CARBOHYDRATE DERIVATIVES AND COMPOSITIONS THEREOF FOR THERAPEUTIC OR DIAGNOSTIC USE, AND METHODS FOR THEIR USE

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 The present invention relates to compounds which are useful for therapeutic treatment of infections as well as profylaxiβ and diagnosis in connection herewith. The inven¬ tion moreover covers a process for targeting of molecules to different organs of mammalβ including man. The technique according to the invention is applicable to microorganisms of different kinds causing diseases, parti¬ cularly pathogenic bacteria, such as cocci, preferably Gram- positive, e.g. streptococci, pneumococci and βtaphylococci. In particular, the invention relates to applications directed to βtaphylococci, such as Staphylococcus βaprophvticus . 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 na¬ tural killer cells (NK-cellβ) . 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 dis¬ closure is intended to cover bacteria, viruses, animal and plant cells. A large number of bacterial infections arise by bacte- rial 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 uri¬ nary tract infections (UTI) mainly in younger women and older men. The bacterium is present on the skin of different ani¬ mals and is a frequent contaminant on meat products .Staphylo¬ coccus saprophvticus has also been connected with mastitis. Studies have shown that adhesion of Staphylococcus saprophvticus tp periurethral cells is caused by a carbo¬ hydrate 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 glycoconjugateβ are absorbed by cells or organs due to the fact that cell membranes contain receptors of a protθinatious character. These receptors can be specific for carbohydrate structures of the glycoconjugateβ and these gly¬ coconjugateβ 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 com- position or compound that can be used for the removal of bac¬ teria from butcher products, contaminated surfaces, for ex¬ ample skin and buildings . Yet another object of the invention is to provide a composition or compound that can be used in diagnosis of bac- teria. A further object of the invention is to provide a compo¬ sition 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 βtruc- tures 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. N -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-cellβ and livercells can be replaced with compositions containing a compound having the formula: wherein R^ and 2 independently are hydrogen, lower alkyl, acyl or together form a cyclic amide or amine; wherein R3 and R4 independently are hydrogen, alkyl, an or¬ ganic residue; and wherein n is _>, 2, the individual monomer residues of the com¬ pound (I) being independent of each other as to structure. In certain preferred compounds of the formula I R^ is hydrogen and R2 has the formula: - R5, 5 and are suitably all fluoro or chloro, particularly fluoro. It is preferred in formula I that ^ is hydrogen and R2 is acyl, particularly acetyl or tri luoroacetyl . R3 and R 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 ho ologueβ 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 4 a further aspect of the present invention there is provided also new compounds of formula II: wherein R^ and R2 independently are hydrogen, alkyl, acyl or together form a cyclic amide or amine; wherein R3 and R4 independently are hydrogen, alkyl, an or- ganic residue, and wherein n is >. 2, with the proviso that when n is 2-4 R3 is hydrogen and R4 is methyl then R_ is different from hydrogen if R2 is acetyl or trifluoroacetyl; and when n __ 2, R3 = R4 = hydrogen then ^ and 2 are different from hydrogen and when R^ is hydrogen then R2 is different from acetyl. With regard to the variables for these new com¬ pounds and preferred embodiments the same conditions apply as given above in connection with compounds of formula I. It may be added that when R^ is hydrogen and R2 is acetyl or triflu- oroacetyl at least one of substituents R2 is trifluoroacetyl . The expression "lower" used in the present disclosure relates to a group- containing i-6 carbon atoms, particularly i-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 rela¬ tion to the application of the invention. Preferred values of R4 are hydrogen, lower alkyl or a macromolecular carrier co- valently bound to the carbohydrate residue, optionally via a coupling arm. As a macromolecular carrier there may be used a synthetically or naturally occurring polypeptide, polysaccha- ride, 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 macromo¬ lecular carrier can be any of the following: -0 (Ό • -NHCSNH-macromolecular carrier -0 (o> -N = N -NH- ( CH 2 > n' -CH 2 <2> N6CSNH- " -NH-<CH 2 > n '-CH 2 ©- N=N -NH-(CH 2 ) n' -CONH- -NH (σ) CONH- In the above examples n' can vary between 1 and 15. The haemagglutination reaction of sheep erythrocytes caused by S.saprophvticus is probably due to interaction between a membrane structure at the surface of the bacterium and receptors on the surface of the sheep erythrocytes con¬ taining 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 the- ories. The active substance constituted by the compound of formula I can be used as such or in combination with a phar¬ maceutically 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 defi¬ nition above in combination with such pharmaceutically acceptable carrier. The active substances according to the present inven- tion can be formulated for use in human or veterinary medi¬ cine for therapeutic, profylactic or diagnostic uses. In cli¬ nical practice the active constituents are normally admi¬ nistered orally or rectally or by injection in the form of a pharmaceutical preparation containing the active conβti- tuents in combination with a pharmaceutically acceptable car¬ rier, which may be solid, semiβolid or liquid, or as a cap¬ sule, 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 men¬ tioned tablets, drops, solutions and suppositories. The ac¬ tive substance usually constitutes from 0.05 to 99% by weight of the preparation, for example from 0.1 to 50% for prepara¬ tions intended for oral administration. To manufacture pharmaceutical preparations in the form of dose units for oral application containing a compound ac¬ cording to the invention the active constituents can be ad- mixed with a solid pulverulent or other carrier, for example lactose, saccharose, βorbitol, mannitol, starch, such as potatoe starch, corn starch, amylopectin, a cellulose de¬ rivative or gelatin and may also include lubricants, such as magnesium or calcium βtearate, 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 ob¬ tained. Liquid preparations for oral application can be in the form of elixires, syrups or suspensions, for example solu¬ tions containing from 0.1 to 20% by weight of active sub¬ stance, sugar and a mixture of ethanol, water, glycerol, propylene, glycol and optionally other additives of a con¬ ventional character. The dose by which the active constituents are admi¬ nistered 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 consti- tuents that may be administered per day there may be mentio¬ ned 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 iden¬ tification or quantification of the compound I or residues of r. i 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 perfor¬ ming desinfection on surfaces, a compound I being applied on to the surface and then removed with bacteria adhering there¬ to. The invention will in the following be described by non-limiting examples. Example 1 Synthesis of compounds β -D-GNAcp_-<l-4>- [3-D-GNAcp-(l-4)-] n -D-GNAc (I-VIII) Chitosan hydrochloride was prepared f om chitin. by alkaline N-deacetylation according to Barker et al . (Ref.2). Chitosan was partially hydrolyzed using aqueous hydrochloric acid and their IT-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_-.i-4)-D-GNAc I &-D-GNAcp-<l-4 - β -D-GNAcp-<l-4)-D-GNAc II a*-D-GNAcp-<l-4 - [ β -D-GNAcp-<l-4> -] 2-D-GNAc III B-D-GNAcp-<l-4 - [ø-D-GNAcp-Cl-4)- 3-D-GNAc IV 0-D-GNAcp-<l-4 - [ D-GNAcp-<l-4> -] 4-D-GNAc V e-D-GNAcp-<l-4 - [β-D-GNAcp-<l-4)-] 5 -D-GNAc VI β-D-GNAcp-(l-4 - [β-D-GNAcp-<l-4)-J 6 -D-GNAc VII 3-D-GNAcp-<l-4 - [0-D-GNAcp-(l-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. SUBSTITUTE SHEET Bruker) and correct sugar and methylation analyses (Refβ. 3,4) in accordance with structure. B-D-GNTFp-<l-4)- [g-D-GNTF p -(l-4)-] n -D-GNTF (IX-XVI) These oligosaccharides were prepared using trifluoroaceto- lyβis (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 waβ evaporated to dryneββ . The residue was dissolved in methanol (20 ml) and the resulting solution was evaporated to dryneββ. 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 waβ gel chrόmatographed on Sephadex G15 (2x100 cm) using water as eluant.. Fractions containing oligosaccharide were pooled and lyophilized. The product waβ 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 F ^ n.m.r. as well as sugar and methylation analyses. Some pertinent p.m.r. and F *- n.m.r. data are given below for compound IX. £ -D-GNTFp-(i-4)-GNTF B A p.m.r. proton <_. (ppm) J(Hz) Multiples HAiα 5.263 2.6 d HAlβ 4.796 8.2 m (virtually) HAiα 4.723 8.6 d HB1S 4.713 8.6 d F 19 n.m.r. Fluorine 6(ppm) J(Hz) Multiples ACF30- • -77.640 - - S ACF 3 3 ■ -78.665 - S SUBSTITUTE B A BCF 3 α -78 . 463 - S BCF 3 3 -78 . 517 - S Shiftβ are given relative to acetone (2.225 ppm. 20°C) for H 1 and relative to external TFA (-78.500 ppm, 20°C) . -D-GNAcp-(l-4)- 3-D-GNAcp-(l-4)-] Q -1-D-GNTF (XVII-XVIII) Short time trifluoroacetolyβiβ of oligoβaccharideβ I and II uβing TFA/TFAA 1:100 (v/v) at 100°C gave a good yield of XVII-XVIII . The oligoβaccharide (I-II) (100 mg) waβ 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 waβ 40 mg. The purity and identity of the productβ waβ corraborated by p.m.r. and F 9 n.m.r. Some pertinent p.m.r. and F 19 n.m.r. data are given below for compare XVII. 3-D-GNAcp-(l-4)-GNTF B p.m.r proton δ(ppm) J(Hz) Multiple HAiα 5.264 3.6 d HA13 4.804 8.1 m (virtually) HBlα 4.561 8.6 d HB1 4.593 8.2 d HBCH3 2.072 - S F i9 n.m.r. fluorine δ(ppm) J(Hz) Multiple A-CF 3 α -77.624 - S A-CF 3 3 -78.667 — S 3-D-GNAcp-(i-4)- [S-D-GNAcp-(l-4)-J 0 _ 1 D-GNH 2 (XIX-XX) Treatment of XVII and XVIII with 1M ammonia in methanol/water for 18h reβulted in quantitative SUB 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 dryneββ . The yield waβ 80% . The purity and identity waβ verified by p.m.r. 0 II 3-D-GNAcp-(l-4)- [3-D-GNAcp-(l-4)] 0 _ 1 -D-GNHCR (XXI-XXIV) XIX and XX waβ treated with different anhydrideβ in pyridine to substitute all hydrocyl and aminogroups . The __- and N.-acylated compoundβ were de-O-acylated with ammonia in methanol/water. The oligoβaccharide (XIX-XX) (50 mg) was treated with (RCO)2θ (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 waβ purified uβing hplc (C-18 column, CH 3 CN/H 2 0) . Starting material anhydride product XIX (CC1 3 C0> 2 0 XXI XX XXII XIX (CH 3 CH 2 CO) 2 0 XXIII XX XXIV B-D-GNAcp-(l-4)-D-GNPHT (XXV) The phtalamide derivative waβ prepared according to the method of Baker et al . (Ref.7). In thiβ manner XXV waβ obtained from XIX. B-D-GN(Me)Acp-(l-4)-_3-D-GN(Me)Acp(l-4)-] 0 - 1 -D-GN(Me)Ac (XXVI- XXVII) I and II (100 mg) waβ treated with methylvinylether (25 ml) at 15°C together with p-tolueneβulphonic acid for 2 h. 'BSTITUTE S SHt I ? s» » -p- The reaction mixture waβ then passed through a Sephadex LH20 column (5x50 cm) and fractions containing oligosaccharide were pooled and concenrated to dryneββ. The acetalized oligo¬ saccharide waβ methylated using dimethylβulphinyl βodium and methyl iodide. Deacetalization of the product waβ achiev¬ ed uβing 50% aqueous acetic acid (100°C for 1 h) . The crude product waβ purified by hplc (C-18 column, CH3CN/H2-) aβ eluant). The purity and identity waβ demonβtrated by p.m.r. Example 2 Inhibition of haemagσlutina ion of sheep ervthorcvteβ with Staphylococcuβ saprophvticus bv the addition of oligosaccha¬ rides In the teβtβ the bacteria S.βaprophvticuβ βtrainβ, both isolated from urine from patients having urinary tract in¬ fection, 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 per¬ formed using erythrocytes from sheep obtained from βheep blood and washed twice with 0.85% saline. In the haemagglutination-indication experiments mic- rotitre plates (Limbo Sc .Comp. Inc . ) were used. 25/ul of bac¬ terial suspension were titrated in PBS by two-step titre steps, 25 / ul 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 (50/ul/well) and 25/ul of bacterial βuβ- penβion diluted to contain 2 HU haemagglutinating unite of bacteria were used. The plateβ were incubated for 30 minutes at 37°C, 25/ul of the erythrocyte suβpenβion being then added to each well. The reβultβ are presented in Table 1. SUBSTITUTE SHEET Table 1 Inhibition of haemagglutination between sheep erythoryctes and S .saprophvticuβ bacteria (Mc 2 and Mc 194) . Inhibitor Minimum inhibitorv cone, .uσ/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 SUBSTITUTE SHEET Example 3 Inhibition of adherence of S . saprophvticus to urinary epithelial cells bv the addition of oliqosaccharideβ . Cells from urinary epithelium were suspended in cell cultivation medium RPMI 1640 (Flow Ltd) to a concentration of 10 9 bacteria/ml. Oligoβaccharideβ which were tested for their ability of preventing adhesion of S.saprophvticuβ to urinary epithelial cells were dissolved in RPMI 1640 to a concen¬ tration of 5 mg/ l. In the experiments 1 ml of the oligosaccharide solution waβ incubated with 0.1 ml bacterial suspenβion at 37°C for 30 minuteβ. Urinary epithelial cellβ were incubated with the bacterial suspension incubated in advance as described above with oligosaccharide solution for 45 minuteβ at 37°C. Aβ a control there waβ used a bacterial suspension preincubated with 1 ml RPMI 1640. Incubated βampleβ were filtrated through a 12/U filter and waβhed 3 timeβ with PBS. The filter waβ pressed against microscope slides for a few seconds, the slides being then fixed in ethanol for- 10 minutes. The slides were dried, in air and coloured with acridine orange for 2 minutes. The slideβ were washed, dried in air and studied in fluorescence microscope. The number of bacteria per cell was counted. In each experiment ' 50 cellβ were counted. In these teβtβ 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 covalentlv linked to a macro¬ molecular carrier. The composition was made starting from oligoβaccharideβ having a free reducing end. The reactionβ used are well known and, therefore, they are only diagrammatically illuβtrated (in the scheme SR repreβentβ the βtructural element without the βugar residue constituting the reducing terminal, and MB represents a macromolecular carrier) . 14 ne R CH 2 OH R 16 ont. e . Example 5 Preparations of compositions containing the βtructural ele¬ ment according to the invention in at least bivalent asso¬ ciation without covalent bond. 5 A glycolipid containing the βaid βtructural element iβ linked by a hydrophobic interaction to lipophilic (hydro- phobic) gels, polymers or particles, for example octyl- βepharoβe, plastics and latex surfaces. 10. Example 6 Preparation of antibodieβ having βpecificitv against the βaid βtructural element. a. Manufacture of monoclonal antibodieβ by hybridoma technique. 15 I . Balb/c-mice are immunized with a compoβition according to the invention. The βpleen 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 20 of polyethylene glycol (PEG, average molecular weight 1500) with establiβhed B-myeloma cell lineβ from Balb/c-mice according to known technique. After cloning the hybridoma cells generating the antibody sought, the cells are propa¬ gated on a large scale, the culture medium supernatants 25 being harvested and the antibodies thereof being purified in a conventional manner. For identification of antibody gene¬ rating clones there is used βo-called enzyme-linked immuno- sorbent assay (ELISA). II. Mammals are immunized with an oligoβaccharide protein or polymer compoβition according to 30 the invention. Antibodieβ are isolated from the hyperimmune serum of the mammal and purified in accordance with conven¬ tional techniques. Example 7 35 Diagnostic test for identification of bacteria having accep¬ tor βtructures 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 βheep erythorycteβ and complete inhibition of haemagglutination after addition of oligoβaccharide verifieβ the fact that the bacteria possess acceptor structure. b. Bacteria are incubated with a composition wherein the βaid βtructural element iβ covalently or by other asβociation in multivalent form linked to a particular matrix according to Example 3 or 4. Incubation iβ 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 struc¬ tural 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 structureβ being maintained by interaction with the receptor structures of the column. After rinβing the column can be eluted with buffer containing a receptor-active βtructural element according to the invention and thiβ reβultβ in elu- βion 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 iβ 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 iβ prepared and applied by uβing a cotton pad on a βurface infected by S.βaprophvticus . The solution resultβ in effec¬ tive removal of the bacterium from the βurface. Example 10 Inhibition of NK-cellβ activity uβing oligoβaccharideβ 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 cellβ in' cul¬ ture. 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 ra¬ dioactive chromium are incubated with effector cells (lympho¬ cytes) 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 Malmstrbm, P., Jonβson, A. and Sjόgren, 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 βtudying inhibiting βubβtanceβ effector cellβ (6x10^ cells/ml) are preincubated with the respective oligo¬ saccharides diluted in cultivating medium for half an hour at 37°C . After washing with centrifugation the effector cells are transferred directly to the target cells. The results obtained are presented in Table 2 below. SUBSTITUTE SHEET Tab l e 2 Inhibition of specific cytotoxic activity of NK-cells. Inhibitor Minimum inhibitory cone, /ug/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 ^^-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 3xl0 6 rabbit he- patocytes in modified Dulbecco's Eagle's medium and lxl0~^^M 25i-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 sampleβ (200 /ul) were taken, tranβferred into 400 /ul centrifuge tubeβ containing 150 /ul of βilicone/mineral oil (4:1, v/v) and centrifuged for 10β in an Eppendorf centrifuge. The cell pellet at the bottom of the tube was counted for radioactivity in a Packard-counter. The 50% in¬ hibition value was determined from the observed inhibition curve and the values are shown in Table 3. SUBSTITUTE ^H ; Tabl e 3 Inhibition of binding of 125 1-asialofetuin to rabbit hepato- cytes 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 X XVVIIII 0.5 XVIII 0.6 XX 1.9 XXI 0.2 In the instant disclosure the following abbreviations have been used: Abbrevia ions GNAcp 2-acetamiαo-2-deoxy-D-glucopyranosyl GNAc » 2-acetamido-2-deoxy-D-glucoβe GNTFp 2-deoxy-2-trifluoracetamido-D-glucopyranosyl GNTF 2-deoxy-2-trifluoracetamido-D-glucose NI-2 2-amino-2-deoxy-D-glucoβe 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 BSTITUTE SHEET -R_-_-_e----f---_-e____r__-__e---_n_-__c_____e____s__» 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. An l.Chem. 12 (1965) 1602 4. Bjδrndal, H., Hellerquist, C.G., Lindberg, fi. and Sven- sson, 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.O., Schaub, R.F. and Williams, J.M. J.Org.Chem 19(19S4) 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. Lflnngren, J. et al (1976) Arch.Biochem.Biophys. 175, 661. 11. Gray, G.R. (1978). In Meth.enzymol.L. , part' C, 155. 12. McBroo , C.R., Sa anen, C.H., Goldstein, I.J. Imftethods in Enzymology, Vol. 28B, ed. V. Ginsburg, p. 212. Academic Press, New York (1972).

Claims: CLAIMS 1. A compound for therapeutic or diagnostic use, chara¬ cterized by having the formula (I): wherein R^ 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 iβ 2.2 , the individual monomer reβidueβ of the compound (I) being independent of each other aβ to structure. 2. A compound according to claim 1, wherein R^ is hyd¬ rogen and R2 has the formula: υ It / R 5 C ■ " C * R 6 R 7 wherein R5, R and R7 independently are hydrogen, lower alkyl or halogen. 3. A compound according to claim 2, wherein R5, R β and R7 all are fluoro r chloro. 4. A compound according to claim 3, wherein R5, R5 and R7 all are fluoro. 5. A compound according to any preceding claim, charac¬ terized thereby that R_ is hydrogen and R2 is acyl . 6. A compound according to claim 5, characterized the¬ reby that R2 iβ acetyl or trifluoroacetyl. 7. A compound according to any preceding claim, charac¬ terized thereby that R3 and R4 independently are hydrogen or (C 1 -C 6 )alkyl. 8. A compound according to any preceding claim, charac¬ terized thereby that n iβ 2-100, preferably 2-10. 9. A compound according to any preceding claim, charac¬ terized thereby that R^ iβ hydrogen, R2 is acetyl, R3 = R = hydrogen and n iβ 2-6. 10. A compound having the formula (I): wherein R j and R2 independently are hydrogen, alkyl, acyl or together form a cyclic amide or amine; wherein R3 and R4 independently are hydrogen, alkyl, an orga¬ nic reβidue, and wherein n is __2, with the proviβo that when n iβ 2-4 R3 iβ hydrogen and R4 is methyl then R^ is different from hydrogen if R2 is acetyl or trifluoroacetyl; and when n __ 2, R3 = R4 = hydrogen then RJL and 2 are different from hydrogen and when R^ iβ hydrogen then 2 iβ different from acetyl; and when R_ 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 R_ is hyd¬ rogen and R2 has the formula: wherein R5, Rg and R7 independently are hydrogen, lower alkyl or halogen. 12. A compound according to claim 11, wherein R5, Rg and R7 all are fluoro or chloro. 13. A compound according to claim 3, wherein R5, Rg and R7 all are fluoro. 14. A compound according to any of claims 10-13, charac¬ terized thereby that R^ iβ hydrogen and R 2 iβ acyl. 15. A compound according to claim 14, characterized the¬ reby that R2 iβ acetyl or trifluoroacetyl . 16. A compound according to any of claimβ 10-15, charac¬ terized thereby that R3 and R4 independently are hydrogen or (C 1 -C 6 )alkyl. 17. A compound according to any of claimβ 10-16, charac¬ terized thereby that n iβ 2-100, preferably 2-10. 18. A compound according to any of claimβ 10-17, charac¬ terized thereby that R3 and R4 both are hydrogen and n iβ 2-6. 19. A compound according to claim 18, characterized thereby that n iβ 2. 20. A compound according to claim 10, characterized thereby that R j iβ hydrogen, R2 iβ acetyl or trifluoroacetyl, at leaβt one R2 being trifluoroacetyl, and n iβ 2-6. 21. A compound according to claim 20, characterized thereby that n iβ 2. 22. A compoβition for therapeutic treatment of mammalβ including man, characterized by a compound according to any preceding claim in combination w th a pharmaceutically acceptable carrier. 23. A method for therapeutic treatment of mammalβ inclu¬ ding man, characterized by adminiβtering to the mammal a the- rapeutically effective amount of a compound according to any of claimβ 1-21 or a compoβition according to claim 22. 24. A method for determining the preβence of pathogenic bacteria in a sample taken from a mammal including man, cha¬ racterized by determining the grade of interaction between the bacteria of the sample and the compound according to any of claimβ 1-21. 25. A method for identification or quantification of βaid βtructure element in native biological material from mammalβ including man, characterized by uβing antibodieβ, the production of which being induced by the compound according to any of claimβ 1-21. 26. A method for purification of acceptor structures in bacteria, characterized thereby that in thiβ purification the S Γ< _26-_> affinity between the compound according to claimβ 1-21 and the acceptor structures of the bacteria iβ 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 βurface of the compound then being removed with bacteria adhering thereto.

Inventors: KALLIN ELISABETH, LINDH FRANK, LOENN HANS, MAONSSON OLLE, MAORDH PER ANDERS, NILSSON BO, SVENSSON SIGFRID, KALLIN, ELISABETH, LINDH, FRANK, LOENN, HANS, MAONSSON, OLLE, MAORDH, PER, ANDERS, NILSSON, BO, SVENSSON, SIGFRID
Applicants: BIOCARB AB
Application year: 1986
Main classification: C07H5
IPC codes: A61K47, C07H3, C07H21, C08B37