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

Carbohydrate derivatives and compositions thereof for therapeutic or diagnostic use, and methods for their use Download PDF

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Publication number
WO1986005789A1
WO1986005789A1 PCT/SE1986/000131 SE8600131W WO8605789A1 WO 1986005789 A1 WO1986005789 A1 WO 1986005789A1 SE 8600131 W SE8600131 W SE 8600131W WO 8605789 A1 WO8605789 A1 WO 8605789A1
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Prior art keywords
hydrogen
compound according
bacteria
compound
independently
Prior art date
Application number
PCT/SE1986/000131
Other languages
French (fr)
Inventor
Sigfrid Svensson
Per Anders MA^oRDH
Frank Lindh
Hans LÖNN
Elisabeth Kallin
Bo Nilsson
Olle MA^oNSSON
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Biocarb Ab
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Publication of WO1986005789A1 publication Critical patent/WO1986005789A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof

Definitions

  • 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.
  • the invention relates to applications directed to staphylococci, such as Staphylococcus saprophvticus, and bacteria of the species Bordetella pertussis causing hooping cough.
  • the compositions according to the invention have the ability of inhibiting the activity of so called natural killer cells (NK-cells).
  • NK-cells natural killer cells
  • the technique according to the invention relating to targeting is applicable to cells and organs of mammals including man.
  • microorganism used in the present disclosure is intended to cover bacteria, viruses, animal and plant 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.
  • 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.
  • 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:
  • R 1 and R 2 independently are hydrogen, lower alkyl, acyl or together form a cyclic amide or amine; wherein R 3 and R 4 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.
  • R 1 is hydrogen and R 2 has the formula:
  • R 5 , R 6 and R 7 are suitably all fluoro or chloro, particularly fluoro.
  • R 1 is hydrogen and R 2 is acyl, particularly acetyl or trifluoroacetyl.
  • R 3 and R 4 may suitably independently be hydrogen or lower alkyl.
  • 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.
  • the same conditions apply as given above in connection with compounds of formula I.
  • R 1 is hydrogen and R 2 is acetyl or trifluoroacetyl at least one of substituents R 2 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.
  • substituent R 4 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 R 4 are hydrogen, lower alkyl or a macromolecular carrier covalently bound to the carbohydrate residue, optionally via a coupling arm.
  • 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.
  • the coup ling arm between the carbohydrate residue and the macromolecular carrier can be any of the following:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, CH 3 CN/H 2 O system). The following oligosaccharides were isolated and characterized:
  • 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.
  • 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 chr ⁇ matographed on Sephadex G15 (2x100 cm) using water as eluant.
  • TFA/TFAA trifluoroacetic acid/trifluoracetic anhydride
  • Shifts are given relative to acetone (2.225 ppm. 20°C) for H 1 and relative to external TFA (-78.500 ppm, 20°C).
  • 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, CH 3 CN/H 2 O) . Typical yield of purified product was 40 mg. The purity and identity of the products was corraborated by p.m.r. and F 19 n.m.r.
  • 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% .
  • 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) 2 O (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, CH 3 CN/H 2 O) .
  • XXV ⁇ -D-GNAcp-(1-4)-D-GNPHT
  • microtitre plates (Limbo Sc. Comp. Inc. ) were used. 25 ⁇ l of bacterial suspension were titrated in PBS by two-step titre steps, 25 ⁇ l of 1% erythrocyte suspension being then added. The haemagglutination titre was determined after incubation of the plates for 2-4 hours in room temperature.
  • RPMI 1640 Flow Ltd
  • 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.
  • 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 12 ⁇ 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.
  • Example 4 Preparation of compositions containing the structural element in at least bivalent state and covalently linked to a macromolecular carrier.
  • 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).
  • 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.
  • a Manufacture of monoclonal antibodies by hybridoma technique.
  • 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.
  • PEG polyethylene glycol
  • the cells 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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.
  • Bacteria are incubated with sheep erythrocytes to agglutinate same.
  • 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 sheep erythrocytes to agglutinate same.
  • 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
  • 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.
  • 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.
  • 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.
  • composition for use for desinfection Composition for use for desinfection
  • 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.
  • 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.
  • a cytotoxic i.e. cell killing activity against certain types of cells in culture.
  • K-562 a human leukemia cell line called K-562 as a target cell for such cytolytic activity.
  • 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.
  • the assays were carried out in capped polystyren tubes (12x75 mm) at 4°C . To each tube was added 3x10 6 rabbit hepatocytes in modified Dulbecco's Eagle's medium and 1x10 -10 M 125 I-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 ⁇ l) were taken, transferred into 400 ⁇ l centrifuge tubes containing 150 ⁇ l 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

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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:
Figure imgf000005_0001
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:
Figure imgf000005_0002
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:
Figure imgf000006_0001
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:
Figure imgf000007_0001
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-GNTFp-(1-4)- [β-D-GNTFp-(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)
Figure imgf000012_0001
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).
Figure imgf000016_0001
Figure imgf000016_0002
Figure imgf000016_0003
Figure imgf000017_0001
Figure imgf000017_0002
Figure imgf000017_0003
Figure imgf000017_0004
Figure imgf000017_0005
Figure imgf000017_0006
Figure imgf000018_0001
Figure imgf000018_0002
Figure imgf000018_0003
Figure imgf000018_0004
Figure imgf000018_0005
Figure imgf000018_0006
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):
Figure imgf000025_0001
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:
Figure imgf000025_0002
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):
Figure imgf000026_0001
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:
Figure imgf000026_0002
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.
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