Process for producing polyvalent and physiologically degradable carbohydrate receptor blockers by enzymatic glycosylation reactions, and uses thereof for manufacturing carbohydrate components

The present invention relates to a production process by enzymatic glycosylation reactions for polyvalent carbohydrate receptor blockers on a polymer basis, which do not cause intolerance reactions in vivo, neither in their entirety nor in the form of degradation products, and to their use for the production of carbohydrate building blocks.

The importance of carbohydrates as information carriers in physiologically relevant recognition processes has been investigated and decoded in recent years. The arrangement on the cell surface in the form of ligands enables them, due to their binding to specific receptors, to play a decisive role in intercellular communication and thus in intercellular recognition processes. Carbohydrate ligands on cell surfaces are recognition domains for viruses, bacteria, toxins and lectins. Therefore, they play a crucial role, for example in bacterial and viral infections and the initiation of inflammatory processes, such as rheumatoid arthritis, allergies, post-infarct syndrome, shock, stroke, sepsis. Studies have shown that in inflammatory processes, the selectin expressed by endothelial cells mediates in vivo the attachment of leukocytes to the focus of inflammation via a carbohydrate ligand.

Sialylated and / or fucosylated carbohydrates such as Sialyl-Lewis X and Sialyl-Lewis A are of particular importance for cell adhesion.

The therapy of inflammatory diseases with free oligosaccharides, which are supposed to bind to receptors instead of the natural ligands, fails due to the very high amounts of the oligosaccharide to be administered, since the affinity between receptor and oligosaccharide is low (K._D ~ 10^-4M in the interaction between a monovalent galactoside and the corresponding lectin DT Connolly et al. J. Biol. Chem.<u>257</u>, 939, (1982)).

Divalent structures with better binding to the respective receptor are described by Wong et al. (J. Am. Chem. Soc.<u>115</u>, 7549 (1993) and in US 5,254,676.

It is also known that an increased interaction between receptor and ligand is achieved by coupling several ligands on one surface. Using the example of the viral protein hemaglutinin, which binds to neuraminic acid on the cell surface, it was possible to show how, by using a polymer, this polyvalent effect has a significant effect on the ligand-receptor interaction (monovalent K_D = 2x20^-4M, polyvalent K_D = 3x10^-7M, A. column stone et al. J. Am. Chem. Soc.<u>113,</u> 686 (1991)).

Liposomes (N. Yamazaki, Int. J. Biochem. <u>24,</u> 99 (1991); WO 91/19501; WO 91/19502), polyacrylamides (RC Rathi et al. J. Polym. Sci .: Part A: Polym. Chem.<u>29,</u> 1895 (1991), S.-I. Nishimura et al. Macromolecules<u>24·</u> 4236 (1991)), polylysine or sulfated polysaccharides. These polyvalent structures either have little disadvantage in vivo or are incompatible in vivo due to their breakdown into toxic metabolites. With polylysine or sulfated polysaccharides, non-specific interactions with cell surface structures occur. The European laid-open publications 0 089 938, 0 089 939 and 0 089 940 describe carbohydrate compounds of different chain lengths which are identical to the ligands located on cell surfaces or the receptors located on microorganisms. The idea of ​​the invention is to block the receptors on the microorganisms by the carbohydrate compounds in vitro and in vivo in order to be able to diagnose and treat diseases. The carbohydrate compounds can be coupled to a carrier. This is used, among other things, for the production of antibodies. WO 92/02527 also discloses an oligosaccharide component which is coupled to a solid support and which serves to diagnose inflammatory processes. The solid carrier is inert towards physiological systems, so it is not degraded physiologically.

In contrast, EP 0 601 417 A2 discloses a physiologically degradable polyvalent carbohydrate receptor blocker on a polymer basis which carries oligosaccharide components on the polymer surface. Improved effectiveness as a medicament is brought about by the increased interaction of the carbohydrate building blocks which are polyvalent on the polymer surface with receptors and by blocking specific structures.

The carbohydrate receptor blocker described in said EP 0 601 417 A2 is physiologically compatible and preferably has a molecular weight of &lt;70 kD.

Specifically, the physiologically compatible and physiologically degradable polymer-based carbohydrate receptor blocker disclosed in EP 0 601 417 A2 has the following structure:

Carbohydrate side chains - spacers - hydrophilic, biodegradable polymer (if necessary) effect enhancer, wherein the carbohydrate side chains consisting of 1 to 20 naturally occurring same or different monosaccharide building blocks are coupled to a hydrophilic, biodegradable polymer via one or more bifunctional spacers of natural or synthetic origin, the hydrophilic, biodegradable polymer possibly also being linked to an action enhancer, which consists of one or more groups with hydrophobic, hydrophilic or ionic properties, is a crosslinker or a solubility improver.

The carbohydrate portion of the carbohydrate receptor blocker according to EP 0 601 417 A2 can consist, for example, of the following sugar residues:<ul id="ul0001" list-style="none" compact="compact"><li>Galβ1-4GlcNAc-;</li><li>Galβ1-3GlcNAc-;</li><li>SAT<i>α</i>2-6Galβ1-4GlcNAc-;</li><li>SAT<i>α</i>2-3Galβ1-4GlcNAc-;</li><li>SAT<i>α</i>2-3Galβ1-3GlcNAc-;</li><li>Galβ1-4 (Fucα1-3) GlcNAc-;</li><li>Galβ1-3 (Fucα1-3) GlcNAc-;</li><li>SAα2-3Galβ1-3 (Fucα1-4) GlcNAc-;</li><li>SAα2-3Galβ1-4 (Fucα1-3) GlcNAc-;</li></ul>

Further examples of preferred embodiments of the carbohydrate portion are: Sialyl-Lewis X, Sialyl-Lewis A, VIM-2 and the following blood group determinants Lewis A, B, X, Y and A-type¹, A type², B-type¹, B-type² and H type¹, H type² (RU Lemieux, Chem.Soc.Rev., 1978, p. 423 and 1989, p. 347)

Examples of the particularly preferred embodiment of the carbohydrate portion are sialyl Lewis X, sialyl Lewis A or VIM-2.

The formula of Sialyl-Lewis X is: NeuNAcα2-3Galβ1-4 (Fucα1-3) GlcNAc and of Sialyl-Lewis A: NeuNAcα2-3Galβ1-3 (Fucα1-4) GlcNAc. The formula of VIM-2 is: NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4 (Fucα1-3) GlcNAc;

The aforementioned EP 0 601 417 A2 also discloses a method for producing the carbohydrate receptor blocker.

The carbohydrate receptor blocker is synthesized on a laboratory scale using the method described. This means that the carbohydrate receptor blocker according to the invention is synthesized in milligrams to grams, while the intermediates necessary for its synthesis, ie the hydrophilic biodegradable polymer, the bifunctional spacer and the activity enhancer can be produced in grams to kilograms. An exception is the carbohydrate content, it can only be synthesized in milligrams up to one gram. The synthesis concepts for the production of oligosaccharides which are known from the literature are designed here in such a way that after a reaction which usually does not proceed with quantitative yield, the product mixture obtained is purified by column chromatography on silica gel. This cleaning process is generally too expensive and complex to produce quantities which correspond to industrial requirements and is used at most for cleaning end products or valuable intermediate products. In addition, heavy metal compounds are very often used for the synthesis of oligosaccharides. Their use for the synthesis of substances with a pharmaceutical effect is very questionable with regard to the future approval of the carbohydrate receptor blocker as a drug.

In the process described, the desired oligosaccharide is only linked to the biodegradable polymer by means of the spacer after it has been built up via numerous chemical and / or chemoenzymatic synthesis steps. This synthesis is very lengthy and difficult because of the problems with the construction of oligosaccharides known to the person skilled in the art (protective groups, formation of anomers, poor yields in glycosylation reactions, non-stereoselective glycosylation, numerous steps).

In view of the very high costs of successive reaction and purification steps in the chemical and / or chemoenzymatic synthesis of carbohydrate building blocks, some solid-phase syntheses have also recently been proposed.

In contrast to the established solid phase syntheses of oligonucleotides and peptides, the chemical synthesis of oligosaccharides on polymeric solid phases is very difficult due to the multitude of functionalities and the need for stereoselective formation of the glycosidic bond. Danishefsky et al. (Science,<u>260</u>, 1307 (1993)) link 3,4-protected glycal via silyl ether bonds with a polystyrene copolymer. This is activated as an epoxy and can be linked to other glycal acceptors to form the oligosaccharide.<u>Douglas</u> et al. (J. Am. Chem. Soc. 113, 5095 (1991) describe the synthesis of di- and trisaccharides on a PEG-bound glucose building block.<u>Zehavi</u> (J. Am. Chem. Soc. 95, 5673 (1973)) uses a photosensitive styrene-divinylbenzene copolymer as the polymeric solid phase. The protected oligosaccharide is split off from the polymer by irradiation.

Disadvantages of the chemical solid phase synthesis of oligosaccharides are:<ul id="ul0002" list-style="dash" compact="compact"><li>e.g. T. incomplete glycosylation reactions</li><li>only a few glycosylation units on both the donor and the acceptor side are suitable.</li><li>The need for protective groups adapted to the respective reaction.</li></ul>

These disadvantages of the chemical synthesis of oligosaccharides on polymer matrices are avoided by the enzymatic glycosylation. The reactions are absolutely stereoselective without protective groups and can be used very widely as glycosyl donors due to the large number of available glycosyltransferases and nucleotide-activated sugars. Nunez and Barker (Biochemistry <u>19,</u> 489 (1980) the enzymatic galactosylation of N-acetylglucosamine bound to agarose via a hexanolamine spacer. However, very large amounts of enzyme galactosyltransferase are used. U. Zehavi describes the enzymatic galactosylation with galactosyltransferase on light-sensitive, both water-insoluble and water-soluble polymers. The transfer yields are very low with &lt;1 to a maximum of 36% (U. Zehavi et al., Carbohydrate Res.<u>124,</u> 23 (1983), U. Zehavi et al. Carbohydrate Res.<u>128</u>, 160 (1984), U. Zehavi Reactive Polymers <u>6,</u> 189 (1987) U. Zehavi et al. Glycoconjugate J.<u>7,</u> 229 (1990), U. Zehavi Innovation Perspect. Solid phase synthesis collect. Paper, Int. Symp. 1990, 389 to 396). The disaccharide obtained on the polymer is exposed to light or via an enzyme (U. Zehavi et al. Carbohydrate Res.<u>133,</u> 339 (1984)) split off from the polymer. Nishimura (Nishimura et al. Biochemical and Biophysical Research Comm. <u>199</u>, 249-254 (1994))) describes the enzymatic preparation of a water-soluble polyacrylamide with 3'-sialyl-N-acetyllactosamine side chains, a water-soluble polyacrylamide carrying N-acetylglucosamine being enzymatically glycosylated in stages. With low overall yields, however, low coverage densities are achieved with this method. A recent work (Wong et al. J. Am. Chem. Soc. 116, 1135 (1994)) describes the enzymatic synthesis of oligosaccharides on a modified silica gel. Due to the insolubility of the silica gel in the aqueous buffers required for the enzymatic carbohydrate synthesis and the low occupancy density with the GlcNAc building block linked via a peptide, only low glycosylation yields are achieved in all three reaction steps. After enzymatic cleavage of the peptide anchor, product mixtures with only 20% of the desired product and 45% of the starting material used are obtained.

WO 92/22661, WO 92/22565 and WO 92/22563 propose enzymatic glycosylations with a sialytransferase of disaccharides bound to an "unnatural carrier" (artificial carrier). A "non-natural carrier" is usually a high or low molecular weight carrier with antigenic properties, for example bovine serum albumin, KLH, HSA, diphtheria or tetanus toxin etc. or a solid carrier which is inert to physiological systems.

Starting from the prior art mentioned, it is an object of the present invention to provide a process for producing the polyvalent, physiologically compatible and physiologically degradable carbohydrate receptor blocker described at the outset, which process is characterized in that the carbohydrate portion of the receptor blocker is built up by enzymatic glycosylation reactions in aqueous buffer systems and homogeneous phase takes place directly on the biodegradable polymer, the yields of the glycosylation reaction are significantly improved compared to the yields of known processes and generally run quantitatively and the occupancy densities of oligosaccharide on the polymer are significantly increased, and to suggest the use of the carbohydrate receptor blocker prepared according to the invention for the production of the free oligosaccharides.

The object is achieved according to the invention by a method for producing a physiologically tolerable and physiologically degradable carbohydrate receptor blocker based on polymer<ul id="ul0003" list-style="none" compact="compact"><li>a) a hydrophilic, biodegradable polymer unit,</li><li>b) at least one di- or oligosaccharide unit and</li><li>c) at least one bifunctional spacer, via which the di- or oligosaccharide units are linked to the polymer unit,</li></ul> characterized in that an acceptor is first produced by chemically linking a mono- or oligosaccharide, the spacer and the hydrophilic, biodegradable polymer, after which one or more further monosaccharide units are added by enzymatic glycosylation.

The enzymatic glycosylation reaction proceeds stereoselectively and with surprisingly high yields directly on the polymer and, since each gycosylation step proceeds quantitatively, can be repeated as often as desired with any donor. In this way, in contrast to the prior art, where the yields are low and product mixtures are formed in further glycosylation steps, the generation of polyvalent carbohydrate compounds by direct construction of the oligosaccharide structures on the polymer is very simple and possible in very high yields. Another advantage is the simple isolation and uniformity of the polyvalent carbohydrate compound.

The acceptor for the enzymatic glycosylation is produced by forming a covalent bond between a mono- or oligosaccharide and the bifunctional spacer, followed by covalent linkage of the mono- or oligosaccharide-spacer complex with the polymer.

The enzymatic glycosylation at the acceptor takes place in a homogeneous aqueous phase, preferably by means of nucleotide-activated carbohydrates as donors and glycosyltransferases.

The aqueous medium should consist of a buffer system which is adapted to the particular glycosyltransferase; the buffer system is preferably present in a concentration of 0.01 M to 1 M and advantageously contains the cations necessary for activating the particular glycosyltransferase.

The pH is between 6.0 and 8.5, preferably between 6.0 and 8.5, very particularly preferably between 7.0 and 7.5.

If the donor is added in equimolar amounts or in excess, alkaline phosphatase should be added to the reaction medium.

0.01 to 10 units of the glycosyltransferase are added to the reaction mixture.

The enzymatic glycosylation is carried out for 1 to 5 days at 10 to 40 ° C, preferably at 20 to 37 ° C, more preferably at 25 to 37 ° C.

The invention is explained in detail below:

1. SYNTHESIS OF THE ACCEPTOR FOR THE ENZYMATIC GLYCOSYLATION REACTION

The acceptor for the enzymatic glycosylation reaction consists of a mono- or oligosaccharide which is covalently linked to a biodegradable hydrophilic polymer via a spacer. The polymer can be provided with an activity enhancer. The acceptor is synthesized by methods known to those skilled in the art. The individual components of the acceptor are described below.

Biodegradable hydrophilic polymer:

By definition, the polymer consists of at least two identical or different monomer units which are linked linearly or branched to one another and can have a molecular weight distribution.

The polymer is preferably a polyamino acid as a polyamide or anhydride linked with a molecular weight less than or equal to 70 kD. The polymer preferably has a minimum size of 2 kD in order to achieve an increased residence time in the blood compared to low molecular weight carriers.

Polyamino acids which are particularly preferably suitable for the production of polymer-based carbohydrate receptor blockers are polyaspartamides, polysuccinimides, polyglutamates and polylysine fumaramides, such as, for example, poly-α, β- (2-hydroxyethyl) -D, L-aspartamide, poly-D, L-succinimide, polyglutamate, Poly-L-lysine methyl ester fumaramide, and their copolymers.

Biodegradable, hydrophilic polymer is produced by processes known to those skilled in the art. These are described, for example, in: HG Elias, Macromolecules, Vol. 1 and 2, Hüthig &amp; Wepf Verlag, Basel, Switzerland, 1991/92 or D. Braun, H. Cherdron, W. Kern, Internship in Macromolecular Organic Chemistry, Hüthig Verlag 1979.

For example, poly-D, L-succinimide (PSI) is produced according to the instructions by Neri et al., J. Med. Chem., 16, 893 (1973) by the action of 85% phosphoric acid on aspartic acid at temperatures of 160 ° C. - 180 ° C won. Polymer-analogous reaction of PSI with hydroxyethylamine at room temperature or slightly elevated temperature gives poly-α, β- (2-hydroxyethyl) -D, L-aspartamide (PHEA) (Neri et al., Ibid). The alcohol groups of the PHEA can be esterified using conventional methods (US 5,041,291). If PSI is partially reacted with ethanolamine, corresponding copolymers are obtained (US 5,229,469). The basic hydrolysis of PSI leads to polyaspartic acid (analogously to Giammona et al., Chem. Pharm. Bull. 37 (8), 2245 (1989)).

Analogously to the reaction with hydroxyethylamine, PSI can also be reacted with other amines (EP 0 548 794), as a result of which additional functional groups can be introduced which can act as enhancers.

Poly-L-lysine methyl ester fumaramide, as a further starting polymer, is produced by boundary phase polycondensation from L-lysine methyl ester and fumaric acid dichloride (US 4,834,248). The methyl ester groups can be reacted directly or after partial hydrolysis and subsequent activation, for example as p-nitrophenyl ester, with the mono-, di- and oligosaccharides containing amino groups.

Analogously, ie using p-nitrophenyl ester, polymeric carbohydrate receptor blockers based on polyglutarmates can be produced (polymer synthesis analogously: Anderson in "Macromolecules as Drugs and as Carriers for Biologically Active Materials" (Ed: DA Tirell), NY Acad. Sci., NY, 1985, pp. 67-75).

Spacer:

The covalent linkage of the polymer with the spacer or with a compound consisting of a covalently linked spacer and carbohydrate and a covalent connection between polymer and activity enhancer with the spacer or with a compound consisting of covalently linked spacer and carbohydrate takes place by reaction between a reactive group and an activated group. Both the reactive group at the end of the spacer or a compound consisting of a covalently linked spacer and carbohydrate and the activated group on the polymer side or a compound consisting of covalently linked polymer and effect enhancer as well as the activated group at the end of the spacer or a compound of covalently linked spacer and carbohydrate and the reactive Group on the side of the polymer or a compound consisting of covalently linked polymer and enhancer. The reaction between reactive and activated groups takes place according to methods known to those skilled in the art for alkylation, acylation or addition to a double bond. These methods are known to the person skilled in the art from the literature. (Larock, RC Comprehensive Organic Transformations, 1989, VCH publishing company Weinheim).

The spacer preferably has the formula I.         (Mono- or oligosaccharide) -O- [Q¹ - (CH₂)_p-Q²]_r- (polymer unit) I, in what mean<dl id="dl0001" compact="compact"><dt>Q¹</dt><dd>-CH₂- or<chemistry id="chem0001" num="0001"><img file="EP0804608B1_D0001.tif" /></chemistry></dd><dt>Q²</dt><dd>-NH- or<chemistry id="chem0002" num="0002"><img file="EP0804608B1_D0002.tif" /></chemistry></dd><dt>p</dt><dd>an integer from 1 to 6 and</dd><dt>r</dt><dd>1 or 2</dd></dl>

Carbohydrate portion of the acceptor

The carbohydrate portion of the acceptor for the enzymatic glycosylation reaction can come from natural sources or can be produced chemically, chemoenzymatically or enzymatically. Suitable natural sources of carbohydrates are known to the person skilled in the art and can be found in the biochemical literature. Established processes for the purification of oligosaccharides known to the person skilled in the art are also described there.

Methods for the chemical, enzymatic or chemoenzymatic synthesis of carbohydrates which are recognized by cell surface receptors are known to the person skilled in the art from the chemical literature and from review articles. For chemical synthesis eg Carbohydrate Research, Elsevier Science Publishers BU Amsterdam; Journal of Carbohydrate Chemistry, Marcel Dekker Inc. New York; H. Paulsen, Angew. Chem.<u>94.</u> 184 (1982) and <u>102,</u> 851 (1990); RR Schmidt Angew. Chem. 98, 213 (1987); H. Kunz Angew. Chem.<u>98</u>, 247 (1987). For enzymatic synthesis, eg Carbohydrate Research, Elsevier Science Publishers BU Amsterdam; Journal of Carbohydrate Chemistry, Marcel Dekker Inc. New York; Bednarski u. Simon Enzymes in Carbohydrate Synthesis, ACS Symposium Series 466 (1991); KGI Nilsson, Applied Biocatalysis<u>1991</u>, 117; S. David et al. Adv. Carbohydr. Chem. Biochem.<u>49</u>, 175: 1991; Y. Ichikawa et al. Anal. Biochem.<u>202</u>, 215 (1992); DG Drueckhammer et al. Synthesis<u>1991</u>, 499; EJ Toone et al. Tetrahedron<u>45</u>, 5365 (1989).

The mono- or oligosaccharides produced in this way can be obtained both with a free reducing end and in a spacer-linked form. The spacer is introduced by methods known to those skilled in the art for chemical or enzymatic glycosylation.

2nd ENZYMATIC GLYCOSYLATION

The process according to the invention for producing a polymeric carbohydrate receptor blocker by enzymatic glycosylation is described below:

The acceptor for the enzymatic glycosylation reaction obtained according to 1. consists of a mono- or oligosaccharide which is covalently linked to a biodegradable hydrophilic polymer via a spacer. The polymer can be provided with an activity enhancer. The enzymatic glycosylation of the acceptor is preferably carried out in a homogeneous, aqueous phase. Nucleotide-activated carbohydrates were preferably used as donors and glycosyltransferases as enzymes.

The acceptor is dissolved in an aqueous buffer system. The buffer is adapted to the particular glycosyltransferase and can consist, for example, of 0.01M to 1M cacodylate, HEPES, PIPES, MOPS, citrate, hydrogen carbonate, etc. It contains the cations necessary to activate the respective glycosyltransferase, for example Mn.

The pH is also adapted to the particular glycosyltransferase, it is between 6.0 and 8.5, preferably between 6.5 and 7.8, very particularly preferably between 7.0 and 7.5.

After the acceptor has been dissolved in the aqueous buffer system, the donor is added. This is a nucleotide activated sugar or an analog of a nucleotide activated sugar. Some of the nucleotide-activated sugars are commercially available, but they can also be prepared by methods known to those skilled in the art by chemical or enzymatic synthesis or isolated from natural sources. This also applies to the analogues. The donor is either added in a 1.1 to 2-fold excess or regenerated in situ by known methods (for example Y. Ichikawa et al. J. Am. Chem. Soc. 114, 9283 (1992), CH Wong et al. J. Org. Chem 57, 4343 (1992), Y. Ichikawa et al. J. Am. Chem. Soc. 113, 6300 (1991), CH Wong et al. J. Org. Chem. 47, 5416 (1982).

In enzymatic galactosylation, UDP-galactose is generally used as the donor. However, UDP-glucose can also be assumed, which can be epimerized enzymatically in situ to UDP-galactose by the enzyme UDP-galactose-4-epimerase (J. Thiem et al. Angew. Chem.<u>102</u>, 78 (1990)).

If the donor is used in the enzymatic glycosylation in an equimolar amount or in excess, it is necessary to enzymatically decompose the UDP released in the reaction by adding alkaline phosphatase in order to prevent inhibition of the glycosyl transferase (C. Unverzagt et al. J. Am. Chem Soc. <u>112</u>, 9308 (1990)).

Nucleotide-activated sugars are, for example, UDP-glucose, UDP-galactose, UDP-N-acetyl-glucosamine, UDP-N-acetylgalactosamine, UDP-glucuronic acid, CMP-neuraminic acid, GDP-fucose, GDP-mannose, dTDP-glucose, dUDP- Galactose. Methods known to those skilled in the art for the production of nucleotide-activated sugars are, for example: M. Kittelmann et al. Annals of the New York Academy of Sciences Vol.<u>672</u> Enzyme Engineering, pp. 444 to 450 (1992), S. Makino et al. Tetrahedron Lett. 34, 2775 (1993), TJ Martin et al. Tetrahedron Lett.<u>34</u>, 1765 (1993), European Patent Application 0 524 143 Al; K. Ikeda, Carbohydrate Res.<u>224</u>, 1992 (123), EL Kean Glycobiology 1, 441 (1991); Y. Ichikawa et al. J. Org. Chem.<u>57</u>, 1992, 2943, K. Adelhorst et al. Carbohydrate Research<u>242</u>, 69 (1993), RR Schmidt et al. Dear Ann. Chem.<u>1991</u>. 121, R. Stiller et al. Dear Ann. Chem.<u>1992</u>, 467; JE Heidlas et al. J. Org. Chem.<u>57</u>, 146 (1992), JE Heidlas Acc. Chem. Res.<u>25</u>, 307 (1992) ES Simon et al. J. Org. Chem.<u>55</u>, 1834 (1990), CH Wong et al. J. Org. Chem.<u>57</u>, 4343 (1992), JE Pallanca et al. J. Chem. Soc. Perkin Trans. 1<u>1993</u>, 3017.

0.01 to 10 units of the glycosyltransferase, which can transfer the respective nucleotide-activated sugar to the acceptor, are added to the acceptor and the nucleotide-activated sugar dissolved in the aqueous buffer system. Some of the glycosyltransferases are commercially available, can be isolated from natural sources or are available recombinantly. Glycosyltransferases that can be used for the enzymatic glycosylation in the sense of the method according to the invention are, for example<ul id="ul0004" list-style="none" compact="compact"><li>β-1,4Galactosyltransferase [R. Barker et al. J. Biol. Chem.<u>247</u>, 7135 (1972), CH Krezhorn et al. Eur. J. Biochem.<u>212</u>, 113 (1993),</li><li>Gal-β-1-4-GlcNAc-α-2-6 sialyltransferase [JC Paulson et al. J. Biol. Chem.<u>252</u>, 2363 (1977), H. Higa et al. J. Biol. Chem.<u>260</u>, 8838 (1985), J. Weinstein et al. J. Biol. Chem.<u>257</u>, 13835 (1982)],</li><li>Gal-β-1-3GalNAc-α-2-3-sialyltransferase [W. Gillespie et al. J. Biol. Chem.<u>267</u>, 21004 (1992)],</li><li>Gal-β-1-3 (4) -GlcNAc-α-2-3-sialyltransferase [J. Weinstein et al. J. Biol. Chem.<u>257</u>, 13835 (1982), M. Nemansky et al. Glycoconjugate J.<u>10</u>, 99 (1993)],</li><li>GalNAc-α-2-6 sialytransferase [HJ: Gross et al. Biochemistry<u>28</u>, 7386 (1989), N-acetylglucosaminyl transferases [R. Oehrlein et al. Carbohydrate Res.<u>244,</u> 149 (1993), T. Szumilo et al. Biochemistry<u>26</u>, 5498 (1987), O. Hindsgaul et al. J. Biol. Chem.<u>266</u>, 17858 (1991) GC Look et al. J. Org. Chem. 58, 4326 (1993)],</li><li>α-1-3-fucosyltransferase [BW Weston, J. Biol. Chem. <u>267</u>, 4152 (1992)],</li><li>α-1-2-fucosyltransferase [TA Beyer, J. Biol. CHem. <u>255</u>, 5364 (1980)],</li><li>α-3/4 fucosyltransferase [PH Johnson Glycoconjugate J. <u>9</u>, 241 (1992)],</li><li>α-1-2 mannosyltransferase [P. Wang, J. Org. Chem.<u>58</u>, 3985 (1993)], general: TA Beyer et al. Advances in Enzymology Vol. 52, 23 to 175 (1981), WO 93/13198</li></ul>

The enzymatic glycosylation is carried out for 1 to 5 days at 10 to 40 ° C., preferably at 20 to 37 ° C., particularly preferably at 25 to 37 ° C.

For working up, the product solution is after the reaction, recognizable by chromatographic methods (TLC, HPLC), against bidistilled. Water dialyses. The carbohydrate receptor blocker can then be further purified by chromatographic methods such as gel chromatography.

The method according to the invention is particularly suitable for producing carbohydrate receptor blockers with the following oligosaccharide or disaccharide units:<ul id="ul0005" list-style="none" compact="compact"><li>Galβ1-4GlcNAc-;</li><li>Galβ1-3GlcNAc-,</li><li>SAT<i>α</i>2-6Galβ1-4GlcNAc-;</li><li>SAT<i>α</i> 2-3Galβ1-4GlcNAc-;</li><li>SAα2-3Galβ1-3GlcNAc-;</li><li>Galβ1-4 &lt;Fucα1-3) GlcNAc-;</li><li>Galβ1-3 &lt;Fucα1-3) GlcNAc-;</li><li>SAα2-3Galβ1-3 (Fucα1-4) GlcNAc-;</li><li>SAα2-3Galβ1-4 (Fucα1-3) GlcNAc-;</li></ul>

Sialyl-Lewis X, Sialyl-Lewis A, VIM-2 and the following blood group determinants Lewis A, B, X, Y and A-type¹, A type², B-type¹, B-type² and H type¹, H type² (RU Lemieux, Chem. Soc. Rev., 1978, p. 423 and 1989, p. 347) and fernder Sialyl-Lewix X, Sialyl-Lewis A or VIM-2.

The formula of Sialyl-Lewis X is: NeuNAcα2-3Galβ1-4 (Fucα1-3) GlcNAc and of Sialyl-Lewis A: NeuNAcα2-3Galβ1-3 (Fucα1-4) GlcNAc. The formula of VIM-2 is: NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4 (Fucα1-3) GlcNAc;

In the following, the rules for the synthesis are described as an example.

Implementation of the polymer with the carbohydrate portion with a bifunctional spacer on the one hand and the enhancer on the other hand with the formation of covalent bonds:

Example 1: 1- (6-aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside

2-Amino-2-deoxyglucose hydrochloride is according to the instructions of RU Lemieux et al. (ACS Symp. Ser. 39, 90 (1976) by reaction with phthalic anhydride and subsequent reaction with acetic anhydride / pyridine in 1,3,4, 6-tetraacetyl-2-N-acetyl-2-deoxyglucose. Treatment with tin tetrachloride / thiophenol, according to Nicolaou et al. (J. Am. Chem. Soc. 112, 3695 (1990)) the corresponding 1-thiophenyl derivative. This is done according to the instructions of BA Silwanis et al. (J. Carbohydr. Chem. 10, 1067 (1991)) with 6- (N-benzyloxycarbonyl) aminohexanol. Analogous to the regulation by KC Nicolaou et al. (J. Am. Chem. Soc. 114, 3127 (1992)) the acetyl and phthaloyl protective groups are cleaved with hydrazine hydrate. Before the benzyl protective groups (H2 / Pd (OH) 2, MeOH) are split off, the free amino group is selectively acetylated in the presence of the free hydroxyl groups with excess acetic anhydride. 1- (6-Aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside is obtained

Example 2: Poly-D, L-succinimide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-β-D-glucopyranose (poly-D, L-succinimide- co-α, β-D, L-aspartamido-C

6

-GlcNAc)

PSI (500 mg, MW 24,000) is dissolved in 2 mL DMF and treated with 225 mg (0.71 mmol) 1- (6-aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside (GlcNAc-C₆-NH₂) added in 2.5 ml DMF. The mixture is 5.5 hours at room temperature under N₂ touched. It is then precipitated with 40 ml of 1-butanol and the polymer obtained is washed with methanol. After a second precipitation from DMF in 1-butanol, the mixture is washed again with methanol and then dried in an oil pump vacuum. Yield: 490 mg Degree of substitution according to NMR: 12.5%

Example 3: Poly-D, L-succinimide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-β-D-glucopyranose (poly-D, L-succinimide- co-α, β-D, L-aspartamido-C

6

-GlcNAc)

Analogously to Example 2, 320 mg of PSI (MW 24,000) are mixed with 300 mg of 1- (6-aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside (GlcNAc-C₆-NH₂) implemented and worked up by double precipitation from DMF with 1-butanol. Yield: 383 mg Degree of substitution according to NMR: 18.5%

Example 4: Poly-D, L-succinimide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-β-D-glucopyranose (poly-D, L-succinimide- co-α, β-D, L-aspartamido-C

6

-GlcNAc)

Analogously to Example 2, 480 mg PSI (MW 9 600) are mixed with 210 mg 1- (6-aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside (GlcNAc-C₆-NH₂) implemented and worked up by double precipitation from DMF with 1-butanol. Yield: 485 mg Degree of substitution according to NMR: 12.5%

Example 5: Poly-D, L-succinimide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-β-D-glucopyranose (poly-D, L-succinimide- co-α, β-D, L-aspartamido-C

6

-GlcNAc)

Analogously to Example 2, 300 mg PSI (MW 9 600) with 280 mg 1- (6-aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside (GlcNAc-C₆-NH₂) implemented and worked up by double precipitation from DMF with 1-butanol. Yield: 331 mg Degree of substitution according to NMR: 18%

Example 6: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 2 (200 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (81 mg) is added. After stirring for 16 hours under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 180 mg Degree of substitution according to NMR: 87.5% HEA, 12.5% ​​GlcNAc-C₆-NH₂

Example 7: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 3 (100 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (41 mg) is added. After stirring for 16 hours under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 112 mg Degree of substitution according to NMR: 81.5% HEA, 18.5% GlcNAc-C₆-NH₂

Example 8: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 4 (150 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (61 mg) is added. After stirring for 16 hours under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 147 mg Degree of substitution according to NMR: 87.5% HEA, 12.5% ​​GlcNAc-C₆-NH₂

Example 9: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 5 (150 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (61 mg) is added. After stirring for 16 hours under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 127 mg Degree of substitution according to NMR: 82% HEA, 18% GlcNAc-C₆-NH₂

Example 10: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 2 (170 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (43 mg) is added. After 4 hours stirring under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 160 mg Degree of substitution according to NMR: 61.5% HEA, 12.5% ​​GlcNAc-C₆-NH₂

Example 11: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 3 (120 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (30 mg) is added. After 4 hours stirring under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 126 mg Degree of substitution according to NMR: 45.5% HEA, 18.5% GlcNAc-C₆-NH₂

Example 12: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 4 (150 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (38 mg) is added. After 4 hours stirring under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 146 mg Degree of substitution according to NMR: 56% HEA, 12.5% ​​GlcNAc-C₆-NH₂

Example 13: Poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

PSI-co-aspartamido-C₆-GlcNAc from Example 5 (150 mg) is dissolved in 2 ml of DMF and freshly distilled hydroxyethylamine (38 mg) is added. After 4 hours stirring under N₂ is precipitated with 1-butanol at room temperature. The polymer is washed with methanol, in H₂O dissolved and freeze-dried. Yield: 143 mg Degree of substitution according to NMR: 50% HEA, 12.5% ​​GlcNAc-C₆-NH₂

Example 14: Poly-D, L-succinimide-α, β- (5-carboxypentyl) -D, L-aspartamide

500 mg PSI (MG 9 600) are dissolved in 2 ml DMF and treated with 1.38 g 6-aminohexanoic acid, dissolved in 8 ml formamide, and 1 ml triethylamine. The mixture is stirred at 45 ° C. for 13 hours and then precipitated with 1-butanol. After washing the polymer with methanol, it is taken up in H20 and freeze-dried. Yield: 350 mg Degree of substitution according to NMR: 8% aminohexanoic acid

Example 15: Poly-D, L-succinimide-co-α, β- (5-carboxypentyl) -D, L-aspartamide

500 mg PSI (MG 24,000) are dissolved in 2 ml DMF and 690 mg 6-aminohexanoic acid, dissolved in 4 ml formamide, and 1 ml triethylamine are added. The mixture is stirred for 3 d at room temperature and for 5 hours at 45 ° C. and then precipitated with 1-butanol. After washing the polymer with methanol in H₂O picked up and freeze-dried. Yield: 470 mg Degree of substitution according to NMR: 12.5% ​​aminohexanoic acid

Example 16: Poly-D, L-succinimide-co-α, β- (5-carboxypentyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2 -acetamido-β-D-glucopyranose (PCPA-co-aspartamido-C

6

-GlcNAc)

100 mg PCPA from Example 14 are in 2 ml H₂O dissolved and with 20 mg of 1. (6-aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside (GlcNAc-C₆-NH₂) offset. 4 times 20 mg 1 ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) are added over 36 hours. After dialysis and freeze-drying, the product remains. Yield: 112 mg Degree of substitution according to NMR: 8% GlcNAc-O (CH₂)₆NH₂

Example 17: Poly-D, L-succinimide-co-α, β- (5-carboxypentyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2 -acetamido-β-D glucopyranose (PCPA-co-aspartamido-C

6

-GlcNAc)

100 mg PCPA from Example 15 are in 2 ml H₂O dissolved and with 20 mg of 1- (6-aminohexyl) -2-deoxy-2-acetamido-β-D-glucopyranoside (GlcNAc-C₆-NH₂) offset. 20 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) are added 4 times over a period of 36 hours. After dialysis and freeze-drying, the product remains. Yield: 137 mg Degree of substitution according to NMR: 12.5% ​​GlcNAc-O (CH₂)₆NH₂

Enzymatic galactosylation

Example 18: Enzymatic galactosylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-GlcNAc

50 mg of the polymer from Example 2 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 55 mg UDP-glucose and 1 mg lactalbumin. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 8 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 74 mg Degree of substitution LacNAc according to NMR: 12.5%

Example 19: Enzymatic galactosylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-GlcNAc

50 mg of the polymer from Example 3 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 55 mg UDP-glucose and 1 mg lactalbumin. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 8 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 76 mg Degree of substitution LacNAc according to NMR: 18.5%

Example 20: Enzymatic galactosylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-GlcNAc

50 mg of the polymer from Example 4 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 55 mg UDP-glucose and 1 mg lactalbumin. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 8 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 77 mg Degree of substitution LacNAc according to NMR: 12.5%

Example 21: Enzymatic galactosylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-GlcNAc

50 mg of the polymer from Example 5 are dissolved in 10 ml 0.05M HEPES buffer pH 7.5 and with 2 mg MnCl₂ and 55 mg UDP-glucose and 1 mg lactalbumin. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 8 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 78 mg Degree of substitution LacNAc according to NMR: 18%

Example 22: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-a, bD, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β-D- Glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 6 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 70.6 mg Degree of substitution LacNAc according to NMR: 12.5%

Example 23: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β- D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 7 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 69 mg Degree of substitution LacNAc according to NMR: 18.5%

Example 24: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β- D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 8 are dissolved in 10 ml 0.05M HEPES buffer pH 7.5 and with 2 mg MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 55 mg Degree of substitution LacNAc according to NMR: 12.5%

Example 25: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β- D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 9 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 55 mg Degree of substitution LacNAc according to NMR: 18%

Example 26: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β- D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 10 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 54 mg Degree of substitution LacNAc according to NMR: 12.5%

Example 27: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β- D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 11 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 71 mg Degree of substitution LacNAc according to NMR: 18.5%

Example 28: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-2-deoxy-2-acetamido-β- D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 12 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 66 mg Degree of substitution LacNAc according to NMR: 12.5%

Example 29: Enzymatic galactosylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-β- D-glucopyranose (PHEA-co-aspartamido-C

6

-GlcNAc)

50 mg of the polymer from Example 13 are dissolved in 10 ml of 0.05M HEPES buffer pH 7.5 and with 2 mg of MnCl₂ and 40 mg UDP-glucose and 1 mg lactalbumin were added. After adding 2 U UDP-galactose-4-epimerase, 2 U galactosyltransferase and 40 U alkaline phosphatase (from calf intestine), the mixture is incubated at 25 ° C. for 7 days. After dialysis against water, it is freeze-dried and then chromatographed on Sephadex G 10. After freeze drying again, the product remains. Yield: 64 mg Degree of substitution LacNAc according to NMR: 18%

Enzymatic sialylation

Example 30: Enzymatic sialylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-LacNAc

35 mg of the polymer from Example 18 are dissolved in 2 ml 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 36.6 mg Degree of substitution 2,6-sialyl-LacNAc: 12.5% ​​(0.65 µmol / mg polymer)

Example 31: Enzymatic sialylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-LacNAc

30th mg of the polymer from Example 19 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 32 mg Degree of substitution 2,6-sialyl-LacNAc: 18.5% (0.77 µmol / mg polymer)

Example 32: Enzymatic sialylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-LacNAc

30th mg of the polymer from Example 20 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 21 mg Degree of substitution 2,6-sialyl-LacNAc: 12.5% ​​(0.65 µmol / mg polymer)

Example 33: Enzymatic sialylation of poly-D, L-succinimide-co-α, β-aspartamido-C

6

-LacNAc

35 mg of the polymer from Example 21 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 38 mg Degree of substitution 2,6-sialyl-LacNAc: 18% (0.76 µmol / mg polymer)

Example 34: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

35 mg of the polymer from Example 22 are dissolved in 2 ml 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 29 mg Degree of substitution 2,6-sialyl-LacNAc: 12.5% ​​(0.51 µmol / mg polymer)

Example 35: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

35 mg of the polymer from Example 23 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 29 mg Degree of substitution 2,6-sialyl-LacNAc: 18.5% (O, 64 µmol / mg polymer)

Example 36: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

35 mg of the polymer from Example 24 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 34 mg Degree of substitution 2,6-sialyl-LacNAc: 12.5% ​​(0.51 µmol / mg polymer)

Example 37: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

25th mg of the polymer from Example 25 are dissolved in 2 ml 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg bovine serum albumin, 2 mg MnCl₂ and 5 rng CMP-neuraminic acid added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 25 mg Degree of substitution 2,6-sialyl-LacNAc: 18% (0.63 µmol / mg polymer)

Example 38: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

35 mg of the polymer from Example 26 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 28 mg Degree of substitution 2,6-sialyl-LacNAc: 12.5% ​​(0.54 µmol / mg polymer)

Example 39: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

35 mg of the polymer from Example 27 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 33 mg Degree of substitution 2,6-sialyl-LacNAc: 18.5% (O, 69 µmol / mg polymer)

Example 40: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

35 mg of the polymer from Example 28 are dissolved in 2 ml 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 31 mg Degree of substitution 2,6-sialyl-LacNAc: 12.5% ​​(0.55 µmol / mg polymer)

Example 41: Enzymatic sialylation of poly-α, β- (2-hydroxyethyl) -D, L-aspartamide-co-α, β-D, L-aspartamido-6 -hexyl-O- (β-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-β-D-Glu copyranose (PHEA-co-aspartamido-C

6

-LacNAc)

35 mg of the polymer from Example 29 are dissolved in 2 ml of 0.05M sodium cacodylate buffer pH 7.8 and with 1.5 mg of bovine serum albumin, 2 mg of MnCl₂ and 5 mg of CMP neuraminic acid were added. After adding 20 mU 2-6-sialyltransferase and 20 U alkaline phosphatase, the mixture is incubated at 25 ° C. for 8 days. After dialysis against water it is freeze-dried and then chromatographed on Biogel P2. After freeze drying again, the product remains. Yield: 15 mg Degree of substitution 2,6-sialyl-LacNAc: 18% (0.68 µmol / mg polymer)

Example 42

A: PRIMARY ASSAYS FOR EXAMINING THE EFFECT OF POLYMER CARBOHYDRATE RECEPTOR BLOCKERS ON CELL ADHESION TO RECOMBINANT SOLUBLE SELECTECT FUSION PROTEINS

This assay demonstrates the effect of polymer-bound carbohydrate building blocks on the cell attachment of promyelocytic cells using selectins:

In order to test the effectiveness of polymer-bound carbohydrate building blocks on the interaction between the E- and P-selectins (old nomenclature ELAM-1 or GMP-140) with their ligands, an assay is used which is only specific for one of these interactions. The ligands are offered in their natural form as surface structures on promyelocytic HL60 cells. Since HL60 cells have ligands and adhesion molecules of different specificity, the desired specificity of the assay can only be achieved via the binding partner. Genetically engineered soluble fusion proteins from the extracytoplasmic domain of E- or P-selectin and the constant region of a human immunoglobulin of the IgG1 subclass were used as binding partners.

A1. PREPARATION OF L-SELECTECT IGG1

The genetic construct "ELAM-Rg", published by Walz et al., In 1990, was used to produce soluble L-selection-IgG1 fusion protein.

For expression, the plasmid DNA was transfected into COS-7 cells (ATCC) using DEAE dextran (molecular biological methods: see Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Struhl, K. and Smith, JA 1990. Current Protocols in Molecular Biology, John Wiley, New York.). Seven days after the transfection, the culture supernatant is obtained, freed from cells and cell fragments by centrifugation and brought to 25 mM Hepes pH 7.0, 0.3 mM PMSF, 0.02% sodium azide and stored at + 4 ° C.

Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M. and Seed, B. 1990. Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells. Science 250, 1132-1135.

A2. PREPARATION OF P-SELECTECT IGG1

The genetic construct "CD62Rg", published by Aruffo et al., 1991, is used to produce the soluble P-selectin-IgG1 fusion protein. The further procedure corresponds to the production of L-selectin-IgG1 shown under A1.

Aruffo, A., Kolanus, W., Walz, G., Fredman, P. and Seed, B. 1991. CD62 / P-Selectin recognition of myeloid and tumor cell sulfatides. Cell 67, 35-44.

A3. PREPARATION OF CD4-IGG1

The genetic construct "CD4: IgG1 hinge", published by Zettlemeissl et al., 1990, is used to produce the soluble CD4-IgG1 fusion protein. The further procedure corresponds to the production of L-selectin-IgG1 shown under A1.

Zettelmeissl, G., Gregersen, J.-P.-, Duport, JM, Mehdi, S., Reiner, G. and Seed, B. 1990. Expression and characterization of human CD4: Immunoglobulin Fusion Proteins. DNA and Cell Biology 9, 347-353.

A4. PERFORMING THE HL60 CELL ADHESION ASSAY ON RECOMBINANT, SOLUBLE ADHESIVE MOLECULES

<ul id="ul0006" list-style="none"><li>1. 96-well microtiter test plates (Nunc Maxisorb) are incubated with 100 μl of a goat anti-human IgG antibody (Sigma) diluted in 50 mM Tris pH 9.5 (1+ IOO) for 2 hours at room temperature. After removal of the antibody solution, it is washed once with PBS.</li><li>2nd 150 µl of the blocking buffer are left in the wells for 1 hour at room temperature. The composition of the blocking buffer is: 0.1% gelatin, 1% BSA, 5% calf serum, 0.2 mM PMSF, 0.02% sodium azide. After the blocking buffer has been removed, it is washed once with PBS.</li><li>3rd 100 μl of cell culture supernatant from appropriately transfected and expressing COS cells are pipetted into the wells. Incubation takes place for 2 hours at room temperature. After removing the cell culture supernatant, it is washed once with PBS.</li><li>4th 20 μl of binding buffer are added to the wells. The binding buffer has the composition: 50 mM Hepes, pH 7.5; 100 mM NaCl; 1 mg / ml BSA; 2mM MgCl2; 1mM CaCl2; 3mM MnCl2; 0.02% sodium azide; 0.2 mM PMSF. For this purpose, 5 µl of the test substance is pipetted, mixed by swirling the plate and incubated for 10 minutes at room temperature.</li><li>5. 50 ml of an HL60 cell culture with 200,000 cells / ml are centrifuged for 4 minutes at 350 g. The pellet is resuspended in 10 ml RPMI 1640 and the cells centrifuged again. To label the cells, 50 µg BCECF-AM (Molecular Probes) are dissolved in 5 µl anhydrous DMSO; 1.5 ml of RPMI 1640 are then added to the BCECF-AM / DMSO solution. The cells are resuspended with this solution and incubated at 37 ° C. for 30 minutes. After centrifugation at 350 g for two minutes, the labeled cell pellet is resuspended in 11 ml of binding buffer and the resuspended cells are distributed in 100 μl aliquots in the microtiter plate wells. The plate is left at room temperature for 10 minutes to allow the cells to sediment on the bottom of the test plate. The cells have the opportunity to adhere to the coated plastic.</li><li>6. To stop the test, the microplate is completely immersed in the stop buffer at a 45 ° angle (25 mM Tris, pH 7.5; 125 mM NaCl; 0.1% BSA; 2 mM MgCl2; 1 mM CaCl2; 3 mM MnCl2; 0, 02% sodium azide). The stop buffer is removed from the wells by inverting and the procedure is repeated two more times.</li><li>7. The BCECF-AM-labeled cells adhering to the wells are measured in a cytofluorimeter (Millipore) with a sensitivity setting of 4, an excitation wavelength of 485/22 nm and an emission wavelength of 530/25 nm.</li></ul>

IC⁵⁰ of HL60 cell attachment to E-selectin IgG: <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">Connection from Ex. 30</entry><entry namest="col2" nameend="col2" align="left">60 µM</entry></row><row><entry namest="col1" nameend="col1" align="right">31</entry><entry namest="col2" nameend="col2" align="left">60 µM</entry></row><row><entry namest="col1" nameend="col1" align="right">32</entry><entry namest="col2" nameend="col2" align="left">100 µM</entry></row><row><entry namest="col1" nameend="col1" align="right">33</entry><entry namest="col2" nameend="col2" align="left">60 µM</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">34</entry><entry namest="col2" nameend="col2" align="left">150 µM</entry></row></tbody></tgroup></table></tables> Comparative value: IC⁵⁰ HL60 cell attachment to E-selectin IgG: Sialyl-Le^x-O (CH₂)₆NH₂: 1 mm (see EPA 93 119 098.7)

B. SECONDARY ASSAY FOR EXAMINING THE EFFECT OF POLYMER CARBOHYDRATE RECEPTOR BLOCKERS ON CELL ADHESION TO STIMULATED HUMAN ENDOTHEL CELLS

The test of the effectiveness of polymeric carbohydrate receptor blockers on cell adhesion to recombinant, soluble fusion proteins is a highly specific assay which is based on the interaction of a type of adhesion molecule with the corresponding ligand. To simulate the in vivo situation of cell-cell interactions, we use an assay in which HL60 cells adhere to stimulated human umbilical cord endothelial cells.

1. Extraction of human umbilical cord endothelial cells (HUVEC).

Umbilical cords are picked up after birth in PBS with 100,000 IU / L penicillin, 100 mg / L streptomycin, 50 mg / L gentamycin, 50,000 IU / L mycostatin at + 4 ° C until further processing. The longest undamaged pieces are cut out of the hub cord with a scalpel and placed on fresh aluminum foil. One end of the umbilical cord is closed with a clamp. At the other end, insert a suitable tube into the umbilical vein and fix it by tying the umbilical cord end.

The vein is filled through the tube with collagenase solution (50 mg collagenase / 100 ml 25 mM Hepes, pH 7.0) and incubated for 15 minutes at 37 ° C.

In order to increase the cell yield, the umbilical cord is gently massaged after the incubation in order to detach the attached endothelial cells.

The cell suspension is then allowed to flow out of the vein into a culture tube containing cell culture medium and 10% fetal calf serum. The vein is washed with PBS to preserve the remaining cells.

The cell suspension is centrifuged at 500 g for 5 minutes; the cell pellet was then resuspended in 4 ml of culture medium and the cells were plated out. After 3-4 days, the cells have grown confluently and can be passed through.

To check the purity of the endothelial cell culture, the culture is stained with an antibody against factor VIII for immunofluorescence. Only endothelial cells, but not contaminating fibroblasts, show a positive reaction.

2nd Carrying out the assay

20,000 endothelial cells are plated out per well of a 96-well microtiter plate and incubated at 37 ° C. for 24 hours. The endothelial cells must not have been passaged more than 5-6 times. Four hours before the assay, the endothelial cells are stimulated by adding II-1 (final concentration: 15 U / ml). After removing the culture medium, the cells are washed once with RPMI medium without serum. After removing and pipetting 20 µl of RPMI medium again, test substances are added.

3rd The further steps of the assay, labeling of the HL60 cells, application of the HL60 cells, are as under A4. Points 5 - 7 carried out.

C. SECONDARY ASSAY FOR EXAMINING THE EFFECT OF POI YME-REN CARBOHYDRATE RECEPTOR BLOCKERS ON CELL ATTACHMENT TO FREEZER SECTIONS OF LYMPHATIC TISSUE

The extent to which leukocytes bind to endothelial cells on frozen sections of lymphoid tissue can be investigated in vitro. These cell-cell interactions are based on the interaction between adhesion molecules on the surface of the freeze-cut endothelial cells and the corresponding ligands on the surface of leukocytes. HL60 cells whose surface ligands are well described in the scientific literature can be used to represent primary leukocytes. The number of HL60 cells bound can determine whether HL60 cells attach to lymph node frozen sections.<ul id="ul0007" list-style="none"><li>1. Axillary, cervical or mesenteric lymph nodes are prepared from freshly killed rats and quickly frozen in liquid nitrogen.</li><li>2nd 10 µm thick cryostat sections are made from the frozen lymph nodes, transferred to round coverslips (diameter 18 mm) and dried for 2 hours at room temperature.</li><li>3rd 20 µl binding buffer is pipetted onto the sections. The test substances are added and incubated for 10 minutes at room temperature. HL60 cells are like A4. Marked item 5. 200,000 labeled HL60 cells in 100 µl binding buffer are added to each coverslip and left for 10 minutes. The sedimenting cells reach endothelial cells to which they partially attach.</li><li>4th The coverslips are immersed in stop buffer at a 45 ° angle to rinse off the cells that have not been attached. The coverslips are then fixed in 4% formaldehyde in PBS for 10 minutes at room temperature.</li><li>5. Cross sections of lymphatic blood vessels are documented photographically in the immunofluorescence microscope (FITC excitation). The attached HL60 cells stand out clearly from the non-stained background. The result is expressed as bound HL60 cells per unit area of ​​endothelium.</li></ul>

D. TERTIARY ASSAY FOR EXAMINING THE EFFECT OF POLYMER CARBOHYDRATE RECEPTOR BOCKERS ON LEAKOCYTE ADHESION IN RAT IN VIVO

The following procedure is able to determine the effectiveness of substances in vivo which inhibit the attachment of leukocytes to the inner walls of the vessels.

It is known that some of the circulating white blood cells tend to adhere to the inner walls of the blood vessels. This tendency increases significantly in inflammatory processes. Leukocytes normally bump against the walls of the blood vessels, but this collision is elastic, so that the cells bounce back and return to the circulation. In inflammatory processes, biochemical changes in both the leukocytes and the endothelial cells lining the blood vessels lead to changes in the surface properties of both cell types. The cells behave more adhesively. This adhesiveness is initially expressed in a tendency of the leukocytes to roll on the endothelial cells after colliding with the endothelium. This rolling of the leukocytes on the endothelium induces further biochemical reactions on both binding partners, as a result of which cell attachment is increased. This further increase in adhesiveness slows down the rolling of the leukocytes until they firmly adhere to the endothelium. As a further step, the firm adherence is followed by the emigration of the leukocytes from the blood vessel.

The rolling of the leukocytes, the firm attachment and the emigration from the blood vessels can be induced with leukocyte-stimulating factors such as FMLP (f-Met-Leu-Phe), LPS (lipopolysaccharides) or TNF (tumor necrosis factor). Microscopic documentation of these processes is possible on the prepared mesenteric tissue, eg the rat. Substances injected into the bloodstream can therefore be examined to determine whether they are able to influence the induced leukocyte adhesions.

Rats are also anesthetized for the practical implementation of this study. The belly height is opened and a section of the small intestine is pulled out. The small intestine section is kept constantly moist on a heatable microscope table. A section of mesenteric tissue is covered with paraffin oil for microscopic observation. As a control, all adhering - not stimulated - leukocytes are counted in this area every 5 minutes over a period of 30 minutes. At the same time, blood pressure, body temperature and blood flow rate are recorded. The test substance is applied by continuous venous infusion throughout the test. After the application of leukocyte stimulants which are dripped onto the mesenteric tissue, the adherent leukocytes are counted every 5 minutes over a period of 90 minutes.

The examination is carried out in test groups, each consisting of three animals. The first animal only receives the vehicle to determine spontaneous adhesion. The second animal only receives leukocyte stimulation to determine the pathogenic control. The third animal receives leukocyte stimulation and test substance.

The number of adhesive leukocytes in the pathogenic control is set equal to 100%. The percentage change in the leukocyte adhesion when administered to the test substance compared to the pathogenic control indicates the effectiveness of a test substance.