PROCESS FOR PREPARING GD2 GANGLIOSIDE AND O-ACETYL-GD2 GANGLIOSIDE

Abstract: The invention relates to a process for isolating and purifying economically worthwhile quantities of GD2 ganglioside and O-acetyl-GD2 ganglioside from fish liver, in particular from the salmon (Salmonidae) and cod (Gadidae) families.

Description: Process for preparing GD2 ganglioside and 0-acetyl-GD2 ganglioside P 42 42 890 - 18.12.1992 The invention relates to a novel process for preparing GD2 and 0-acetyl-GD2 gangliosides from the liver cells of fish, especially from the salmon (Salmonidae) and cod (Gadidae) families. The invention further relates to the use of fish liver for obtaining these gangliosides for use in the diagnosis and therapy of tumours. Gangliosides represent an important class of carbohydrate-rich glycolipids of exceptional size and complexity. Gangliosides are usually found on the surface of cell membranes. Examples of such gangliosides are GM1, GM2, GM3, GD3, 0-acetyl-GD3, GD2, 0-acetyl-GD2, GDla or GDlb (Fig. 1). Although they can also be found on normal tissue, they are nevertheless significantly increased in number in tumour tissue. GD2 ganglioside, for example, can be found in concentrated form in tissue from brain tumours, neuroblastomas, retinoblastomas, small-cell lung carcinomas and a series of types of melanomas (e.g. Cheung et al. (1985), Cancer Res. 45, 2642; Hakomori et al. (1989), Adv. Cancer Res. 52, 257). 0-Acetyl-GD2 was recently detected in human tumours (Ye et al. (1992), Int. J. Cancer 50, 197; Sjoberg et al. (1992), J. Biol. Chem. 267, 16200). Owing to their preferred occurrence in carcino¬ genic tissue, these gangliosides are also of considerable importance in the therapy and diagnosis of tumours and in immunology. Thus, a series of antibodies which are directed against individual representatives of these gangliosides are already known. For example, the murine monoclonal antibody ME 361 (EP 0280 209) is directed, in particular, against GD2 and 0-acetyl-GD2 antigen. For the said uses, and also for characterizing antibodies which are to be developed, it is necessary to have available sufficient quantities of the corresponding tumour-associated antigens. Such compounds can only be isolated with difficulty from the appropriate tumour tissue and then only in small quantities. In addition to this, tumour tissue is in scarce supply. Thus, GD2 ganglioside is only available in very small quantities at very high prices or has to be obtained from tumour tissue. In principle, chemical synthesis represents a further method for obtaining the desired gangliosides. However, such syntheses have the disadvantage that they take place by way of many intermediate steps, some of which are elaborate, with attention also having to be paid to the stereochemistry. This has the consequence that the yields are for the most part modest and do not repay the high level of input involved: in addition, there has thus far been no report of a completely synthetic preparation of GD2 ganglioside. GD2 ganglioside can be prepared send-synthetically or enzymically, with relatively low yields, from GDlb ganglioside, which in turn can be obtained from tumour tissue. Recently, there have been suggestions that it might be possible to isolate some of the gangliosides mentioned from non-tu orous tissue, e.g. from brain tissue, in useful quantities. Thus, the isolation of four neutral and some acidic glycosphingolipids (GM3, GM4, GM2 and GDla) from the liver of the Dover sole (Parophrys vetulus) has been reported (Ostrander et al. (1988), J. Biol. Chem. 263, 3103 and 18716). The same group also succeeded in isolating GM3, GM2, GDla and 9-0-acetyl-GD3, but not GD2 ganglioside or O- acetyl-GD2 ganglioside, from the liver of rainbow trout (Ostrander et al. (1991), Arch. Biochem. Biophys. 284, 413). The object therefore was to make available gangliosides of the GD2 type in a simple and cost-effective manner and in economically interesting quantities. It has now been found that GD2 ganglioside and O- acetyl-GD2 ganglioside (in addition to 0-acetyl-GD3 ganglioside) can be obtained, by methods known per se, from the liver of fish in a simple manner and in economically worthwhile yields. This is to be adjudged as being very surprising, since, as cited above, it was not possible to isolate these gangliosides from fish liver using a process which was only slightly modified. The invention consequently relates to a process for preparing GD2 ganglioside and 0-acetyl-GD2 ganglioside by extracting ganglioside-containing animal cells and purifying by means of chromatographic methods, which process is characterized in that fish liver cells are employed. In this context, the qualitative and quantitative yields vary from species to species. The invention consequently relates, in particular, to a corresponding process in which liver cells of fish from the salmon (Salmonidae) and cod (Gadidae) families are employed. The process according to the invention can be carried out very simply and rapidly. The invention thus relates to a process which is characterized in that the isolation and purification are carried out in accordance with the following steps: (a) extraction of the liver tissue with at least one organic solvent or solvent mixture, preferably with a solvent mixture which forms two phases, and (b) purification of the resulting extract using at least one chromatographic step, preferably ion-exchange chromatography. In a preferred embodiment, an additional extraction with acetone is introduced before the extraction step and the ion-exchange chromatography is followed by molecular sieving chromatography or adsorption chromatography. The invention additionally relates to the use of liver tissue from the salmon or cod fish families for obtaining GD2 ganglioside and 0-acetyl-GD2 ganglioside. The figures included in the application are explained below. Fig. 1: Structural formulae of some important tumour- associated gangliosides. Fig. 2: (a) TLC Plates: HPTLC silica gel, Eluent: Chloroform/methanol/O.02% CaCl2 in water: 55/45/10, Staining: Resorcinol (gangliosides), 5 naphthol (sugars) ; (A) : Extract after ionexchanger (B) : GD2 ganglioside, standard (Rf = 0.11) (C): 0-Acetyl-GD3, standard (Rf = 0.28) One of the bands between GD2 and 0- 10 acetyl-GD3 represents 0-acetyl-GD2 ganglioside. (b) TLC immunostaining (MAb ME 361-antigen complex) 15 The monoclonal antibody ME 361 (EP 0280209) binds specifically to GD2 ganglioside and 0-acetyl-GD2 ganglioside (as well as to GD2 lactone) . Conditions: as in (a); 20 (A) : Mixture containing 0-acetyl-GD3 (reaction negative) , (B) : Extract after ionexchanger (1): GD2 complex (Rf = 0.085), (2) : 0-acetyl-GD2 complex 25 (R f = 0.32), (3) : GD2 lactone complex . (R f = 0.32) , (C): Standard, GD2 complex (Rf = 0.085). 30 (c) TLC immunostaining (MAb ME 311-antigen complex) The monoclonal antibody ME 311 binds specifically to GD2 ganglioside and O- acetyl-GD2 ganglioside (as well as to GD2 35 lactone) . Conditions: as in (b) ; (A) : Standard 0-acetyl-GD3 complex (R f = 0.23) (B) : Extract after ion exchanger (1): 0-acetyl-GD3 complex (R f = 0.23) (C) : Mixture containing GD2 standard (reaction negative) . Fig. 3 HPLC chromatography Column: Lichrosorb-NH2® (E. Merck, Darmstadt), Conditions: Buffer A: 20 mM phosphate/acetonitrile Buffer B: 50 mM phosphate/acetonitrile Flow: 1 ml/min A = GD2, B = 0-acetyl-GD2-containing fraction, C = 0-acetyl-GD3 + GD2 lactone. The starting material for the process according to the invention is fish liver. The liver can be used fresh or in the deep-frozen state. Preferably, fresh fish liver is employed. In principle, .all conventional fish species are suitable, with the ganglioside content varying in depen¬ dence on family or species. Those fish species are preferred which have a high content of gangliosides in the liver. Fish from the salmon (Salmonidae) and cod (Gadidae) families are preferred. Trout, which have a particularly high content of glycolipids in their liver tissue, are particularly preferred. The process according to the invention is prefer¬ ably carried out using 500 to 5000 g of fish liver. For this purpose, liver is homogenized in a customary manner. Those solvents or solvent mixtures are expediently employed as extracting agents which accord both with the polar and the non-polar character of the gangliosides. Preferably, mixtures of chlorinated hydrocarbons, such as, for example, dichloromethane, chloroform, carbon tetrachloride, dichloroethane or trichloroethane, and lower alcohols, such as, for example, methanol, ethanol, n- or iso-propanol or n-, sec- or tert-butanol, are employed, if desired in the presence of water. However, appropriate mixtures are also suitable which, instead of the chlorinated hydrocarbons, contain liquid hydrocarbons, such as, for example, hexane or cyclohexane, or ethers, such as, for example, diethyl ether, methyl ethyl ether or methyl tert-butyl ether (MTB ether) . In this context, the composition of the solvent mixture is preferably to be selected such that a two-phase partition can be achieved. Particularly preferred extraction mixtures are chloroform/methanol/water, for example in a ratio of 4:8:3, or else MTB ether/n- butanol/water, for example in a ratio of 6:4:5. In accordance with the invention, the extraction is carried out once to three times. In a preferred embodiment of the process, the described extraction is preceded by an extraction of the liver cells with acetone. For this, fish liver is homo¬ genized with acetone. The supernatant is then separated off and discarded and the tissue residue is extracted as described above. When extraction has taken place, the proportion of water is preferably increased in accordance with the Folch partition (Folch-Pi et al. (1951), J. Biol. Chem 191, 833). A two-phase-forming mixture having the composition chloroform/methanol/50 mM CaCl2 in water (4:8:5-6) is particularly favourable. The more polar upper phase contains gangliosides such as GM3, GD2, GD3, GDla, O- acetyl-GD2 and 0-acetyl-GD3 and is accordingly used for further purification. If ether-containing or hydrocarbon- containing extraction agents are used, it is the lower (more polar) phase which is used for further processing. The ganglioside-containing phases, obtained in this way, are concentrated, in accordance with the invention, and then desalted by standard methods. Further purification is preferably effected by means of ion-exchange chromatography, for example using DEAE. The ganglioside mixture, which has been loaded on the column, is fractionated and/or eluted by means of a salt gradient. An example of a step-wise salt gradient which may be mentioned is (1) 0.04 M ammonium acetate/methanol, (2) 0.15 M ammonium acetate/methanol and (3) 0.45 M ammonium acetate/methanol. The appropriate fractions are collected and analysed (see below). In accordance with a preferred embodiment, the fraction which contains the desired gangliosides and which has previously been desalted by known methods is further purified by means of molecular sieve chromatography or adsorption chromatography. For example, a silica gel column, which is eluted, for example, by means of a chloroform/methanol/water gradient, can be employed. After this second separation, the desired gangliosides are obtained in pure form. If required, the individual fractions can be purified to an even higher degree by (preparative) HPLC, for example on silica gel coated with amino groups. The gangliosides which have been isolated and purified in this way can be characterized in more detail using the. following standard techniques, which are described in the literature: (1) Chemical characterization: periodate-resorcinol assay for determining bound sialic acid, (2) TLC (thin-layer chromatography): resorcinol staining (staining of gangliosides), naphthol staining (sugar staining) , (3) TLC immunostaining and (4) HPLC. More precise information can be found in the corresponding places in the text. The above-described process according to the invention provides the following advantageous results: On average, 500 to 750 mg of purified ganglioside are obtained from 1 kg of fish liver. Of this amount, 25 to 50 mg are due to 0-acetyl-GD3 ganglioside (5-10%), 4 to 7 mg to GD2 ganglioside (1-3%) and 1 to 3 mg to O-acetyl- GD2 ganglioside (< 1%), inter alia. The yields vary depending on the species of fish used. The highest yields of GD2 and 0-acetyl-GD2 are obtained by using fish from the ' salmon family, in parti¬ cular trout. The liver of these fish is thus currently the only truly worthwhile source of GD2 ganglioside and 0- acetyl-GD2 ganglioside, apart from the existence (in very small quantities) of these compounds in scarce tumour tissue. Since, in practice, it is almost impossible to prepare monoacetylated gangliosides by chemical means, the described process is also a very simple and cost-effective way of preparing 0-acetyl-GD2. Example 1: 500 g of fresh trout liver are homogenized together with 1.5 1 of acetone. The homogenate is centri- fuged and the supernatant is decanted off. The remaining tissue is treated a further two times with acetone in a corresponding manner. The tissue residue is then extracted twice with a mixture consisting of 5 1 of chloroform/methanol/water in the ratio 4:8:3. A 50 mM CaCl2 solution is then added such that a two-phase mixture of chloroform/methanol/water in the ratio 4:8:5.6 (v/v) is finally obtained. The upper phase, which mainly consists of methanol and water, is separated off. It contains gangliosides and long-chain glycosphingolipids. The combined upper phases are concen¬ trated, dialysed and, where appropriate, freeze-dried. Example 2: 1 kg of trout liver is extracted in the manner described with the solvent mixture mentioned in Example 1, but the prior extraction of the liver tissue with acetone is not carried out. Example 3 : About 5 kg of cod liver are homogenized in 7 litres of acetone in accordance with Example 1. The mixture is then filtered with suction and the residue is washed with a further 15 litres of acetone. 8.35 1 of MTB ether and 5.57 1 of n-butanol in the ratio 6:4 (v/v) are added to the remaining residue and the mixture is stirred for 1 hour. Subsequently, 7 1 of a 50 mM solution of NaCl in water are added, such that a final ratio of 6:4:5 (v/v) is obtained. The extraction is repeated twice again. The combined lower phases, which contain gangliosides, are desalted and concentrated as described above. Example 4: 0.25 kg of carp liver is homogenized and extracted in analogy with Example. 3. Example 5: The freeze-dried residue according to Examples 1 to 4 is taken up in 45 ml of chloroform/methanol/water (30:60:8 v/v) and loaded onto a DEAE column (DEAE Sepharose CL-6B, from Pharmacia, 50 x 3 cm) . The sample is washed in with about 1 1 of the same mixture. Subsequently, the column is washed with about 1,200 ml of methanol. During this process, unbound neutral glycolipids are eluted. There then takes place a step-wise elution of the gangliosides using (a) 1,200 ml of 0.04 M ammonium acetate/methanol, (b) 1,200 ml of 0.15 M ammonium acetate/methanol and (c) 1,200 ml of 0.45 M ammonium acetate/methanol. The gangliosides GD2, GD3, 0-acetyl-GD2 and 0-acetyl-GD3 are contained in the 0.15 M fraction. The fractions are desalted and concentrated as described. Subsequently they are subjected to analytical characterization as indicated above, Example 6: The residues of the fractions according to Example 5 are dissolved in 0.5 ml of chloroform/methanol/water (65:2:3 v/v) and loaded onto a silica gel column (Si 60, 70 x 1.5 cm). Elution takes place step-wise: (a) 95 ml of chloroform/methanol/water (65:32:3) (b) 100 ml of chloroform/methanol/water (60:37:3) (c) 160 ml of chloroform/methanol/water (40:57:3). The eluate is collected in fractions and the individual fractions are subjected to analytical characterization, as indicated. The purified gangliosides are determined quantitatively using standard methods.

Claims: Patent Claims 1. Process for preparing GD2 ganglioside and 0- acetyl-GD2 ganglioside by extracting ganglioside-containing animal cells and purifying by means of chromatographic methods, characterized in that fish liver cells are employed. 2. Process according to Claim 1, characterized in that liver tissue of fish from the salmon family (Salmonidae) is employed. 3. Process according to Claim 1,' characterized in that liver tissue of fish from the cod family (Gadidae) is employed. 4. Process according to Claim 2, characterized in that liver tissue of trout is employed. 5. Process according to one of Claims 1 to 4, characterized in that the isolation and purification are carried out in accordance with the following steps: (a) extraction of the liver tissue with at least one organic solvent or solvent mixture, (b) purification of the extract from (a) using at least one chromatographic step. 6. Process according to Claim 5, characterized in that a treatment of the liver tissue with acetone precedes steps (a) and (b) . 7. Process according to Claim 5 or 6, characterized in that step (a) is carried out at least once using a two- phase-forming solvent mixture, if desired in the presence of water. 8. Process according to Claim 7, characterized in that a chloroform/methanol/water mixture or a methyl tert- butyl ether/n-butanol/water mixture is employed. 9. Process according to one of Claims 5 to 8, characterized in that step (b) comprises an ion-exchange chromatography step and a molecular sieve chromatography step. 10. Use of liver tissue from the salmon or cod families of fish for obtaining GD2 ganglioside and 0- acetyl-GD2 ganglioside.

Inventors: AVEMARIE NORBERT, KORDOWICZ MARIA, AVEMARIE, NORBERT, KORDOWICZ, MARIA
Applicants: AVEMARIE NORBERT, KORDOWICZ MARIA, MERCK PATENT GMBH, AVEMARIE, NORBERT, KORDOWICZ, MARIA
Application year: 1993
Main classification: C07H1
IPC codes: C07H1, C07H15