AU2012203519A1 - Treatment of cytokine mediated conditions - Google Patents

Abstract

Abstract The present invention relates to the usefulness of a sulfated glycosaminoglycan (GAG) selected from the group consisting of heparan sulfate (HS), depolymerised heparan sulfate, dermatan sulfate (DS), depolymerised dermatan sulfate, low molecular weight heparin (LMWH), and depolymerised heparin having an anti-factor Xa activity of 200 IU/mg or less in treatment or prevention of at least one cytokine mediated condition of the endothelial or epithelial linings.

Description

A ustralian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title Treatment of cytokine mediated conditions The following statement is a full description of this invention, including the best method of performing it known to me/us: P/00/0 II S107 Field of invention The present invention refers to the use of certain sulfated glycosaminoglycans for the prevention and treatment of cytokine mediated conditions of the epithelial and endothelial linings. Background Glycosaminoglycans (GAGs) constitute a highly heterogeneous class of large macromolecules composed of repeating disaccharide units. In general, each of the repeating units comprises an aminosugar, either glucosamine or galactosamine, and an uronic acid or a hexose, either glucuronic acid, iduronic acid or galactose. The hydroxyl group at C2 of the uronic acid and the hydroxyl groups at C3, C4, C6 and the amino group on C2 on the aminosugar may be substituted by sulfate groups. The GAGs comprise the following polymers: heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate and hyaluronan. Heparin is a GAG isolated on a commercial basis from animal tissues and used in the clinic as an antithrombotic drug. Naturally occuring heparin consists of unbranched, negatively charged polysaccharide chains of varying lengths, and molecular weights. Chains of molecular weight from 5 to over 40 kilodaltons (kDa), make up polydisperse pharmaceutical-grade heparin. Heparin derived from natural sources, mainly porcine intestine and bovine lung, can be used to prevent thrombosis. However, the effects of natural, or unfractionated heparin can be difficult to predict. After a standard dose of unfractionated heparin, coagulation parameters must be monitored very closely to prevent over- or under anticoagulation. Numerous brands of heparins and low molecular weight heparins (LMWH), such as tinzaparin and dalteparin, are available for the treatments that rely on their anti coagulant activity. LMWHs have been demonstrated to be at least as effective and safe as unfractionated heparin. Beside therapies relying on the anticoagulant effect of 2 heparin, other uses can be envisioned. Heparin and LMWH have been suggested to be important agents in the treatment of cancer and the anti-tumor effect appears to be partially unrelated to their anticoagulant effect. In Pathophysiology Haemost Thromb, 2007-08; 36; 195-203, B Casu et al. describe a modified non-anticoagulant heparin and explain its dominant anticancer mechanism as exerted through competition with functions of HS chains in HS proteoglycans associated with cell surfaces and extracellular matrices. In this context it is discussed that these heparins also effectively would inhibit tumor cell heparanases in experimental metastasis. Further they exert antimetastic action by disrupting selectin mediated tumor cell-vasular interactions and inhibition of growth factors. The authors also suggest that the heparin mediated release of tissue factor pathway inhibitor (TFPI) may explain their anti-angiogenetic and anti-metastatic effect, while attributing this effect to NS regions of heparin and suggesting that a minimum chain length of heparins is expected to accomplish the effect. In Clinical and Applied Thrombosis/Hemostasis, 2012; 000(00); 1-7, SA Mousa compares the antiangiogenesis activity of the two LMWHs tinzaparin and dalteparin. It is discussed that the molecular weight profile is important for the release of TFPI and concluded that fragments within 2-8 kDa appear to primarily be responsible for increases in TFPI plasma levels. It is further concluded that tinzaprin induces higher levels of TFPI due to its relatively higher molecular weight fractions and higher sulfate/carboxylate ratio. It remains, however, obvious that heparin, LMWH and non-coagulant modified heparins exert its anti-tumor effects through different mechanisms and that the field is far from elucidated. US 8,071, 569 (Mousa) describes a supersulfated, oxidized heparin fraction with angiogenesis inhibition characteristics that retains certain anticoagulant properties. Another type of modified heparins with an anti-tumour effect is described by JW Park et al in Journal of Controlled Release, 2010; 148; 317-326. These heparin derivatives are prepared by conjugating a LMWH (Fraxiparin 4,5 kDa) with deoxycholic acid (DOCA) in order to reduce their anticoagulant activity and establish an orally available preparation. The derivatives are suggested to retain the capacity of heparin and HS bind to factors important for the angiogenic process and specifically to reduce the angiogenic activity of FGF2 and VEGF. It is further generally discussed 3 herein that there are structural requirements necessary for obtaining the interactions between heparins and the growth factors related to the capability of electrostatic and hydrophobic interactions. EP 710483 describes low molecular weight heparins and other sulfated glycosaminoglycans for prevention of structural and functional alterations of the peritoneal membrane in patients suffering from renal insufficiency who thereby risk peritoneal loss of proteins and compromised transport of urea and creatinine. Heparin and LMWH also have well documented anti-inflammatory properties and efficacy against sepsis and shock which suggest direct anti-inflammatory activity rather than anticoagulant properties, see AD Cornet et al in Thromb Heaemost 2007; 98; 579-586. This article refers to studies that indicate that a non-anticoagulant heparin prevents from vascular endothelial dysfunction during hyperdynamic sepsis. The non-anticoagulant heparins are further described in US 5,583,121 (Chaundry et al) who describe 0-desulfated heparins or heparin fragments in the treatment of hypovolemic shock. Heparin is reported to protect against protein-losing enteropathy (PLE), a life threatening complication developed from different unrelated diseases, such as Crohn's diseases and complications following the Fontan procedure, see L Bode et al The Journal of Biological Chemistry, 2006; 281; 7809-7815. It is reported that loss of HS from the basolateral surface of intestinal epithelial cells has an important role in the development of PLE, while an increased availability of cytokines appears to further contribute to aggravate the complication. Heparin therapy is concluded to compensate for the HS loss and also to inactivate cytokines like IFNy and TNFa and restore the intestinal epithelium from protein leakage. KM Bhagirath et al in Ann Thorac Surg, 2007; 84; 2110-2 compares LMWH with unfractionated heparin for the treatment in PLE in the context of an effective treatment with LMWH (dalteparin). It is discussed that LMWH is less ionic than unfractionated heparin and has less propensity to incorporate itself into epithelial cells which may question the need of heparin sulfate binding to epithelial cells of the intestine.

4 Furthermore, N Brodszki et al in Acta Paediatrica, 2011; 100; 1448-1453, report a successful treatment of Gorham-Stout syndrome with the low anticoagulant heparin tafoxiparin. In the report comparisons are drawn with earlier therapies with heparin for treating PLE. It is apparent that the role of heparin and LMWH in treating or preventing from protein leakage in the different discussed complications is not fully understood and it is not clarified to what extent they exert similar mechanisms as in the context of inhibiting angiogenesis in tumor treatment or when counteracting inflammation. It is also evident that there is no clear picture of the requirements of a heparin derivative that effectively would replace heparin, being an even more suitable therapy with less of the side-effects normally associated with heparin. For the treatment or prevention of protein leakage from the endothelium or the epithelium as precipitated by the underlying discussed diseases, it would be highly advantageous if an effective LMWH agent was available with low or negligible anticoagulant effect. Such an agent should also exhibit a suitable bioavailability, at least comparable to that of tinzaparin or dalteparin, and retain other usual advantages compared to unfractiona'ted heparin. It would also be highly desirable to obtain such a heparin derivative that is perfected to both treat protein leakage and inhibit endothelial proliferation (such as angiogenesis) with consideration to the multiple outlined mechanisms involved in the activity-of heparin or LMWH. Accordingly, there is demand for a low anticoagulant heparin with a highly favorable molecular weight distribution of the polysaccharide chains and with a favorable content of charged groups on the chains for the purpose of treating both protein leakage and treat abnormal endothelial proliferation associated with tumors and also other degenerative diseases wherein inhibition of endothelial proliferation is required, such as diabetic retinopathy and macular degeneration. Description of the invention ) Before the present invention is described, it is to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

5 It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Also, the term "about" is used to indicate a deviation of +/- 2 % of the given value, preferably +/- 5 %, and most preferably +/- 10 % of the numeric values, where applicable. In the context of the present invention the term "cytokine mediated condition of the epithelial or endothelial linings" relates to a complication or condition wherein the integrity or functionality of the epithelium or endothelium is impaired and the activity of cytokines place an important role in the deteriorative process which becomes manifested for example as leakage of proteins. Alternatively, the cytokine mediated complication relates to abnormalities in the endothelium generation including abnormal proliferation, expressed for example as angiogenesis associated with tumor growth and also other diseases such as diabetic retinopathy and macular degeneration, especially the wet form of age-related macular degeneration. In the context of the present invention the term "protein leakage" relates to extravasation of fluid and proteins over the endothelial cell layer, as for example is seen in sepsis, or transepithelial leakage of proteins and fluid, as for example is seen in PLE. The invention generally relates to the usefulness of least one sulfated GAG selected from the group consisting of HS, depolymerised HS, DS, depolymerised DS, LMWH, and depolymerised heparin having an anti-factor Xa activity of 200 IU/mg or less for treating of or preventing at least one cytokine mediated condition of the endothelial or epithelial linings. The present invention further relates to a method for treating or preventing at least one cytokine mediated condition of the endothelial or epithelial linings comprising administering a sulfated GAG selected from the group consisting of HS, depolymerised HS, DS, depolymerised DS, LMWH, and depolymerised heparin having an anti-factor Xa activity of 200 IU/mg or less.

6 The cytokine-mediated condition of the endothelial or epithelial linings in particular relates to abnormalities in such issues, as expressed by loss of integrity and leakage. Thereby, present invention aims at preventing or treating such complications which have severe consequences for surrounding organs or tissues. In one aspect, protein leakage is linked to vascular dysfunctions related to endothelial proliferation (such as angiogenesis). Protein leakage from endothelial and epithelial linings occurs in many different disorders. Non-limiting examples of disorders wherein protein leakage is a complication are Gorham Stout Syndrome, lymphangioma, sepsis, PLE, and nephrosis. Likewise endothelial proliferation (e.g. angiogenesis) occurs in several different disorders. Non-limiting examples of disorders wherein endothelial proliferation leading to dysfunctional neovascularization are cancer, diabetic retinopathy, Gorham Stout syndrome and lymphangioma and macular degeneration. Accordingly the presently invented treatments with sulfated GAGs is in an aspect directed to treatment of complications wherein proliferating vessels are dysfunctional as manifested by defective retaining capacity The present invention also relates to the prevention and treatment of protein leakage as a part of a treatment of Gorham stout syndrome, lymphangioma, sepsis, and PLE. The present invention further relates to the prevention and treatment ofcomplications of cancer arriving from neovascularization and dysfunctional vessels, treatment of diabetic retinopathy, Gorham Stout syndrome and lymphangioma. HS, DS and CS, are composed of alternating hexosamine and uronic acid residues. 5 The presence of D-glucuronic acid (GIcA) and its C-5 epimer L-iduronic acid (IdoA) and the specific sulfation of hexosamines and uronosyl residues endow the polymer an extreme structural variation. The structure is built on repeating disaccharides containing from none or very few to nearly 100% iduronic acid-containing disaccharides. The organization of GIcA-and ldoA-N-hexosamine containing 7 disaccharides can vary from long blocks to an alternating disaccharide pattern. The variation of sulfation and the degree of IdoA sulfate generates a wide variety of biological activity. There are different well-defined polysaccharides of DS, CS, HS and depolymerised heparin. The glucosamine of the repeating building blocks of HS is either N-acetylated or N sulfated, and their presence is arranged mainly in a segregated manner. HS has ubiquitous distribution on cell surfaces and in the extracellular matrix. It is generally less sulfated and has a lower IdoA content than heparin and has a more varied structure. Interactions between HS and proteins are implicated in a variety of physiological processes, such as cell adhesion, cell proliferation, enzyme regulation, cytokine action, virus entry and anticoagulant properties. HS possesses anticoagulant activity depending on the presence of a specific anticoagulant pentasaccharide, however considerably less than heparin. Heparan sulfate is a linear polysaccharide which can be prepared from porcine intestinal mucosa or from bovine lung, from heparin side fractions using cetylpyridinium chloride fractions and sequential salt extraction as described by Fransson et al., Structural studies on heparan sulphates, Eur. J. Biochem. 106, 59-69 (1980). CS is a linear polysaccharide consisting of alternating glucuronic acid and N-acetyl galactosamine residue, the latter being sulfated at either C4 or C6 or both. They can be prepared from bovine trachea or nasal cartilage. CS is of importance for the organization of extracellular matrix, generating an interstitial swelling pressure and participating in recruitment of neutrophils. DS is a linear polysaccharide consisting of alternating uronic acid and N-acetyl galactosamine residue. The uronic acids are either GIcA or IdoA and the disaccharide can be sulfated at C4 and/or C6 on the galactosamine and at C2 of the IdoA. DS can be prepared from porcine skin and intestinal mucosa and possesses biological activities such as organization of extracellular matrix, interactions with cytokines, anti-coagulant activities and recruitment of neutrophils. LMWH or depolymerized heparins are linear oligosaccharides mainly consisting of alternating N-sulfated glucosamine and IdoA residue and often containing the 8 anticoagulant pentasaccharide. They can be prepared from heparin by specific chemical cleavage. Their main clinical function is to inhibit factor Xa, resulting in an antithrombotic effect. It is proposed to have antimetastatic properties. Fragmin@ (Pfizer, USA) is an example of a LMWH obtained by controlled depolymerisation of heparin and having an antithrombotic effect owing to inhibition of factor Xa. Heparin fragments having selective anticoagulant activity, as well as methods for the preparation thereof, are described in US patent number 4,303,651. According to the European Pharmacopoeia (PharmEur) a heparin, in order to be called a LMWH (low molecular mass heparin), should have an antifactor Xa activity not less than 70 IU(International Unit)/mg and an M, of less than 8 kDa. Accordingly, the GAGs administered in methods and in compositions of the present invention preferably have a weight average molecular weight (Mw) of 30 kDa or less, preferably less than 20 kDa, more preferably 10 kDa or less, even more preferably not higher than 8 kDa and most preferable not higher than 7 kDa. Preferably, the GAG belongs to the group consisting of depolymerized heparins having an average molecular weight below 20 kDa, preferably below 10 kDa, more preferably not higher than 8 kDa and most preferably not higher than 7 kDa. For the purpose of treatment or prevention of at least one cytokine mediated condition of the endothelial or epithelial linings, the GAG of the present invention has an anti-factor Xa activity of 200 IU/mg or less, preferably less than 150 IU/mg, more preferably less than 100 IU/mg, more preferably less than 70 IU/mg, more preferably less than 30 lU/mg, and most preferable less than 10 lU/mg. The anticoagulant activity of heparin, LMWH and other heparin derivatives is often measured as their ability to potentiate the inhibition of coagulation factor Xa and factor Ila by antithrombin. Methods for measuring anti-factor Xa- and anti-factor Ila activity are well known to the skilled person and are also described in pharmacopoeias such as the European pharmacopoeia (Pharm Eur) and the United States pharmacopoeia (USP).

9 The anticoagulant activity can be abrogated by for example selective periodate oxidation (see e.g. Fransson LA, and Lewis W, Relationship between anticoagulant activity of heparin and susceptibility, to periodate oxidation, FEBS Lett. 1979, 97 119-23; Lindahl et al.and Proc Natl Acad Sci USA, 1980; 77(11):6551-6555). In one embodiment the disaccharide structure of the GAG is essentially devoid of non-sulfated monosaccharide glucuronic and iduronic units and having an antifactor Xa activity of 10 lU/mg or less. In another embodiment the GAG is a LMWH with an antifactor Xa activity of 10 IU/mg or less and an average molecular weight not higher than 8 kDa, and preferably not higher than 7 kDa. A suitable such LMWH has an anticoagulant activity (< 10 IU/mg by pharmacopoeial anti-factor Xa- and anti-factor Ila assays) and an weight average Mw of 5-7 kD, while having a distribution of polysaccharides and their corresponding molecular mass in cumulative % according to the table below. Molecular mass, Cumulative weight, kDa % >15 0 >10 6-15 >9 7-20 >8 13-23 >7 18-32 >6 26-42 >5 40-53 >4 52-68 >3 >70 >2 >85 It is of desirable that the GAGs to be used in the present invention comprise a suitable distribution of chains with molecular weight in the range of 4-7 kDa while retaining chains differently sized outside the range. This can preferably be obtained by purposeful depolymerisation and fractionation.

10 It is also important to retain the distribution of clusters of negatively charged groups of the chains as have been found important in the mode of action in native unfractionated heparin. This is preferably accomplished by a selective process to eliminate the pentasaccaharide sites of the chain associated with the anticoagulant effect. Without being bound to theory, it has been hypothesized that the GAGs as defined above and administered according to the present invention will bind to the cell surface and decrease the ability of proteins to pass through the cellular barrier and thereby treat or prevent protein leakage. It is further hypothesized that the GAGs as defined above bind factors such as cytokines and growth factors (such as VEGF) and thereby modulate the activity of these factors. Another such factor, heparin-binding protein (HBP, azurocidin) is involved in endothelial leakage and was recently suggested to be the prime marker of early sepsis. Given the fact that HBPs seem to be involved in both the pathological angiogenic process in for example Gorham stout syndrome and in conditions with leaky vessels (HBP), and that loss of heparan sulfate can lead to leakage of proteins over the intestinal epithelium, the present inventors postulate that giving heparin in conjunction with more conventional therapies, would be beneficial and that the GAG will, for example, bind to the proliferating factors (eg VEGF), and thereby slow the process down. One non-limiting way of obtaining a sulfated GAG to be used according to the invention is to treat a sulfated GAG, such as heparin (e.g. pig intestinal mucosa heparin) by periodate oxidation followed by alkaline p-elimination of the product. This process leads elimination of the anticoagulant activity and depolymerization. The process disclosed in US Patent 4,990,502 (Lormeau et al) demonstrates one way of treating native heparin to selectively cleave residues of the pentasaccharide residues responsible for the anticoagulant effect and a following depolymerization that results in a low anticoagulant, LMWH with a an average molecular weight 5,8 to 7,0 kDa. The invention further relates to a parenteral composition of least one sulfated GAG as described above for treating of or preventing at least one cytokine mediated condition of the endothelial or epithelial linings.

11 In the described parenteral compositions the active compounds can be incorporated into a solution or suspension, which also contain one or more adjuvants such as sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial agents, antioxidants, chelating agents, buffers and agents for adjusting the osmolarity. The compositions can be delivered in ampoules, vials, disposable syringes or as infusion arrangements, also for self administration. Encompassed by the present invention is any combination with the disclosed embodiments. The invention will be further disclosed in the following non-limiting examples Examples Tafoxiparin is a depolymerised heparin that is essentially deprived of its anticoagulant activity (< 10 IU/mg by pharmacopoeial anti-factor Xa- and anti-factor Ila assays). The weight average Mw is 5-7 kDa. Table 1. Distribution of polysaccharides and their corresponding molecular mass in cumulative % of weight for several batches of Tafoxiparin Molecular mass, Cumulative weight, kDa % >15 0 >10 6-15 >9 7-20 >8 13-23 >7 18-32 >6 26-42 >5 40-53 >4 52-68 >3 >70 12 >2 >85 The pharmaceutical preparation of DF01 is a solution for subcutaneous injection, 8 mL dispensed in glass vials sealed with a rubber stopper and covered with a tear-off aluminum cap. CASE DESCRIPTIONS OF TREATMENT OF GORHAM STOUT SYNDROME P1, a 2.5-year-old boy, with an unremarkable family history and previously healthy except for nonallergic asthma, was referred to the clinic for the chest tube drainage of an extensive and progressive pleural effusion detected when the patient was hospitalized for pneumonia. Computed tomography (CT) had shown extensive amounts of pleural effusion, atelectasis and emphysematous areas in the right lung. A tube thoracostomy was performed with direct exchange of 700 mL of whitish fluid, with triglyceride levels at 6 mM (reference value for plasma 1.8 mM), indicative of chylothorax. A new chest CT was performed the next day showing regression of the pleural effusion but, in addition, it detected multiple skeletal, possibly lytic, abnormalities in several vertebrae, the pelvis, and in the right clavicle as well as rounded changes within the spleen. During the following days, extensive pleural drainage, infection, malignancy and immunodeficiency were ruled out. A bone marrow biopsy and a fine needle biopsy of one of the lytic skeletal changes were inconclusive. The patient was started on total parenteral nutrition (TPN). Open biopsy of one of the lytic bone changes revealed lymphatic vessels dissecting the bone, resulting in the zones of bone resorption and marrow hypoplasia. The pathological changes were consistent with Gorham-Stout disease, or disseminated lymphangiomatosis. Treatment with interferon a-2b (IntronA, Schering-Plough Sweden, Stockholm, Sweden) was started at a dose of 2 million units/tn2 body surface, every second day. A prompt response with reduced pleural fluid drainage could be seen, within a few days. A repeat chest CT showed clearing of the chylothorax, but a pericardial effusion appeared and was confirmed by echocardiography. The pericardial effusion was successfully managed conservatively with diuretic therapy. The patient was discharged but returned after 15 days owing to increasing pericardial and pleural fluid, despite continued interferon a-2b and diuretic therapy. His clinical condition quickly deteriorated, despite continuous positive airway 13 pressure treatment. The patient was sedated and intubated. The lymph leakage continued, some days exceeding 6 1124 h or 545 mL/kg/24 h. TPN was restarted and the interferon a-2b therapy was intensified with doses daily instead of every second day. Explorative thoracoscopy was performed revealing large amounts of gelatinous fluid and thick mesh-like tissue in the pleural compartments, and a pleural biopsy was taken. The large drainage volumes continued and tafoxiparin (low anticoagulant, LMWH) was started. The patient received a total of 3 weeks treatment with tafoxiparin. Because no changes in the drainage volumes were observed, chest radiation therapy (RT) was performed with 15 Gy given in 10 fractions over 2 weeks, as suggested by the current literature. Tafoxiparin and interferon a-2b were paused during the RT. In spite of all the aforementioned efforts, the chest tube drainage volumes remained unaltered and ultimately surgical intervention was attempted after thoracoscopy revealed, surprisingly, that the mesh-like tissue had completely resolved by this time. A resection of the right middle lobe with concomitant pleurodesis and thoracic duct ligation was performed; the lung specimen showed lymphangiogenesis. This operation finally stemmed the fluid leakage, and the patient could subsequently return to his home clinic for continued interferon a-2b therapy. Four years after the treatment, he is clinically stable with no breathing problems and without any new symptoms. P2, a 4-year-old girl with an unremarkable family history and in good prior health, started complaining about nonspecific back pain approximately 8 months before her first admission. She was admitted to the local hospital after mild trauma to the thorax, when an X-ray of her chest showed substantial bilateral pleural effusions with the compression of the inferior vena cava and several fractured ribs. Routine blood tests and analysis of the pleural exudates (shown to be chylous) revealed no infection or malignant cells. A CT scan confirmed, besides massive chylothorax, several rib fractures, fracture of the sternum and diffuse osteolytic changes in the humerus, femur, pelvis, sacrum and multiple vertebrae. Also, poorly defined changes were noted in the spleen. A chest tube was inserted and the amount of drained pleural fluid averaged between 1400 and 2000 ml/24 h. The patient was started on TPN. The clinical picture suggested lymphangiomatosis, and treatment with pegylated interferon a-2b (Peglntron, Schering-Plough Sweden, Stockholm, Sweden) was initiated (weekly injections of 1 Ig, later 0.5 Ig/kg bodyweight). She received a 7-day 14 course of continuous intravenous infusion of octreotide, 1 Ig/kg bodyweight/h. Despite the treatment, no remission of the chylothorax was observed during 2 months of treatment and P2 was referred to our unit. Biopsies from pleurae and ribs confirmed the diagnosis of lymphangiomatosis. A cytokine profile of the fluid was determined. PegIntron was switched to subcutaneously administered IntronA at a dose of 2 million unitsAn2 body surface, every second day. Furthermore, tafoxiparin was included in the treatment. The patient underwent RT directed towards all affected areas; 15 Gy was given in 10 fractions over 2 weeks in two sessions, 4 weeks apart to allow bone marrow recovery. Interferon treatment was paused during RT. During the 4 weeks between the RT blocks, she was treated with filgrastim (Neupogen, Amgen AB, Solna, Sweden) for the purpose of collecting CD34+ cells, should an autologous rescue stem cell transplant be necessary. She responded adequately to this treatment regimen. After the second round of RT, interferon was administered at an intensified dosage, with daily injections instead of every other day. Her clinical status improved steadily. After approximately 2.5 months, the pleural drain could be removed. Tafoxiparin was stopped after a total of 3 months' treatment. She continues with daily injections of IntronA. An MR control at 3 months after discharge showed clear improvement on the previously noted changes in the lungs and sen, and moreover, a reduction in the lytic changes in the bones was seen. Six months postdischarge, the patient is doing remarkably well. Measurements of selected cytokine levels are shown in Table 2.

15 Table 2 Measurement of cytokines, using specific ELISA kits, from serum or pleural effusion (PE) taken from P1 and P2 at different states of disease activity values outside the reference intervals are indicated in bold State of disease Source Cytokine Maximal activity Remission Reference interval P1 - serum VEGF-A (ng/L) 730 570 62-707 VEGF-C(ng/L) 4050 3910 2459-6651 P2 - serum VEGF-A (ng/L) 1200 370 62-707 VEGF-C(ng/L) 6930 2260 2459-6651 IL-5 (ng/L) <8 16 <8 1L-12 (ng/L) <7,8 <8 <16 IL-18(ng/L) 140 630 140-500 IL-10(ng/L) <5 <5 <9 IL-1 # (ng/L) <5 <5 <5 IL-6 (ng/L) 7 15 <8 IL-8 (ng/L) <5 12 <60 slL-2R (kU/L) 479 1030 <700 TNF-a (ng/L) 1 1 21 <15 IL-4 (ng/L) <16 - <100 P2 - PE VEGF-A (ng/L) 220 nk VEGF-C (ng/L) 200 - nk IL-5 (ng/L) <8 - nk IL- 12 (ng/L) <7,8 - nk IL-10 (ng/L) <5 - nk IL-1# (ng/L) <5 - nk IL-6 (ng/L) 2828 - nk IL-8 (ng/L) 559 - nk sIL-2R (kU/L) 530 - nk TNF-a (ng/L) 23 - nk IL-4 (ng/L) <16 - nk Discussion 16 Given the fact that heparin binding proteins seem to be involved in both the pathological lymphangiogenetic process in GSS (e.g. IL- 8, VEGF) and in conditions with leaky vessels (HBP), and that the loss of HS or HSPG ??? can lead to the leakage of proteins over the intestinal epithelium, we postulated that giving the patients with GSS heparin in conjunction with more conventional therapies (discussed later on) would be beneficial. However, our initial patient lost up to 5 L of lymph daily, corresponding to approximately 50% of his body weight, which had to be replenished by the massive infusions of fresh frozen plasma (FFP), blood and albumin. We thus chose not to give any drug that could possibly further worsen his coagulation status. Heparin and LMWH can, however, be enzymatically or chemically modified to selectively almost completely lose its anticoagulant properties while keeping most of its charge and hence ability to bind cationic proteins. One such preparation is tafoxiparin, a periodate-oxidized, LMWH currently used in a clinical obstetric study The Swedish Medical Products Agency (Lakemedelsverket) approved its usage in our patients owing to the extreme situation, despite no available data on its use in children. The anticipated risks were HIT (heparin-mediated thrombocytopenia), an allergic reaction or coagulation disturbances. We first administered a test dose and then choose to give a dose corresponding to 2500 F'n2 twice daily, subcutaneously, in accordance with the doses of heparin used in PLE. The mortality of patients afflicted with Gorham Stout syndrome and the condition chyclothorax is high, about 70 % (although based on a small number of patients). Thus, there is of great importance to improve or find alternative treatments for these patients. The case reports exemplified herein present an interesting alternative or supplemental therapy to the currently applied treatment regimen. Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is contemplated by the inventor that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

17 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (32)

1. A sulfated glycosaminoglycan (GAG) selected from the group consisting of heparan sulfate (HS), depolymerised heparan sulfate, dermatan sulfate (DS), depolymerised dermatan sulfate, low molecular weight heparin (LMWH), and depolymerised heparin having an anti-factor Xa activity of 200 IU/mg or less for use in treatment or prevention of at least one cytokine mediated condition of the endothelial or epithelial linings.

2. The sulfated GAG for use according to claim 1 wherein the cytokine mediated condition is protein leakage.

3. The sulfated GAG for use according to claim 1, wherein the cytokine mediated condition is endothelial proliferation.

4. The sulfated GAG for use according to claim 3, wherein the cytokine mediated condition is angiogenesis.

5. The sulfated GAG for use according to any one of the preceding claims, wherein the cytokine mediated condition is a complication being a part of an underlying disease selected from the group consisting of Gorham Stout syndrome, lymphangioma, sepsis, protein losing enteropathy (PLE), cancer, diabetic retinopathy, and macular degeneration.

6. The sulfated GAG for use according to claim 2, wherein prevention and or treatment of protein leakage is part of treatment of Gorham stout syndrome, lymphangioma, sepsis, and PLE.

7. The sulfated GAG for use according to claim 3, wherein prevention and or treatment of endothelial proliferation is part of treatment of complications from cancer, treatment of diabetic retinopathy, macular degeneration, Gorham Stout syndrome and lymphangioma.

8. The sulfated GAG for use according to any one of the preceding claims, wherein said GAG has a weight average molecular weight below 30 kDa. 19

9. The sulfated GAG for use according to claim 8, wherein said GAG has a weight average molecular weight below 10 kDa.

10.The sulfated GAG for use according to any one of the preceding claims, wherein said at least one sulfated GAG having an anti-factor Xa activity of 30 IU/mg or less.

11. The sulfated GAG for use according claim 10, wherein said at least one sulfated GAG having an anti-factor Xa activity of 10 IU/mg or less.

12. The sulfated GAG for use according to any one of claims 1-11, wherein said GAG is LMWH or depolymerised heparin.

13. The sulfated GAG for use according to claim 12, wherein said the GAG is LMWH or depolymerised heparin having a weight average molecular weight below 10 kDa.

14. The sulfated GAG for use according to claim 12 or 13, wherein the at least one glycosaminoglycan is LMWH or depolymerised heparin and has an antifactor Xa activity of 10 IU/mg or less.

15.The sulfated GAG for use according to any one of claims 1-14, wherein the at least one GAG is administered in a parenteral pharmaceutical preparation.

16. The sulfated GAG for use according to claim 14, wherein said at least one sulfated GAG is a depolymerised heparin with a weight average Mw of 5-7 kDa and having an anticoagulant activity of less than1O lU/mg by pharmacopoeial anti factor Xa- and anti-factor Ila assays.

17.A method for treating of or preventing at least one cytokine mediated condition of the endothelial or epithelial linings comprising administering a sulfated GAG selected from the group consisting of HS, depolymerised HS, DS, depolymerised DS, LMWH, and depolymerised heparin having an anti-factor Xa activity of 200 IU/mg or less.

18. The method according to claim 17, wherein the cytokine mediated condition is protein leakage. 20

19. The method according to claim 17, wherein the cytokine mediated condition is endothelial proliferation.

20. The method according to claim 19, wherein the cytokine mediated condition is angiogenesis.

21. The method according to any one of claims 17-20, wherein the cytokine mediated condition is a condition being a part of an underlying disease selected from the group consisting of Gorham Stout syndrome, lymphangioma, sepsis, PLE, cancer, diabetic retinopathy, and macular degeneration

22. The method according to claim 17, wherein prevention and or treatment of protein leakage is part of treatment of Gorham stout syndrome, lymphangioma, sepsis and PLE.

23. The method according to claim 19, wherein prevention and or treatment of endothelial proliferation is part of tumor treatment, treatment of diabetic retinopathy, macular degeneration, Gorham Stout syndrome and lymphangioma.

24. The method according to any one of claims 17-23, wherein said GAG has a weight average molecular weight below 30 kDa.

25.The method according to claim 24, wherein said GAG has a weight average molecular weight below 10 kDa.

26. The method according to any one of claims 17-25, wherein said at least one sulfated GAG having an anti-factor Xa activity of 30 IU/mg or less.

27.The method according claim 26, wherein said at least one sulfated GAG having an anti-factor Xa activity of 10 IU/mg or less.

28. The method according to any one of claims 17-27, wherein said GAG is LMWH or depolymerised heparin.

29.The method according to claim 28, wherein said GAG is LMWH or depolymerised heparin having a weight average molecular weight below 10 kDa. 21

30.The method according to claim 28 or 29, wherein the at least one GAG is LMWH or depolymerised heparin and has an antifactor Xa activity of 10 IU/mg or less.

31.The method according to any one of claims 17-30, wherein the at least one GAG is administered in a parenteral pharmaceutical preparation.

32.The method according to claim 30, wherein said at least one sulfated GAG is a depolymerised heparin with a weight average Mw of 5-7 kDa and having an anticoagulant activity of less than1O IU/mg by pharmacopoeial anti-factor Xa- and anti-factor Ila assays.