WO1986005789A1

WO1986005789A1 - Carbohydrate derivatives and compositions thereof for therapeutic or diagnostic use, and methods for their use

Info

Publication number: WO1986005789A1
Application number: PCT/SE1986/000131
Authority: WO
Inventors: Sigfrid Svensson; Per Anders MA^oRDH; Frank Lindh; Hans LÖNN; Elisabeth Kallin; Bo Nilsson; Olle MA^oNSSON
Original assignee: Biocarb AB
Current assignee: Biocarb AB
Priority date: 1985-04-01
Filing date: 1986-03-25
Publication date: 1986-10-09
Anticipated expiration: 1987-10-01
Also published as: AU5668286A; EP0217912A1; SE8501613D0

Abstract

A compound for therapeutic or diagnostic use, characterized by having formula (I): wherein R1 and R2 independently are hydrogen, lower alkyl, acyl or together form a cyclic amide or amine; wherein R3 and R4 independently are hydrogen, alkyl, an organic residue; and wherein n is >=2, the individual monomer residues of the compound (I) being independ of each other as to structure; a composition containing such compound, methods for use of the compound and the composition, respectively.

Description

"Carbohydrate derivatives and compositions thereof for therapeutic or diagnostic use, and methods for their use".

The present invention relates to compounds which are useful for therapeutic treatment of infections as well as profylaxis and diagnosis in connection herewith. The invention moreover covers a process for targeting of molecules to different organs of mammals including man.

The technique according to the invention is applicable to microorganisms of different kinds causing diseases, particularly pathogenic bacteria, such as cocci, preferably Grampositive, e.g. streptococci, pneumococci and staphylococci. In particular, the invention relates to applications directed to staphylococci, such as Staphylococcus saprophvticus, and bacteria of the species Bordetella pertussis causing hooping cough. Moreover, the compositions according to the invention have the ability of inhibiting the activity of so called natural killer cells (NK-cells). The technique according to the invention relating to targeting is applicable to cells and organs of mammals including man.

The expression "microorganism" used in the present disclosure is intended to cover bacteria, viruses, animal and plant cells.

A large number of bacterial infections arise by bacterial attack on mucous membranes. In the initial stage of the course of infection it is essential to the bacteria to have the capability of binding to epithelial cells. The infectious ability of bacteria is often directly related to the ability of the bacteria to adhere to epithelial cells. Staphylococcus saprophvticus is a bacterium which is frequent cause to urinary tract infections (UTI) mainly in younger women and older men. The bacterium is present on the skin of different animals and is a frequent contaminant on meat products. Staphylococcus saprophvticus has also been connected with mastitis. Studies have shown that adhesion of Staphylococcus saprophvticus tp periurethral cells is caused by a carbohydrate component and that the same carbohydrate component is found in erythrocyte membranes of sheep. This also explains the fact why Staphylococcus saprophvticus agglutinates sheep erythrocytes.

Many glycoconjugates are absorbed by cells or organs due to the fact that cell membranes contain receptors of a proteinatious character. These receptors can be specific for carbohydrate structures of the glycoconjugates and these glycoconjugates can specifically be directed to certain cells or organs.

The present invention has for its purpose to provide a composition or substance having the ability of replacing the normal receptor function in vivo and in vitro in relation to pathogenic bacteria inclined to cause infections in human beings and animals.

Another object of the invention is to provide such composition or compound that can be used for the removal of bacteria from butcher products, contaminated surfaces, for example skin and buildings.

Yet another object of the invention is to provide a composition or compound that can be used in diagnosis of bacteria.

A further object of the invention is to provide a composition or compound that can form the basis for a process for therapeutic or prophylactic treatment of mammals including man. Still another object of the invention is to provide a process for identification and/or quantification of receptor structures in biological material from mammals including man. Another object of the invention is to provide a process for isolation and purification of bacteria or receptor structures of bacteria.

Still a further object of the invention is to provide a composition or substance that can be used for visualization of cells or organs. The invention moreover provides a process for targeting of molecules to different cells or organs. The invention is particularly applicable to receptor structures for Staphylococcus saprophvticus. NK-cells and li-vercells, but it should be observed that the invention is not limited to these particular cells. Through studies and experiments it has been found that the receptor for Staphylococcus saprophvticus. NK-cells and livercells can be replaced with compositions containing a compound having the formula:

wherein R₁ and R₂ independently are hydrogen, lower alkyl, acyl or together form a cyclic amide or amine; wherein R₃ and R₄ independently are hydrogen, alkyl, an organic residue; and wherein n is ≥ 2, the individual monomer residues of the compound (I) being independent of each other as to structure. In certain preferred compounds of the formula I R₁ is hydrogen and R₂ has the formula:

R₅ , R₆ and R₇ are suitably all fluoro or chloro, particularly fluoro.

It is preferred in formula I that R₁ is hydrogen and R₂ is acyl, particularly acetyl or trifluoroacetyl. R₃ and R₄ may suitably independently be hydrogen or lower alkyl.

In connection to the definition of the compounds of formula I above it is stated that n is 2 or more and n is preferably 2-100, particularly 2-20 and especially 2-10. Lower homologues are preferred in that n is preferably 2-6 and particularly 2.

Within the scope of the general formula I as stated above new as well as known compounds are found. According to a further aspect of the present invention there is provided also new compounds of formula II:

wherein R₁ and R₂ independently are hydrogen, alkyl, acyl or together form a cyclic amide or amine; wherein R₃ and R₄ independently are hydrogen, alkyl, an organic residue, and wherein n is ≥ 2, with the proviso that when n is 2-4 R₃ is hydrogen and R₄ is methyl then R₁ is different from hydrogen if R₂ is acetyl or trifluoroacetyl; and when n ≥ 2 , R₃ = R₄ = hydrogen then R₁ and R₂ are different from hydrogen and when R₁ is hydrogen then R₂ is different from acetyl. With regard to the variables for these new compounds and preferred embodiments the same conditions apply as given above in connection with compounds of formula I. It may be added that when R₁ is hydrogen and R₂ is acetyl or trifluoroacetyl at least one of substituents R₂ is trifluoroacetyl. The expression "lower" used in the present disclosure relates to a group containing 1-6 carbon atoms, particularly 1-4 carbon atoms and especially 1 or 2 carbon atoms.

In regard to substituent R₄ this may be of any type as long as it does not negatively affect the conditions in relation to the application of the invention. Preferred values of R₄ are hydrogen, lower alkyl or a macromolecular carrier covalently bound to the carbohydrate residue, optionally via a coupling arm. As a macromolecular carrier there may be used a synthetically or naturally occurring polypeptide, polysaccharide, other polymer or particle. This is conventional in the art, see for example references 8-12, the contents of which are incorporated herein by reference. When present the coup ling arm between the carbohydrate residue and the macromolecular carrier can be any of the following:

In the above examples n' can vary between 1 and 15. The haemagglutination reaction of sheep erythrocytes caused by S . saprophyticus is probably due to interaction between a membrane structure at the surface of the bacterium and receptors on the surface of the sheep erythrocytes containing compound I above. The adhesion of S . saprophvticus to periurithral cells is probably due to interaction between a membrane structure at the surface of a bacterium and receptors of the surface of the epithelial cells containing the above-mentioned compound I. However, the invention is not bound by any of these theories.

The active substance constituted by the compound of formula I can be used as such or in combination with a pharmaceutically acceptable carrier. According to another aspect of the invention there is thus provided also a composition for therapeutic treatment of mammals including man, said composition containing a compound I according to the definition above in combination with such pharmaceutically acceptable carrier.

The active substances according to the present invention can be formulated for use in human or veterinary medicine for therapeutic, profylactic or diagnostic uses. In clinical practice the active constituents are normally administered orally or rectally or by injection in the form of a pharmaceutical preparation containing the active constituents in combination with a pharmaceutically acceptable carrier, which may be solid, semisolid or liquid, or as a capsule, and such compositions constitute a further aspect of the invention. The compounds may also be used as such without carrier and in a form of an aqueous solution for injection. As examples of pharmaceutical preparations there may be mentioned tablets, drops, solutions and suppositories. The active substance usually constitutes from 0.05 to 99% by weight of the preparation, for example from 0.1 to 50% for preparations intended for oral administration.

To manufacture pharmaceutical preparations in the form of dose units for oral application containing a compound according to the invention the active constituents can be admixed with a solid pulverulent or other carrier, for example lactose, saccharose, sorbitol, mannitol, starch, such as potatoe starch, corn starch, amylopectin, a cellulose derivative or gelatin and may also include lubricants, such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to form tablets or cause for dragees.

By using several layers of the active drug separated by slowly dissolving layers tablets of delayed release are obtained.

Liquid preparations for oral application can be in the form of elixires, syrups or suspensions, for example solutions containing from 0.1 to 20% by weight of active substance, sugar and a mixture of ethanol, water, glycerol, propylene, glycol and optionally other additives of a conventional character. The dose by which the active constituents are administered may vary within wide limits and depend on different factors, such as the severity of the disorder, the age and the weight of the patient and can be individually adjusted. As a conceivable range for the quantity of active constituents that may be administered per day there may be mentioned from 0.1 to 2000 mg or from 1 mg to 2000 mg.

The present invention has also for an object to provide a method for therapeutic treatment of mammals including man, and in this treatment a therapeutically active amount of a substance or a composition in accordance with the invention is administered.

The present invention also includes a process for identification or quantification of the compound I or residues of native biological material from mammals including man. In this process antibodies are used the generation of which has been induced by the compound I as defined above.

The invention furthermore includes a process for the purification of acceptor structures of bacteria, and in this purification the affinity between compound I and the acceptor structures of the bacteria is utilized.

Furthermore, the invention covers a process for performing desinfection on surfaces, a compound I being applied on to the surface and then removed with bacteria adhering thereto.

The invention will in the following be described by non-limiting examples.

Example 1

Synthesis of compounds β-D-GNAcp-<1-4>-[β-D-GNAcp-(l-4)-]_n-D-GNAc (I-VIII)

Chitosan hydrochloride was prepared from chitin by alkaline N-deacetylation according to Barker et al. (Ref.2). Chitosan was partially hydrolyzed using aqueous hydrochloric acid and their N-acetylated (Ref. 2). N-acetylated oligosaccharides were separated using gel chromatography (Saphadex G15) and hplc (C-18 column, CH₃CN/H₂O system). The following oligosaccharides were isolated and characterized:

β-D-GNA-cp-(1-4)-D-GNAc I β-D-GNAcp-(1-4)-β-D-GNAcp-(1-4)-D-GNAc II β-D-GNAcp-(1-4)- [β-D-GNAcp-(1-4)-]₂-D-GNAc III B-D-GNAcp-(1-4)- [β-D-GNAcp-(1-4)-] ₃-D-GNAc IV β-D-GNAcp-(1-4)- [β-D-GNAcp-(1-4)-]₄-D-GNAc V β-D-GNAcp-(1-4)- [β-D-GNAcp-(1-4)-]₅-D-GNAc VI β-D-GNAcp-(1-4)- [β-D-GNAcp-(1-4)-]₆-D-GNAc VII β-D-GNAcp-(1-4)- [β-D-GNAcp-(1-4)-]_≥7-D-GNAc VIII

The purity of the oligosaccharides I-VII was checked by hplc. The oligosaccharides gave expected p.m.r. spectra (500 Mhz . Bruker) and correct sugar and methylation analyses (Refs. 3,4) in accordance with structure.

β-D-GNTF_p-(1-4)- [β-D-GNTF_p-(1-4)-]_n-D-GNTF (IX-XVI)

These oligosaccharides were prepared using trifluoroacetolysis (Refs. 5,6) from the oligosaccharides I-VIII according to the following general technique.

The oligosaccharides (I-VIII) (100 mg) were treated with trifluoroacetic acid/trifluoracetic anhydride (TFA/TFAA) (1:100; v/v; 20 ml) at 100°C for 48 hours. After cooling the reaction mixture was evaporated to dryness. The residue was dissolved in methanol (20 ml) and the resulting solution was evaporated to dryness. The residue was dissolved in globial acetic acid (10 ml) and water (10 ml) was added. The mixture was heated at 100°C for 4 hours and evaporated to dryness. The crude product was gel chrόmatographed on Sephadex G15 (2x100 cm) using water as eluant. Fractions containing oligosaccharide were pooled and lyophilized. The product was further purified using hplc (C-18 column, CH₃CN/H₂O) . Typical yield of N-trifluoroacetylated oligosaccharide was 50 mg.

Analysis of IX-XVI was performed using p.m.r. and F¹⁹ n.m.r. as well as sugar and methylation analyses.

Some pertinent p.m.r. and F¹⁹ n.m.r. data are given below for compound IX.

β -D-GNTFp-(1-4)-GNTF

B A p.m.r. proton δ(ppm) J(Hz) Multiples

HA1α 5.263 2.6 d

HA1β 4.796 8.2 m (virtually)

HA1α 4.723 8.6 d

HB1β 4.713 8.6 d

F¹⁹ n.m.r. Fluorine 6(ppm) J(Hz) Multiples

ACF₃α -77.640 - S

ACF₃β -78.665 - S B A

BCF₃ α -78.463 - S

BCF₃ β -78.517 - S

Shifts are given relative to acetone (2.225 ppm. 20°C) for H¹ and relative to external TFA (-78.500 ppm, 20°C).

β-D-GNAcp-(1-4)-[β-D-GNAcp-(1-4)-]_0-1-D-GNTF (XVII-XVIII)

Short time trifluoroacetolysis of oligosaccharides I and II using TFA/TFAA 1:100 (v/v) at 100°C gave a good yield of XVII-XVIII.

The oligosaccharide (I-II) (100 mg) was treated with TFA/TFAA (1:100, v/v, 20 ml) at 100°C for 30 min. After work-up (see above) the product was purified by hplc (C-18 column, CH₃CN/H₂O) . Typical yield of purified product was 40 mg. The purity and identity of the products was corraborated by p.m.r. and F¹⁹ n.m.r.

Some pertinent p.m.r. and F¹⁹ n.m.r. data are given below for compare XVII.

3-D-GNAcp-(1-4)-GNTF

B A p.m.r proton δ (ppm) J(Hz) Multiple

HA1α 5.264 3.6 d

HA1β 4.804 8.1 m (virtually)

HB1α 4.561 8.6 d

HB1β 4.593 8.2 d

HBCH₃ 2.072 - S F¹⁹ n.m.r. fluorine δ(ppm) J(Hz) Multiple

A-CF₃α -77.624 - S A-CF₃β -78.667 - S

β-D-GNAcp-(1-4)-[β-D-GNAcp-(1-4)-]_0-1 D-GNH₂ (XIX-XX)

Treatment of XVII and XVIII with 1M ammonia in methanol/water for 18h resulted in quantitative N-detrifluoroacetylation.

The oligosaccharide (XVII-XVIII) (50 mg) was dissolved in 1M ammonia in methanol/water (1:4, v/v) and was left at 20°C for 18 h. The reaction mixture was evaporated to dryness. The yield was 80% .

The purity and identity was verified by p.m.r.

β-D-GNAcp-(1-4)- [β-D-GNAcp-(1-4)]_0-1-D- (XXI-XXIV)

XIX and XX was treated with different anhydrides in pyridine to substitute all hydrocyl and aminogroups. The O- and N-acylated compounds were de-O-acylated with ammonia in methanol/water.

The oligosaccharide (XIX-XX) (50 mg) was treated with (RCO)₂O (10-fold excess) in pyridine (20 ml) at 100°C for 2h. The reaction mixture was evaporated to dryness. The residue was treated with 1M ammonia in methanol/water (3:1, v/v) for 18h arid the reaction mixture was evaporated to dryness. The crude product was purified using hplc (C-18 column, CH₃CN/H₂O) .

Starting material anhydride product

XIX (CCl₃CO)₂O XXI

XX " XXII

XIX (CH₃CH₂CO)₂O XXIII

XX " XXIV

β-D-GNAcp-(1-4)-D-GNPHT (XXV) The phtalamide derivative was prepared according to the method of Baker et al. (Ref.7). In this manner XXV was obtained from XIX.

β-D-GN(Me)Acp-(1-4)-[β-D-GN(Me)Acp(1-4)-]₀-₁-D-GN(Me)Ac (XXVI-XXVII)

I and II (100 mg) was treated with methylvinylether (25 ml) at 15°C together with p-toluenesulphonic acid for 2 h.

'BSTITUTE S SHtI ?rs»^»-p- The reaction mixture was then passed through a Sephadex LH20 column (5x50 cm) and fractions containing oligosaccharide were pooled and concenrated to dryness. The acetalized oligosaccharide was methylated using dimethylsulphinyl sodium and methyl iodide. Deacetalization of the product was achieved using 50% aqueous acetic acid (100°C for 1 h) . The crude product was purified by hplc (C-18 column, CH₃CN/H₂O as eluant). The purity and identity was demonstrated by p.m.r.

Example 2

Inhibition of haemagglutination of sheep ervthorcvtes with Staphylococcus saprophyticus bv the addition of oligosaccharides

In the tests the bacteria S.saprophyticius strains, both isolated from urine from patients having urinary tract infection, were used. The bacteria were cultivated in tryptone broth over night, pelletized by means of centrifugation and washed twice with PBS, pH 7.2. Haemagglutination was performed using erythrocytes from sheep obtained from sheep blood and washed twice with 0.85% saline.

In the haemagglutination-indication experiments microtitre plates (Limbo Sc. Comp. Inc. ) were used. 25μl of bacterial suspension were titrated in PBS by two-step titre steps, 25μl of 1% erythrocyte suspension being then added. The haemagglutination titre was determined after incubation of the plates for 2-4 hours in room temperature.

In the inhibition experiments serial dilutions of the relevant inhibitor (50μl/well) and 25μl of bacterial suspension diluted to contain 2 HU haemagglutinating unite of bacteria were used. The plates were incubated for 30 minutes at 37°C, 25μl of the erythrocyte suspension being then added to each well.

The results are presented in Table 1. Table 1

Inhibition of haemagglutination between sheep erythoryctes and S.saprophyticus bacteria (Mc 2 and Mc 194).

Inhibitor Minimum inhibitory cone μg/ml

I 200

II 200

III 200

IV 175

V 175

VI 175

VII 150

IX 75

X 75

XI 60

XII 75

XIII 50

XIV 50

XV 50

XVI 50

XVII 100

XVIII 125

XIX 125

XX 150

XXI 600

XXII 700

XXIII 100

XXIV 75

XXV 75

XXVI 300

XXVII 400 Example 3

Inhibition of adherence of S,saprophyticus to urinary epithelial cells by the addition of oligosaccharides.

Cells from urinary epithelium were suspended in cell cultivation medium RPMI 1640 (Flow Ltd) to a concentration of 10⁹ bacteria/ml. Oligosaccharides which were tested for their ability of preventing adhesion of S.saprophyticus to urinary epithelial cells were dissolved in RPMI 1640 to a concentration of 5 mg/ml. In the experiments 1 ml of the oligosaccharide solution was incubated with 0.1 ml bacterial suspension at 37°C for 30 minutes. Urinary epithelial cells were incubated with the bacterial suspension incubated in advance as described above with oligosaccharide solution for 45 minutes at 37°C. As a control there was used a bacterial suspension preincubated with 1 ml RPMI 1640.

Incubated samples were filtrated through a 12μ filter and washed 3 times with PBS. The filter was pressed against microscope slides for a few seconds, the slides being then fixed in methanol for 10 minutes. The slides were dried in air and coloured with acridine orange for 2 minutes. The slides were washed, dried in air and studied in fluorescence microscope. The number of bacteria per cell was counted. In each experiment 50 cells were counted.

In these tests it was found that I, XVII and IX reduced the number of adhering bacteria by more than 95%, whereas lactose did not reduce the number of adhering bacteria in comparison with the control test without oligosaccharides.

Example 4 Preparation of compositions containing the structural element in at least bivalent state and covalently linked to a macromolecular carrier.

The composition was made starting from oligosaccharides having a free reducing end. The reactions used are well known and, therefore, they are only diagrammatically illustrated (in the scheme SR represents the structural element without the sugar residue constituting the reducing terminal, and MB represents a macromolecular carrier).

Example 5

Preparations of compositions containing the structural element according to the invention in at least bivalent association without covalent bond. A glycolipid containing the said structural element is linked by a hydrophobic interaction to lipophilic (hydrophobic) gels, polymers or particles, for example octylsepharose, plastics and latex surfaces.

Example 6

Preparation of antibodies having specificity against the said structural element. a. Manufacture of monoclonal antibodies by hybridoma technique. I. Balb/c-mice are immunized with a composition according to the invention. The spleen from hyper immunized animals is harvested and a cell suspension is prepared by mechanical comminution of the tissue. After gradient centrifugation to obtain a pure cell preparation the cells are fused by means of polyethylene glycol (PEG, average molecular weight 1500) with established B-myeloma cell lines from Balb/c-mice according to known technique. After cloning the hybridoma cells generating the antibody sought, the cells are propagated on a large scale, the culture medium supernatants being harvested and the antibodies thereof being purified in a conventional manner. For identification of antibody generating clones there is used so-called enzyme-linked immunosorbent assay (ELISA).

II. Mammals are immunized with an oligosaccharide protein or polymer composition according to the invention. Antibodies are isolated from the hyperimmune serum of the mammal and purified in accordance with conventional techniques.

Example 7 Diagnostic test for identification of bacteria having acceptor structures showing specificity towards the structural element according to the invention. a. Bacteria are incubated with sheep erythrocytes to agglutinate same. In a parallel test bacteria are incubated with sheep erythrocytes together with the structural element according to the invention at such concentration as to totally inhibit haemagglutination. Haemagglutination and inhibition of haemagglutination is performed according to Example 1. Positive haemagglutination of sheep erythoryctes and complete inhibition of haemagglutination after addition of oligosaccharide verifies the fact that the bacteria possess acceptor structure. b. Bacteria are incubated with a composition wherein the said structural element is covalently or by other association in multivalent form linked to a particular matrix according to Example 3 or 4. Incubation is carried out on microscope slides for 10-15 minutes, the preparation being then studied in a microscope. If the bacteria posses acceptor structure the particles are covered by bacteria. Where the reaction is negative the particles are free from bacteria. c. Bacteria are mixed with a composition according to claim 4 or 5 on microscope slides positive reaction resulting in agglutination of particles covered with the said structural element.

Example 8 Purification of bacteria or acceptor structures a. A composition according to Example 3 or 4 above is arranged in the form of a column. A mixture of bacteria is then passed through the column, bacteria possessing acceptor structures being maintained by interaction with the receptor structures of the column. After rinsing the column can be eluted with buffer containing a receptor-active structural element according to the invention and this results in elusion of bacteria having acceptor structures in a pure form. b. By a process fully analogous with a. the acceptor structure can be obtained in a pure form.

Bacteria or acceptor structures can be used for the manufacture of vaccines or for determination of antibody in for example body fluids, such as blood, urine or mother's milk.

Example 9

Composition for use for desinfection

By desinfection there is meant herein primarily removal of bacteria from a surface, for example a wound.

A 0.1% by weight aqueous solution of the compounds I or XV is prepared and applied by using a cotton pad on a surface infected by S.saprophvticus. The solution results in effective removal of the bacterium from the surface.

Example 10 Inhibition of NK-cells activity using oligosaccharides

Lymphocytes isolated from peripheral blood drained from normal donors have the ability of exerting a cytotoxic, i.e. cell killing activity against certain types of cells in culture. In the instant example there is used a human leukemia cell line called K-562 as a target cell for such cytolytic activity.

Method : K-562 cells intracellularly labelled with radioactive chromium are incubated with effector cells (lymphocytes) for 4 hours at 37°C. The cytotoxic effect is measured as the quantity of isotope released in relation to remaining isotope in the target cells. For details, see

Malmström, P., Jönsson, A. and Sjögren, H.O. "Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. II. Separation into subsets of lymphocytes with distinctive functions". Cell Immunol. 53:51-64, 1980. For studying inhibiting substances effector cells (6x10⁶ cells/ml) are preincubated with the respective oligosaccharides diluted in cultivating medium for half an hour at 37°C . After washing with centrifugat ion the effector cells are transferred directly to the target cells. The results obtained are presented in Table 2 below.. Table 2

Inhibition of specific cytotoxic activity of NK-cells.

Inhibitor Minimum inhibitory cone, μg/ml. I 3000

V 3000

IX 2000

XV 1500

XVII 2000 XVIII 2500

XX 4000

XXI 1500

XXII 1500

XXIII 1500 XXV 1500

XXVI 4000

Example 11

Inhibition of binding of ¹²⁵-I-asialofetuin to rabbit heoatoevtes uaing oligosaccharides.

The assays were carried out in capped polystyren tubes (12x75 mm) at 4°C . To each tube was added 3x10⁶ rabbit hepatocytes in modified Dulbecco's Eagle's medium and 1x10^-10M ¹²⁵I-asialofetuin and different concentrations of oligosaccharide in a total volume of 1 ml. The tubes were rotated slowly at 4°C for 4h, after which time duplicate samples (200 μl) were taken, transferred into 400 μl centrifuge tubes containing 150 μl of silicone/mineral oil (4:1, v/v) and centrifuged for 10s in an Eppendorf centrifuge. The cell pellet at the bottom of the tube was counted for radioactivity in a Packard-counter. The 50% inhibition value was determined from the observed inhibition curve and the values are shown in Table 3. Table 3

Inhibition of binding of ¹²⁵I-asialofetuin to rabbit hepatocytes at 4°C by different oligosaccharides.

Inhibitor Minimum cone. for 50% inhibition mM

I 1.2

V 0.8

IX 0.4

XV 0.2 XVII 0.5

XVIII 0.6

XX 1.9

XXI 0.2

In the instant disclosure the following abbreviations have been used:

Abbreviations

GNAcp 2-acetamido-2-deoxy-D-glucopyranosyl

GNAc 2-acetamido-2-deoxy-D-glucose GNTFp 2-deoxy-2-trifluoracetamido-D-glucopyranosyl

GNTF 2-deoxy-2-trifluoracetamido-D-glucose

GNH2 2-amino-2-deoxy-D-glucose

GNPHT 2-deoxy-2-N-phtalimido-D-glucose

GN(Me)Ac 2-(N-methyl)-acetamido-2-deoxy-D-glucose GN(Me)Acp 2- (N-methyl)-acetamido-2-decxy-D- glucopyranosyl

References

1. Gibbons, R.J. and Hoυte. Ann.Rev. Microbiol.29 (1975)19

2. Barker, S.A., Foster, A.B., Stacey, M. and Webber, J.M. J.Cheat.Soc. (1958) 2218

3. Sawardeker, J.S., Sloneker and Jeanes, A.R. Anal.Chem. 12 (1965) 1602

4. Björndal, H., Hellerquist, C.G., Lindberg, B. and Svensson, S. Angew.Chem. Int.Ed. 9 (1970) 610

5. Nilsson, B. and Svensson, S. Carbohyd.Res. 62 (1978) 377

6. Nilsson, B. and Svensson, S. Carbohyd.Res. 69 (1979) 292

7. Baker, B.R., Joseph, J.D., Schaub, R.F. and Williams, J.M. J.Org.Chem 19(1954) 1786

8. Svensson, S.B. and A.A. Lindberg (1979) J. Immunol.Meth. 25, 323.

9. Zopf, D. et al (1978) Immunol.Meth.enzymol.L, part C, 163.

10. Lönngren, J. et al (1976) Arch.Biochem.Biophys. 175, 661.

11. Gray, G.R. (1978). In Meth. enzymol.L., part C, 155.

12. McBroom, C.R., Samanen, C.H., Goldstein, I.J. In:Methods in Enzymology, Vol. 28B, ed. V. Ginsburg, p. 212. Academic Press, New York (1972).

Claims

1. A compound for therapeutic or diagnostic use, characterized by having the formula (I):

wherein R₁ and R₂ independently are hydrogen, lower alkyl, acyl or together form a cyclic amide or amine; wherein R₃ and R₄ independently are hydrogen, alkyl, an organic residue; and wherein n is ≥2, the individual monomer residues of the compound (I) being independent of each other as to structure.

2. A compound according to claim 1, wherein R₁ is hydrogen and R₂ has the formula:

wherein R₅ , R₆ and R₇ independently are hydrogen, lower alkyl or halogen.

3. A compound according to claim 2, wherein R5 , R6 and R₇ all are fluoro br chloro.

4. A compound according to claim 3, wherein R₅ , R₆ and R₇ all are fluoro.

5. A compound according to any preceding claim, characterized thereby that R₁ is hydrogen and R₂ is acyl.

6. A compound according to claim 5, characterized thereby that R₂ is acetyl or trifluoroacetyl.

7. A compound according to any preceding claim, characterized thereby that R₃ and R₄ independently are hydrogen or (C₁-C₆)alkyl.

8. A compound according to any preceding claim, characterized thereby that n is 2-100, preferably 2-10.

9. A compound according to any preceding claim, characterized thereby that R₁ is hydrogen, R₂ is acetyl, R₃ = R₄ = hydrogen and n is 2-6.

10. A compound having the formula (I):

wherein R₁ and R₂ independently are hydrogen, alkyl, acyl or together form a cyclic amide or amine; wherein R₃ and R₄ independently are hydrogen, alkyl, an organic residue, and wherein n is ≥2 , with the proviso that when n is 2-4 R₃ is hydrogen and R₄ is methyl then R₁ is different from hydrogen if R₂ is acetyl or trifluoroacetyl; and when n ≥ 2 , R₃ = R₄ = hydrogen then R₁ and R₂ are different from hydrogen and when R₁ is hydrogen then R₂ is different from acetyl; and when R₁ is hydrogen, R₂ is trifluoroacetyl and n is 3 then one of R₃ and R₄ is different from hydrogen.

11. A compound according to claim 10, wherein R₁ is hydrogen and R₂ has the formula:

wherein R₅, R₆ and R₇ independently are hydrogen, lower alkyl or halogen.

12. A compound according to claim 11, wherein R₅ , R₆ and R₇ all are fluoro or chloro.

13. A compound according to claim 3, wherein R₅, R₆ and R₇ all are fluoro.

14. A compound according to any of claims 10-13, characterized thereby that R₁ is hydrogen and R₂ is acyl.

15. A compound according to claim 14, characterized thereby that R₂ is acetyl or trifluoroacetyl.

16. A compound according to any of claims 10-15, characterized thereby that R₃ and R₄ independently are hydrogen or (C₁-C₆)alkyl.

17. A compound according to any of claims 10-16, characterized thereby that n is 2-100, preferably 2-10.

18. A compound according to any of claims 10-17, characterized thereby that R₃ and R₄ both are hydrogen and n is 2-6.

19. A compound according to claim 18, characterized thereby that n is 2.

20. A compound according to claim 10, characterized thereby that R₁ is hydrogen, R₂ is acetyl or trifluoroacetyl, at least one R₂ being trifluoroacetyl, and n is 2-6.

21. A compound according to claim 20, characterized thereby that n is 2.

22. A composition for therapeutic treatment of mammals including man, characterized by a compound according to any preceding claim in combination with a pharmaceutically acceptable carrier.

23. A method for therapeutic treatment of mammals including man, characterized by administering to the mammal a therapeutically ef f ect ive amount of a compound according to any of c laims 1-21 or a composition according to claim 22.

24. A method for determining the presence of pathogenic bacteria in a sample taken from a mammal including man, characterized by determining the grade of interaction between the bacteria of the sample and the compound according to any of claims 1-21.

25. A method for identification or quantification of said structure element in native biological material from mammals including man, characterized by using antibodies, the production of which being induced by the compound according to any of claims 1-21.

26. A method for purification of acceptor structures in bacteria, characterized thereby that in this purification the affinity between the compound according to claims 1-21 and the acceptor structures of the bacteria is utilized.

27. A method for the removal of bacteria from surfaces, characterized thereby that a compound according to any of claims 1-21 is applied on the surface of the compound then being removed with bacteria adhering thereto.