WO2006039709A1 - Methods using glycosaminoglycans for the treatment of kidney disease - Google Patents

Abstract

The present invention relates to methods for the treatment of kidney disease by the administration of at least one glycosaminoglycan to a patient in need of such treatment. In particular, the kidney disease is focal segmental glomerulosclerosis (FSGS) and the glycosaminoglycan is sulodexide.

Description

METHODS USING GLYCOSAMINOGL YCANS. FOR THE TREATMENT OF KIDNEY DISEASE

The present application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/615,472 filed October 1, 2004 and U.S. Provisional Application No. 60/622,332 filed October 26, 2004, the disclosure of each of which is incorporated by reference herein in its entirety.

This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe' the state of the art as known to those skilled therein as of the date of the invention described herein.

1. FIELD OF THE INVENTION

The present invention relates to methods for the treatment of kidney disease by the administration of at least one glycosaminoglycan to a patient in need of such treatment. In particular, the kidney disease is focal segmental glomerulosclerosis (FSGS) and the glycosaminoglycan is sulodexide.

2. BACKGROUND OF THE INVENTION

Focal segmental glomerulosclerosis (FSGS) is a kidney disorder involving formation of scar tissue in some of the glomeruli. The pathogenesis of the sclerosing lesions and their progressive nature are debated. Humans with idiopathic focal segmental glomerulosclerosis initially have a high glomerular filtration rate and evidence of hyperfiltration, suggesting that hyperfiltration and increased intracapillary glomerular pressure may be mediators of this disease.

The cause of focal segmental glomerulosclerosis is usually unknown. A minority of cases result from reflux nephropathy, infections, obesity and other conditions. Some of the glomeruli become scarred. It affects about 1 out of 10,000 people. The main result of focal segmental glomerulosclerosis is nephrotic syndrome. It causes about 10 to 15% of cases of l nephrotic syndrome. Protein is persistently excreted in the urine, especially urine albumin. Most cases will progress to chronic renal failure. Although the disorder may be in part immune-system related, response to corticosteroid or immunosuppressive medications is inconsistent. The goal of current treatment of FSGS is control of the symptoms associated with nephrotic syndrome and chronic renal failure. Treatment may be chronic and lifelong. Corticosteroids and immunosuppressive medications may be prescribed to reduce the imune response. Antihypertensive and diuretic medications may help control symptoms such as high blood pressure and edema. Antibiotics may be needed to control infections. The treatment of high blood cholesterol and triglyceride levels, which are also common with this disorder, may be recommended to reduce the development of atherosclerosis. Dietary limitation of cholesterol and saturated fats may be of only limited benefit as the high levels seem to result from overproduction of cholesterol and triglycerides by the liver rather than the excessive intake of fats. Medications to reduce cholesterol and triglycerides may be recommended. High-protein diets are of debatable value. In many patients, reducing the amount of protein in diet produces a decrease in urine protein. In most cases, a moderate- protein diet (1 gram of protein per kilogram of body weight per day) is usually recommended. In cases in which renal failure is present, a low-protein diet may be preferred. The sodium in the diet and/or fluids may be restricted to help control swelling. Vitamin D may need to be replaced if nephrotic syndrome is chronic and unresponsive to therapy.

Dialysis or kidney transplantation may be necessary to control renal failure when end-stage renal failure occurs.

The pathological manifestations of FSGS are distinctive. The kidney biopsy in focal segmental glomerulosclerosis demonstrates focal and segmental glomerular sclerosis, hyaline and lipid deposition and often adhesion of the glomerulus to Bowman's capsule. The remainder of the glomerular tuft is normal in appearance; thus the term 'segmental'. The lesions are considered to begin or to be more common near the corticomedullary junction. Focal segmental glomerulosclerosis may be primary (idiopathic) or secondary to a number of etiologic agents including: unilateral renal agenesis; renal ablation; sickle cell disease; morbid obesity (with or without sleep apnea); congenital cyanotic heart disease; heroin nephropathy; aging kidney. Glycosaminoglycans ("GAG"), such as heparin, are routinely used in anticoagulant and antithrombotic therapies. Heparin is a heterogeneous glycosaminoglycan compound and its function may include not only its role as an anticoagulant (through its potentiation of anti-thrombin III and factor Xa) but other functions as well. The range of functions or activities of heparin are directed to reversing the endothelial dysfunction resulting in anti¬ inflammatory action, endothelial cell interactions, cytokine interaction, radical oxygen metabolites, and intestinal repair. Heparin also reduces ROM generation by stimulated neutrophils and binds superoxide dismutase (SOD) to the endothelial cell. Compared to animal studies, human studies regarding heparin's anti-inflammatory activity are limited. Korzenik, Inflammatory Bowel Diseases 3 : 87-94 ( 1997).

Sulodexide is a glycosaminoglycan of natural origin extracted from mammalian intestinal mucosa that posses an anticoagulant activity. Sulodexide has a sulfation degree lower than those of heparin. Radhakrishnamurthy et al. , Atherosclerosis 31:217-229 (1978). In pharmacological models, sulodexide has been shown to be a strong anticoagulant, antithrombotic, and profibrinolytic agent. Callas et al. , Semin. Thromb. Hemost. 19:49 (1993). A study has also demonstrated that sulodexide therapy is accompanied by less bleeding than when heparin is used. Barbanti et al, Int. J. Clin. Lab. Res. 22:179 (1992); Iacoviello et al, Thromb. Haemostas. 76:1002 (1996). Sulodexide also offers additional advantages such as its ability to improve blood viscosity, lower fibrinogen levels, and increase fibrinolytic activity. Harenberg, Med. Res. Rev. 18:1-20 (1998). In addition, studies indicate a high sulodexide bioavailability after intramuscular and oral administration. Id. The preparation of sulodexide is described in U.S. Patent 3,936,351, which is incorporated herein by reference in its entirety.

Sulodexide comprises about 80% iduronylglycosaminoglycan sulfate (IGGS), which is a fast-moving heparin fraction, and about 20% dermatan sulfate. The fast moving component, which is determined by its electrophoretic mobility in the barium- propanediamine system, is found in commercial heparin along with a slower moving component. IGGS has a low to medium molecular weight of about 7 kD and a lower anticoagulant activity than the slow moving heparin fraction and unfractionated heparin. Compared to heparin, IGGS has the same dimeric component but with lower amount of iduronic acid-2-O-sulfate and a different amount of glucosamine-acetylated-glucuronic acid dimer.

Sulodexide is marketed in Europe under the trademark VESSEL DUE F® and is prescribed for the treatment of vascular pathologies with thrombotic risk such as peripheral occlusive arterial disease (POAD), healing of venous leg ulcers and intermittent claudication, as described by Harenberg, Med. Res. Rev. 18:1-20 (1998) and Crepaldi et al,

Atherosclerosis 81 :233 (1990), cardiovasculopathies, as described by Tramarin et al.

Medical Praxis 8:1 (1987), cerebrovasculopathies as described by Sozzi, Eur. Rev. Med. Pharmacol Sci. 6:295 (1984), and venous pathologies of the lower limbs, as described by

Cospite et al., Acta Therapeutica 18:149 (1992).

Kanway et al, Sent. Nephrol. 5:307 (1985) and Groggel et al, Kidney int. 33:517

(1988) produced evidence of the probable role of glycosaminoglycans in helping the integrity and the functioning of the renal cells. Canfield et al, Lab. Invest. 39:505 (1978), showed a decrease of membranal glycosaminoglycans in conditions of diabetic nephropathy. (Jensen, Diabetes 46 (Suppl. 2):S98-S100 (1997). This decrease may be mediated by decreased heparan sulfate production and/or sulfation (Raats et al, Kidney Int.

57:385-400 (2000).

U.S. Patent No. 5,236,910 disclose the use of glycosaminoglycans for the treatment of diabetic nephropathy and diabetic neuropathy. U.S. Patent No. 5,496,807 discloses a method of treatment of diabetic nephropathy by the administration of sulodexide.

Citation or identification of any reference in Section 2 or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention is directed to a method for preventing, reducing or eliminating a symptom or complication of focal segmental glomerulosclerosis ("FSGS") comprising: administering to a subject, in need thereof, at least one glycosaminoglycan in an amount effective to inhibit, reduce or eliminate one or more symptoms or complications of FSGS.

The present invention also is directed to a method for preventing, reducing or eliminating a symptom or complication of focal segmental glomerulosclerosis ("FSGS") comprising: administering to a subject, in need thereof, Elmiron® (ALZA Corporation, Mountain View, CA) in an amount effective to inhibit, reduce or eliminate one or more symptoms or complications of FSGS. Elmiron® is pentosan polysulfate sodium.

The present invention is further directed to use of at least one glycosaminoglycan for the preparation of a medicament for the prevention, reduction or elimination of a symptom or complication of FSGS. The present invention is further directed to a pharmaceutical composition for the prevention, reduction or elimination of a symptom or complication of FSGS, which composition comprises as an active ingredient at least one glycosaminoglycan together with a pharmaceutically acceptable carrier. In an embodiment of the invention, the subject is a mammal, preferably a human. In a preferred embodiment, the subject is not diabetic. In yet another preferred embodiment, the subject is not infected with a human immunodeficiency virus (HIV). In a more preferred embodiment of the invention, the subject is a human subject who is not diabetic and is not infected with HIV. In a preferred embodiment, the glycosaminoglycan is sulodexide. hi an aspect of this preferred embodiment, sulodexide is administered in an amount of 10-1000 mg/day, preferably 50-500 mg/day, more preferably 100-400 mg/day.

The mode of administration may be oral, mucosal, parenteral, or transdermal.

4. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic of the research schedule used in Example III, infra, in determining the effect of sulodexide administration in an experimental mouse model of focal segmental glomerulosclerosis.

Figure 2 is a chart showing the urine albumin/creatinine ration in adriamycin-treated mice alone (Adr/V), adriamycin-treated mice given sulodexide (Ard/Sul) at day 14 and day 48, control mice given saline (V/V), and control mice given sulodexide (V/Sul) at day 14 and day 48.

Figure 3 shows PAS stain histology of kidney tissue at day 48 in adriamycin-treated mice with (ADR/S) or without (ADR/V) sulodexide administration.

Figure 4 shows Masson Trichrome stain of kidney tissue at day 48 in adriamycin- treated mice with (ADR/S) or without (ADR/V) sulodexide administration.

Figure 5 is a chart summarizing the histological changes in the kidney at day 48 in adriamycin-treated mice alone (Adr/V), adriamycin-treated mice given sulodexide (Ard/Sul), control mice given saline (V/V) and control mice given sulodexide (V/Sul).

Figure 6 is a chart summarizing the mRNA expression levels of TFG-βl, CTGF and FGF-2 in kidney tissue at day 48 in adriamycin-treated mice alone (Adr/V), adriamycin- treated mice given sulodexide (Ard/Sul) at day 14 and day 48, control mice given saline (V/V), and control mice given sulodexide (V/Sul) at day 14 and day 48. 5. DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a method for the prevention, reduction or elimination of a symptom or complication of focal segmental glomerulosclerosis ("FSGS") by administration to a patient, in need thereof, an effective amount of at least one glycosaminoglycan. In a preferred embodiment, the glycosaminoglycan is sulodexide. Examples of glycosaminoglycans (GAG) are those acceptable in the therapeutic field, such as heparin and its pharmaceutically acceptable salts; low molecular weight heparins obtained by chemical or enzymatic depolymerization; chemically modified heparins, for instance through reactions of O and/or N sulfation or desulfation; dermatan sulfate and its low molecular weight fractions; hyaluronan; chondroitin sulfate; heparan sulfate; and keratan sulfate and their low molecular weight fractions. The glycosaminoglycans may also comprise a combination or mixture of two or more of the above.

The term "sulodexide" in the context of the invention refers to a composition comprising from about 60% to about 90% iduronylglycosaminoglycan sulfate and between about 10% to about 40% dermatan sulfate. This term in the context of the present invention refers also to a pharmaceutically acceptable salt, solvate, hydrate, or clathrate of sulodexide.

Preferably, sulodexide comprises about 80% iduronylglycosaminoglycan sulfate (IGGS), which is a fast-moving heparin fraction, and about 20% dermatan sulfate. The fast moving component, which is determined by its electrophoretic mobility in the barium- propanediamine system, is found in commercial heparin along with a slower moving component. IGGS has a low to medium molecular weight of about 7 kD and a lower anticoagulant activity than the slow moving heparin fraction and unfractionated heparin. Compared to heparin, IGGS has the same dimeric component but with lower amounts of iduronic acid-2-O-sulfate and a different amount of glucosamine N-acetylated-glucuronic acid dimer.

Use of Elmiron® (ALZA Corporation, Mountain View, CA), which is chemically related to glycosaminoglycans, is also encompassed within the invention. Thus, the present invention also is directed to a method for the prevention, reduction or elimination of a symptom or complication of FSGS by administration to a patient, in need thereof, an effective amount of Elmiron®.

The term "prevention, reduction or elimination of a symptom or complication of FSGS" in the context of the present invention refers to at least one of the following: diminution of albuminuria or proteinuria; slowing in rate of rise of serum creatinine or fall in glomerular filtration rate (GFR); fall in serum creatinine or rise in GFR; slowing of the appearance of histological kidney abnormalities characteristic of FSGS such as fibrosis, inflammation, etc. ; regression of such abnormalities; slowing of the time interval until the development of end stage renal failure as manifested by the need for renal replacement therapy; elimination or reduction of one or more medications used in treating the subject, such as corticosteroids and immunosuppresive medications, antibiotics, antihypertensive and diuretic medications (ACE-inhibitors), medications used to reduce cholesterol and triglycerides (statins), and Vitamin D.

The above term "focal secmental glumerolusclerosis " refers both to idiopathic FSGS and secondary FSGS, including FSGS not otherwise specified (NOS), perihilar, cellular, tip, and collapsing variants thereof.

In particular this term refers to FSGS not otherwise specified (NOS), perihilar, cellular, tip, and collapsing variants thereof excluding HIV-associated nephropathy (HIVAN). Most preferably this term refers to FSGS not otherwise specified (NOS), perihilar, cellular, and tip.

The method of administration, according to the present invention, may be oral, mucosal, parenteral, intramuscular or transdermal. The dosage of the active ingredient will vary considerably depending on the mode of administration, the patient's age, weight and the patient's general condition, as well as the severity of the disease, and can be determined by standard clinical techniques. In addition, animal assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed should be decided according to the judgment of the practitioner and each patient's circumstances.

Preferably the pharmaceutical composition is in a form acceptable for oral administration. Because of their ease of administration, tablets and capsules are preferred and represent the most advantageous oral dosage unit form where solid pharmaceutical excipients are employed. If desired, tablets may be coated by standard aqueous or non¬ aqueous techniques.

Preferably, the oral pharmaceutical composition used in the methods of the invention may be administered in a single or divided dosage from to 1 to 4 times per day. Preferably, the therapeutically or prophylactically effective amount of an active ingredient ranges from about 20 L.R.U. (lipoprotein lipase releasing unit) to about 100,000 L.R.U. daily, depending on the type of administration. For oral administration, the therapeutically effective amount of the active ingredient may be several times greater than that for parenteral administration. The amount of the orally administered active ingredient may range from about five to ten times greater than that for intravenous or subcutaneous administration. Preferably, the amount of pharmaceutical composition used daily in the present invention ranges from about 20 L.R.U. to about 1,000 L.R.U. for parenteral administration, about 200 L.R.U. to about 10,000 L.R.U. for oral administration, and about 2,000 L.R.U. to about 100,000 L.R.U. for rectal administration. The pharmaceutical composition can comprise any commercially available form of sulodexide, for example, VESSEL DUE F® commercially available form Alpha Wassermann, SpA in Italy. Preferred solid dosage forms of the pharmaceutical compositions are tablets or capsules, which are either coated or uncoated. The oral pharmaceutical compositions may be administered in single or divided dosage from one to four times a day, and preferably range from 50 milligrams ("mg") (500 L.R.U.) a day to about 1000 mg (10,000 L.R.U.) a day such as, for example, 50 mg/day, 100 mg/day, 150 mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day, etc. Preferably, when the pharmaceutical compositions are administered rectally, the dosage should be about 10 times higher than that used for oral administration, i.e., from about 500 mg to about 10 grams. Other solid dosage forms of the pharmaceutical compositions are tablets or capsules which are coated or uncoated, and the preferred dosage forms range from about 10 mg per day to about 1,000 mg per day, preferably from about 50 mg to about 500 mg per day, more preferably from about 100 mg to about 400 mg per day. Preferably 400 mg per day is administered. The methods of the invention include the use of a glycosmainoglycan, e.g., sulodexide, or its pharmaceutically acceptable salt, solvate, hydrate, or clathrate, which can be administered in combination with one or more additional active ingredients. The method of the invention includes the simultaneous or sequential administration of two or more active ingredients. If the administration is sequential, the separate administrations may be temporally spaced apart by about at least two minutes or more. Temporal spacing of the action or effects of the active ingredients may also be achieved by using, for each active ingredient, at least one different carrier, excipient, or solid dosage coating, so as to cause a differential rate of release of each of the active ingredient into the body. Alternatively, at least one of the active ingredients may be administered in a controlled-release preparation, or each of the ingredients may be administered in a different controlled-release preparation each of which has its own release rate. Pharmaceutical Dosage Forms

Pharmaceutical compositions used in the method of the present invention suitable for oral administration may be presented as discrete pharmaceutical unit dosage forms, such as capsules, cachets, soft elastic gelatin capsules, tablets, caplets, or aerosol sprays, each containing a predetermined amount of the active ingredient, such as a powder or granules, or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Dosage forms such as oil-in-water emulsions typically comprise surfactants such as anionic phosphate ester or lauryl sulfates, but other types of surfactants such as cationic or nonionic surfactants may be used in the compositions of the present invention. See generally, Remington's Pharmaceutical Sciences, 18^th ed., Mack Publishing, Easton PA (1990).

Pharmaceutical compositions of the present invention suitable for oral administration may be formulated as a pharmaceutical composition in a soft elastic gelatin capsule unit dosage form by using conventional methods well known in the art. See, e.g. , Ebert, Pharm. Tech. l(5):44-50 (1977). Pharmaceutical compositions in the form of capsules or tablets coated by an enterosoluble gastro resistant film and which contains a lyophilisate consisting of glycosaminoglycan, a thickening agent, and a surfactant have been previously described in U.S. Patent No. 5,252,339, which is incorporated herein by reference in its entirety. Soft elastic gelatin capsules have a soft, globular gelatin shell somewhat thicker than that of hard gelatin capsules, wherein a gelatin is plasticized by the addition of plasticizing agent, e.g., glycerin, sorbitol, or a similar polyol. Varying the type of gelatin used and the amounts of plasticizer and water may change the hardness of the capsule shell. The soft gelatin 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, triglycercides, surfactants, such as polysorbates, or a combination thereof.

Typical oral dosage forms of the invention are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL® PH-101, AVICEL® PH-103 AVICEL® RC-581, AVICEL® PH- 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL® RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL® PH-103 and Starch 1500 LM.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to talc, calcium carbonate (e.g. granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

Pharmaceutical stabilizers may also be used to stabilize the compositions of the invention. Acceptable stabilizers include but are not limited to L-cysteine hydrochloride, glycine hydrochloride, malic acid, sodium metabisulfϊte, citric acid, tartaric acid and L- cysteine dihydrochloride.

Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally after the release of the active ingredients should be used to form solid oral dosage forms of the invention. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, preferably from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, MD), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, TX), CAB-O-SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated. In addition to the common dosage forms set out above, 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 U.S. Patent Nos.: 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.

These pharmaceutical compositions can be used to provide slow or controlled- release of one or more of the active ingredients therein using, for example, hydropropylmethyl 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 controlled-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.

Active ingredients of the invention can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 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, each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients of the invention. The invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled-release. 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 include extended activity of the drug, reduced dosage frequency, and increased patient compliance. 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. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects. Controlled-release of an active ingredient can be stimulated by conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds. Further, when it is desired to temporally space the release, and hence the effect, of two or more active ingredients used in the method of the invention, it is possible to use: (a) a controlled-release preparation for the release of at least one of the active ingredients; or (b) two or more controlled-release preparations having different release coefficients, for the separate release of each active ingredient.

Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the invention.

Transdermal, topical, and mucosal dosage forms of the invention include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, enemas, suppositories, or other forms known to one of skill in the art. See, e.g., Remington 's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton PA (1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Further, transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients. Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms encompassed by this invention are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1, 3 -diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non-toxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired! Examples of such additional ingredients are well known in the art. See, e.g., Remington 's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton PA (1980 & 1990). Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of the invention. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate). The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more active ingredients, particularly for the tissues of the colon. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

The pharmaceutical compositions may be prepared by any methods well known in the art of pharmacy, but all methods include the step of bringing into association one or more active ingredient(s) with the carrier. In general, the compositions are prepared by uniformly and intimately admixing the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. Oral solid preparations are preferred over oral liquid preparations. One preferred oral solid preparation is tablets, but the most preferred oral solid preparation is capsules.

Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water so that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific types of active ingredients in a dosage form may differ depending on factors such as, but not limited to, the route by which it is to be administered to patients. Other factors include the patient's general condition, age, and weight, as well as the severity of the disease.

Co-Administration

The method of treatment of the present invention may also include co-administration of another therapeutically effective agent for the treatment of FSGS, together with the administration of the at least one glycosaminoglycan (GAG), e.g., sulodexide. Additionally, the method may also include co-administration of another therapeutic agent for the treatment of another disease(s) afflicting the subject. Examples of such agents that can be co-administered with the GAG and/or Elmiron® (ALZA Coporation, Mountain View, CA) are corticosteroids and immunosuppressive medications; antibiotics; antihypertensive and diuretic medications (such as ACE-inhibitors); medications used reduce cholesterol and triglycerides (statins); and Vitamin D.

Kits

The active ingredients of the invention may or may not be administered to a patient at the same time or by the same route of administration. Therefore, the methods of the invention encompass kits which, when used by the medical practitioner, can simplify the administration of appropriate amounts of active ingredients to a patient.

A typical kit of the invention comprises a unit dosage form of a glycosmainoglycan, e.g., sulodexide, or a pharmaceutically acceptable salt, solvate, hydrate, or clathrate thereof, and a unit dosage form of at least one additional active ingredient. Examples of additional active ingredients that may be used in conjunction with the glycosaminoglycans, include, but are not limited to, Elmiron® (ALZA Corporation, Mountain View, CA) are corticosteroids and immunosuppressive medications; antibiotics; antihypertensive and diuretic medications (such as ACE-inhibitors); medications used reduce cholesterol and triglycerides (statins); and Vitamin D.

The invention also provides a method for preventing, reducing or eliminating a symptom or complication of focal segmental glomerulosclerosis comprising administering, to a subject in need thereof, a first pharmaceutical composition comprising at least one glycosaminoglycan and a second pharmaceutical composition comprising one or more additional active ingredients, wherein all active ingredients are administered in an amount sufficient to inhibit, reduce, or eliminate one or more symptoms or complications of focal segmental glomerulosclerosis. In on e aspect, the administration of the first and second pharmaceutical composition is temporally spaced apart by at least about two minutes.

Kits of the invention can further comprise devices that are used to administer the active ingredients. Examples of such devices include, but are not limited to, syringes, drip bags, patches, inhalers, enemas, and dispensers for the administration of suppository formulations.

Kits of the invention can further comprise pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients. For example, if an active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit can comprise a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particulate-free sterile solution that is suitable for parenteral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Assessment of renal function

In order to assess the efficacy of the method of the invention, serial measurements of renal function of the patients can be determined. Quantitative assessment of renal function, and parameters of renal dysfunction are well known in the art and can be found, for example, in Levey, 1993, Assessing the effectiveness of therapy to prevent the progression of renal disease, Am J Kidney Dis. 22(l):207-214.

Examples of assays for the determination of renal function/dysfunction are:

Serum creatinine level; Creatinine clearance rate;

24-hour urinary protein secretion;

Glomerular filtration rate (GFR);

Urinary albumin creatinine ratio (ACR);

Albumin excretion rate (AER); and Renal biopsy.

The following series of examples are presented by way of illustration and not by way of limitation on the scope of the invention.

6. EXAMPLES

Example I Puromycin aminonucleoside (PAN) is a podocyte toxin that induces acute nephrotic syndrome in rats, with proteinuria peaking at day 7, and subsequent glomerulosclerosis at 2- 6 months. PAN on day 0 is administered to the rats with the following treatments: control (no active ingredient), sulodexide daily from day 1-14, or sulodexide daily from days 7-14. Each group contains 4 rats.

On days 7 and 14, 24 hr urine is collected for measurement of protein. Sulodexide therapy is stopped at day 14 and the rats are sacrificed at month 4.

Pathologic assessment is performed which includes evaluation for glomerulosclerosis and interstitial fibrosis using morphometric techniques.

Example II

The effects of sulodexide in the adriamycin model of focal segmental glomerulosclerosis (FSGS) have been studied as set forth below.

BALB/c mice received a single 9.5 mg/kg intravenous dose of adriamycin (ADR) or vehicle (V) injected on day 0. Mice received saline vehicle (V) or sulodexide (15 mg/kg, S) by daily subcutaneous injection 6 days/week from day -1 to day 48. Assessment included measurement of serum creatinine by HPLC, measurement of urine albumin/creatinine ratio (A/C) by ELISA (expressed as mg/mg), and enumeration of the number of glomeruli showing segmental or global glomerulosclerosis (expressed as percent). The results are set forth in Table I.

TABLE I

Data are presented as mean±SE

* denotes significantly different from V/V (negative control) (P<0.05)

? denotes significantly different from ADR/V (positive control) (P<0.05)

Compared to positive control (ADR/V), sulodexide reduced proteinuria at day 14 but not at day 48 (when proteinuria was returning toward normal), and largely prevented glomerulosclerosis at day 48. These findings suggest that sulodexide reduces proteinuria and slows the progression of renal fibrosis.

Example III The following example demonstrates that Sulodexide reduces proteinuria and glomerular scarring in experimental focal segmental glomerulosclerosis. Sulodexide is an orally-available glycosaminoglycan that has anti-thrombotic and fibrinolytic effects in a rat venous thrombosis model (Barbanti et al., Int. J. CHn. Lab Res. 22(3): 179- 184 (1992)). A recent randomized controlled trial in patients with diabetic nephropathy showed that sulodexide reduces proteinuria by 50%, in patients with diabetes type 1 and type 2, in patients with microalbuminuria and macroalbuminuria, and in patients taking angiotensin converting enzyme inhibitors and in patients not taking these medications (Gambaro et al., J. Am. Soc. Nephrol. 13 :1615-1625 (2002)). It has been marketed since 1982 in Europe for vascular pathologies with thrombotic risk. Additional controlled trials are now planned in diabetic nephropathy and focal segmental glomerulosclerosis (FSGS).

The following experimental protocol was designed to determine if sulodexide reduces proteinuria, glomerulosclerosis, and interstitial fibrosis in an experimental model of focal segmental glomerulosclerosis (FSGS) induced by adriamycin in mice; and to investigate the mechanism of renal protection and to measure the expression of pro-fibrotic cytokines such as TGFb-I, CTGF and FGF2 in the kidneys. Adriamycin nephropathy in mice was chosen as a model of focal segmental glomerulosclerosis (FSGS) (Wang et al., Kidney Int. 58(4): 1797-1804 (2000)). In this model, on day 48 after administration of adriamycin, extensive focal glomerulosclerosis and interstitial fibrosis occur in this model. 9.5mg/kg of adriamycin was administered intravenously to 8-week old 20-25 g BALB/c male mice on day 0 as a single dose. Sulodexide (15 mg/kg) was administered 6 days/week by subcutaneous injection 5 mg/kg beginning on day-1. On day 14 and day 48, blood was obtained by retro-orbital bleeding and 24-hour urine samples were obtained. See Figure 1 for a schematic of the research schedule. Mice were sacrificed on day 48 and kidney tissues were fixed with zinc formalin, embedded in paraffin, and stained with periodic acid Schiff and Masson trichrome. Tissues were also immersed in RNAlater

(Qiagen, Valencia, CA) at 4⁰C overnight, transferred to the new sample tubes without RNA later on the following day, and stored at -8O⁰C. RNA was extracted from kidneys using Rneasy Mini Kit (Qiagen, Valencia, CA), treated with DNase (Qiagen, Valencia, CA), and reverse-transcribed with first strand cDNA synthesis kit for RT-PCR (Roche). Serum and urine creatinine were measured by HPLC (Yuen et al., Am. J. Physiol. Renal. Physiol.

286(6):F1116-1119 (2004)), and urine albumin was measured by ELISA, using AlbuwellM (Exocell Inc., Philadelphia, PA).

Serum levels of creatinine, the ratios of urine protein levels to urine creatinine levels on day 14 and day 48 were compared among the groups (Figure 2). The percentage of segmental and global glomerulosclerosis was calculated by counting the number of all glomeruli in the samples, and the areas of interstitial fibrosis were assessed by analyzing 10 captured images of 200X magnification per slide with ImageJ (NIH) (Figures 3-5). Quantitative PCR was performed by TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) with ABI Prism 7900. Negative controls included water and RNA without reverse transcriptase. The following primer sets were purchased from ABI (Foster City, CA): TGF-bl (MmOl 178820_ml), CTGF (Mm00515790_gl) and FGF2 (Mm00433287_ml). 18S rRNA primer pairs and VIC-labeled probe were used as an endogenous control. The relative fold changes were calculated by the DDCt method. Statistical testing was by ANOVA (parametric data) or Kruskal- Wallace test (nonparametric data).

Male BALB/c mice received a single 9.5 mg/kg intravenous dose of adriamycin (ADR) or vehicle (V) injected on day 0. Mice received saline vehicle (V) or sulodexide (15 mg/kg, S) by daily subcutaneous injection 6 days/week from day -1 to day 48. Assessment included measurement of serum and urine creatinine by HPLC, measurement of urine albumin by ELISA (expressed as albumin/creatinine ratio (A/C), mg/mg), and enumeration of the glomeruli showing segmental or global glomerulosclerosis (expressed as percent). Data are presented as mean±SE. Compared to positive control (ADR/V), sulodexide reduced proteinuria at day 14 but not at day 48 (when proteinuria was returning toward normal), and largely prevented glomerulosclerosis at day 48. See Figures 2-5

As shown by the data in Figures 2-6, in a mouse model of FSGS, prophylactic treatment with sulodexide showed a trend toward reducing proteinuria on day 14, reduced glomerulosclerosis and interstitial fibrosis at day 48,and did not affect expression of renal mRNA encoding for TGFbI, CTGF and FGF2. Thus, Sulodexide reduced proteinuria, slowed glomerulosclerosis and interstitial fibrosis in experimental FSGS, demonstrating that treatment with Sulodexide is a therapy to slow progressive functional loss in patients with glomerular disease.

7. REFERENCES CITED

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. Such modifications are intended to fall within the scope of the appended claims.

All references, patent and non-patent, cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method for preventing, reducing or eliminating a symptom or complication of focal segmental glomerulosclerosis comprising administering, to a human subject in need thereof, at least one glycosaminoglycan in an amount effective to inhibit, reduce or eliminate one or more symptoms or complications of focal segmental glomerulosclerosis.

2. The method of claim 1, wherein the glycosaminoglycan is sulodexide, or a pharmaceutically acceptable salt, hydrate, or clathrate thereof.

3. The method of claim 1, wherein the glycosaminoglycan is Elmiron®.

4. The method of claim 1, 2 or 3, wherein the composition is administered orally, parenterally, transdermally, or rectally.

5. The method of claim 1, 2 or 3, wherein the composition further comprises an active ingredient selected from the group consisting of a corticosteroid, an immunosuppressive medication; an antibiotic; an antihypertensive or diuretic medication; a medication used to reduce cholesterol or triglycerides; and Vitamin D.

6. The method of claim 5, wherein the antihypertensive or diuretic medication is an

ACE inhibitor.

7. The method of claim 5, wherein the medication used to reduce cholesterol or triglycerides is a statin.

8. The method of claim 1, 2 or 3, wherein the administration is oral.

9. The method of claim 1, 2 or 3, wherein the amount of composition administered per day ranges from about 20 L.R.U. to about 100,000 L.R.U.

10. The method of claim 2, wherein the amount of sulodexide, or a pharmaceutically acceptable salt, solvate, hydrate, or clathrate thereof, administered per day ranges from about 20 L.R.U. to about 100,000 L.R.U.

11. The method of claim 1, 2 or 3, wherein the composition is parenterally administered and ranges in amount from about 20 L.R.U. to about 1,000 L.R.U. per day.

12. The method of claim 1, 2 or 3, wherein the composition is orally administered and ranges in amount from about 200 L.R.U. to about 10,000 L.R.U. per day.

13. The method of claim 1, 2 or 3, wherein the amount of rectally administered composition is about 2000 L.R.U. to 100,000 L.R.U. per day.

14. The method of claim 1, 2 or 3, wherein the composition is parenterally administered and ranges in amount from about 2 mg/day to about 100 mg/day.

15. The method of claim 1, 2 or 3, wherein the composition is orally administered and ranges in amount from about 20 mg/day to about 1000 mg/day.

16. The method of claim 1, 2 or 3, wherein the composition is in the form of tablet, capsule, suppository, solution, emulsion, suspension, aerosol spray, cachet, powder, transdermal patch , topical cream, lotion, ointment or gel.

17. The method of claim 1, 2 or 3, wherein the composition comprises one or more excipients.

18. The method of claim 1, wherein the glycosaminoglycan is selected from the group consisting of heparin; low molecular weight heparin obtained by chemical or enzymatic depolymerization; chemically modified heparin; dermatan sulfate; low molecular weight fractions of dermatan sulfate; hyaluronan; chondroitin sulfate; heparan sulfate; keratan sulfate; and a mixture of two or more of the foregoing.

19. A method for preventing, reducing or eliminating a symptom or complication of focal segmental glomerulosclerosis comprising administering, to a mammal in need thereof, at least one glycosaminoglycan in an amount effective to inhibit, reduce or eliminate one or more symptoms or complications of focal segmental glomerulosclerosis.