MXPA01007279A - Organoprotective solutions. - Google Patents

Abstract

The invention relates to organoprotective solutions containing polysulfated glycosaminoglycanes.

Description

ORGAN PROTECTIVE SOLUTIONS DESCRIPTION The invention relates to the use of poly-sulfated glycosamino-glycans with a sulfur content of at least 12 5% for the manufacture of pharmaceutical preparations to inhibit up-and-down regulation of inter-feron proteins MHC Class I, MHC Class II and ICAM 1 The invention also relates to protective solutions of organs containing polysulino-glycans and a method for the ex vivo protection of transplant organs. The use of glycosaminoglycans and particularly of heparins and heparinoids for the manufacture of pharmaceutical preparations for the treatment of circulatory disturbances is well known. More recently, the use of glycosamino glycans has been described for a series of additional cures. In this way, E.U.A. 5,236,910 claims the use of glycosamino-qlicans in the treatment of diabetic nephropathy and neuropathy. The use of low molecular heparins for the same indication has been described by van der Pijl et al (J Arneric Soc, Hephrol, 1997, 8. 456-462) E.U.A 5,032,679 claims the use of glycosamino glycans to inhibit the proliferation of mild muscle cells and related diseases. 5 In E.U.A. 4,966,894 Polysulfatized hepae are claimed for the treatment of diseases caused by retroviruses. Gralinski et al have described the modulation of the complement system with poly-sulphonated hepapins. 10 In Clin Exp Immunol (1997, 107 573-534) the antagonization of the inflammatory promoter effect of interferon and with heparin, heparan sulfate or molecules are previously heparin is studied by Douglas et al. Heparin is in a position to influence the immunogenic effect of interferon and. When transplanting organs, undesirable rejection reactions are a frequent occurrence. To prevent such rejection reactions, a number of different trajectories have been run. In this way and mainly, the histocompatibility antigens of donor and recipient have been compared. Only those organs are transplanted when the donor and recipient are maximally identical or who have very similar histo-compatible antigens, 25 However, rejection of undesirable organ t > ».-., -«. ^ ^ £ ^^ í & j ^ ^ ^ results persistently. Here, for example, the rejection of acute renal allograft occurs when the recognition of allo-MHC antigens by T lymphocytes is the main effect that involves lysis of tubular cells. If it also involves a so-called "graft-versus-host reaction", a strong reaction to the recipient of the immune cells of the donor, transplanted with the organ. Cytotoxic T cells and antibodies are formed against the host organism. In order to further reduce the risk of transplant rejection, the organs are cooled immediately after their removal and stored under an organ protection solution. In addition, the recipient receives medication that suppresses the defense 15 immune from the container. In the literature, a complete series of protective organ solutions has been described. In this way, Collins et al (Lancet, vol 2, 1969: 1219 j describe intracellular electrolyte solutions for 20 Conserving organs Sacks SA (Lancet, vol.1, 1973, 1024) described solutions that have an osmotically stabilizing effect. ATP-MgCl2, AMP-MgCl2, and inosine have been described as suitable agents in such solutions (Siegle, NJ et al .. Am J Physiol. 254: F530, 1983, 25 Belzer et al., Transpl Proc 16: 161, 1984) USA ^. * - ^ - l- »fcL ...: ^ 4,920,004 claims a solution containing mannitol, adenosine and ATP-MgCl2 E.U.A. 4,798,824 and E.U.A 4,873,230 claim an organ protective solution containing hydroxyethyl starch, in E.U.A. No. 4,879,283, a solution containing KH2P04 -MgS04, adenosine, alopironol, raffinose and hydroxyethyl cellulose is claimed. It is also known as the solution of the University of Wisconsin (= UW solution). This solution was described for the successful preservation of liver, kidney and heart (Jamieson et al, Transplantat i on, vol, 46, 1938: 517; Ploeg et al., Transplantation, vol. 46, 1988: 191; and Wicomb W. Transplantation, vol. 47. 1988: 733). In E.U.A. 5,200,398 describes an additional additive in said protection solutions with glucuronic acid, its salts and esters. Despite the success obtained in protecting organs for transplantation and the suppression of undesirable organ rejection, the need for further improvements remains. Therefore, the object was to achieve a further improvement both in the protection of organs before transplantation as well as during the same and also to further reduce the danger of organ rejection. This object has been achieved by using poly-sulphated glycosamino-glycans with a sulfur content of at least 12.5% (by weight) for the manufacture of pharmaceutical preparations to inhibit up-regulation of interferon-induced and MHC Class I, MHC proteins. Class II and ICAM l. The invention is further related to protective solutions of organs containing an amount of a poly-sulfated glycosamino-glycan with a sulfur content of at least 12 5% for appropriate pharmaceutical preparations which effectively maintain the 10 cell integrity and vitality Pharmaceutical preparations manufactured for use in accordance with the invention, including for example organ protective solutions can also contain the above-mentioned compounds as compounds 15 free in the form of their physiologically active salts or esters, their tautomer and / or isomer forms or in the form of the combination of free compounds and various salts. They are notable as physiologically effective propitious salts, for example, the Na salts, 20 Ca or Mg. Likewise, salts with organic bases such as diethylane, triethylane or triethanolamine are appropriate. The pharmaceutical preparations may advantageously contain at least one free substance or at least one compound in the form of its salt or 25 mixtures thereof. jM < The poly-sulfated glycosamino glycans (= mucopolysaccharides) used according to the invention are negatively charged poly saccharides (= glycans) consisting of variously linked units of di-saccharides in which, for example, a molecule of a so-called uronic acid such as glucuronic acid D or iduronic acid L is glycosidically combined with the third or fourth position of an amino sugar such as glucosamine or qalacoamine. When At least one of the sugars in the di-saccharide possesses a negatively charged carboxylate or sulfate group which may occur via an oxygen or nitrogen atom. With uronic acids as well as with sulfuric acid ester groups, glucosamino-glycans react 15 strongly acidic. These acidic reaction groups are already partly present in nature but are valuable in order to achieve the degree of sulfatization required by the invention by synthetically introducing, for example, sulphation towards the compound, as In the case of sulfation methods, the literature describes, as examples, sulfation with sulfuric acid and sulfur-chloric acid (E.U.A 4.72'7.063, E.U.A. 4.948.881). Sulfatization with sulfur-Doric acid in pyridine (Wolfrom et al., J Am Chem Soc, 1953, 75,1519) or 25 Sulfatization with nitrous acid (Shively et al. &v? uá aÁÁi.

Biochemistry, vol. 15, o. 18, 1976. 3932) The additional methods are well known to those skilled in the art. Examples are the use, for sulfation, of natural glycosamino-glycans such as heparin, heparan sulfate, keratan sulfate, determatan sulfate, chondroitin or chondroitin sulfate. The structure of heptane sulfate corresponds to that of hepapin with the Unlike that in contrast to the latter, it has fewer N and O sulfate groups and more acetyl n groups. The glycosaminoglycans can be easily isolated from animal tissue such as intestinal mucosa or from the ears of pigs and cattle. The tissue used for isolation of the mucosal glands is, for example, autolized and extracted with alkali. TO Then the protein is allowed to coagulate and precipitates, v. Gr, acidifying it. After administering the precipitate in a polar non-aqueous solution similar to ethanol or acetone, the fats are eliminated by extraction with an organic solvent. Through 20 protein digestion, the proteins are finally separated and in this way the cosamipo-glycans are recovered Charles et al (Biochem J, vol 30, 1936 1927-1933) and Coyne E in Chemistry and Biology of Hepapn (Elsevier Publishers, North Holland, NY, Lunblad RL, ed 25 1981) describe methods for isolating heparin, by For example, these glycosamino-glycans isolated from natural sources can preferably receive another poly-sulphide derivative, as described, by example, in US 5,013,724 or as described above By means of said poly-sulfation, the ylicosa moglycans then show a sulfur content of 6-15% (by weight) .For use according to the invention or for pharmaceutical preparations, the polysulfinated glycosamino glycans are selected to have a sulfur content of at least 12.5% (by weight). Preferably, said poly-sulfated glycosamino glycans have a sulfur content of 13-16. % (by weight), preferably 13-15% (by weight, particularly good is 13.5-14.5% (by weight) These substances are used for the manufacture of pharmaceutical preparations which are suitable for inhibiting ascending regulation induced by interferon and of the MHC Class I, MHC Class II and ICAM 1 proteins. Preference is given to using said substances in a physiologically effective amount for treatment and prevention of diseases associated with up-regulation induced by myferferone and MHC Class I proteins, MHC Class II and ICAM 1 Among the derivatives of the substances also includes compounds that enhance the application properties of the poly-sulphated glycosamino-glycans used with respect to their stabilizing effect. and its elimination, particularly of the body. Preferably, heparins and / or dermatan sulfate with average molecular weight of about 1000 to 2000 Dalton should be used, preferably between 1500 and 9000 Dalton and particularly between 2000 and 9000 Dalton and, optimally, between 2000 and 6000 Dalton Heparins are particularly advantageous Low molecular poly-sulphates and / or sulfates of determatane in the free acid form or in the form of a salt with physiologically tolerable bases or mixtures made of these compounds. These substances have a slight anti-coagulation effect and, therefore, are Particularly suitable for treatment and prevention when used in accordance with the invention. Preferred salts of poly-sulphated glycosamino glycans are, for example, sodium, calcium and magnesium salts. Low molecular glycosamino-glycans, eg, low molecular heparins and / or dermatan sulfates can be manufactured by a number of methods. The production of low molecular heparins through depolymerization with the aid of nitrous acid is 25 describes, for example, in EP-B-0 037 319 or in Biochemistry, vol. 15, 1976: 3932. The manufacture of low molecular heparin or low molecular glycosamino-glycans can also be achieved with enzymes (Biochem J. Vol. 108, 1968: 647), with sulfuric acid and sulfuric acid (FR No. 2,538,404, simultaneous sulfatization), with periodate or with physical methods such as radiation and (EP-A-0 269 937) or ultrasound (Fuchs et al Lebensrn Unters Forsch, vol 198: 436-490). The additional uses according to the invention reside in protective solutions of organs By means of propitious administration of the poly-sulphated glycosamino-glycans to protective solutions of organs the storage of the organs after the removal of the organism from the donor, i.e. , ex vivo can be further enhanced by inhibiting the upregulation induced by interferon and of the MHC Class I, MHC Class II and ICAM proteins. Preferably, the organs should be refrigerated, as is known to those skilled in the art. A number of these solutions as described above are known from the literature. The solutions generally contain salts, buffers, substances supposed to stabilize the organs osmotically or which are supposed to prevent oxidation such as sugar or sugar alcohols, proteins, amino acids, lower carboxylic acids, purines, pyrimidines or pharmaceutical agents. As examples of these substances, the following could be mentioned: raffinose, glucose, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, potassium chloride, potassium hydrogen carbonate, sodium hydrogen carbonate, magnesium sulfate, chloride of magnesium, adenosine, albumin, mannitol, citrate, verapamil, allopurinol. insulin, dexmetasone, hydroxy-ethyl starch, glutathione or glucuronic acid, The intended use of the invention in protective organ solutions leads to making it possible to transplan the organs in better condition or to store them for a longer period of time than it has been usual so that rejection reactions can be reduced. In order to further reduce the risk of organ rejection, the poly-sulphated glycosammo glycans can be administered to transplant patients or transplant donors, when possible, orally or parenterally before transplantation. The treatment after the operation of the patients with the substances is also feasible. The polyunsaturated glycosamino glycans are contained in the protective solutions of organs or other pharmaceutical preparations in amounts of 10 mg / l to 10,000 mg / l, preferably in amounts of 10 mg / l to 5000 mg / l preferably still in amounts of 50 mg / l to 3000 mg / l and most preferably in amounts of 100 mg / l to 3000 mg / l additionally, 5 to 100 g / l of a substantial osmotically stabilizing hydroxy-containing hydrochloride -on One additional protective formulations of advantageous organs have the following composition: a) 10 mg / l to 10 000 mg / l of polysulino-gl isotonic glycosaminoglycans with a sulfur content of at least 12 5% (in weight), 5 to 100 g / l of ethyl hydroxy-starch and 5 to 100 mmoles of rafmosa or b) 10 mg / l to 10 000 mg / l of polyacrylated glycosamino-glycans with a sulfur content of at least 12 5% by weight), 5 to 100 g / l of hydroxy-ethyl starch 5 to 100 mmoles of rafmosa, 5 to 40 mmoles of KH2P04 1 to 50 mmoles of MgSQ, 1 to 50 mmoles of adenosm, 0 5 to 5 mmoles of alopunnol or 1 to 10 mmol of glutathione For treatment of patients, the poly-sulfated glycosammo glycans can be used together with usual process substances of the formula The pharmaceutical preparations intended for use according to the invention can be administered in the usual way orally or parenterally * * * * - (subcutaneously, intravenously, intramuscularly, intraperitoneally) with oral or intravenous applications being preferred Dosage depends on the age of the patient, condition and weight and the type of application. They are applied more advantageously in a dosage of 0.1 to 500 mg / kg of body weight per day.In case of parenteral application, glycosaminoglycans are best administered in a 10 dosage of 0.1 to 30 mg / kg of body weight per day, in case of oral application in a dosage of 0.2 to 500 mg / kg of body weight per day when the dosage administered can be applied in one dose or in several doses. Also mixes, for example, at least one Low molecular heparin and / or its poly-sulfated derivative and / or at least one low molecular dermatan sulfate and / or its polarized fat derivative are administered in a dosage of 0.1 to 30 mg / kg body weight per day in parenteral application or in a dosage of 0.2 to 20 500 mg / kg of body weight per day in case of oral application. Among the pharmaceutical preparations containing poly-glycan glycosylated glycosides for treatment and prevention of diseases in connection with 25 organ transplantation one in principle includes the usual galenic application forms for oral or parenteral application, either solid or liquid, such as tablets, coated tablets, capsules, powder, granules, dragees, suppositories, solutions or suspensions. They are manufactured in the usual way. The active agents can be processed with the usual galenic process materials similar to tablet binders, fillers, preservatives, tablet explosives, flow regulating substances, softeners, 10 humectants, dispersing agents, emulsifiers, solvents, retarders, antioxidants and / or propellant gases (H Sucker et al Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991). The forms of applications obtained in this way contain the The active agent is usually in an amount of 0.1% to 90% by weight. For the production of tablets, coated tablets, dragees and hard gel capsules, the poly-sulphated glycosamino glycans can also be 20 process with pharmaceutically inert, inorganic or organic excipients. As said excipients for tablets, dragees and gel capsules last, lactose, corn starch, or derivatives thereof, talc, stearic acid or its salts can be used. For 25 soft gel capsules, vegetable oils, waxes, fats, . ^? Am e *** -. ^ ~.

Semi-solid and liquid polyols are suitable as excipients, For manufacture of solutions and syrups, suitable excipients are, for example, water, polyols, sucrose, invert sugar, glucose and so on, For injection solutions, water, alcohols, polyols, Glycerin, vegetable oils are suitable as excipients. For suppositories. Suitable excipients are natural or hardened oils, waxes, 10 fats, semiliquid or liquid polyols and so on. The pharmaceutical preparations may additionally contain preservatives, stabilizing solvents, humectants, emulsifiers, 15 sweeteners coloring agents, aromatic agents, salts for modification of osmotic pressure, buffers, protective layers and / or antioxidants.

EXAMPLES 20 For experiments, PTEC (= proximal tubular epithelial cells) and HUVEC cells (= human umbilical vein endothelial cells) can be used. CETP cells are cultured with the method described by Detrisac et al (Kidney Int 25, 1984: 383) 25 PTEC cells were pelleted in culture bottles of tissue coated with Collagen I (Sigma, St Louis MO) and fetal calf serum (= FC?, Gibco BRL) - The culture medium consisted of "? agle's Medium" modified according to Dulbecco and Ham 's F12 Medium ( both of Gibco BRL) in a 1: 1 ratio, supplemented with insulin (5 μm / ml), transferrin (5 μg / ml), selenium (5 ng / ml), hydrocortisone (36 ng / ml), tri-idot ironin (4 pg / ml), epidermal growth factor (10 ng / ml) and penicillin / estraptomycin [(5IU / mlo, 5ug / ml) all from Sigma]. The cell lines were obtained from various sources such as pre-transplant tissue biopsies, allografts not usable for transplants and normal surgical kidney samples. The experiments were carried out with cells from passages 1 to 4 CETP were characterized by positive labeling with an epithelial membrane antigen (- • • MA, Dako Glostrup, Denmark) and with adenosine-diaminase-binding protein ( graciously provided by Dr Dinjens, University Hospital, Maastricht, The Netherlands). HUVEC cells (= endothelial cells of human umbilical cord veins were recovered from fresh umbilical cords with the method described by Jaffe et al (Culture of Human Endothelial Cells Derived from Umbilical Veams. Identification by Morphologic and Immunologic Criteria. 52. 1973: 2745-2746) The procedure is briefly described as follows: Endothelial cells were isolated from umbilical cord veins by digestion with Collagenase V (Sigma, St Louis MO, 20 minutes at 37 ° C.). rinsed with sterile culture medium and the endothelial cells were collected.The culture medium consists of Medium 199 (Gibco BRL) supplemented with 15% fetal calf serum (= FCS), endothelial cell growth factor and antibiotics from penicillin and estroptomycin The cells were grown in 25 cm2 bottles coated with 1% gelatin (Sigma, St Louis MO 1. All the experiments were carried out with cells of the third and sixth cell culture passage. HUVEC cells were characterized by positive staining with Factor VIII-related antigen (Dako, High Wycombe, UK) and endothelial marker EN4 (CD31). IFN and stimulation, treatment with heparin and sodium chlorate Confluent mono-layers of PTEC cells and HUVEC were treated with tipsin and disseminated in 24-well dishes. When confluence was reached, the cells were stimulated with IFN and Sigma. St Louie MO) for 72 hours in the presence or absence of different heparinoids in varying concentrations contained in different heparins- such as Heparin-Braun (K) from Braun-Melsungen, Melsungen, Germany, Heparin Fragrain "" molecular Ba to P of 5 Pfrímmer Kabi, Erlangen, Germany, and modified low molecular heparin from Knoll AG, Ludwigshafen, Germany. In some experiments, the culture medium is supplemented with sodium chlorate in order to inhibit sulfation with proteoglycan of heparan sulfate. 10 ligated with cell (= HSPG). Chlorate was used in concentrations of 50-150 mM. It was added to the medium 24 hours before the administration of IFM and. The stimulation with IFN and was carried out in the presence of chlorate. Sodium chloride was used as an osmolar control. The 15 cells cultured after cultivation were recovered with a trypsin EDTA treatment for flow cytometry.

Flow cytometry The cells of the various deposits are 20 collected and subsequently divided into two test tubes and washed. Antibodies that do not bind to cell isotopes and bind to RPE and FITC (from Dako, Glostrup, Denmark) and Cy-5 (from Dianowa, Hamburg, Germany) are administered to the first tube of 25 test. This deposit was used as a negative control * M -: -.¿ »s -« *. ^, .. ^ .., .--. for the FACS background, cells in the second reservoir were labeled with antibodies against MHC Class I (conjugated RPE, W6 / 32, Dakoj, against MCH Class II (conjugated Cy-5, CR3 / 43, Dianova) and against ICAM 1 ( Conjugate FITC, Dianova) in concentrations as indicated by the manufacturers After incubation of the cells for 30 minutes at 4SC, the latter were washed and analyzed with flow cytometry (FACScan, Becton Dickinson) when at least 10,000 positive events were analyzed. The results were expressed as mean fluorescence intensity (= mean fluorescence intensity = MFI).

Spot Spot Analysis For spot spot analysis, narrow strips of a nitrocellulose membrane were prepared to which 1 ul of heparin, Fragrnin and various other N-acetylated N-de-atomized glycosamino-glycans (= GAGs) are applied, all in one concentration. of 1 mg / l). After drying, the strips were fixed with 1% glutaraldehyde + 0.5% ceti chloride-ppdinimium in order to prevent GAG losses and subsequently wash with Tris buffer. The finished strips were finally exposed in a Kodak film.

Statistic analysis - '' - ^ i * ri The significance of the changes in antigen expression was determined with the help of Student's T test values <0.05 were considered significant, Results- The expression of MHC Class I proteins. Class II and ICAM 1 were modulated in PTEC using IFN and depending on the dosage. The expression MHC Class I and ICAM 1 was regulated by a concentration of 50 ng / ml IFN and In addition, with the same concentration of IFN and. MHC Class II expression in PTEC was elevated (see Figure 1). The corresponding results were obtained with the cultured HUVEC cells In order to study the influence of heparin on the capacity of IFM and to modulate MHC and ICAM 1 expression, both cultures of HUVEC and CETP were stimulated with IFN and in the presence or absence of heparin. The administration of heparin in concentrations of 0.03 to 3 mg / ml in HUX '? C cultures completely prevents up-regulation of MHC Class I and ICAM 1 proteins caused by 100 ng / ml of IFN y. Likewise, the induction of MHC Class I proteins was suppressed in cells by the administration of heparin. Heparin itself had in the absence of IFN and and no influence on the expression of the three antigens studied (Figure 2). In comparable experiments with CETP it could also be shown that with 100 ng / ml of IFN and the 5"up-regulation" induced of ICAM 1 and the induction of MHC Class II proteins can be inhibited by heparin. Up-regulation of MHC Class I proteins with IFN and, however, could not be influenced with heparin. In order to test whether the upward regulation of 10 MHC Class I proteins induced by omisible IFN concentration and can be blocked by heparin, the same experiments were carried out with 10 ng / ml of cells stimulated with IFN and it turned out that heparin itself at concentrations of 3 mg / ml only has 15 a marginal influence of up-regulation of MHC Class I with IFN y (see Figure 3a j) In contrast, both the MHC Class II induction and up-regulation of ICAM 1 are significantly inhibited by 0.03 mg / ml heparin (p. 0.01) (see 20 Figures 3b and c). In order to study the influence of the degree of sulfatization of heparin on the inhibition of MHC expression and ICAM 1 after stimulation with IFN and, various heparinoids were investigated in this test system (Table 1). The hepapnoids were all tested 25 at a concentration of 3 mg / ml for its capacity ^ «.-.« - 4 ,; ..rtaa. inhibit MHC Class I and ICAM 1 after stimulation with IFN and. The MHC Class II tests were carried out with a stimulation with 10 ng / ml of IFN y. Compared with normal and low molecular heparins, super-sulfatized GAG (GAG 6-8) inhibits the expression of MHC and ICAM 1 in both HUVEC and CETP after stimulation with IFN and. In contrast, acetylated fatigued GAG N (GAG 1-5) could not influence the MHC and ICAM 1 expression in these cells after stimulation with y (Figures 4a-c and 5a-c). A clear trend was evident that super-sulfatized GAGs were more clearly effective in inhibiting than normal and low molecular heparins. This was more pronounced with the PTEC cultures (Figure 5a), in addition the tests related to dosing with low molecular heparins and GAG 6-8 and with PTEC cells after stimulation with 10 ng / ml of IFN and showed that with the administration of GAG 6-8 in concentrations of 0.03 mg / ml in cultures-stimulated with IFM and significantly inhibited MHC and ICAM 1 expression (p 0.05). Heparin did not show significant impact on the MHC Class I and II expression under these conditions, ICAM 1 was also inhibited by hepapna under these conditions; however, significantly less than with super sulfatized GAG (Figure 6a-c). These results clearly showed that super-sulfatized GAGs are more effective than comparable heparin in inhibiting MHC and ICAM 1 expression. In order to study whether the degree of glycosaminoglycan sulfatization has a significant influence on the GAG inhibition effect on the expression MHC and ICAM 1 after stimulation with IFM and, PTEC cells were incubated in the presence of NaCl03, In this way, the sulfatization of HSPG was supposed to be blocked. As a control, the cells were treated with equimolar concentrations of NaCl. Then both deposits were stimulated with IFN and in concentrations of 0 to 10 ng / ml. Even when IFN was still in a position to modulate the MHC and ICAM 1 expression in the PTEC cells treated with NaC103, however this modulation was considerably less than with the NaCl treated cells (see Figures 7a-c). This means that the degree of sulfatization of HSPG plays an important role in modulating the expression of these antigens with IFN and in order to clarify if the GAGs should be fatigated for IFN binding, and 125IFN and linkage studies were carried out. The results showed that both heparma and super sulfatized GAGs can bind 151FN and. This can no longer be done by the acetylated fatigued GAGs N which show only a few sulphate groups (GAG 3-5). Acetylated desulforated GAGs N with an upper portion of sulfate (GAG 1 and 2) still bind 12SIFM and, but markedly less than heparin or super sulphated GAGs. The binding of l25IFN and heparin or GAG bound to trocellulose filters can be blocked by a 3000-fold excess of heparin in the binding solution. Under these conditions, no further binding occurred to GAG 1 and 2 while the binding to heparin, Fragmin and eugen sulfatized GAGs was markedly reduced (Figure 81. Table 1. Properties of the various heparins and glycosamino-glycans *) used in this study Name Sulfate Fxa Row M "M. 15 [%] [IU / mg] [IU / rng] Acetylated heparinoids Desul fatized N GAG 1 7 .3 C 0 3289 3802 20 G GAAGG 22 6 6,. 44 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 25 Table 1 (continued) S-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 BBrraauunn NNDD NMDD NNDD 14,000 18,000 Fragmin P ND 360 70 4900 6000 *) The properties of the various heparins and glycosamino-glycans used in this study. Mn total weight divided by the number of molecules, M2 molecular weight, ND - not determined, Row and Fxa activity was determined according to the method described by Handeland et al (Assay of Unfractionated and LMW Heparin with Chromogenic Substrates: Twin Methode with Factor Xa and Thrombin, Thrombosis Res 1984, 35: 627) with the first international standard for a low molecular hepanna (introduced in 1987, code no 85/600).

Figure 1 Expression of MHC Class I, Class II and ICAM 1 related to dosing in PTEC after stimulation with IFN and. The cells were stimulated for more than 72 hours with various concentrations of IFN and then the expressions of MHC Class I (left scale), MHC Class II (right scale) and ICAM 1 (left scale) FACS were determined (= cytometry flow). The results are given in the Figure as mean fluorescence intensity of a representative experiment.

Figure 2 The effect of hepapna on MHC and ICAM 1 expression of HUVEC cells stimulated with IFN and. HUVEC stimulated with I FN and (100 ng / ml, for 72 hours, gray bar) or unstimulated (white bar) were incubated during the stimulation with various concentrations of heparin. The expression of MHC and ICAM 1 below was determined with the FACS. The results are given in the Figure as mean fluorescence intensity of a representative experiment.

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

Figure 4 Effect of various hepapins and glycosa ino-glycans on the MHC and ICAM 1 expression of cells stimulated with IFM and HUVEC cells stimulated with IFN and (10 ng / ml for 72 hours, solid bar) or unstimulated (striped bar ) were incubated with various heparins or glycosamino-glycans at a concentration of 3 mg / ml during the stimulation period The expression MHC and ICAM 1 was then determined with the FACS. Figure 4a shows the MHC Class 1 expression, Figure 4b shows the MHC Class II expression, and Figure 4c shows the ICAM I expression. The results are given in the figure as mean fluorescence intensity +/- 2 SD.

Figure 5 The effect of various heparmas and glycosaminoglycans on the MHC and ICAM 1 expression of PTEC cells stimulated with IFN and PTEC cells stimulated with IFN y (10 ng / ml, for 72 hours, solid bar) or unstimulated (striped bar ) were incubated with various heparins or glycosamino-glycans at a concentration of 3 mg / ml during the stimulation period The MHC expression and ICAM 1 was then determined with the FACS. Figure 5a shows the MHC Class I expression, Figure 5b shows the MHC Class II expression and Figure 5c shows the ICAM expression 1. The results are given in the Figure as mean fluorescence intensity of a representative experiment.

Figure 6 15 Comparison of GAG 6-8 and heparin in its effect of inhibiting the MHC and ICAM 1 expression of FTEC cells stimulated with IFN y (10 nq / ml, for 72 hours) The PTEC cells were incubated with various concentrations of GAG 6 (), GAG 7 (), GAG 8 () and heparin () during the 20 period of stimulus. The expression MHC and ICAM 1 was then determined with the FACS The expression of these antigens was also determined in the absence of GAG (-GAG) or in the absence of IFN and (-IFN) Figure 6a shows the MHC Class I expression, the Figure 6B shows the MHC Class II expression and Figure 6c shows the - ,,,,, ^., ^ »^^» -. , "ICAM expression 1. The results are given in the Figure as mean fluorescence intensity +/- 2 SD.

Figure 7 Effect of NaCl03 on the extension of the MHC expression stimulus and ICAM 1 in PTEC cells caused by IFN and. The PTEC cells were treated one day before the stimulation with IFN and with 15 mM NaCl03 (solid bar) or 150 mM NaCl (striped bar) as control os olar.Then, the cells with the various concentrations of IFN and stimulated for 72 hours in the presence of the same concentration of NaC103 or NaCl The MHC and ICAM 1 expressions of three replicate cultures were then determined with the FACS Figure 7a shows the MHC Class I expression, Figure 7b shows the MHC Class expression II Figure 7c shows the expression ICAM 1. The results are given in the figure co or mean fluorescence intensity +/- 2 SD An asterisk means p <0.05 and two asterisks mean p <0.01 in the Student T test.

Figure 8 Linkage of 1SIFN and heparin, Fragmin and various other glycosamino-glycans. Hepapna (Hep), glycosami.no-glycan (GAG) 1 to 8 and Fragmin iFragm) were stained on a nitrocellulose filter which was produced as described above. The nitrocellulose strips were incubated with I35IFN and in the presence or absence of a 3000-fold excess of heparin.

Claims (2)

1. - Protective solutions of organs containing a quantity of the following components, 5 effective to maintain cell integrity and cell vitality: a) 10 mg / l to 10,000 mg / l of glycosaminoglycans pol i-sulfated with a sulfur content of at least 12.5% by weight, 10 b) 5 to 10 g / L of ethyl hydroxy-starch.

2. The organ-protecting solution according to claim 1, which contains: 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 ethyl hydroxy-starch, c) 5 to 100 mmol of raffinose. 3 - The organ protecting solution according to claims 1 and 2 containing: a) 10 mg / l to 10,000 mg / l of polysulino glycans pol i-sulfated with a sulfur content of at least 12.5% in weight, b) 5 to 100 g / l of hydroxy-ethyl starch, c) 5 to 100 mmoi of raffinose 25 d) 5 to 40 mmol of KH2P04 . ^^. «« ¡¡¡¡¡¡¡¡¡¡^ ^ ^ ^ ^ -jachauma-e) 1 to 50 mmol of MgSO "f) 1 to 50 mmol of adenosine g) 0.5 to 5 mmol of allopurinol h) 1 to 10 mmol of glutathione 4. The application of protective organ solutions according to claims 1 to 3, for the manufacture of pharmaceutical preparations for inhibiting up-regulation of MHC Class I and MHC Class II proteins and ICAM 1 induced by interferon and. 5. The application of protective organ solutions according to claim 4 for the treatment and prevention of diseases associated with ascending uptake of MHC Class I and MHC Class II proteins and ICAM 1 induced by methylferon and, 6. The application of protective solutions of organs according to claim 4 or claim 5 for treatment of transplant patients 7.- The application of protective solutions of 20 organs according to claim 4 or claim 5 for protection and storage of organs to be used for transplants. 25