention and at least one other pharmaceutically or biologically active ingredient. Said present active substance(s) and said pharmaceutically or biologically active ingredient(s) may be administered in either the same or different pharmaceutical formulation(s), simultaneously or sequentially in any order.

The dosage or amount of the present active ingredients used, optionally in combination with one or more other active compound to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect.

Thus, it depends on the nature and the severity of the disorder to be treated, and also on the sex, age, body weight, general health, diet, mode and time of administration, and individual responsiveness of the human or animal to be treated, on the route of administration, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the agent(s) of the invention.

Preferably, the pharmaceutical compositions may be administered in single or divided doses, from one to four times a day, or for example once in 2, 7, 14, 21 or 28 days. The oral dosage forms may be conveniently presented in unit dosage forms and prepared by any methods well known in the art of pharmacy.

In embodiments, the formulation may be provided in unit dosage form comprising between 10 mg and 1000 mg per unit dosage, more preferably between 25 and 1000 mg per unit dosage or between 25 and 250 mg per unit dosage or between 25 and 200 mg per unit dosage, of GAGs preferably sulodexide. In embodiments, the unit dosage may comprise about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, or 1000 mg of GAG preferably sulodexide. Said amount may be administered to subjects preferably human subjects one or more times per day.

Without limitation, depending on the type and severity of the disease, a typical daily dosage might range from about 0.05 mg/kg to about 100 mg/kg of body weight, more typically between about 1 mg/kg and about 20 mg/kg of body weight, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. Thus, by means of example one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 10 mg/kg or 20 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every three weeks. The above aspects and embodiments are further supported by the following non-limiting examples.

The present examples demonstrate that sulphated glycosaminoglycans and particularly sulodexide restore glycocalyx barrier properties of cultured endothelial cells in hyperglycaemia, thereby substantiating its applicability to reduce pathologically increased vascular permeability particularly in hyperglycaemic disorders.

EXAMPLE 1

M199 media, L-glutamine, antibiotic-antimycotic and trypsin were obtained from Gibco-BRL, PBS pH: 7.4 from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker. The following chemicals were obtained from Sigma; heparin, endothelial cell growth supplement (ECGS). D(+)glucose was obtained from Merck.. Fibronectin was a kind gift from Central Laboratory for Blood transfusion (CLB), Amsterdam, The Netherlands. Semi-permeable inserts 12 wells 3 μm pores were purchased from Greiner. HUVEC cells were isolated from human umbilical cords from the department of obstetrics of the AMC in Amsterdam. Briefly, umbilical veins were canulated and rinsed with PBS before applying trypsin solution. The trypsin solution was incubated for 37⁰C to detach the endothelial cells from the venous vessel wall. The trypsin solution was collected and the vein was rinsed with PBS. The cell solution was centrifuged for lOmin at llOOrpm, supernatant was removed and the cell pellet was resuspended in 5ml M199 medium. The cells were grown on 10 μg/ml fibronectin- coated cell culture flasks in M199 media supplemented with 20% heat-inactivated fetal bovine serum, 50 μg/ml heparin and 12.5 μg/ml endothelial cell growth supplement, 0.2mmol/l L- glutamine and lOOU/ml Penicillin-G, lOOU/ml Streptomycin sulfate, 25 μg/ml Amphotericin-B at 37⁰C in 5% CO₂. Sodium sulphate inhibits sulphate donor PAPS and decreases sulphation of glycosaminoglycans and 4methylumbelliferone acts as substrate analogue in hyaluronan synthesis, and to lesser extent heparan and keratan sulphate. Cells were incubated for 24 hours in the presence of inhibitors at concentration 5OmM sodium chlorate and 200μm 4methylumbelliferone and endothelial permeability was assessed. To measure the permeability human umbilical vein endothelial cells (HUVECs) were cultured on semi-permeable inserts and exposed to normo- (5mM) or hyperglycaemia (25mM) for 5 days, the last 24 hours in the presence of the GAG mixture at concentrations ranging up to 60 μg/ml. Endothelial permeability was assessed by determining FITC-labelled albumin transfer over the monolayer. Top compartment with HUVEC cells was incubated with 400 μg/ml FITC-labelled albumin in 1% BSA/RPMI1640 media without phenol red. After 3 hours the media from the bottom well was removed and FITC-Albumin content was measured by fluorescent spectrometry in Fluostar.

After 5 days incubation, HUVEC cells were washed two times with ice cold RPMI1640 and fixed in 4% para-formaldehyde for 30 min at room temperature. The cells were rinsed three times in lectin buffer (0.1% BSA in HBSS) and incubated with 67 μg/ml FITC-LEA, which is directed against glucosamine residues and 69 μg/ml TRITC-BSI, which is directed against galactosamine residues and lOug/ml HOECHST, which stains cell nuclei, for 30min at room temperature. After 30min the cells were washed with lectin buffer and imaged with fluorescence microscope. Statistical analysis, two-way unpaired t-tests were used. A value of P < 0.05 was considered statistically significant And the values are means ± SE.

EXAMPLE 2

Determination of the baseline parameters

The isolated HUVECs were cultured for 5 days under normoglycaemic (5mM) conditions on a semi-permeable membrane. After 5 days, the formation of the monolayer was assessed by measuring endothelial transfer of FITC-labelled albumin over the layer. The baseline permeability of FITC-albumin was very low which was indicative of a solid formation.

EXAMPLE 3

Determination of the permeability of the monolayer after normo, and hvperqlvcaemia The HUVECs were also cultured for 5 days under hyperglycaemic (25mM) conditions to investigate whether or not this condition would affect endothelial permeability. Figure 1 shows the permeability of the HUVECs grown under normo- and hyperglycaemia. The albumin transfer rate of the HUVECs grown under normoglycaemic conditions was set to 100%. Hyperglycaemia increased permeability of albumin by 22% (p<0.01), indicating a deterioration of the barrier. EXAMPLE 4

Determination of the permeability of the monolayer after qlvcocalyx inhibitors

To determine the barrier function of the glycocalyx in albumin permeability, we incubated HUVECs grown under normoglycaemic conditions in the last 24 hours with the glycocalyx inhibitors sodium chlorate and 4-methylumbelliferone (Figure 2). Fluorescent spectrometry showed a 4-fold increase of albumin permeability in the presence of glycocalyx inhibitors compared to baseline conditions after 3 hours of incubation with FITC-albumin. As the inhibitors prevent the incorporation of certain GAGs and by doing so, cause an imperfect glycocalyx layer, these findings prove the importance of the glycocalyx barrier function for large negatively charged molecules. EXAMPLE 5

Determination of the permeability of the monolayer after supplementation of the compound Sulodexide

In an attempt to restore the hyperglycaemic induced increase in permeability, increasing amounts of Sulodexide were added to the model to determine the optimal dose which could restore endothelial barrier properties. The cultured cells were incubated in the last 24 hours with Sulodexide at a dose of 0.06, 0.6 and 6 μg/ml. The dose-range pointed out in Figure 3 shows that the lowest concentration sulodexide of 0.06 μg/ml sulodexide has a very beneficial effect on the restoration of hyperglycaemic-induced increase in permeability. GAG incubation restored permeability in hyperglycaemia (122±8% vs. 85±6%; p<0.05) even resulting in a decrease of baseline albumin leakage.

FITC-LEA lectin staining was used to determine the heparan sulfate/hyaluronan content of the glycocalyx layer of the HUVECs. LEA-lectin, which is directed against glucosamine residues in heparin sulfate and hyaluronan, revealed a 21% decrease in glucosamine staining in hyperglycemic cells which was restored following GAG mix incubation, pointed out in Figure 4. The increased staining correlates with the restored barrier function after incubation with Sulodexide 0.06 μg/ml.

Claims

1. Use of a glycosaminoglycan or a mixture of more than one glycosaminoglycan in the preparation of a medicine for restoring glycocalyx functions or preventing glycocalyx damage.

2. Use as described in claim 1 characterized in that the glycosaminoglycan is sulodexide.

3. Use as described in claim 2 in the preparation of a medicine administrable by oral, parenteral, intranasal, pulmonary, transdermal, topical, rectal, vaginal route for restoring glycocalyx functions or preventing glycocalyx damage.

4. Use as described in claim 3 in the preparation of medicine for restoring glycocalyx integrity in a subject with hyperglycaemia.

5. A pharmaceutical composition for restoring or preventing glycocalyx damage comprising a therapeutically effective amount of glycosaminoglycan or a mixture of more than one glycosaminoglycans with pharmaceutical acceptable excipients.

6. The pharmaceutical composition according to claim 5 characterized in that the glycosaminoglycan is Sulodexide.

7. The pharmaceutical composition according to claim 5 characterized in that the administration is oral, parenteral, intranasal, pulmonary, transdermal, topical, rectal, vaginal.

8. Use of pharmaceutical composition according to claim 5 to lower proteinuria value, albuminuria including microalbuminuria or macroalbuminuria values.

9. Use of a pharmaceutical composition according to claim 6 characterized in that the Sulodexide is administered in a quantity from about 25 mg to 1000 mg per unit dosage.