HRP20010605A2 - Organoprotective solutions - Google Patents

Description

This invention relates to the use of poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% by weight in the production of pharmaceutical preparations for inhibiting interferon γ-induced regulation of MHC Class I, MHC Class II proteins and ICAM 1. The invention further relates to organ protection solutions containing poly-sulfated glycosaminoglycans and on the process of ex vivo protection of organs for transplantation.

The use of glycosaminoglycans, especially heparin and heparinoids in the production of pharmaceutical preparations for the treatment of circulatory disorders is well known.

But in recent times, the use of glycosaminoglycans for the treatment of some additional diseases has also been described. Thus, US 5,236,910 states the use of glycosaminoglycans in the treatment of diabetic nephropathy and neuropathy. The use of low molecular weight heparins for the same indication is described by van der Pijl et al (J Americ Soc Nephrol, 1997, 8:456-462).

US 5,032,679 outlines the use of glycosaminoglycans to inhibit the proliferation of smooth muscle cells and similar diseases.

US 4,966,894 polysulfated heparins are reported for the treatment of diseases caused by retroviruses.

Gralinski et al describes the modulation of the complement system with polysulfated heparins.

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

When organs are transplanted, unwanted rejection reactions often occur. To prevent such rejection reactions, a number of different courses of action are taken. The histocompatibility antigens of the donor and the recipient are most often compared. Only those organs are considered for transplantation where the donor and recipient are maximally identical or when they have very similar histocompatible antigens.

But despite what has been said, unwanted organ rejection still occurs in a large number of cases. Thus, for example, acute rejection of a kidney allograft occurs where the recognition of allo-MHC antigens by T lymphocytes is the main effect that leads to the lysis of tubular cells. Furthermore, with the so-called "graft against host cell reaction" is a strong reaction to the recipient by the donor's immune cells, which are transplanted together with the organ. Cytotoxic T cells and antibodies are produced against the host organism.

In order to further reduce the risk of transplant rejection, the organs are cooled after their removal from the donor organism and stored with a solution to protect the organs. Furthermore, the recipient is given a medication that suppresses the reaction of his immune response.

Many solutions for organ protection are described in the literature - Thus Collins et al (Lancet, vol 2, 1969:1219) describes intracellular electrolytic solutions for organ preservation. Sacks SA (Lancet, vol 1, 1973: 1024) describes solutions which have a stabilizing osmotic effect. ATP-MgCl 2 , AMP-MgCl 2 and inosine are described as suitable agents in such solutions (Siegel, NJ et al, Am J Physiol, 254: F530, 1983; Belzer et al, Transpl Proc 16:161, 1984). US 4,920,004 discloses a solution containing mannitol, adenosine and ATP-MgCl 2 . US 4,798,824 and US 4,873,230 disclose an organ protection solution containing hydroxyethyl starch. US 4,879,283 discloses a solution containing KH 2 PO 4 , MgSO 4 , adenosine, allopurinol, raffinose and hydroxyethyl cellulose. It is also known as the University of Wisconsin solution (=UV solution). This solution is described for successful liver, kidney and heart preservation (Jamieson et al, Transplantation, vol 46, 1988: 517; Ploeg et al, Transplantation vol 46, 1988: 191; and Wicomb WN, Transplantation, vol. 47, 1988 :733). US 5,200,398 describes an additional additive with glucuronic acid for such protective solutions, its salts and esters.

Despite the success that has been achieved in preserving organs for transplantation and suppressing unwanted organ rejection, there still remains a need for further improvements and improvements.

Thus, the goal of further improvement was set both in organ protection before transplantation and during transplantation, as well as in reducing the risk of organ rejection.

This was achieved by using poly-sulfated glycosamino-glycans with a sulfur content of at least 12.5% (weight fraction) for the production of pharmaceutical preparations for inhibiting interferon γ-induced upregulation of MHC Class I, MHC Class II proteins and ICAM 1.

The invention further relates to organ protection solutions that contain a certain amount of poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% for suitable pharmaceutical preparations that effectively maintain cellular integrity and vitality.

Pharmaceutical preparations produced for use in the context of this invention, which also contain, for example, solutions for protecting organs, may contain the above-mentioned substances in the form of their physiologically active salts or esters, their tautomeric and/or isomeric forms, or may be in the form of a combination of free substances and different salts. Suitable physiologically effective salts include, for example, Na, Ca, or Mg salts. Salts with organic bases such as diethylamine, triethylamine or triethanolamine are also suitable. Pharmaceutical preparations may also contain at least one free substance or at least one substance in the form of its salts or their mixture.

Poly-sulfated glycosamino-glycans (= mucopolysaccharides) used in the context of this invention are negatively charged polysaccharides (= glycans) consisting of differently linked disaccharide units in which, for example, one molecule of the so-called uronic acid, such as D glucuronic acid or L iduronic acid, is attached to the 3rd or 4th position of an amino sugar similar to glucosamine or galacamine by a glycosidic bond. At least one of the sugars in the disaccharide has a negatively charged carboxylate or sulfate group that can be attached with an oxygen or nitrogen atom. Glycosamino-glycans react extremely acidic with uronic acids and sulfuric acid ester groups. Such acid-reactive groups are partially already present in nature, but their special value is that they maintain the level of sulfation, which is normally performed in the context of this invention by synthetic introduction, such as sulfation in a substance. As sulphation methods, the literature mentions as examples sulphation with sulfuric acid and sulphur-chloric acid (US 4,727,063; US 4,948,881), sulphation with sulphur-chloric acid in pyridine (Wolfrom et al, J Am Chem Soc, 1953, 75 1519) or sulphation with nitric acid (Shively et al, Biochemistry, vol 15, no 18, 1976: 3932). Further methods are well known to those skilled in the art. Examples are the use of natural glycosaminoglycans such as heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin or chondroitin sulfate for sulfation. The structure of heparan sulfate corresponds to the structure of heparin with the difference that it contains fewer N and O sulfate groups and more n acetyl groups.

Glycosaminoglycans can be easily isolated from animal tissues such as intestinal mucosa or from the ears of pigs and cattle. The tissue used for glycosaminoglycan isolation is, for example, autolyzed and extracted with an alkaline substance. Next, the protein is allowed to coagulate and then precipitated, for example by adding acid. After introducing the precipitate into a polar non-aqueous solution such as ethanol or acetone, the fats are removed by extraction with an organic solvent. With the help of proteolytic digestion, proteins are immediately removed, and glycosaminoglycans are obtained. 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 of heparin isolation.

Such glycosaminoglycans isolated from natural tissues can be further derivatized by polysulfating them as described for example in US 5,013,724 or as stated above. With the help of such polysulfation, glycosaminoglycans show a sulfur content of 6-15% (weight fraction). For use in the context of this invention or for pharmaceutical preparations, poly-sulfated glycosaminoglycans containing at least 12.5% (weight fraction) of sulfur are selected. Preferably, such polysulfated glycosaminoglycans have a sulfur content of 13-16% (weight ratio), preferably 13-15% (weight ratio), and in the best case 13.5-14.5% (weight ratio). Such substances are used for the production of pharmaceutical compositions which are suitable for inhibiting interferon γ-induced regulation of MHC Class I, MHC Class II proteins and ICAM 1. It is preferred to use such substances in an amount that is physiologically effective for the treatment and prevention of diseases associated with with interferon γ-induced regulation of MHC Class I, MHC Class II proteins and ICAM1. Among the various derivatives, there are also substances that improve the application properties of poly-sulfated glycosaminoglycans, used because of their effect, their stability and the way of their elimination, especially the elimination from the body.

Primarily, heparins and/or dermatan sulfates with an average molecular weight of 1000 to 2000 Daltons, preferably 1500 to 9000 Daltons, in an even better case 2000 to 9000 Daltons, should be used, and the optimal use of molecules weighing 2000 and 6000 Daltons. Particularly preferred are low molecular polysulfated heparins and/or dermatan sulfates in the form of free acids or in the form of salts with physiologically tolerable bases or mixtures of such components. Such substances have a small anti-coagulation effect, and are thus particularly suitable for treatment and prevention when used in the context of the present invention. Preferred salts of polysulfated glycosaminoglycans are, for example, sodium, calcium and magnesium salts.

Low molecular weight glycosaminoglycans, such as low molecular weight heparins and/or dermatan sulfates, can be produced in a number of ways. The production of low-molecular-weight heparins via de-polymerization with the help of nitric acid is described, for example, in EP-B-0 0 37 319 or in Biochemistry, vol 15, 1976: 3932. The production of low-molecular-weight heparin or low-molecular-weight glycosamine-glycans can also be achieved by enzymes (Biochem J , vol. 108, 1968: 647), with sulfuric acid and sulphur-chloric acid (FR No 2,538,404, simultaneous sulphation), physical methods such as γ radiation (EP-A-0 269 937) or ultra sound (Fuchs et al , Lebensm Unters Forsch, vol 198, 1994: 486-490).

Further use in the context of the present invention is in the form of organ protection solutions. With the help of a convenient method of application of poly-sulfated glycosaminoglycans in organ protection solutions, the storage of organs after their removal from the donor organism, for example ex vivo, can be further improved by inhibiting the interferon γ-induced upregulation of MHC Class I, MHC Class II proteins and ICAM 1 Organs should be hypothermic, as is well known to those skilled in the art.

Many such solutions mentioned above are described in the literature. Solutions generally contain salts, buffers, substances that have the task of stabilizing organs osmotically or that should prevent the oxidation of sugar or sugar alcohols, proteins, amino acids, lower carboxylic acids, purines, pyrimidines or pharmaceutical agents. As examples of such substances, the following substances are mentioned: raffinose, glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride, potassium hydrogen carbonate, sodium hydrogen carbonate, magnesium sulfate, magnesium chloride, adenosine, albumin, mannitol, citrate, verapamil, allopurinol, insulin, dexmethasone, hydroxyethyl starch, glutathione or glucuronic acid.

The use that is primarily envisaged by this invention leads to the possibility of transplanting organs in better conditions of their storage for a longer period of time, which was previously common. Thus, the rejection reaction can be reduced to the smallest possible extent.

In order to further reduce the risk of organ rejection, poly-sulfated glycosaminoglycans are introduced into the donor's body or the patient's body, where possible orally or parenterally before the actual transplantation. Post-operative treatment with the mentioned substances is also useful.

Poly-sulfated glycosaminoglycans are contained in solutions for organ protection or in other pharmaceutical preparations in amounts from 10 mg/l to 10,000 mg/l, preferably in amounts from 10 mg/l to 5000 mg/l, in an even better case in in the amount of 50 mg/l to 3000 mg/l, and in the best case in amounts from 100 mg/l to 3000 mg/l. Furthermore, it is an advantage when such solutions contain 5 to 100 g/l of an osmotically stabilizing substance, which also includes the content of hydroxy ethyl starch.

Furthermore, it is suitable if the organ protection solutions have the following composition:

a) 10 mg/l to 10,000 mg/l of poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% (weight fraction), 5 to 100 g/l of hydroxy ethyl starch and 5 to 100 mmol of raffinose, or

b) 10 mg/l to 10,000 mg/l poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% (weight fraction), 5 to 100 g/l hydroxy ethyl starch and 5 to 40 mmol KH2PO4, 1 to 50 mmol MgSO4 , 1 to 50 mmol adenosine, 0.5 to 5 mmol allopuronil or 1 to 10 mmol glutathione.

For the treatment of patients, polysulfated glycosaminoglycans can be used together with the usual formula for process substances.

Pharmaceutical preparations intended for use in the context of this invention can be applied in the usual way, orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally), with oral or intravenous application being preferred.

The dosage will depend on the patient's age, fitness and body weight, and on the type of application.

Glycosaminoglycans are primarily applied in doses of 0.1 to 500 mg/kg of body weight per day. In the case of parenteral application, glycosaminoglycans are palliated in doses from 0.1 to 30 mg/kg of body weight per day, and in the case of oral administration in doses from 0.2 to 500 mg/kg of body weight per day, where the applied dose can be taken in the form of a single dose or in several portions. Also in the case of mixtures, for example, at least one low-molecular heparin molecule and/or its poly-sulfated derivative and/or at least one low-molecular dermatan sulfate and/or its poly-sulfated derivative are administered in doses of 0.1 to 30 mg/kg of body weight per day in the case of parenteral application or in doses from 0.2 to 500 mg/kg of body weight per day in the case of oral application.

Among the pharmaceutical preparations containing poly-sulfated glycosaminoglycans for the treatment and prevention of diseases associated with organ transplantation, one in principle contains the usual galenic form of application for oral or parenteral administration, be it solid or liquid, such as tablets, coated pills, capsules, powders, granules, dragees, suppositories, solutions or suspensions. They are produced in the usual way. Active ingredients may be processed with common galenic processing materials such as tablet binders, fillers, preservatives, tablet explosives, flow control substances, emollients, wetting agents, dispersing agents, emulsifiers, solvents, retardants, antioxidants and/or propellant gases ( cf H Sucker et al: Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991). Application forms obtained in this way contain the active ingredient in the amount of 0.1 to 90% by weight.

In the production of tablets, coated pills, dragees and solid gel capsules, polysulfated glycosaminoglycans can also be processed with pharmaceutically inert, inorganic or organic excipients. As such excipients for tablets, dragees and solid gel capsules, lactose, corn starch or their derivatives, talc, stearic acid or their salts can be used. In the case of soft gel capsules, vegetable oils, waxes, fats; semi-solid and liquid polyols are suitable as excipients.

For the production of solutions and syrups, suitable excipients are, for example, water, polyols, sucrose, invert sugar, glucose, etc. For injection solutions, water, alcohols, polyols, glycerin, vegetable oils will be used as suitable excipients. Natural and solid oils, waxes, fats, semi-liquid and liquid polyols, etc. will be used for suppositories.

Pharmaceutical preparations may further contain preservatives, solubilizing agents, stabilizers, humectants, emulsifiers, sweeteners, color, flavoring agents, salts for modifying osmotic pressure, buffers, protective layers and/or antioxidants.

Examples

For experimental purposes, PTEC (= proximal tubular epithelial cells) and HUVEC cells (= human umbilical vein endothelial cells) can be used. PTEC cells are cultured by the method described by Detrisac et al (Kidney Int 25, 1984: 383). PTEC cells are introduced into tissue culture vials coated with collagen I (Sigma, St Louis MO) and fetal bovine serum (= FCS, Gibco BRL). Culture medium consisting of "Eagle's Medium" modified according to Dulbecco and Ham's F21 medium (both from Gibco BRL) in a 1:1 ratio, supplemented with insulin (5 μg/ml), transferrin (5 μg/ ml), selenium (5 ng/ml), hydrocortisone (36 ng/ml), tri-idothyronine (4 pg/ml), epidermal growth factor (10 ng/ml) and penicillin/streptomycin [(5 IU/ml, 5 μg/ml) all from Sigma]. Cell lines are obtained from various sources, such as tissue biopsies before transplantation, allografts that are not usable for transplants, and from normal surgical kidney specimens. Experiments were performed with cells from lines 1 to 4. PTECs were characterized with the help of positive labeling with epithelial membrane antigen (= EMA, Dako Glostrup, Denmark), and with adenosine-deaminase-binding protein (donated by Dr Dinjens, University Hospital, Maastricht, Netherlands).

HUVEC cells (= endothelial cells from human umbilical cordial vein) were obtained from fresh umbilical veins by the method described by Jaffe et al (Culture of Human Endothelial Cells Derived from Umbilical Veins. Identification by Morphologic and Immunologic Criteria. J Clin Invest 52, 1973: 2745-2756). The procedure is further briefly described:

Endothelial cells were isolated from umbilical cordial veins by digestion with Collagenase V (Sigma, St Louis MO, 20 minutes at 37°C). Then the veins are washed with sterile culture medium, and the endothelial cells are collected. The culture medium consists of Medium 199 (Gibco BRL) supplemented with 15% fetal bovine serum (= FCS), endothelial cell growth factor and the antibiotics penicillin and streptomycin. Cells are cultured in 25 cm2 vials coated with 1% gelatin (Sigma, St Louis MO).

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

IFN γ stimulation, treatment with heparin and sodium chlorate

Confluent monolayers of PTEC and HEVEC cells are treated with trypsin, and then seeded into 24-well plates. When confluence is achieved, the cells are stimulated with IFN γ (Sigma, St Louis MO) for 72 hours, in the presence or absence of different heparinoids at different concentrations, contained in 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 inhibit sulfation with cell-bound heparan sulfate proteoglycan (= HSPG). Chlorate was used in concentrations of 50 to 150 mM. Add s eu medium 24 hours before application of IFN γ. Stimulation with IFN γ is performed in the presence of chlorate. Sodium chloride is used as an osmotic control. Cultured cells are treated with trypsin EDTA after cultivation for flow cytometry

Flow cytometry

Cells from different deposits are brought into contact and then separated into two test tubes and washed. Antibodies that do not bind to cellular isotopes and that are bound to RPE and FITC (from Dako, Glostrup, Denmark) and Cy-5 (from Dianow, Hamburg, Germany) are applied to the first test tube. This deposit is used as a negative control for the FACS background. Cells in the second deposit are labeled with antibodies labeled with antibodies against MHC Class I (RPE conjugated, W6/32, Dako), against MHC Class II (Cy-5 conjugated, CR3/43, ianova) and against ICAM 1 (FITC conjugated , Dianova) in concentrations as specified by the manufacturer. After incubating the cells for 30 minutes at 4°C, they are washed and analyzed by flow cytometry (FACScan, Becton Dickinson). At least 10,000 positive results were analyzed. The results are expressed as fluorescence intensity (= mean fluorescence intensity = MFI).

Dot-blot analyses

To perform this type of analysis, thin strips of nitro-cellulose membrane are prepared to which 1 μg of heparin, Fragmin and other different N de-sulfated acetylated glycosamino-glycans (= GAGs, all in concentrations of 1 mg/l) are applied. After drying, the strips are fixed with 1% glutaraldehyde + 0.5% cetyl-pyridine chloride to prevent GAG losses, followed immediately by washing with Tris buffer. The prepared strips are finally exposed to Kodak film.

Statistical analyses

The significance of changes in antigen expression is determined using the Student's T test. P values <0.05 are taken as significant.

the results

The expression of MHC Class I, MHC Class II and ICAM 1 proteins was modulated in PTEC by IFN γ in a dose-dependent manner. The expression of MHC Class I and ICAM 1 proteins is regulated by a concentration of 50 ng/ml IFN γ. Furthermore, with the same concentration of IFN γ, the expression of MHC Class II in PTEC is increased (see Figure 1). Corresponding results were obtained with cultured HUVEC cells.

To study the effect of heparin on the ability of IFN γ to modulate MHC and ICAM 1 expression, HUVEC and PTEC cultures were stimulated with IFN γ in the presence or absence of heparin. The application of heparin in HUVEC cultures in concentrations from 0.03 to 3 mg/ml completely prevents the regulation of MHC Class I and ICAM 1 proteins caused by 100 ng/ml IFN γ. Thus, with the application of heparin, the induction of MHC Class I protein is suppressed in the cells. Heparin alone, in the absence of IFN γ, has no effect on the expression of the three studied antigens (Figure 2).

In comparable experiments with PTEC, it could also be shown that with 100 ng/ml IFN γ, induced upregulation of ICAM 1 protein and induction of MHC Class II protein could be inhibited with heparin. The regulation of MHC Class I proteins by IFN γ, however, cannot be influenced by the action of heparin. In order to test the fact, whether the regulation of MHC Class I proteins induced with insignificant concentrations of IFN γ can be blocked with heparin, the same experiments were performed with 10 ng/ml IFN γ-stimulated cells. It turned out that heparin, by itself, at concentrations of 3 mg/ml has only a marginal effect on the regulation of MHC Class I proteins by IFN γ (see Figure 3a). In contrast, induction of MHC Class II and upregulation of ICAM 1 protein were significantly inhibited by 0.03 mg/ml heparin (p<0.01) (see Figures 3b and c). In order to study the influence of the degree of heparin sulfation on the inhibition of MHC and ICAM 1 expression after stimulation with IFN γ, different heparinoids were tested within this test system (Table 1). All heparinoids were tested at concentrations of 3 mg/ml for their ability to inhibit MHC Class I and ICAM 1 proteins after stimulation with IFN γ. MHC Class II tests were performed by stimulation with 10 ng/ml IFN γ. In comparison with normal and low molecular weight heparins, super-sulfated GAG (GAG 6-8) inhibits the expression of MHC and ICAM 1, both in HUVEC and in PTEC after stimulation with IFN γ. In contrast, desulfated N acetylated GAG (GAG 1-5) could have no effect on the expression of MHC and ICAM 1 after stimulation with IFN γ (Figures 4a-c and 5a-c). A clear tendency is evident that super-sulfated GAG molecules are significantly more efficient in inhibition than normal and low molecular weight heparins. This is even more clearly shown with PTEC cultures (Figure 5a). Further experiments with different dosages with low molecular weight heparins, with GAG 6-8 molecules, and with PTEC cells after stimulation with 10 ng/ml IFN γ, showed that application of GAG 6-8 in concentrations of 0.03 mg/ml in IFN γ stimulated cultures , significantly inhibits the expression of MHC and ICAM 1 (p<0.05). Under these conditions, heparin did not show a significant effect on the expression of MHC Class I and Class II, while under these conditions ICAM 1 was inhibited by heparin; however, to a significantly lesser extent than is the case with super-sulfated GAG (Figure 6a-c). These results clearly showed that super-sulfated GAG molecules are much more efficient than compared heparins in inhibiting the expression of MHC and ICAM 1 proteins.

In order to study whether the degree of glycosaminoglycan sulfation has an important influence on the inhibitory effect of GAG on the expression of MHC and ICAM 1 proteins after stimulation with IFN γ, PTEC cells were incubated in the presence of NaClO3. In this way, sulfation of HSPG was expected to be blocked. As a control, cells were treated with equimolar concentrations of NaCl. Then both amounts were stimulated with IFN γ in concentrations from 0 to 10 ng/ml. Although IFN γ was still able to modulate the expression of MHC and ICAM 1 proteins in PTEC cells treated with NaClO3, such modulation was significantly less than in the case of cells treated with NaCl (see Fig. 7a-c). This means that the degree of HSPG sulfation plays an important role in the modulation of the expression of these antigens by IFN γ.

In order to clarify the fact, whether GAG molecules must be sulfated for IFN γ binding, binding experiments with 125IFN γ were performed. The results showed that both heparin and super-sulfated GAG molecules can bind 125IFN γ. However, this is not possible with de-sulfated N acetylated GAG molecules showing only a few sulfate groups (GAG 3-5). De-sulfated N acetylated GAG molecules with a higher sulfate content (GAG 1 and 2) still bind 125IFN γ, but to a significantly lower extent than do heparin or super-sulfated GAG molecules. Binding of 125IFN γ to heparin or GAG, which are bound to nitrocellulose filters, can be blocked with a 3000-fold excess of heparin in the binding solution. Under such conditions, binding to GAG 1 and 2 does not occur, while binding to heparin, Fragmin and super-sulfated GAG molecules is significantly reduced (Figure 8).

Table 1: Properties of different heparins

and glycosamino-glycans*) that were used in this study ;Name Sulphate Fxa FIIa ;[%] [IU/mg] [IU/mg] Mp Mr ;De-sulfated 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; SULFATIZED; HEPARINOIIDI; GAG 6 14.2 26.1 39 7800 8360; GAG 7 14.2 21.5 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 ;*) Properties of different heparins and glycosamine-glycans used in this study: Mp: total weight divided by number of molecules; MW molecular weight, ND – not determined, FIIa 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, number 85/600).

Picture 1

Dose-dependent expression of MHC Class I, Class II and ICAM 1 in PTEC after stimulation with IFN γ. These cells were stimulated for 72 hours with different IFN γ concentrations. Thus, the 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 reported in the figure as the fluorescence intensity of a representative experiment.

Figure 2

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

Figure 3

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

Figure 4

Effects of different heparins and glycosaminoglycans on the expression of MHC Class I and ICAM 1 proteins in IFN γ-stimulated cells. IFN γ stimulated HUVEC cells (10 ng/ml, for 72 hours, smooth bar) and unstimulated cells (dashed bar) were incubated with different heparins at a concentration of 3 mg/ml during the stimulation period. The expression of MHC and ICAM 1 is then determined by FACS. Figure 4a shows MHC Class I expression, Figure 4b shows MHC Class II expression, and Figure 4c shows ICAM 1 expression. Results are shown as fluorescence intensity +/- 2 SD.

Figure 5

Effects of different heparins and glycosaminoglycans on MHC and ICAM 1 protein expression in IFN γ-stimulated PTEC cells. IFN γ stimulated PTEC cells (10 ng/ml, for 72 hours, smooth line) and unstimulated cells (dashed line) were incubated with different heparins or glycosaminoglycans at a concentration of 3 mg/ml during the stimulation period. The expression of MHC and ICAM 1 is then determined by FACS. Figure 5a shows MHC Class I expression, Figure 5b shows MHC Class II expression, and Figure 5c shows ICAM 1 expression. Results are shown as fluorescence intensity of a representative experiment.

Figure 6

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

Figure 7

Effect of NaClO3 on stimulation of MHC and ICAM 1 expression in PTEC cells stimulated with IFN γ. PTEC cells were treated one day before IFN γ stimulation with 150 mM NaClO3 (smooth line) or with 150 mM NaCl (dashed line) which served as an osmolar control. The cells were then stimulated for 72 hours with different concentrations of IFN γ in the presence of the same concentration of NaClO3 or NaCl. The expression of MHC and ICAM 1 in three replicate cultures was then determined by FACS. Figure 7a shows MHC I expression, Figure 7b shows MHC II expression and Figure 7c shows ICAM 1 expression. Results are plotted as fluorescence intensity +/- 2 SD. An asterisk indicates p<0.05 and two asterisks indicate p<0.01 results obtained using the Student T test.

Figure 8

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

Claims (7)

1. Solutions for the protection of organs, characterized by the fact that they contain a certain amount of the following components that are effective for maintaining cellular integrity and vitality: a) 10 mg/ml to 10,000 mg/ml of poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% by weight b) 5 to 100 g/l of hydroxy ethyl starch 2. Solutions for the protection of organs, in accordance with claim 1, indicated that they contain a) 10 mg/l to 10,000 mg/l of poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% by weight b) 5 to 100 g/l of hydroxy ethyl starch c) 5 to 100 mmol of raffinose 3. Solutions for the protection of organs, in accordance with requirements 1 and 2, indicated that they contain a) 10 mg/l to 10,000 mg/l of poly-sulfated glycosaminoglycans with a sulfur content of at least 12.5% by weight b) 5 to 100 g/l of hydroxy ethyl starch c) 5 to 100 mmol of raffinose d) 5 to 40 mmol KH2PO4 e) 1 to 50 mmol MgSO4 f) 1 to 50 mmol adenosine g) 0.5 to 5 mmol allopurinol h) 1 to 10 mmol of glutathione 4. Use of solutions for organ protection, in accordance with claims 1 to 3, indicated to be used in the production of pharmaceutical preparations for inhibiting γ-interferon-induced regulation of MHC Class I, MHC Class II and ICAM 1 proteins. 5. Application of organ protection solutions, in accordance with claim 4, characterized by the fact that they serve for the treatment and prevention of diseases associated with γ-interferon induced regulation of MHC Class I, MHC Class II and ICAM 1 proteins. Drawings 6. Application of organ protection solutions, in accordance with claims 4 or 5, characterized by the fact that they are used for the treatment of patients undergoing transplantation. 7. Application of solutions for organ protection, in accordance with claim 4, characterized by the fact that they serve to protect and store organs that will be used for transplantation.