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"Dendritic Polyglycerol Sulfates And Sulfonates And Their Use For Inflammatory Diseases"

Abstract: The present invention relates to the novel compound classes of dendritic polyglycerol sulfates and sulfonates as well as to their production and use for the treatment of diseases, particularly inflammatory diseases, and to their use as selectin inhibitors and selectin indicators. The dendritic polyglycerol sulfates and sulfonates are also suitable for imaging diagnostics, particularly with respect to inflammatory diseases.

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Patent Information

Application #
Filing Date
02 February 2009
Publication Number
21/2009
Publication Type
INA
Invention Field
CHEMICAL
Status
Email
Parent Application

Applicants

FREIE UNIVERSITÄT BERLIN
KAISERWETHER STRASSE 16-18, 14195 BERLIN GERMANY

Inventors

1. HAAG RAINER
STANZER ZEILE 82, 12209 BERLIN GERMANY
2. DERNEDDE JENS
NIEBUHRSTRASSE 72, 10629 BERLIN GERMANY
3. TAUBER RUDOLF
KLEISTSTRASSE 26A, 14163 BERLIN GERMANY
4. BERNHARD GESCHE
SENNJISTRASSE HAUS RÖNEBLICK CH-3937 BALTSCHIEDER SWITZERLAND
5. ENDERS SVEN
BRAHESTRASSE 33, 10589 BERLIN GERMANY
6. WEINHART HEIDEMARIC
WARSCHAUER STRASSE 28, 10246 BERLIN GERMANY
7. ZÜGEL ULRICH
EDELHOFDAMM 29A, 13465 BERLIN GERMANY
8. TÜRK HOLGER
Q5, 9-10 68161 MANNHEIM GERMANY
9. VON BONIN ARNE
ROSE-LUXEMBURG-STRASSE 6A, 16548 GLIENICKE/NORDBAHN GERMANY

Specification

Dendritic polyglycerol sulfates and sulfonates and their use for inflammatory diseases The present invention relates to the novel compound classes of dendritic polyglycerol sulfates and sulfonates as well as to their production and use for the treatment of diseases, particularly inflammatory diseases, and to their use as selectin inhibitors and selectin indicators. The dendritic polyglycerol sulfates and sulfonates are also suitable for imaging diagnostics, particularly with respect to inflammatory diseases. Background of the invention Inflammation is a fundamental response to the damage of tissue and the invasion of pathogens, wherein leukocytes play a key role due to their antimicrobial, secretory and phagocytosis activities. Recruiting of leukocytes to the vascular endothelium and subsequent migration into the surrounding tissue are observed in all forms of the inflammatory reaction. The migration of leukocytes into tissue is initiated by the adherence of leukocytes onto the vascular wall. This allows the leukocytes to accumulate in the source of infection and to effect defence reactions. A variety of vascular cell adhesion molecules on leukocytes and on endothelium cells mediate and control the adhesion of blood cells onto the vascular wall. This process takes place in a cascade of series connected molecular interactions. At first, selectins, a family of lectin-like adhesion molecules, mediate the "docking" and rolling of the leukocytes on the surface of the endothelium. This leads to a slowdown of the leukocytes and allows the contact with signalling molecules on the surface of the endothelium, like e.g. chemokins. These signalling molecules stimulate the activation of integrins on the surface of leukocytes which than in turn mediate the efficient binding of these cells onto the surface of the endothelium. Members of the superfamily of immunoglobulins (Ig) act as ligands of the integrins. The now stably adherent leukocytes can move in a directed manner and can actively move through the endothelium cell layer. As already stated, the initial contact and the rolling of the leukocytes on the endothelium is mediated by transient receptor-ligand interactions between the (three) selectins and their ligands [1]. Close contact of the leukocytes with the endothelium is subsequently guaranteed via the interaction of activated integrins with adhesion molecules of the immunoglobuline (Ig) super family [2]. In addition to the desired defence action and the repair of tissue damages the uncontrolled migration of leukocytes from the bloodstream can be of pathological relevance and lead to the damage of tissue [3]. The general attendance of endothelial cell adhesion molecules in acute and chronic inflammatory processes renders them suitable target structures for diagnostics and therapy [for a review see 4]. Selectins are carbohydrate binding adhesion molecules, which contribute to the increased adhesion of leukocytes onto the vascular endothelium of the inflamed tissue during the process of immune defence. According to their cells of origin, they are divided into L-(leukocytes), E-(endothelium) and P-selectin (platelets and endothelium). Due to their protein structure and their special molecular bindung characteristics selectins initiate leukocyte adhesion; after temporarily binding of the corresponding ligands the leukocytes experience a "rolling slowdown" from the fluent bloodstream alongside the vascular wall. Afterwards other adhesion molecules mediate the close binding of the leukocytes onto the endothelium as well as their extravasation for accomplishing their defence function. Shortly after the discovery of selectins and after the elucidation of their structure at the beginning of the nineties the selectins became attractive target structures in the field of pharmaceutical research. In addition to their physiological function in immune response, a dysregulation of the selectin expression during pathological processes, such as rheumatoid arthritis, asthma, diabetes mellitus and ischemia/reperfusion, as well as an attendance during the tissue invasion of metastasizing cancer cells was observed. This motivated an intensive search for selectin inhibiting compounds. E- and P-selectin, and L-selectin ligands are expressed on microvascular endothelium in an inflammation-dependent manner, L-selectin is presented on leukocytes [1, 2]. Only several highly affine ligands are known for the reported selectins. In principle, these are mucin-like structures, i.e. long elongated glycoproteins, which have many carbohydrate side chains glycosidically attached on their serine or threonine rich protein scaffold as the actual binding epitopes. Via fast formation and dissociation of receptor bindings on the highly flexible ligands cell rolling is mediated in the shearing stream of the vessels. The carbohydrate epitopes essential for binding are N-acetyl lactosamin based oligosaccharides with a specifically attached fucose and a terminal sialic acid (N-acetyl neuraminic acid). The tetrasaccharide sialyl LewisX (sLeX) is an outstandingly relevant binding epitope. sLeX is used as a standard ligand for structure-function relations in order to characterize binding characteristics as well as for searching selectin inhibitors. The findings of sLex as an important binding partner of selectins and findings of polyvalence as a key for targeted blockage of leukocyte adhesion are known for quiet some time and are the basis for the development of diverse selectin inibitors [for a review see 5]. As of yet, the target selectin has not led to the development of market-ready therapeutics, even though highly affine inhibitors are available [5]. At present therapeutic intervention in the case of rheumatoid arthritis and psoriasis is achieved by employing blockers of the inflammatory cytokin TNFa (infliximab, etanercept, [6,7]). In addition, with efalizumab [7] an anti-integrin antibody is on the market, which is approved for the systemic therapy of psoriasis. Further compounds are tested in clinical trials, such as the Pan-selectin antagonist bimosiamose (l,6-bis[3-(3-carboxymethylphenyl)-4-(2-alpha-D-mannopyranosyloxy)phenyl]hexane) which belongs to the class of small molecule drugs and which is a selectin inhibiting compound with a glycoside structure having a substantially higher affinity to selectins than sLeX (trials performed by Revotar AG, Hennigsdorf). Bimosiamose is supposed to be employed for asthma, psoriasis, atopical dermatitis and reperfusion damages [8]. Linear neoglycopolymes carrying sulfated sLex structures have been described and can reach IC50 values in the low nanomaolcular range [5, 9], as well as dendritic polyethylene oxide (PEO) glycopolymes, which are sulfated [10]. Therefore, it is an object of the present invention to provide compounds and compound classes, which are easy to be synthesized and which are suitable for the treatment of diseases, particularly inflammatory diseases. The object is solved by the present invention by providing dendritic polyglycerol sulfonates. A dendritic polyglycerol sulfonate according to the present invention is characterized by a) a poJvmeric polyglycerol core, composed of repeated units of glycerin with the fornwa (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, preferably 1 to 4 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 %, preferably 60 %, and an average molecular weight of 100 to 1,000,000 g/mol, preferably 1,000 to 20,000 g/mol, b) the substitution of one or more OH groups of the glycerin units with -SO3H or - SOsNa groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -(CH2)„- or -[(CH2)m-0)]n- wherein m is 1 to 100 and n is 1 to 50,000, and bound thereto -SO3H or -SOsNa groups, so that a degree of sulfonation of 1 to 100 %, preferably 1 to 30 %, is obtained, and c) a molecular weight of 110 to 1,500,000 g/mol, preferably 1,100 to 30,000 g/mol. The polymeric polyglycerol core is produced by using a (multi)functional starter molecule or initiator, respectively, during the ring-opening polymerization of glycidol. The starter molecule or initiator, respectively, is a polyhydroxy compound, having 1 to 1,000, preferably 1 to 100 and more preferably 1 to 4 OH groups. The starter molecule has the generic formula R-(OH)x, wherein R can be any molecule, which is stable under the conditions of the anionic polymerization, and x is 1 to 1,000; preferably 1 to 100 and more preferably 1 to 4. Preferably the used initiators are tris- or tetrafunctional inititators, such as 1,1,1-trishydroxymethylpropane (TMP) as preferred trisfunctional initiator or pentaerythrol (PE) as preferred tetrafunctional initiator. The starter molecule or the initiator, respectively, can carry further heterofunctionalities, such as particularly SH groups, NH2 groups. In a particular embodiment the starter molecule contains OH groups and/or further heterofunctionalities (like SH, NH2). Further suitable initiators are known to the person of skill in the art. Depending on the choice of the initiator and the polymerization conditions the polymeric polyglycerol core reaches a branching degree and an arbitrarily adjustable molecular weight with narrow polydispersities. According to the present invention polymeric polyglycerol cores with a branching of 0 to 100 % are used. Preferably, highly branched structures are used, preferably with a branching degree of 30 to 80 %, particularly preferably with a branching degree of 60 %. The average molecular weight of the polymeric polyglycerol core according to the present invention is preferably 100 to 1,000,000 g/mol, more preferably 500 to 100,000 g/mol, wherein 1,000 to 20,000 g/mol are particularly preferred. The polymeric polyglycerol cores according to the present invention are subjected to a sulfonation. Preferably sodium salt of vinylsulfonic acid in presence of catalytic amounts of a base, such as potassium hydroxide, is used as sulfonation reagent. The degree of sulfonation reached is preferably 1 to 100 %, particular preferably 10 to 30 %, more particular preferably 30 to 100%. „Degree of sulfonation" according to this invention means the percentage of functionalized OH groups of the glycerine units of the polymeric polyglycerol core. The functionalization results either from the substitution of one or more OH groups of the glycerin units with -SO3H or -SOsNa groups or from the attachment of an oligomeric spacer at one or more OH groups of the glycerin units. The oligomeric spacer has the generic formula: -(CH2)n-or -[(CH2)m-0)]„-wherein m is 1 to 100, preferably 1 to 50, more preferably 1 to 10 and even more preferably 2, and n is 1 to 50,000, preferably 1 to 5,000, more preferably 1 to 100 and has bound thereto -SO3H or -SOsNa groups. An oligomeric spacer is e.g. a oligoethylene glycol (OEG) chain, a polyethylene glycol (PEG) chain, aliphatic carbohydrate chains or also other linear polyethers. Depending on the choice of the polymeric polyglycerol cores according to the present invention and the sulfonation conditions, i.e. the degree of sulfonation, the molecular weight of a dendritic polyglycerol sulfonate according to the present invention is preferably 110 to 1,500,000 g/mol, more preferably 600 to 150,000 g/mol and particular preferably 1,100 to 30,000 g/mol. Particularly preferred embodiments of a dendritic polyglycerol sulfonate according to the present invention have a) a polymeric polyglycerol core with an average molecular weight (M„) of 2,500 to 20,000 g/mol and a branching degree of 60 %, which corresponds to a dendritic branching degree, and b) a degree of sulfonation of 10 to 30 %, which is obtained by sulfonation with sodium salt of vinylsulfonic acid. A particularly preferred embodiment of a dendritic polyglycerol sulfonate according to the present invention has a polymeric polyglycerol core with an average molecular weight of 5,000 g/mol, a degree of sulfonation of 4 % and a molecular weight of 5,200 g/mol, such as compound 3b of Example 2 (see Table 2). A further particularly preferred embodiment of a dendritic polyglycerol sulfonate according to the present invention has a polymeric polyglycerol core with an average molecular weight of 20,000 g/mol, a degree of sulfonation of 8 % and a molecular weight of 21,800 g/mol, such as compound 3d of Example 2 (see Table 2). The object is furthermore solved by the present invention by providing dendritic polyglycerol sulfates. A dendritic polyglycerol sulfate according to the present invention is characterized by: a) a polymeric polyglycerol core, composed of repeated units of glycerin with the formula (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, preferably 1 to 4 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 %, preferably 60 %, and an average molecular weight of 100 to 1,000,000 g/mol, preferably 1,000 to 20,000 g/mol, more preferably 2,000 to 7,500 b) the substitution of one or more OH groups of the glycerin units with -OSO3H or -OSOsNa groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -3/pyridine complex was purchased from Fluka (Buchs, Switzerland). The reagent was used without further purification. The solvent N,N-dimethyl formamide (DMF, p.a. quality, purchased from Roth, Karlsruhe, Germany) was dried over CaH2 and stored over molecular sieve 4 A prior to further use. Dialysis was carried out with tubing of regenerated cellulose (SpectraPore 6/Roth) in distilled water in a 5 1 beaker, wherein the solvent was changed three times over a period of 48 hours. 1. Polymeric polyglycerol cores Polyglycerol 1 is a readily available, well defined polymer with dendritic (tree-like) branching, which is obtained by controlled anionic polymerization of glycidol [12-14]. The degree of branching of 1 (60%) is lower than that of a perfect glycerol dendrimer (100%) [15]. However, the physico-chemical characteristics are similar [16]. The molecular weight (1,000-30,000 g/mol) and the degree of polymerization (DP) can readily be tailored via the ratio of monomer and initiator and narrow polydispersities are obtained (typically < 2.0) [14]. Due to the biocompatible characteristics of the aliphatic polyether polyol (e.g. polysaccharides, poly(ethyleneglycol)s) in general similar characteristics are anticipated of polyglycerol [13]. In addition, oligoglycerols (with 2-10 monomer units) were studied in detail with respect to their toxicological characteristics and were approved as nutritional and pharmacological additives [16,17]. Thus, the dendritic polyglycerol 1 should be suitable as a highly functional carrier for drugs, and for the generation of dendritic polyanions (polysulfates, polycarboxylates, polysulfonates). Furthermore, the polygtycerols (PG) la (Mn = 2,500 g/mol, Mw/M„ = 1.6), lb (M„ = 5,000 g/mol, Mw/Mn = 1.6), lc (M„ = 10,000 g/mol, Mw/Mn = 1.8) and Id (Mn = 20,000 g/mol, Mw/Mn < 2.0) were prepared using l,l,l-tris(hydroxymethyl)propane (TMP) as initiator in case of la-c and pentaerythrol (PE) as initiator in case of Id, as previously described [14]. 2. Analysis lH NMR and 13C NMR spectra were recorded in D2O at concentrations of 100 mg/ml in a Bruker ARX 300 spectrometer, which operates at 300 or 75.4 MH, respectively. IK measurements were performed at a Bruker IFS 88 FT-IR spectrometer using a KBr plate. The degree of sulfation (ds) (Table 1) of compounds 2a-d was determined using elemental analysis. 3. Synthesis of the polyglycerol sulfates The synthesis of the polyglycerol sulfates was carried out by modifying an established method for the sulfation of P-l,3-glukanes which was described by Alban et al. [18], using dendritic polyglycerols with different molecular weights (la-d) as core polymers and the SCVpyridine complex as sulfation reagent in dry DMF as solvent (see Scheme 1). The concentration of the SCVpyridine complex in DMF as well as its molar ratio (SO3 per OH groups) was kept constant in all cases. For a synthesis scheme see Figure 1. To a stirred solution of 5.0 g polyglycerol (la, lb, lc, Id) (67.5 mmol OH groups) in 25 ml DMF a solution of 10.75 g (67.5 mmol) S03/pyridine complex in 67.5 ml DMF was added drop-wise for 4 hours at 60°C under an argon atmosphere. After stirring the reaction mixture for additional 2 hours at 60°C and 18 hours at room temperature, 50 ml distilled water were added. To the aqueous solution immediately 1 M NaOH were added until reaching a pH of 11. Concentration in vacuum resulted in the raw product, which was further purified by dialysis in water. After evaporating the solvent polyglycerol sulfates 2a-d were obtained as light yellow solids, which were further dried over P2O2. The polyglycerol sulfates (2a-d) were obtained in good yields (50-75%) and high purities (>98% according to !H NMR) after dialysis in distilled water. Yields: 7.49 g (2a); 8.96 g (2b); 7.01 g (2c); 6.86 g (2d). 'H NMR (300 MHz, D20): 8 (ppm) 0.98 [t, 3H, CH3CH2C(CH2O)-PG-OSO3Na], 1.48 [m, 2H, CH3CH2C-(CH20)3-PG-0S03Na], 3.40-4.00 [m, CH3CH2C(CH2O)3-.PG-OS03Na], 4.19, 4.33,4.38 [PG-OS03Na], 4.72 [PG-OCH2CH(OS03Na)CH2OS03Na]. Note: in case of 2d the peaks at 0.98 and 1.48 do not apply. 13C NMR (D20, 75.4 MHz): 5 (ppm) 66.9, 67.6, 68.2, 69.4, 70.3, 75.8, 77.2, 78.3 [PG-OS03Na]. IR (KBr): v (cm'1) 3470 [OH], 2930 [CH], 1260 [S=0], 780 [C-O-S]. Sulfur content after elemental analysis: 2a: 15.38% S, 2b: 14.28% S, 2c: 15.20% S, 2d: 13, 96%. By 'H-NMR spectroscopy no degradation of the polyglycerol core was observed. Using a S03/pyridine concentration which was equimolar to the OH groups about 85% of all free OH groups were sulfated (Table 1). This high degree of sulfation shows that polyglycerols are more easily accessible to sulfation than polysaccharides (24). Table 1 Characterization of the dendritic polyglycerol sulfates 2a-c Table a determined by NMR and/od GPC (DMF). b degree of sulfation (ds) obtained by elemental analysis; c calculated using M„ of the polymer core and the experimental measure of functionalization. Mn = average molecular weight of the polyglycerol core; DPn = degree of polymerization of the polyglycerol core; The detailled analyis of all starting materials la-d and products 2a-d by NMR, IR and elemental analysis confirmed the structure and the degree of functionalization of these dendritic polyglycerol sulfates. The molecular weights of the non-functionalized polyglycerols were determined by using 'H NMR data after precipitation. Example 2: Synthesis of the dendritic polyglycerol sulfonates Materials The sodium salt of vinylsulfonic acid (25 % solution by weight in water) was commercially obtained from the company Sigma-Aldrich and used without further purification. For the dialysis of the synthesized sulfonates in water dialysis tubing made of regenerated cellulose from the company Roth (SpectraPor6) with a MWCO of 1,000 g/mol was used. /. Polymeric polyglycerol cores See Example 1. 2. Analytics NMR spectroscopy: 'H-NMR and 13C-NMR spectra were recorded with a Bruker ARX 300 spectrometer at 300 MHz or 75.4 MHz, respectively, in D2O at concentrations of 100 mg/ml. The degree of sulfonation was determined using elemental analysis. 3.Synthesis of the polyglycerol sulfonates For a synthesis scheme see Figure 2. 10 g polyglycerol lb, Id (2.0 mmol; approx. 135 mmol OH groups) were dissolved in 20 ml water and a solution of 757 mg (13.5 mmol) potassium hydroxide in 1 ml water were added reaching a 10 % deprotonation of the OH groups of the polyglycerol. The reaction solution was cooled to approx. 5°C with the aid of an ice bath. Then, sodium salt of vinylsulfonic acid (26.347 g; 202.5 mmol) in form of a 25 % by weight, aquenous solution were slowly added for 4 hours via a dropping funnel. After the addition was completed the reaction mixture was heated to RT and stirred for another 3 days. After removing the solvent in vacuum, the obtained raw product was further purified by dialyzing in water for 24 hours, wherein the water was changed three times. Afterwards the raw product was concentrated in vacuum and dried for removing the remaining water in an exsiccator over phosphor pentoxide. The synthesized polyglycerol sulfates 3b, 3d were obtained in form of a slightly yellow colored highly viscous liquid with a degree of functionalization of 3 to 10%. Yiels: 3b 6,58 g, 3d: 5,48 g. 'H-NMR (D20, 300 MHz): 5 (ppm) = 0.88 [t, 3H, CH3CH2(CH2CO)3-PG-CH2CH2SO3Na], 1.42 [m, 2H, CH3CH2CHO)3-PG-CH2CH2SO3Na], 3.21 [t, 2H, CH3CH2C(CH20)3-PG-CH2CH2SO3Na] 3.35-4.05 [m, CH3CH2C(CH2O)3-PG-CH2CH2SO3Nal: l3C-NMR (D2O, 75.4 MHz): 8 (ppm) = 53.0 [PG-CH2CH2S03Na], 63.3, 65.1 [PG-CH2CH2S03Na], 68.2 [PG-CH2CH2S03Na], 71.4, 72.9, 74.7, 80.5, 82.0 [PG-CH2CH2S03Na]. Results of the elemental analysis: 3b: 0,58 % S, 3d: 1,30% S. By 'H-NMR spectroscopy no degradation of the polyglycerol core was observed. Table 2 Characterization of the dendritic polygylcerol sulfonates 3b and 3d Table Example 3: Binding of the dendritic polyglycerol sulfates to selectin in vitro In a competitive binding assay the binding of the polyglycerol sulfates to L-, P- and E-selectin was analyzed by surface plasmon resonance in Biacore X. In this approach the selectins are at first immobilized on colloidal gold beads. Then, the binding of the analyte to the selectin ligand sLeX-tyrosine sulfate which is coupled to the sensor chip is measured. By preincubating the analyte with the polyglycerol sulfates the binding of the analyte to the chip-coupled ligand is decreased in a concentration-dependent manner when the interaction of the polyglycerol sulfates with the binding domain of the ligand of the selectins is specific. In this case a decrease of the binding signal is observed. Figure 3 shows the concentration-dependent inhibition of L-selectin ligand binding by selected polyglycerol sulfates. With increasing molecular weight the polyglycerol sulfates show an increasing inhibitory potential with a comparable degree of sulfation. As apparent from Figure 3, compound 2d has an IC50 value of about 10 nM. For a further characterization of selectin-specific binding inhibition curves of L-, P- and E-selectin after preincubation with the polyglycerol derivative 2c were obtained (see Figure 4). Here it appears, that L-selectin is inhibited best by the derivative 2c (IC50 =10 nM), for P-selectin the compound has an IC50 of 30 nM, whereas E-selectin is not inhibited. The influence of the degree of sulfation of the dendritic polyglycerols on the L-selectin binding was investigated for the example of derivative 2d (Mn of the PG core = 20,000 [g/mol]). The derivative 2d was used with a concentration of 10 nM and sulfation degrees of 10 %, 38 % and 76 %. Again, the influence of the polyglycerol sulfates on the interaction between the analyte L-selectin and the immobilized ligand sLeX-tyrosine sulfate was measured (competitive binding assay, see above). The control value was set at 100 %, which corresponds to the binding signal which is generated by the interaction between L-selectin and the chip-coupled ligand sLeX-tyrosine. By preincubating the analyte L-selectin with 10 nM of the differently sulfated polyglycerol derivatives a reduction of the L-selectin-binding signal is measured with an increasing degree of sulfation, which is shown in Figure 5 as percental value compared to the control value. The 10 % sulfation of 2d obviously appears to be not sufficient to interact with L-selectin during the preincubation phase; the binding signal corresponds to the control value. The 38 % sulfation of 2d reduces the L-selectin ligand binding to about 60 % of the binding signal of the control value and the 76 % sulfation of 2d reduces it to about 45 % of the control value. These measurements show that the degree of sulfation and binding affinity correlate positively. Furthermore, a particularly threshold value of the sulfation degree appears to be necessary in order to accomplish an interaction with L-selectin. Example 4: Dendritic polyglycerol (dPG) and sulfated derivatives (dPGS) Dendritic polyglycerols are well defined polymers with treelike branching. The detailed synthesis was carried out as described in Example 1. The degree of polymerisation and branching can easily be tailored and narrow polydispersities can be obtained. We synthesized different core structures with molecular weights (MW) between 240 and 6,000 Da, as described in Example 1. The compounds were further functionalized with the SCVpyridine complex as sulfation reagent. The percentage loading of sulfate (degree of sulfation) on the dendritic polyglycerol scaffold was determined by elemental analysis and ranged from 10-92%. dPG and dPGS were stored at 4°C, aqueous solutions were stable after 6 month storage at -20 °C. For an example of a dPGS see Figure 6. Table 3 Characterization of the dendritic polyglycerol sulfates (dPGS) Table Example 5: Cytotoxicity and immuno-regulating properties of polyglycerol sulfates To test whether the polyanionic dPGS of the invention could be used safely in cell culture and in vivo in mice the compound dPGS6000/76 was characterized exemplarily in detail. To test cellular toxicity, we performed proliferation assays with the monocytic cell line THP-1. The compound dPGS6000/76 showed no inhibition of cellular proliferation up to a concentration of 10 uM (see Figure 7). When peripheral blood mononuclear cells (PBMCs) were cultured in the presence of up to 30 uM dPGS6000/76 for 24 h only a slight increase of apoptotic cells was observed irrespective of cellular stimulation (see Figure 8). We next examined the influence of dPGS on cellular immuno-regulating activity. Cytokine release was characterized on murine dendritic cells (see Figure 9) and the T cell fraction of human PBMCs (see Figure 10). Compared to the control (no dPGS added) the concentration of m TNFa and hu IL-2 did not change significantly. Example 6: Dendritic polyglycerol sulfates block selectin-ligand binding in vitro To evaluate selectin-binding of dPGS in vitro we applied a highly sensitive Biacore-based competitive binding assay, as described in Example 3, which allows to determine 50% inhibitory concentrations (IC50) of inhibitory compounds. The selectin specificity of dPGS was analyzed. Whereas E-selectin binding was not affected by dPGS4000/84, L- and P-selectin were inhibited efficiently and gave IC50 values of 8 and 30 nM (see Figure 11). (These experiments were carried out as described in Example 3 and for reconfirmation.) Sulfate dependency of selectin binding was then confirmed with compounds bearing a different functionalization on the same scaffold. At a defined concentration of 30 nM the inhibitory effect of the derivatives was studied (see Figure 12). Core structure dPG6000 with no or 10% sulfate did not interfere with L-selectin-ligand binding, in contrast 38% and 76% sulfation reduced the relative binding to 55% or 26%, respectively. (These experiments were carried out as described in Example 3 and for reconfirmation.) The influence of the dendrimer core size on selectin inhibition was then characterized (see Figure 13). Dendritic polyglycerols with molecular weights ranging from 240 Da (3 monomer units) to 6,000 Da (80 monomer units) were synthesized and further highly sulfated. The degree of functionalization was in the range from 76 to 92%. The small compound triglycerol (TGS) 240/83 showed no inhibition on L-selectin binding up to the high micromolar range but for compound dPGS2500/85 the IC50 was 80 nM. By increasing the degree of sulfation another 7% the IC50 value of the resulting compound dPGS2500/92 further decreased to 4 nM. It is obvious that selectin binding requires a critical size of the polymer core but density of sulfate groups (degree of sulfation) on the polymeric scaffold seems to be of even greater importance. Further increase of the core structure and equal functionalization did not improve selectin binding. For comparison the L-and P-selectin binding polymer heparin was included to this study. This polysulfated glucosaminoglycan has an average molecular weight of 15,000 Da and carries about 2.4 sulfates per disaccharide. The IC50 value on L-selectin binding of this compound was 15 uM and hence about 4000 fold greater than dPGS2500/92. Table 4 Core size and sulfation rate dependent selectin binding of dPGS. Table dPG = dendritic polyglycerol dPGS = dendritic polyglycerol sulfate TGS = triglycerol n.d. not determined Example 7: dPGS reduce leukocyte recruitment in acute and subchronic skin inflammation model We then investigated the influence of the dendritic polyglycerol sulfates in a murine model for skin inflammation. In an acute TMA-induced inflammatory response the compound dPGS6000/76 prevented edema formation and therefore ear swelling. At a dose of 30 mg/kg the antiinflammatory efficacy was comparable the corticosteroid prednisolone (see Figure 14). This antiinflammatory effect was attributed to the dPGS-mediated reduction of granulocyte emigration (see Figure 15). In a subchronic inflammation model 8 days after TMA challenge the protecting effect of dPGS was still obvious. Ear thickness of dPGS treated mice was reduced but not as effective as in the prednisolone positive control (see Figure 16). In addition still a clear reduction in granulocyte and neutrophil infiltration was measured (see Figure 17) and comparable to the prednisolone standard. We then analysed activation of nai've T cells by measuring cytokine levels in mice ear homogenates. The obvious concentration-dependent decrease of Thl-type IL-2 and the Th2-type IL-4 in dPGS treated mice further indicates that dPGS damp down the T cell dependent skin inflammation in TMA-induced contact hypersensitivity (see Figure 18). References 1. Ley, K. (2003) The role of selectins in inflammation and disease. Trends Mol Med., 9(6): 263 8. 2. Springer, T.A. (1990) Adhesion receptors of the immune system. Nature, 346: 425-434. 3. Lefer, D.F. (2000) Annu Rev. Pharmacol Toxicol., 40: 283-294. 4. Boehncke, WH et al. (2005) Exp. Dermatol, 14(1): 70-80. 5. Simanek et al., (1998) Selectin-carbohydrate interactions: From natural ligands to designed mimics. Chem. Rev.,98(2): 833-862. 6. Boehncke WH et al., (2006) Biologic therapies for psoriasis. A systematic review. J. Rheumatol, 33: 1447-1451. 7. Willburger et al., (2006) Zertifizierte medizinische Fortbildung: Pharmakologische Therapie der rheumatoiden Arthritis. Dtsch Arztebl; 103(1-2): A 48-57 8. Ulbrich H., et al. (2003) Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol Sci., 12: 640-647. 9. Mowery, P et al. (2004) Synthetic glycoprotein mimics inhibit L-selectin-mediated rolling and promote L-selectin shedding. Chem. Biol. 11: 752-732. 10. Rele, SM et al. (2005) Dendrimer-like PEO glycopolymers exhibit anti-inflammatory properties. J. Am. Chem. Soc, 127: 10132-10133. 11. Tiirk, H.; Haag, R. and Alban, S. (2004) Dendritic Polygylcerol Sulfates as New Heparin Analogues and Potent Inhibitors of the Complement System. Bioconjugate Chem. 15: 162-167. 12. Sunder, A., Miilhaupt, R., Haag, R., and Frey, H. (2000) Hyperbranched Polyether Polyols: A Modular Approach to Complex Polymer Architectures. Adv. Mater. 12,: 235-239. 13. Frey, H., and Haag, R. (2002) Dendritic polyglycerol: a new versatile biocompatible material. Rev. Mol. Biotech. 90: 257-267. 14. Sunder, A., Hanselmann, R., Frey, H., and Miilhaupt, R. (1999) Controlled Synthesis of Hyperbranched Polyglycerols by Ring-Opening Multibranching Polymerization. Marcromolecules 32: 4240-4246. 15. Haag, R., Sunder, A., and Stumbe\ J.-F. (2000) An Approach to Glycerol Dendrimers and Pseudo-Dendritic Polyglycerols. J. Am. Chem. Soc. 122, 2954-2955. 16. Wilson, R., Van Schie, B.J., and Howes, D. (1998) Overview of the Preparation, Use and Biological Studies on Polyglycerol Polyricinoleate (PGPR). Food Chem. Toxicol. 36:711-718. 17. Howes, D., Wilson, R., and James, C.T. (1998) The Fate of Ingested Glyceran Esters of Condensed Castor Oil Fatty Acids [Polyglycerol, Polyricinoleate (PGPR)] in Rat. Food Chem. Toxicol. 36: 719-738. 18. Alban, S., Kraus, J., and Franz, G. (1992) Synthesis of Laminarin Sulfates with Anticoagulant Activity. Arzneim.-ForschJDrug Res. 42; 1005-1008. Claims 1. Dendritic polyglycerol sulfonate, characterized by a) a polymeric polyglycerol core, composed of repeated units of glycerin with the formula (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 % and an average molecular weight of 100 to 1,000,000 g/mol, b) the substitution of one or more OH groups of the glycerin units with -SO3H or - SOsNa groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -(CH2)„- or -[(CH2)m-0)]n-, wherein m is 1 to 100 and n is 1 to 50,000, and bound thereto -SO3H or -SOsNa groups, so that a degree of sulfonation of 1 to 100 % is obtained, and c) a molecular weight of 110 to 1,500,000 g/mol. 2. Compound according to claim 1, characterized by a) a polymeric polyglycerol core built on a multifunctional starter molecule having 1 to 4 OH groups. 3. Compound according to claim lor 2, characterized by a) a polymeric polyglycerol core built on a multifunctional starter molecule, which contains further heterofunctionalities, particularly SH groups, NH2 groups. 4. Compound according to any of claims 1 to 3, characterized by a) a polymeric polyglycerol core having a branching degree of 60 %. 5. Compound according to any of claims 1 to 4, characterized by a) a polymeric polyglycerol core having an average molecular weight of 1,000 to 20,000 g/mol. 6. Compound according to any of claims 1 to 5, characterized by b) a degree of sulfonation of 30 %. 7. Compound according to any of claims 1 to 5, characterized by b) a degree of sulfonation of 30 % to 100 %. 8. Compound according to any of claims 1 to 7, characterized by c) a molecular weight of 1,100 to 30,000 g/mol. 9. Compound according to any of claims 1 to 8 loaded with signalling molecules or having signalling molecules bound thereto. 10. Compound according to claim 9, wherein the signalling molecules are selected from the group of radioactively labelled derivatives or the group of dyes, particularly fluorophores and chromophores. 11. Compound according to any of claims 1 to 10 immobilized to a matrix. 12. Compound according to claim 11, wherein the matrix is of inorganic or polymeric nature. 13. Method for producing a compound according to any of claims 1 to 12, comprising the use of a multifunctional starter molecule and a sulfonation reagent. 14. Use of a compound according to any of claims 1 to 8 as a medicament for the treatment of diseases. 15. Use of a compound according to any of claims 1 to 8 as a medicament for the treatment of inflammatory diseases. 16. Use according to claim 15, wherein the inflammatory diseases are chronic inflammatory diseases, particularly rheumatoid arthritis, asthma and psoriasis. 17. Use according to claim 15, wherein the inflammatory diseases are ischemia reperfusion damages or graft repulsion. 18. Use of a compound according to any of claims 1 to 12 as selectin inhibitor. 19. Use of a compound according to any of claims 1 to 12 as selectin indicator. 20. Use according to claim 18 or 19 as inhibitor or indicator of L-selectin and/or P-selectin. 21. Use of a compound according to any of claims 1 to 12 for binding of proteins. 22. Use according to claim 21, wherein the proteins are selectins, chemokines or coagulation factors. 23. Use according to claim 22, wherein the chemokines are selected from the group consisting of proinflammatory cytokines, particularly TNFa, IL-1, IL-6, as well as from IL-8 andMIP-ip. 24. Use according to any of claims 21 to 23 for the purification of proteins from biological samples, particularly bodily fluids, whole blood, serum, cell suspensions and supematants of cell cultures. 25. Use according to any of claims 21 to 24 as capture molecule. 26. Use of a dendritic polyglycerol sulfate, that is characterized by: a) a polymeric polyglycerol core, composed of repeated units of glycerin with the formula (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 % and an average molecular weight of 100 to 1,000,000 g/mol, b) the substitution of one or more OH groups of the glycerin units with -OSO3H or -OSOsNa groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -(CH2)n- or -[(CH2)m-0)]n-, wherein m is 1 to 100 and n is 1 to 50,000, and bound thereto -OSO3H or -OSOsNa groups, so that a degree of sulfation of 1 to 100 % is obtained, and c) a molecular weight of 200 to 5,000,000 g/mol. medicament for the treatment of diseases. Use of a dendritic polyglycerol sulfate, that is characterized by: a) a polymeric polyglycerol core, composed of repeated units of glycerin with the formula (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 % and an average molecular weight of 100 to 1,000,000 g/mol, b) the substitution of one or more OH groups of the glycerin units with -OSO3H or -OS03Na groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -CCH2)„- or -[(CH2)m-0)]n-, wherein m is 1 to 100 and n is 1 to 50,000, and bound thereto -OSO3H or -OSOsNa groups, so that a degree of sulfation of 1 to 100 % is obtained, and c) a molecular weight of 200 to 5,000,000 g/mol. as a medicament for the treatment of inflammatory diseases. 28. Use according to claim 27, wherein the inflammatory diseases are chronic inflammatory diseases, particularly rheumatoid arthritis, asthma and psoriasis. 29. Use according to claim 27, wherein the inflammatory diseases are ischemia reperfusion damages or graft repulsion. 30. Use of a dendritic polyglycerol sulfate, that is characterized by: a) a polymeric polyglycerol core, composed of repeated units of glycerin with the formula (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 % and an average molecular weight of 100 to 1,000,000 g/mol, b) the substitution of one or more OH groups of the glycerin units with -OSO3H or -OSOsNa groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -(CH2)„- or -[(CH2)ra-0)]„- wherein m is 1 to 100 and n is 1 to 50,000, and bound thereto -OSO3H or -OSOsNa groups, so that a degree of sulfation of 1 to 100 % is obtained, and c) a molecular weight of 200 to 5,000,000 g/mol. as selectin inhibitor. 31. Use of a dendritic polyglycerol sulfate, that is characterized by: a) a polymeric polyglycerol core, composed of repeated units of glycerin with the formula (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 % and an average molecular weight of 100 to 1,000,000 g/mol, b) the substitution of one or more OH groups of the glycerin units with -OSO3H or -OSOjNa groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -(CH2)„- or -[(CH2)m-0)]n- wherein m is 1 to 100 and n is 1 to 50,000, and bound thereto -OSO3H or -OSOsNa groups, so that a degree of sulfation of 1 to 100 % is obtained, and c) a molecular weight of 200 to 5,000,000 g/mol. as selectin indicator. 32. Use according to claim 30 oder 31 as inhibitor or indicator of L-selectin und/or P-selectin. 33. Use of a dendritic polyglycerol sulfate, that is characterized by: a) a polymeric polyglycerol core, composed of repeated units of glycerin with the formula (RO-CH2)2CH-OR on a multifunctional starter molecule, which is a polyhydroxy compound having 1 to 1,000 OH groups, wherein R = H or further glycerin units, the core having a branching degree of 0 to 100 % and an average molecular weight of 100 to 1,000,000 g/mol, b) the substitution of one or more OH groups of the glycerin units with -OSO3H or -OS03Na groups or the attachment of an oligomeric spacer at one or more OH groups of the glycerin units, the oligomeric spacer having the generic formula -CCH2)„- or -[(CH2)m-0)]„-, wherein m is 1 to 100 and n is 1 to 50,000, and bound thereto -OSO3H or -OS03Na groups, so that a degree of sulfation of 1 to 100 % is obtained, and c) a molecular weight of 200 to 5,000,000 g/mol. for binding of proteins. 34. Use according to claim 33, wherein the proteins are selectins, chemokines or coagulation factors. 35. Use according to claim 34, wherein the chemokines are selected from the group, consisting of proinflammatory cytokines, particularly TNFa, IL-1, IL-6, as well as from IL-8 andMIP-lp. 36. Use according to any of claims 34 and 35 for the purification of proteins from biological samples, particularly bodily fluids, whole blood, serum, cell suspensions and supernatants of cell cultures. 37. Use according to any of claims 34 to 36 as capture molecule. 38. Use according to any of claims 26 to 37, wherein the dendritic polyglycerol sulfates are characterized by a) a polymeric polyglycerol core built on a multifunctional starter molecule, having 1 to 4 OH groups. 39. Use according to any of claims 26 to 38, wherein the dendritic polyglycerol sulfates are characterized by a) a polymeric polyglycerol core built on a multifunctional starter molecule, which contains further heterofunctionalities, particularly SH groups, NH2 groups. 40. Use according to any of claims 26 to 39, wherein the dendritic polyglycerol sulfates are characterized by a) a polymeric polyglycerol core having a branching degree of 60 %. 41. Use according to any of claims 26 to 40, wherein the dendritic polyglycerol sulfates are characterized by a) a polymeric polyglycerol core having an average molecular weight of 1,000 to 20,000 g/mol, preferably 2,000 to 7,500. 42. Use according to any of claims 26 to 41, wherein the dendritic polyglycerol sulfates are characterized by c) a molecular weight of 2,000 to 50,000 g/mol, preferably 5,000 to 13,500. 43. Use according to any of claims 26 to 42, wherein the dendritic polyglycerol sulfates are loaded with signalling molecules or have signalling molecules bound thereto. 44. Use according to claim 43, wherein the signalling molecules are selected from the group of radioactively labelled derivatives or the group of dyes, particularly fluorophores and chromophores. 45. Use according to any of claims 30 to 44, wherein the dendritic polyglycerol sulfates are immobilized to a matrix. 46. Use according to claim 45, wherein the matrix is of inorganic or polymeric nature.

Documents

Application Documents

# Name Date
1 771-DELNP-2009-Correspondence-Others-(24-07-2009).pdf 2009-07-24
1 771-DELNP-2009_EXAMREPORT.pdf 2016-06-30
2 771-delnp-2009-abstract.pdf 2011-08-20
2 771-DELNP-2009-Petition-138-(28-07-2009).pdf 2009-07-28
3 771-DELNP-2009-Form-3-(28-07-2009).pdf 2009-07-28
3 771-delnp-2009-claims.pdf 2011-08-20
4 771-delnp-2009-correspondence-others.pdf 2011-08-20
4 771-DELNP-2009-Correspondence-Others-(28-07-2009).pdf 2009-07-28
5 771-DELNP-2009-GPA-(02-09-2009).pdf 2009-09-02
5 771-delnp-2009-description (complete).pdf 2011-08-20
6 771-DELNP-2009-Form-1-(02-09-2009).pdf 2009-09-02
6 771-delnp-2009-drawings.pdf 2011-08-20
7 771-delnp-2009-form-1.pdf 2011-08-20
7 771-DELNP-2009-Correspondence-Others-(02-09-2009).pdf 2009-09-02
8 771-DELNP-2009-GPA-(12-10-2009).pdf 2009-10-12
8 771-delnp-2009-form-2.pdf 2011-08-20
9 771-DELNP-2009-Form-1-(12-10-2009).pdf 2009-10-12
9 771-delnp-2009-form-3.pdf 2011-08-20
10 771-DELNP-2009-Correspondence-Others-(12-10-2009).pdf 2009-10-12
10 771-delnp-2009-form-5.pdf 2011-08-20
11 771-DELNP-2009-Form-18-(09-07-2010).pdf 2010-07-09
11 771-delnp-2009-pct-210.pdf 2011-08-20
12 771-DELNP-2009-Correspondence-Others-(09-07-2010).pdf 2010-07-09
12 771-delnp-2009-pct-237.pdf 2011-08-20
13 771-delnp-2009-pct-301.pdf 2011-08-20
13 771-delnp-2009-pct-306.pdf 2011-08-20
14 771-delnp-2009-pct-304.pdf 2011-08-20
15 771-delnp-2009-pct-301.pdf 2011-08-20
15 771-delnp-2009-pct-306.pdf 2011-08-20
16 771-DELNP-2009-Correspondence-Others-(09-07-2010).pdf 2010-07-09
16 771-delnp-2009-pct-237.pdf 2011-08-20
17 771-delnp-2009-pct-210.pdf 2011-08-20
17 771-DELNP-2009-Form-18-(09-07-2010).pdf 2010-07-09
18 771-delnp-2009-form-5.pdf 2011-08-20
18 771-DELNP-2009-Correspondence-Others-(12-10-2009).pdf 2009-10-12
19 771-DELNP-2009-Form-1-(12-10-2009).pdf 2009-10-12
19 771-delnp-2009-form-3.pdf 2011-08-20
20 771-delnp-2009-form-2.pdf 2011-08-20
20 771-DELNP-2009-GPA-(12-10-2009).pdf 2009-10-12
21 771-DELNP-2009-Correspondence-Others-(02-09-2009).pdf 2009-09-02
21 771-delnp-2009-form-1.pdf 2011-08-20
22 771-delnp-2009-drawings.pdf 2011-08-20
22 771-DELNP-2009-Form-1-(02-09-2009).pdf 2009-09-02
23 771-delnp-2009-description (complete).pdf 2011-08-20
23 771-DELNP-2009-GPA-(02-09-2009).pdf 2009-09-02
24 771-DELNP-2009-Correspondence-Others-(28-07-2009).pdf 2009-07-28
24 771-delnp-2009-correspondence-others.pdf 2011-08-20
25 771-DELNP-2009-Form-3-(28-07-2009).pdf 2009-07-28
25 771-delnp-2009-claims.pdf 2011-08-20
26 771-DELNP-2009-Petition-138-(28-07-2009).pdf 2009-07-28
26 771-delnp-2009-abstract.pdf 2011-08-20
27 771-DELNP-2009_EXAMREPORT.pdf 2016-06-30
27 771-DELNP-2009-Correspondence-Others-(24-07-2009).pdf 2009-07-24