SK10232001A3 - Organoprotective solutions - Google Patents

Description

Technical field

The present invention relates to the use of polysulfated glycosaminoglycans with a sulfur content of at least 12.5% for the manufacture of pharmaceutical compositions for the inhibition of interferon γ-induced upregulation of MHC class I, MHC class II and ICAM 1 proteins. poly-sulfated glycosaminoglycans and methods for ex vivo protection of organs for transplantation.

BACKGROUND OF THE INVENTION

The use of glycosaminoglycans and, in particular, heparins and heparinoids for the preparation of pharmaceutical compositions for the treatment of circulatory disorders is well known.

Recently, the use of glycosaminoglycans in a series of minor diseases has been described. Thus, U.S. Pat. No. 5,236,910 protects the use of glycosaminoglycans in the treatment of diabetic nephropathy and neuropathy. The use of low molecular weight heparins in the same indication has been described by van der Pijl et al. (J. Americ. Soc. Nephrol., 1997, 8, 456-462).

U.S. Pat. No. 5,032,679 claims protection of the use of glycosaminoglycans in inhibiting smooth muscle cell proliferation and related diseases.

In U.S. Pat. No. 4,966,894, poly-sulfated heparins are protected for the treatment of diseases caused by retroviruses.

Gralinski et al. described the modulation of the complement system with polysulfatized heparins.

In Clin. Exp. (1997, 107, 578-584) Douglas et al. investigated the antagonism of the inflammatory-promoting effect of interferon γ with heparin, heparan sulfate or heparin-like molecules. Heparin is in a position to influence the immunogenic effect of interferon γ.

- Adverse rejection reactions often occur in organ transplantation. In order to prevent such rejection reactions, research has taken many different paths. Thus, the histo-compatibility of donor and recipient antigens was first compared. Only those organs in which the donor and recipient are as identical as possible or where the histocompatibility antigens are very similar are transplanted.

However, undesirable organ rejection persists. For example, there is an acute renal allograft rejection, where the differentiation of aloMHC antigens by T lymphocytes is a major factor involved in lysis of tubular cells. This further causes a strong reaction to the recipient with the so-called "graft versus host reaction" due to the donor immune cells transplanted with the organ. Cytotoxic T cells and antibodies are generated against the host organism.

In order to further reduce the risk of transplant rejection, organs are immediately frozen after collection and stored under organ preservative solutions. In addition, a treatment is provided to the recipient that suppresses the recipient's immune defense.

A variety of organ protection solutions have been described in the literature. Thus Collins et al (Lancet, vol 2, 1969, 1219) describe intracellular electrolytic solutions for organ preservation. Sacks SA (Lancet, vol 1, 1973, 1024) described solutions having an osmotic stabilizing effect. ATP-MgCL, AMP-MgCl and inosine have been described as suitable reagents in such solutions (Siegel, NJ et al., Am. J. Physiol., 254: F530, 1983; Belzer et al., Transpl. Proc. 16, 161, 198) ). U.S. Pat. No. 4,920,004 discloses solutions containing mannitol, adenosine and ATP-MgCl2. U.S. Pat. No. 4,798,824 and U.S. Pat. No. 4,873,230 claim organoprotective solutions containing hydroxyethyl starch. U.S. Pat. No. 4,879,283 discloses a protected solution comprising KH2PO4, MgSCU adenosine, allopurinol, raffinose and hydroxyethylcellulose. This is also known as the University of Wisconsin solution (= UW solution). This solution has been described for the successful preservation of liver, kidney and heart (Jamieson et al., Transplantation, 46, 1988, 517; Ploeg et al., Transplantation, 46, 1988: 191; and Wicomb WN, Transplantation, 47. 1988: 733). IN

U.S. Pat. No. 5,200,398 discloses another additive to such preservatives, which is glucuronic acid, salts and esters thereof.

Despite the success achieved in protecting organs for transplantation and suppressing unwanted organ rejection, there is still a need for further improvement.

It was therefore an object of the present invention to achieve further improvements both in protecting organs from and during transplantation and also to further reduce the risk of organ rejection.

SUMMARY OF THE INVENTION

This objective was achieved using poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% (w / w) for the manufacture of pharmaceutical preparations for inhibiting interferon γ-induced upregulation of MHC class I, MHC class II and ICAM 1 proteins.

In addition, the present invention relates to organ protection solutions comprising an amount of poly-sulfated glycosaminoglycan having a sulfur content of at least 12.5% for suitable pharmaceutical compositions that effectively maintain cell integrity and vitality.

Pharmaceutical compositions produced for use in the present invention, including, for example, organ protection solutions, may contain the above compounds as free compounds in the form of their physiologically active salts or esters, their tautomeric and / or isomeric forms, or in the form of a combination of free compounds and miscellaneous salts. Notable physiologically active salts are, for example, the Na, Ca or Mg salts. Similarly, salts with organic bases such as diethylamine, triethylamine or triethanolamine are also suitable. The pharmaceutical preparations may advantageously contain at least one free substance or at least one compound in the form of a salt thereof or mixtures thereof.

The poly-sulfated glycosaminoglycans (= muco-polysaccharides) used according to the present invention are negatively charged polysaccharides (= glycans) consisting of differently bound disaccharide units in which, for example,

A single molecule of the so-called uronic acid, such as D-glucuronic acid or L-iduronic acid, is glycosidically linked to a third or fourth position of an aminosaccharide, such as glucosamine or galactamine. At least one of the carbohydrates in the disaccharide has a negatively charged carboxylate or sulfate group, which may be attached via an oxygen or nitrogen atom. Glycosaminoglycans give a strongly acidic reaction with uranium acids as well as sulfuric acid ester groups. These acid-reacting groups are partially already present in nature, but are rare to achieve the sulfatization step required by the present invention by synthetic introduction, for example, sulfation into a compound. As the sulfation methods, the literature describes, for example, sulfation with sulfuric acid and chlorosulphuric acid (US 4,727,063, US 4,948,881), sulfation with chlorosulphuric acid in pyridine (Wolfrom et al., J. Am. Chem. Soc., 1953, 75, 1519) or sulfation with nitrous acid (Shively et al., Biochemistry, vol. 5, No. 18, 1976: 3932). Other methods are well known to those skilled in the art. Examples are uses for sulfating the natural glycosaminoglycans, such as heparin, heparan sulfate, keratansulfate, dermatansulfate, chondroitin or chondroitin sulfate. The structure of heparan sulphate corresponds to that of heparin except that, unlike the latter, it contains fewer N and O sulfate groups and more acetyl groups.

Glycosaminoglycans can be readily isolated from animal tissues such as the small intestine mucosa, or from the ears of pigs and cattle. For example, the tissue used to isolate glycosaminoglycans is autolysed and extracted with a base. The protein is then allowed to coagulate and precipitate, for example by acidifying it. After adding the precipitate to a polar non-aqueous solution such as ethanol or acetone, the fats are removed by extraction with an organic solvent. By proteolytic digestion, the proteins are finally removed and glycosaminoglycans are recovered. Charles et al (Biochem. J., vol. 30, 1936: 1927-1933) and Coyne E in Chemistry and Biology of Heparin (Elsevier Publishers, North Holland, NY, Lunblad RL, ed. 1981) describe, for example, methods for isolating heparin.

These glycosaminoglycans isolated from natural sources may advantageously be further derivatised by polysulfatizing them as described in the example in US Patent No. 5,013,724 or as described above.

Using such polysulfatization, glycosaminoglycans then have a sulfur content of at least 6 to 15% (w / w). Polysulphated glycosaminoglycans having a sulfur content of at least 12.5% by weight are selected for use in the present invention or for pharmaceutical compositions. Preferably such polysulfated glycosaminoglycans have a sulfur content of 13 to 16% (w / w), particularly preferably 13 to 15% (w / w), particularly preferably a content of from 13.5 to 14.5% (w / w). These substances are used for the preparation of pharmaceutical preparations which are suitable for inhibiting interferon γ-induced upregulation of MHC class I, MHC class I and ICAM 1 proteins. It is preferred to use such substances in physiologically effective amounts for the treatment and prevention of interferon-related diseases γ-induced up-regulation of MHC class I, MHC class II and ICAM 1 proteins. Compounds which also improve the application properties of the poly-sulfated glycosaminoglycans used in terms of their effect, stability and elimination thereof, in particular from the body.

Advantageously, heparins and / or dermatan sulfate may be used with an average molecular weight of about 1000 to 2000 Daltons, preferably between 1500 and 9000 Daltons, and particularly preferably between 2000 and 9000 Daltons and, optimally, between 2000 and 6000 Daltons. Particularly preferred are low molecular weight polysulfated heparins and / or dermatan sulfates in free acid or salt form with physiologically acceptable bases or mixtures prepared from such compounds. Such substances have a weak anti-coagulant effect and are therefore particularly suitable for treatment and prevention when used in accordance with the present invention. Preferred salts of poly-sulfated glycosaminoglycans are, for example, sodium, calcium and magnesium salts.

Low molecular weight glycosaminoglycans, for example low molecular weight heparins and / or dermatan sulphates, can be produced by a series of procedures. The preparation of low molecular weight heparins by depolymerization with the addition of nitric acid is described, for example, in European patent document EP-B-0 037 319 or in Biochemistry, vol JJ5, 1976: 3932. The production of low molecular weight heparin or low molecular weight glycosaminoglycans can also be carried out enzymes (Biochem. J., vol. 108, 1968: 647), sulfuric acid and chlorosulphuric acid (FR 2,538,404, simultaneous sulfatization),

Or by physical methods such as γ-radiation (EP-A-0 269 937) or ultrasound (Fuchs et al., Lebensm. Unters. Forsch., Vol. 198, 1994: 486-490).

Another use according to the present invention consists in organ protection solutions. By appropriately adding poly-sulfated glycosaminoglycans to organ protection solutions, organ storage after removal from the donor organism, i. ex vivo, can be further improved by inhibition of interferon γ-induced upregulation of MHC class I, MHC class II and ICAM proteins

1. Preferably, the organs should be frozen as known to those skilled in the art.

A series of such solutions as described above are known in the literature. The solutions generally comprise salts, buffers, substances which are believed to stabilize the organs osmotically or are believed to prevent oxidation such as carbohydrate carbohydrate, sugar alcohols, proteins, amino acids, lower carboxylic acids, purines, pyrimidines, or pharmaceutical agents. Examples of such substances include the following: raffinose, glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride, potassium bicarbonate, sodium hydrogen carbonate, magnesium sulfate, magnesium chloride, adenosine, albumin, mannitol, citrate, verapamil, alopine, insulin , hydroxyethyl starch, glutathione or glucuronic acid.

The use proposed in the organ protection solutions proposed by the invention leads to the possibility of transplanting the organs in better condition or storing them for a longer period of time than previously used so that rejection reactions can be reduced.

In order to further reduce the risk of organ rejection, poly-sulphated glycosamine glycans may be administered to patients undergoing transplantation or transplant donors where possible, orally or parenterally prior to transplantation. Post-operative treatment of patients with these agents is also possible.

Poly-sulphated glycosaminoglycans are contained in solutions for the protection of organs or in other pharmaceutical preparations in amounts ranging from

10mg / l to 10,000mg / l, preferably in amounts from 10mg / l to 5,000mg / l, particularly preferably in amounts from 50mg / l to 3,000mg / l, and most preferably

-7 in amounts from 100 mg / l to 3,000 mg / l. As an addition, 5 to 100 g / l of an osmotic stabilizer containing hydroxyethyl starch are preferred.

Other preferred organ protection solutions have the following composition:

(a) 10 mg / l to 10 000 mg / l of poly-sulphated glycosaminoglycans with a sulfur content of not less than 12,5% by weight, 5 to 100 g / l of hydroxyethyl starch and 5 to 100 mmol of raffinose, or

(b) 10 mg / l to 10 000 mg / l of poly-sulphated glycosaminoglycans with a sulfur content of not less than 12,5% by weight, 5 to 100 g / l of hydroxyethyl starch, 5 to 100 mmol of raffinose, 5 to 40 mmol of KH ₂ PO _4, 1 and 50 mM MgSO _4, 1 and 50 mmol adenosine, from 0.5 to 5 mM allopurinol, or 1 to 10 mM glutathione.

For the treatment of patients, the poly-sulfated glycosaminoglycans can be used together with the usual treatment agents.

The pharmaceutical compositions to be used according to the present invention may be administered in the usual manner, orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally), with oral or intravenous administration being preferred.

The dose depends on the age, condition and weight of the patient and the route of administration.

Glycosaminoglycans are most preferably administered at a dose of from 0.1 to 500 mg / kg of body weight per day. For parenteral administration, glycosaminoglycans are most suitably administered at a dose of from 0.1 to 30 mg / kg of body weight per day, for oral administration at a dose of from 0.2 to 500 mg / kg of body weight per day, and the dose administered may be administered. in a single dose or in multiple doses. Mixtures are also administered, for example at least one low molecular weight heparin and / or its poly-sulfated derivative and / or at least one low molecular weight dermatan sulfate and / or its poly-sulfated derivative, at a dose of from 0.1 to 30 mg / kg body weight per day for parenteral administration or at a dose of 0.2 to 500 mg / kg body weight per day for oral administration.

For pharmaceutical preparations containing poly-sulphated glycosamine glycans for the treatment and prevention of organ transplant diseases, these essentially include conventional galenic dosage forms for oral or parenteral administration, whether solid or liquid, such as tablets, coated tablets, capsules. , powders, granules, dragees, lace, solutions or suspensions. These are produced in the usual way. The active ingredients may be formulated with conventional processing materials such as tablet binders, fillers, preservatives, explosive tablet additives, flow regulators, humectants, dispersants, emulsifiers, solvents, retardants, antioxidants and / or propellants ( cf. H Sucker et al., Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991). The dosage forms thus prepared will normally contain from 0.1% to 90% by weight of the active ingredient.

For the preparation of tablets, coated tablets, dragees and hard gelatin capsules, the polysulphated glycosaminoglycans can also be formulated with pharmaceutically inert, inorganic or organic excipients. Lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof may be used as such excipients for tablets, dragees and hard gelatine capsules. For soft gelatine capsules, suitable excipients are vegetable oils, waxes, fats, semi-solid and liquid polyols.

For the preparation of solutions and syrups, suitable excipients are, for example, water, polyols, sucrose, invert saccharide, glucose and the like. For injection solutions, suitable excipients are water, alcohols, polyols, glycerin, vegetable oils. For lace, suitable excipients are natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like.

The pharmaceutical preparations may additionally contain preservatives, solvents, stabilizers, humectants, emulsifying agents, sweeteners, coloring agents, flavoring agents, salts for modifying the osmotic pressure, buffers, protective layers and / or antioxidants.

BRIEF DESCRIPTION OF THE DRAWINGS

-9Picture 1

Dose-dependent expression of MHC class I, class II and ICAM 1 in PTEC after stimulation with IFN γ. Cells were stimulated for 72 hours at different concentrations of IFN γ. Subsequently, expression of MHC class I (left scale), MHC class II (right scale) and ICAM 1 (left scale) was determined by FACS (= flow cytometry). The results are shown in the figure as the average fluorescence intensity of a representative experiment.

Figure 2

Effect of heparin on the expression of MHC and ICAM 1, HUVEC cells stimulated with IFNγ. IFN γ-stimulated (100 ng / ml, for 72 hours, gray bar) or unstimulated (white bar) HUVEC cells were incubated during stimulation with various concentrations of heparin. MHC and ICAM 1 expression was then determined by FACS. The results are shown in the figure as the average fluorescence intensity of a representative experiment.

Figure 3

Effect of heparin on the expression of MHC and ICAM 1 PTEC cells stimulated with sIFNγ. PTEC cells stimulated with IFN γ (10 ng / ml, for 72 hours) or unstimulated PTEC were incubated with different concentrations of heparin during stimulation. The expression of MHC and ICAM 1 of three repeated cultures was then determined using FACS. Figure 3a shows the expression of MHC class I. Figure 3b shows the expression of MHC class II. Figure 3c shows the expression of ICAM 1. The results are shown in the figure as mean fluorescence intensity +/- 2 SD.

Figure 4 ¹

Effect of various heparins and glycosaminoglycans on the expression of MCH and ICAM 1 cells stimulated with IFN γ. With IFN γ-stimulated (10 ng / ml, for 72 hours, solid bar) or unstimulated (shaded bar) HUVEC cells were incubated with various heparins or glycosaminoglycans at a concentration of 3 mg / ml during the stimulation period. MHC and ICAM 1 expression was then determined with FACS. Figure 4a shows MHC class I expression, Figure 4b shows

-10C MHC class II expression, and Figure 4c shows ICAM 1 expression. Results are shown in the figure as mean fluorescence intensity of +/- 2 SD.

Figure 5

Effect of various heparins and glycosaminoglycans on the expression of MHC and ICAM 1 PTEC cells stimulated with IFN γ. PTEC cells stimulated with IFN γ (10 ng / ml, for 72 hours, solid bar) or unstimulated (shaded bar) were incubated with various heparins or glycosaminoglycans at a concentration of 3 mg / ml during the stimulation period. MHC and ICAM 1 expression was then determined by FACS. Figure 5a shows the expression of MHC class I, Figure 5b shows the expression of MHC class II, and Figure 5c shows the expression of ICAM 1. The results are shown in the figure as the average fluorescence intensity of a representative experiment,

Figure 6

Comparison of GAG 6-8 and heparin effect in inhibiting expression of MHC and ICAM 1 PTEC cells stimulated with IFN γ (10 ng / ml, for 72 hours). PTEC cells were incubated with different concentrations of GAG 6 (), GAG 7 (), GAG 8 () and heparin () during the stimulation period. MHC and ICAM 1 expression was subsequently determined by FACS. Expression of these antigens was also determined in the absence of GAG (-GAG) or in the absence of IFN γ (-IFN). Figure 6a shows the expression of MHC class I, Figure 6b shows the expression of MHC class II, and Figure 6c shows the expression of ICAM 1. The results are shown in the figure as mean fluorescence intensity +/- 2 SD.

Figure 7

Effect of NaCIO ₃ on the extent of stimulation of MHC and ICAM 1 expression in PTEC cells caused by IFN γ. PTEC cells were treated one day before IFN γ stimulation with 150 mM NaCl ₁₀ (solid bar) or 150 mM NaCl (shaded bar) as an osmolar control. Subsequently, cells of different IFN γ concentrations were stimulated for 72 hours in the presence of the same concentration of NaCl ₁₀ or NaCl. The expression of MHC and ICAM 1 of three repeated cultures was then determined by FACS. Figure 7a shows expression of MHC class I, Figure 7b shows expression

-11 MHC Class II. Figure 7c shows the expression of ICAM 1. The results are shown in the figure as mean fluorescence intensity +/- 2 SD. The asterisk indicates p <0.05 and the two asterisks indicate p <0.01 in the Student's T test.

Figure 8

Binding of ¹²⁵ IFN γ to heparin, Fragmin and various other glycosaminoglycans. Heparin (Hep), glycosaminoglycan (GAG) 1 up to 8, and Fragmin (Fragm) were applied to a nitrocellulose filter prepared as described above. Nitrocellulose strips were incubated with ¹²⁵ IFN γ in the presence or absence of 3000-fold excess heparin.

DETAILED DESCRIPTION OF THE INVENTION

PTEC (= proximal tubular epithelial cells) and HUVEC cells (= human umbilical vein endothelial cells) can be used for the experiments. PTEC cells were cultured according to the method described by Detrisac et al. (Kidney Int. 25, 1984: 383) PTEC cells were seeded into vials of tissue culture coated with collagen (Collagen I, Sigma, St. Louis, MO) and fetal calf serum (foetal calf serum = FCS, Gibco BRL). The culture medium consisted of "Eagle Medium" modified according to Dulbecco's and Ham's F12 medium (both from Gibco BRL) at a ratio of 1: 1, supplemented with insulin (5 µg / ml), transferrin (5 µg / ml), selenium (5 µg) / ml), hydrocortisone (36 ng / ml), tri-idotyronine (4 pg / ml), epidermal growth factor (10 ng / ml), and penicillin / streptomycin [(5 IU / ml, 5 pg / ml), all from Cell lines were obtained from various sources, pretran biopsies splants, allografts not used for transplantation, and normal kidney surgical samples. The experiments were performed on cells from passages 1 through 4. PTEC was characterized by positive labeling with epithelial membrane antigen (= EMA, Dak Glostrup, Denmark) and with adenosine deaminase-binding protein (provided by Dr. Dinjens, University Hospital, Maastricht, The Netherlands) .

HUVEC cells (= endothelial cells of human umbilical cord veins) were obtained from fresh umbilical cord using

The procedures described by Jaffe et al. (Culture of Human Endothelial Cell Derived from Umbilical Veins. Identification by Morphologic and Immunologic Criteria. J. Clin. Invest. 52, 1,973: 2745-2756). The method is briefly described below:

Endothelial cells were isolated from umbilical veins by digestion with collagenase (Collagenase V, Sigma, St. Louis MO, 20 minutes at 37 ° C). Subsequently, the veins were washed with sterile culture medium and endothelial cells were harvested. The culture medium consisted of medium (Medium 199, Gibco BRL) supplemented with 15% fetal calf serum (= FCS), endothelial cell growth factor and penicillin and streptomycin antibiotics. Cells were cultured in 25 cm ³ vials coated with 1% gelatin (Sigma, St. Louis, MO).

All experiments were performed with cells from the third to sixth passage of the cell culture. HUVEC cells were characterized by their positive staining with Factor VIII-related antigen (Dako, High Wycombe, UK) and endothelial marker EN4 (CD31).

IFN γ-stimulation, treatment with heparin and sodium chlorate

Flowing monolayers of PTEC and HUVEC cells were strypsin-treated and dispersed in 24-well plates. When fusion was achieved, cells were stimulated with IFN γ (Sigma, St. Louis MO) for 72 hours in the presence or absence of different heparinoids at various concentrations containing different heparins, such as Heparin-Braun® from Braun-Melsungen, Melsungen, Germany , low molecular weight Heparin Fragmin® P from Pfrimmer Kabi, Erlangen, Germany and modified low molecular weight heparin from Knoll AG, Ludwigshafen, Germany.

In some experiments, the culture medium was supplemented with sodium chlorate to achieve inhibition of sulfatization with cell-bound heparan sulfate proteinoglycan (= HSPG). Chlorate was used at concentrations from 50 to 150 mM. It was added to the medium 24 hours before IFN γ addition. Stimulation with IFN γ was performed in the presence of chlorate. Sodium chloride was used as an osmolarity regulating agent. The cultured cells were recovered after culturing by treatment with trypsin EDTA for flow cytometry determination.

- 13Flow cytometry

Cells of different deposits were pooled and then divided into two test tubes and washed. In a first test tube, antibodies that did not bind to the cell isotopes and that bound to RPE and FITC (from Dako, Glostrup, Denmark) and Cy-5 (from Dianow, Hamburg Germany) were added. This deposit was used as a negative control on FACS background. Cells in the second deposit were labeled with anti-MHC class 1 antibodies (RPE conjugated, W6 / 32, Dak), anti-MCH class II (Cy-5 conjugated, CR3 / 43, Dianova) and anti ICAM 1 (FITC conjugated, Dianova) v concentrations as indicated by the manufacturer. After incubating the cells for 30 minutes at 4 ° C, the latter were washed and analyzed by flow cytometry (FACScan, Becton Dickinson). At least 10 000 positive cases were analyzed. Results were expressed as mean fluorescence intensity (MFI).

Dot-blot analysis

For dot-blot analysis, narrow strips of nitrocellulose membrane were prepared, to which 1 μΙ of heparin, Fragmin and various other N -sulfatized-N-acetylated glycosaminoglycans (= GAG, all at 1 mg / L) were applied. After drying, the strips were stabilized with 1% glutaraldehyde + 0.5% cetyl-addinim chloride to prevent loss of GAG and subsequently washed with Tris buffer. The finished strips were finally exposed to Kodak.

Statistical analysis

Significance of changes in antigen expression was determined by Student's T test. P values <0.05 were considered significant.

The results

MHC class I, class II and ICAM 1 protein expression was modulated in PTEC by dose-dependent IFN γ. MHC class I and ICAM 1 expression was regulated up to a concentration of 50 ng / ml IFN γ. In addition, at the same concentration of IFN γ sa

-14 MHC class II expression in PTEC increased (see Figure 1). Corresponding results were obtained with cultured HUVEC cells.

To investigate the effect of heparin on the ability of IFN γ to modulate MHC and ICAM 1 expression, both cultures, HUVEC and PTEC, were stimulated with IFN γ in the presence or absence of heparin. Administration of heparin at concentrations of 0.03 to 3 mg / ml to HUVEC cultures completely prevented upregulation of MHC class I proteins and ICAM 1 caused by the use of 100 ng / ml IFN γ. Similarly, induction of MHC class I proteins in these cells was suppressed by administration of heparin. Heparin alone had no effect on the expression of the three antigens of interest in the absence of IFN γ (Figure 2).

Similar comparative PTEC experiments have similarly shown that with 100 ng / ml IFN γ induced up-regulation of ICAM 1 and induction of MHC class II proteins can be inhibited by heparin. However, up-regulation of MHC class I proteins with IFN γ could not be influenced with heparin. In order to test whether upregulation of MHC class I proteins induced by a low concentration of IFN γ can be blocked by heparin, the same experiments were performed with cells stimulated with ng / ml IFN γ. Heparin alone at concentrations of 3 mg / ml was considered to have minimal effect on upregulation of MHC class I with IFN γ (see Figure 3a). In contrast, both MHC class II induction and ICAM 1 up-regulation are significantly inhibited by 0.03 mg / ml heparin (p <0.01) (see Figures 3b and c). To investigate the effect of the degree of heparin sulphation on the inhibition of MHC and ICAM 1 expression after stimulation with IFN γ, various heparinoids were investigated in this test system (Table 1). All heparinoids were tested at a concentration of 3 mg / ml for their ability to inhibit MHC class I and ICAM 1 upon stimulation with IFN γ. MHC class assays were performed with 10 ng / ml IFN γ stimulation. Compared to normal and low molecular weight heparin, super-sulphated GAG (GAG 6-8) inhibited MHC and ICAM 1 expression in both HUVEC and PTEC, after stimulation with IFN γ. In contrast, desulfated N-acetylated GAG (GAG 1-5) would not affect MHC and ICAM 1 expression in these cells upon stimulation with γ (Figures 4a-c and 5a-c). There was a clear tendency that super-sulfated GAGs were clearly more potent in inhibition than normal and low molecular weight heparins. This was more pronounced in PTEC cultures (Figure 5a). Further dose-related experiments, p

- 15 low molecular weight heparins and GAG 6-8 and PTEC cells stimulated with 10 ng / ml IFN γ showed that administration of GAG 6-8 at 0.03 mg / ml to cultures stimulated with IFN γ significantly inhibited MHC expression and ICAM 1 (p <0.05). Heparin showed no significant effect on MHC class I and II expression under these conditions, ICAM 1 was similarly inhibited by heparin under these conditions; however, significantly less than with super-sulphated GAG (Figure 6a). These results clearly showed that super-sulfated GAGs are more effective than comparable heparin in inhibiting MHC and ICAM 1 expression.

To investigate whether the degree of sulfation of glycosaminoglycans has a significant effect on the inhibitory effect of GAG on MHC and ICAM 1 expression after stimulation with IFN γ, PTEC cells were incubated in the presence of NaCIO ₃ . It was believed that in this way the sulfation of HSPG could be blocked. As a control, cells were treated with equimolar concentrations of NaCl. Then, both deposits were stimulated with IFN γ at concentrations of 0 to 10 ng / ml. Although IFN γ was still in a position to modulate MHC and ICAM 1 expression in NaCIO3-treated PTEC cells, such modulation was already considerably lower than with NaCl-treated cells (see Figures 7a-c). Thus, the degree of sulfation of HSPG plays an important role in modulating the expression of these antibodies with IFNγ.

Binding studies with ¹²⁵ IFN γ were performed to clarify whether GAGs had to be sulfatized to IFN γ binding. The results showed that both heparin and supersulfated GAGs can bind ¹²⁵ IFN γ. This cannot be accomplished with desulfatized N-acetylated GAGs having few sulfate groups (GAG 3-5). Desulfatized N-acetylated GAG with a higher sulfate content (GAG 1 and 2) still bind ¹²⁵ IFN γ, but significantly less than heparin or super-sulfated GAG. Binding of ¹²⁵ IFN γ to heparin or GAG bound to nitrocellulose filters can be blocked by a 3000-fold excess of heparin in the binding solution. Under such conditions, binding to GAGs 1 and 2 is no longer performed, whereas binding to heparin, fragmine and super-sulphated GAG is significantly reduced (Figure 8).

-16Table 1

Properties of the various heparins and glycosaminoglycans *) used in this study

Title sulphate FXa FIIa M _p M _w [%] [U / mg] [U / mg] Desulfatizované N-acetylated heparinoids GAG 1 7.3 0 0 3289 3802 GAG 2 6.4 0 0 2992 3548 GAG 3 5.5 0 0 2869 3382 GAG 4 3.4 0 0 2364 2800 GAG 5 1.2 0 0 1618 2074

Sulfated heparinoids

GAG 6 14.2 26.1 39 7800 8360 GAG 7 14.2 21.5 30 6000 7700 GAG 8 13.7 25.8 28 5500 6300 Commercially available heparinoids Braun ND ND ND 14 000 18 000 Fragmin P ND 160 70 4900 6000 *) Study features: M _p : different total heparins mass and the glycosaminoglycans used in this divided by the number of molecules; M _w molecular

mass, ND - undetermined, Flla and Fxa activity was determined according to the method described by Handeland et al. (Assay of Unfractionated and LMW Heparin with Chromogenic Substrates: Twin Methods with Factor Xa and Thrombin, Thrombosis Res. 1984, 35: 627) with the first international standard for low-molecular-weight heparin (introduced in 1987, code 85/600) .

Claims (6)

PATENT CLAIMS 1. Organ protection solutions, characterized in that they contain a number of the following components effective in maintaining cell integrity and cell vitality (a) 10 mg / l to 10 000 mg / l of poly-sulphated glycosaminoglycans with a sulfur content of not less than 12,5% by weight b) 5 to 100 g / l of hydroxyethyl starch. Organ protection solution according to claim 1, characterized in that it contains (a) 10 mg / l to 10 000 mg / l of poly-sulphated glycosaminoglycans with a sulfur content of not less than 12,5% by weight b) 5 to 100 g / l of hydroxyethyl starch c) 5 to 100 mmol raffinose. An organ protection solution according to claim 1, characterized in that it comprises (a) 10 mg / l to 10 000 mg / l of poly-sulphated glycosaminoglycans with a sulfur content of not less than 12,5% by weight b) 5 to 100 g / l of hydroxyethyl starch c) 5 to 100 mmol raffinose d) 5 to 40 mmol KH 2 PO 4 e) 1 to 50 mmol MgSO ₄ f) 1 to 50 mmol of adenosine g) 0.5 to 5 mmol of allopurinol h) 1 to 10 mmol glutathione. Use of organ protection solutions according to claims 1 to 3, for the preparation of pharmaceutical preparations for the inhibition of γ interferon-induced upregulation of MHC class I and MHC class II proteins and ICAM 1. -185. Use of organ protection solutions according to claim 4 for the treatment and prevention of diseases associated with γ interferon-induced upregulation of MHC class I and MHC class II proteins and ICAM 1. Use of organ protection solutions according to claim 4 or claim 5, for the treatment of transplant patients. Use of organ protection solutions according to claim 4 or claim 5 for the protection and storage of organs to be used for transplantation.