WO1994016714A1

WO1994016714A1 - The use of a composition for the treatment of infections caused by helicobacter pylori

Info

Publication number: WO1994016714A1
Application number: PCT/SE1994/000021
Authority: WO
Inventors: Olle Larm; Torkel Wadström
Original assignee: Medicarb Ab
Priority date: 1993-01-19
Filing date: 1994-01-14
Publication date: 1994-08-04
Also published as: AU5894494A; SE9300139D0; SE9300139L

Abstract

The use of a composition in the preparation of a drug for the treatment or prophylaxis of infections caused by or associated with Helicobacter pylori, said composition containing as an active constituent an immobilized, negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate and dextran sulphate; a method of treating or preventing infections caused by or associated with Helicobacter pylori in mammals including man, comprising administering to a mammal in need of such treatment or prophylaxis an effective amount of a composition containing as an active constituent an immobilized, negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate and dextran sulphate; a protein with an apparent molecular weight of not more than about 120 kDa (on SDS-PAGE) and active subfragments thereof down to a molecular weight of about 20 kDa, said protein and subfragments specifically binding to heparan sulphate and being producible from H. pylori strains 25 and 1139 as a surface protein antigen thereof; mono- and polyclonal antibodies; process for detecting H. pylori; drug compositions; reagent kit; and a vaccine.

Description

The use of a composition for the treatment of infections caused by Helicobacter pylori

The present invention relates to the use of a composition in the preparation of a drug for the treatment or prophylaxis of infections caused by Helicobacter pylori. The invention further involves a method of treating or preventing infections caused by Helicobacter pylori in mammals, new compositions and a process for diagnozing the presence of H. pylori in a specimen, a vaccine and a method for manufacturing the active component of the present composition.

Helicobacter pylori, previously called Campylobacter pylori, is a highly motile organism which penetrates the gastric mucin layer and colonizes the gastric epithelium or ectopic gastric tissues in other parts of the intestinal tract, such as gastric metaplasia in the duodenum.

(Hazell S. L., Lee A., Brady L., and Hennesey W. 1986.

Campylobacter pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factor in colonization of the gastric epithelium. J. Infect.Dis. 153: 658-663; Lingwood C.A., Law, H., Pellizzari A., Sherman P., and Drumm B. 1989.

Gastric glycerolipid as a receptor for Campylobacter pylori. Lancet II: 238-241). The bacterium has recently been reported to express sialic acid-specific agglutinating activity against erythrocytes of various animal species (Robinson, J., Goodwin C.S., Cooper, M., Burke V. and Mee B.J. 1990. Soluble and cell-associated haemagglutinins of Helicobacter (Campylobacter) pylori. J.Med.Microbiol. 33: 277-284; WadstrOm T., Guruge J. L., Wei S., Aleljung

P., and Ljungh Å. 1990. Helicobacter pylori hemagglutinins - possible gut mucosa adhesins, p 96-103. In: P. Malfertheiner and H. Ditschuneit (ed.), Helicobacter pylori Gastritis and peptic ulcer. Springer-Verlag, Berlin).

Helicobacter pylori is a bacterium which frequently infects the human stomach and causes the so called type B gastritis. So far attempts to treat or prevent infections caused by said bacterium have not been successful, and although various approaches have been suggested the problem remains unsolved. US patent 5 116 821 is an example of prior art related to the treatment of gastrointestinal disorders caused by or associated with H. pylori. However, the approach used in said US patent is principally different from that of the present invention in that sulphated glyceroglucolipids are used as an active constituent, whereas the present invention involves the use of immobilized polysaccharides.

Through extensive research and experimentation it has now been surprisingly found, that H. pylori strongly interacts with certain immobilized, negatively charged polysaccharides. This finding is of a pioneer character and constitutes an important step towards solving the problem of treating or preventing infections caused by the bacterium in question.

Accordingly, the present invention has for a main object to provide a composition for use in the preparation of a drug for the treatment or prophylaxis of infections caused by H. pylori.

Another object of the invention is to use in such composition as an active constituent an immobilized, negatively charged polysaccharide.

Yet another object of the invention is to immobilize said polysaccharide to an insoluble carrier by covalent binding thereto.

Another object of the invention is to provide a method of treating or preventing infections caused by H. pylori in mammals including man.

Still another object of the invention is to provide techniques for the treatment or prophylaxis of gastrointestinal disorders, such as gastric ulcer.

A further object of the invention is to provide a protein capable of binding specific glucoseaminoglycans at a high binding strength. A further object of the invention is to provide a process for performing diagnosis to detect the presence of H. pylori in a specimen.

For these and other objects that will follow from the present disclosure the invention provides for the use of a composition in the preparation of a drug for the treatment or prophylaxis of ..infections caused by Helicobacter pylori, said composition containing as an active constituent an immobilized, negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate, and dextran sulphate.

Said insoluble carrier may be selected from the group comprising organic non-polysaccharide polymers and gelforming carbohydrate och protein polymers.

Preferred gelforming polymers are agar, algin, alginic acid, carrageenan, chitin, chitosan, collagen, gelatin, guar gum, locust bean gum, and xanthan gum.

A particularly preferred polysaccharide is heparin or heparan sulphate, and a preferred insoluble carrier is chitin or chitosan or an insoluble substrate coated with chitosan or chitin.

The invention also covers a method for treating or preventing infections caused by or associated with H. pylori in mammals including man, comprising administering to a mammal in need of such treatment or prophylaxis an effective amount of a composition containing as an active constituent an immobilized, negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate, and dextran sulphate, optionally together with a pharmaceutically acceptable carrier or excipient.

One preferred route of administration is oral administration, especially for the treatment or prophylaxis of gastrointestinal disorders, such as gastric ulcer.

In accordance with the present invention a specific cell surface protein antigen has been identified, and the invention also covers such a protein with an apparent molecular weight of not more than about 120 kDa (on SDS- PAGE) and active subfragments thereof down to a molecular weight of about 20 kDa, said protein and subfragments specifically binding to heparan sulphate and being producable from H. pylori strains 25 and 1139 as a surface protein antigen thereof. The binding strength (K_d) to heparan sulphate at a pH of about 4 is preferably more than about 10 ^-7M. In fact, the binding strength of a surface protein originating from H.pylori strains 25 and 1139 to heparan sulphate is about 9x10 ^-9M. (Said bacterial strains are available from the Culture Collection of Gothenburg,

Sweden)

The invention also includes mono- or polyclonal antibodies directed against said cell surface protein.

Another application of the present invention resides in a process for diagnosing a specimen for the presence of H. pylori, wherein said specimen is contacted with the antibody defined above, and where the presence or absence of interaction between antibody and specimen is recorded.

Finally, the invention provides for a composition for medicinal use comprising as an active constituent a negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate, .and dextran sulphate immobilized to an insoluble carrier by covalent binding thereto, said carrier being selected from the group comprising: agar, algin, alginic acid, carrageenan, chitin, chitosan, collagen, gelatin, guar gum, locust bean gum, and xanthan gum, optionally together with a pharmaceutically acceptable carrier or excipient. A preferred embodiment of such composition contains heparin or heparan sulphate as a polysaccharide, the carrier being chitin or chitosan or an insoluble substrate coated with chitin or chitosan.

The present invention thus relates to the finding that a specific suface protein interacts specifically with certain immobilized, negatively charged polysaccharides selected from heparin, heparan sulphate and dextran sulphate. The finding is of immense practical use, both for the manufacture of new drugs useful in preventing and treating H. pylori infections in mammals including humans, in the intestinal tract, such as in the gastroduodenal part thereof and for diagnosis on the presence of H. pylori in a specimen.

The new surface protein according to the present invention is quite specific in its interaction with the selected group of negatively charged polysaccharides in an immobilized state in that it does not recognize other glycose amino glycans of comparable size, such as chondroitin sulphate, dermatan sulphate, highly glycosylated glycoproteins (hog gastric mucin and fetuin), and various carbohydrates (fucose, mannose, galactose, glucosamine and n- acetyl-D-glucosamine. Nor does it interact with dextran but in contrast high- and low-molecular weight dextran sulphates of the same molecular weight.

The strong interaction between the selected polysaccharides and the specific surface protein according to the invention is useful also for immunization of animals to raise antibodies which can be used to perform diagnosi to detect the presence of H. pylori in the specimen.

The invention also involves proteins specifically binding to a heparan sulphate which can be prepared by a process comprising the following steps:

a) preparing a cell extract by suspending H. pylori, such as strain 25 or strain 1139, in a buffer solution of an acid pH, such as a pH of about 2;

b) removing the cell debris from the suspension resulting from step a) above, such as by centrifugation; c) applying the supernatant resulting from step b) above onto a heparin-sepharose column;

d) eluting said column using a solution of a pH abov neutral, such as a pH of about 9 to 10;

e) collecting the fractions having the ability to in hibit binding of H. pylori to heparan sulphate, such as fractions showing absorption at 280 nm; and f) recovering the proteins having such inhibiting capacity from the fractions resulting from step e) above.

The approximate molecular weight of the proteins recovered in step f) above preferably lies within the range about 20 to about 120, such as about 120, 72 and 35.

Finally, the invention includes a vaccine for use in creating resistance to infections caused by or associated with H.pylori. Such vaccine comDrises as an active constituent a protein as defined above or prepared according to the above process, said protein being present in an immunologically active amount and being used in combination with a pharmaceutically acceptable carrier or diluent.

The term "insoluble carrier" as used herein means that the carrier, to which the selected polysaccharide is to be immobilized, preferably by covalent binding thereto, is insoluble in the environment where the present composition is to be used. Such environment may be found in the gastrointestinal tract, such as in the stomach or in the duodenum. The latter two sites are those of major interest since the bacterium H. pylori is frequently found thereat.

Representative of materials which can be used are polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, cellulose acetate, cellulose nitrate, cellulose triacetate, ethylene vinylacetate, polyesters, polyanhydrates, polyothoesters, different cellulose derivatives, such as hydroxiethyl cellulose, acetylated hydroxiethyl cellulose, polyglycolic acids, polylactic acids, polyparalactic-glycolic acids, polyether and polyester uretanes, polyamides and methacrylates.

The insoluble carrier can be made from polymers which have the ability of losing strength via erosion, by dissolution, hydrolysis or enzymatic degradation. Such polymers include but are not limited to polyethylene glycol, polyethylene oxide, polyvinyl alcohol, dextran, gelatin, polyvinyl pyrrolidone, hydroxi propyl methyl cellulose, cellulose acetate phtalate, polysaccharides, gum arabic, copolymers of dimethylamino ethyl methacrylate and methacrylic acid esters, polyorthoesters, polyglycolic acid, polylactic acid, etc. The carrier may also be constituted by cross-linked hydrogels, such as polyhydroxiethyl

methacrylate and the like.

The present compositions for medicinal use containing as an active substituent a negatively charged polysaccharide selected from heparin, heparan sulphate, and dextrane sulphate immobilized to an insoluble carrier by covalent binding, can additionally contain a pharmaceutically acceptable carrier or excipient. Such pharmaceutical formulations may be prepared from a predetermined quantity of the active constituent, and are preferably in solid form suitable for oral administration. The formulations may take the form of powders, elixirs, solutions, pills, capsules, pellets or tablets. Pharmaceutically acceptable carriers or excipients, such as starch, sugar, talc, commonly used synthetic and natural gums, water and the like may be used in such formulations. Binders, such as gelatin, and lubricants, such as sodium stearate, may be used to form tablets. Disintegrating agents, such as sodium bicarbonate may also be included in the tablets.

The invention will now be further illustrated by specific examples which are not to be construed as limiting. In the examples, unless otherwise stated, percentages and parts refer to weight.

The illustration of the invention will be made with reference to the appended drawings, wherein:

Fig. 1 illustrates the saturability of heparan sulphate-binding by H. pylori, strain 25;

Fig. 2, A and B, illustrates the kinetics of heparan sulphate-binding by displacement of bound ¹²⁵I-heparan sulphate from H. pylori, strain 25;

Fig. 3, A and B, illustrates displacement of cell bound ¹²⁵I-heparan sulphate by unlabelled heparan sulphate and heparin; Fig. 4 illustrates binding of the cell surface receptor structure to immobilize heparin on ELISA

plates. EXAMPLE 1

MATERIALS AND METHODS

Strains and culture conditions

The seventeen strains of Helicobacter pylori examined, were from the collection in our laboratory (Emödy L., Carlsson Å., Ljung Å., and Wadstrom T. 1988. Mannose-resistant haemagglutinin by Campylobacter pylori. Scand. J. Infect.Dis. 20: 353-354; Guruge, J. L., Schalen C, Nilsson I., Ljung Å., Tyszkiewicz T., Wikander M. and Wadstrom T. 1990. Detection of antibodies to Helicobacter pylori cell surface antigens. Scan. J. Infect.Dis. 22: 457-465; Wadstrom T., Guruge J. L., Wei S., Aleljuhg P., and Ljungh Å. 1990. Helicobacter pylori hemagglutinins-possible gut mucosa adhesins, p 96-103. In: P. Malfertheiner and H. Ditschuneit (ed.), Helicobacter pylori Gastritis and peptic ulcer.

Springer-Verlag, Berlin). They were grown, under microaerophilic conditions at 37ºC for 3-5 days on Lab M blood agar base #1 (London Analytical & Bacteriological Media Limited) supplemented with 5% defibrinated horse blood. The bacteria were harvested with 0.01 M potassium phosphate buffer (pH 7.2) containing 0.15 M NaCl (PBS) (1 ml/plate), washed once, and suspended to a final density of 10¹⁰ CFU/ml.

Chemicals

Highly purified preparations of glycosaminoglycans /hyaluronan, bovine lung heparan sulphate, bovine cartilage chondroitin sulphate, porcine skin dermatan sulphate, and porcine intestinal heparin (Table 1) were obtained as previously described (Fransson, L. Å., Havsmark B., and Silverberg I. 1990. A method for the sequence analysis of dermatan sulphate. Biochem. J. 269: 381-388; Schmidtchen A., Carlstedt E., Malmstrom A., and Fransson L. Å. 1990. Inventory of human skin fibroblast proteoglycans. Biochem. J. 265: 289-300). Dextran (M_r 500,000) was obtained from Pharmacia-LKB (Uppsala, Sweden) and dextran sulphates (M_r 5,000 and 500,000) were from Sigma Chemical Co. (St.

Louis, MO, USA). All other chemicals were of analytical grade and were purchased from various commercial sources.

Binding assay

Heparan sulphate was derivatized at the amino-terminal serine residue with p-hydroxyphenyl proprionate and radio-iodinated (Fransson, L.Å., Havsmark B., and Silverberg I. 1990. A method for the sequence analysis of dermatan sulphate Biochem. J. 269: 381-388). The binding of

¹²⁵I-heparan sulphate by H. pylori was quantitated as previously described for the binding of fibronectin by Escherichia coli (Froman G., Switalski L.M., Faris A., Wadstrom T., and Höök M. 1984. Binding of Eschirichia coli to fibronectin. J.Biol.Chem. 23: 14899-14905). Briefly, samples of a ¹²⁵I-labelled heparan sulphate solution ( 50 μl containing ca 20 ng [25,000 cpm] of heparan sulphate) in 0.1

M sodium phosphate buffer (PBS, pH7.2) supplemented with

0.05% sodium azide and 0.1% bovine serum albumin were mixed with 100 μl of H. pylori cell suspension (10⁹ cells) in polystyrene centrifuge tubes and kept at room temperature for 1 h. Incubation mixtures without bacteria were used as background cpm controls. After adding 2 ml of ice- cold PBS containing 0.1% Tween 20, the mixtures were centrifuged (4,500 x g, 10 min) and the radioactivity of the pellets was measured in a gamma counter. Streptococcus mutans served as a positive control (Choi S.H., and Stinson M.H. 1989. Purification of Streptococcus mutans protein that binds to heart tissue and glycosaminoglycans. Infect. Immun. 577 3834-3840). Other binding assays were performed in the presence of divalent cations (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Fe⁺⁺) and EDTA (final concentrations of 1 mM ), and in various pH and ionic strength buffers. The bound heparan sulphate was expressed as a percentage of the total radiolabelled heparan sulphate incubated with the cells, and was corrected for the background cpm.

EXAMPLE 2

1²⁵I-heparan sulphate binding-inhibition assay

H. pylori suspensiohs (100 μl containing 10⁹ cells) were incubated at 20°C for 1 h with 100 μg of unlabelled heparan sulphate, chondroitin sulphate, dermatan sulphate, heparin, hyaluronan, fetuin, hog gastric mucin, dextran sulphate (high and low molecular weight), and dextran. The treated cells were washed with 2 ml of PBS, suspended in

100 μl of buffer, and used in the binding assay described above. Enzymatic digestion of bacterial cells.

H. pylori suspensions (100 ul containing 10⁹ cells) were treated with 100 μg of pepsin, pronase E, (from

Streptomyces griseus), proteinase K (from Tritirachium album), trypsin, chymotrypsin, and neuraminidase (from

Vibrio cholerae). The conditions for each enzyme were those described in the Worthington Enzyme Manual (Worthington Biochemical Corporation. 1978. Worthington.

Enzyme and related biochemicals. Worthington Biochemical Corporation, Freehold, NJ). Cells were washed twice with PBS (4 ml), suspended in the same buffer at the original cell concentration, and used in the binding assays described above.

EXAMPLE 3

Purification of heparan sulphate-binding protein(s) from H. pylori.

All purification procedures were done at 4ºC.

H. pylori strain 25 was grown and harvested as described above, washed with PBS, and suspended in 0.2 M glycine buffer ( pH 2.2) containing 10 mM EDTA and 1 mM PMSF, at a final concentration of 0.4 g (wet weight) per 100 ml of buffer. The suspension was incubated with gentle stirring at 20°C for 30 min, the bacteria were removed by centrifugation (8,000 x g, 30 min), and the supernatant fluid was dialyzed against 0.01 M ammonium bicarbonate. The crude extract (100 ml) was applied to a heparin-Sepharose (Pharmacia-LKB) column (2 x 8 cm) equilibrated with PBS, and the column was washed with PBS ( at a flow rate of ca 30 ml/h, and the effluent was collected in 5-ml fractions), until no 280-nm absorbance was detected in the effluent. The column was then eluted with 2 M NaCl and with 0.01 M NaOH, and the effluents were collected in the same conditions as described above. The fractions in each 280 nm-absorbing peak were pooled, dialyzed against 0.01 M ammonium bicarbonate, and assayed for the ability to inhibit binding of ¹²⁵I-heparan sulphate as described above.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography. SDS-PAGE of the pooled fractions comprising the peaks that inhibited heparan sulphate-binding activity was performed according to the method of Laemmli (Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685) under denaturing conditions in 12% polyacrylamide gel, and the protein bands were revealed by silver staining (Bio-Rad Laboratories, Richmond, CA., USA). In western blotting experiments, the separated proteins were electrophoretically transferred to nitrocellulose in a trans-blot cell. Additional binding sites were blocked by incubating the membranes with 1% bovine serum albumin in PBS (pH 7.2) and the membranes were washed with PBS containing 0.05% Tween20, at 22ºC. The membranes were probed with ¹²⁵I-heparan sulphate, 2,000 cpm per 10μl, in PBS (pH 7.2) or in 100 mM sodium acetate buffer (pH 4) for 2 h, at 22°C. After washing with PBS-Tween (pH 7.2) or acetate buffer ( pH 4) containing 0.05% Tween-20, the membranes were dried and examined by autoradiography (-70ºC, 48 h) using X-Omat RR film (Eastman Kodak). RESULTS

H. pylori strains 25 and 1139, which showed moderate binding and which have previously been examined for hemagglutinins (Guruge J.L., Schalen C, Nilsson I., Ljungh Å., Tyszkiewicz T., Wikander M. and Wadstrom T. 1990. Detection of antibodies to Helicobacter pylori cell surface antigens. Scan. J. Infect.Dis. 22: 457-465; Wadstrom T., Guruge J.L., Wei S., Aleljung P., and Ljungh Å. 1990.

Helicobacter pylori hemagglutinins-possible gut mucosa adhesins, p 96-103. In: P. Malfertheiner and H. Ditschuneit (ed.), Helicobacter pylori Gastritis and peptic ulcer. Springer-Verlag, Berlin), were selected for further characterization of heparan sulphate-binding. Increasing amounts of heparan sulphate were incubated with strain 25 and saturation of the binding sites was obtained within the concentration-range used. Fig. 1 shows the saturability of heparan sulphate-binding by H. pylori strain 25.

Bacteria (10⁹ CFU) were mixed with the indicated amounts of ¹²⁵I-heparan sulphate and binding assays were performed as described above. The inset shows a scatchard plot representation of the data. Scatchard plot analysis of the binding data gave a straight line (Fig. 1, inset), which indicated the presence of one class of heparan sulphate receptor, and a K_d of 9x10^-9 M was calculated from the slope of the line.

Fig. 2 shows kinetics of heparan sulphate-binding by, and displacement of bound ¹²⁵I-heparan sulphate from

H. pylori strain 25. Fig. 2A shows the kinetics of binding, whereas Fig. 2B shows the displacement of cell-bound

¹²⁵ I-heparan sulphate by unlabelled heparan sulphate (100 μg/100 μl of bacterial suspension [10^-9 cells]). The unlabelled heparan sulphate was added to bacterial suspensions containing bound ¹²⁵I-heparan sulphate and the suspensions were incubated at 22°C for various intervals of time. The released ¹²⁵I-heparan sulphate was counted in a gamma counter. Fig. 3 shows the displacement of cell-bound ¹²⁵I-heparan sulphate by unlabelled heparan sulphate and heparin. The unlabelled heparan sulphate (panel A) or heparin (panel B) was added to bacterial suspensions containing bound

¹²⁵I-heparan sulphate, and the suspensions were incubated at 22°C for one hour. The released ¹²⁵I-heparan sulphate was counted in a gamma counter.

The binding was time-dependent (Fig. 2A), and cellbound ¹²⁵I-heparan sulphate was displaced by an excess of unlabelled heparan sulphate (Figs. 2B and 3A) and heparin (Fig. 3B). Binding was pH-, and ionic strength-dependent; binding was optimal at pH 4 and was inhibited in high io- nic-strength solutions e.g., 2 M NaCl virtually abolished binding. Divalent cations (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Fe⁺⁺ ) and EDTA did not affect heparan sulphate-binding by the bacterium.

Unlabelled heparan sulphate and heparin inhibited binding of ¹²⁵I-heparan sulphate by K. pylori (see appended Table 2). However, other glycosaminoglycans of comparable size (chondroitin sulphate and dermatan sulphate), highly glycosylated glycoproteins (hog gastric mucin and fetuin), and various carbohydrates (fucose, mannose, galactose, glucosamine, and N-acetyl-D-glucosamine) did not inhibit binding. In contrast, high- and low-molecular weight dextran sulphates, but not dextran of the same molecular weight, inhibited binding.

Heat-treatment (80°C, 10 min) of the bacteria reduced their heparan sulphate-binding activity by ca 90%. Binding was unaffected after treatment of the bacteria with pepsin and neuraminidase, but was reduced by 50% to 70% by exposure to pronase E, proteinase K, trypsin, and chymotrypsin. Results of heat- and enzyme-inactivation studies showed that the heparan sulphate-binding component was proteinaceous. EXAMPLE 4

Isolation of surface protein

Studies were made to isolate heparan sulphate-binding, surface-proteins from H. pylori strain 25. Three proteins, of an approximately M_r 120, 72 and 35 kDa (revealed by SDS-electrophoresis/autoradiography) that inhibited heparan sulphate-binding by H. pylori were isolated by affinity chromatography on heparin-Sepharose, from a cell surface preparation obtained by extracting the bacterium with 0.2 M glycine buffer (pH 2.2).

The binding strength was studied and the low binding at high pH (pH 8) and the high binding at low pH (pH 4), as well as the inhibition of binding at high ionic- strength (2 M NaCl) suggest that charge interactions are important for optimal binding. The proteinaceous binding- site on H. pylori appears to have a strong positive charge at pH 4; heparan sulphate is not expected to have a particularly high negative charge at this pH (Fransson L.Å., 1987. Structure and function of cell associated proteoglycans. Trends Biochem. Sci. 12: 406-411). Other negatively charged glycosaminoglycans, such as dermatan sulphate and chondroitin sulphate, did not inhibit binding (see Table 1), thus suggesting that binding has some specificity for polysaccharides with a particular chain geometry and distribution of negative charges.

Amination of surfaces

EXAMPLE 5

Etching of polymeric surface

A polymeric surface of polyethylene was treated for 2 min at room temperature with a solution of 2% potassium permanganate (KMnO₄) (w/v) in concentrated sulfuric acid H₂SO₄ and carefully rinsed with distilled water. EXAMPLE 6

Amination with polyethyleneimine

The polymeric surface resulting from Ex. 5 above was treated at room temperature with a solution of polyethyleneimine SN (PEI, 0.005% in borate buffer pH 9.0) together with a solution of crotonaldehyde (0.034% in borate buffer pH 9.0). The surface was carefully rinsed with distilled water and then treated with a solution of dextran sulphate (Pharmacia) 0.1 g/L in 0.15 M NaCl, pH 3.0 for 10 min at 55°C. After rinsing with large volumes of water, the surface was treated with a solution of 0.05% PEI at pH 9.0, 10 min at room temperature and washed as above. The presence of amino groups was verified with an indicator (ponceau S, Sigma).

EXAMPLE 7

Amination with chitosan

The surface was treated as in Ex. 2 with the exception that a solution of chitosan containing 15% N-acetyl groups (0.25% w/v) in water was used instead of PEI and that the surface after the first step was rinsed with ethanol (80% v/v) instead of water. The presence of aminogroups was verified with an indicator as in Ex. 2 and on films by FTIR.

EXAMPLE 8

Periodate oxidation of heparin

In a typical example (when ca 10% of the monosaccharide residues should be oxidized), the polysaccharide (heparin) (5 g) was dissolved in water (100 ml) and sodium periodate (0.5 g) was added. The solution was kept in the dark at room temperature for 24 h. The reaction mixture was then dialysed against distilled water and freeze dried. EXAMPLE 9

Nitrous acid degradation of heparin

Heparin (1 g, pig intestinal mucousa (Kabivitrum)) was dissolved in water 300 ml. The solution was kept at 0°C in ice-water. First sodium nitrite (10 mg) and then acetic acid (2 ml) was added to the stirred solution. The reaction mixture was kept at 0°C for 2 hours and then processed by dialysis against distilled water (24 h) and freeze drying.

EXAMPLE 10

End-point attachment of heparin

Polyethylene films (5x5 cm, 0.125 mm) etched as in Ex. 5 and then aminated as in Ex. 6 and treated with dextran sulphate as in Ex. 6. The amination step was repeated and the surface was then treated with 0.05% PEI in water (adjusted to pH 9.0 with M NaOH) for 10 min at room temperature. Finally, the films were reacted for 2 hours at 50°C with nitrous acid degraded heparin and sodium cyanoborohydride (0.0025% and 0.00025% w/v, respectively, in

0.15 M NaCl, pH 3.9) and then rinsed with borate buffer pH 9.0.

EXAMPLE 11

Periodate coupling of heparin

Polyethylene films were heparinized as described in Ex. 10 with the modification that periodate oxidized heparin prepared as in Ex. 8 was used. The coupling yield was 2 ug/cm² as determined by FTIR.

EXAMPLE 12

Binding of H. pylori cellsurface protein (HSBP-4) to ELISA plates

ELISA plates (polystyren, NUNC ) were coated with heparin as in Ex. 10 (end-point attachment, LY A, N Hep) and as in Ex. 11 (multi-point attachment, LY B, p Hep), respectively, and the ability for binding HSBP-4 was evalu ated. The results are summarized in Fig. 4 and Table 3 and show that end-point attached heparin has a higher affinity for HSBP-4 than multi-point attached heparin.

TABLE 3 Effect of the heparin immobilization procedure on ELISA plates for binding of H.pylori heparinbinding proteins (HpBPs).

Claims

1. The use of a composition in the preparation of a drug for the treatment or prophylaxis of infections caused by or associated with Helicobacter pylori, said composition containing as an active constituent an immobilized, negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate and dextran sulphate.

2. The use according to claim 1, wherein said polysaccharide has been immobilized to an insoluble carrier by covalent binding thereto.

3. The use according to claim 2, wherein said insoluble carrier is selected from the group comprising:

organic non-polysaccharide polymers and gelforming carbohydrate or protein polymers.

4. The use according to claim 3, wherein said gelforming polymers are selected from the group comprising:

agar, algin, alginic acid, carrageenan, chitin, chitosan, collagen, gelatin, guar gum, locust bean gum, and xanthan gum.

5. The use according to any preceding claim, wherein said polysaccharide is heparin or heparan sulphate.

6. The use according to any of claims 2 to 5, wherein said insoluble carrier is a non-polysaccharide carrier coated with chitin or chitosan.

7. A method of treating or preventing infections caused by or associated with Helicobacter pylori in mammals including man, comprising administering to a mammal in need of such treatment or prophylaxis an effective amount of a composition containing as an active constituent an immobilized, negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate and dextran sulphate.

8. A method according to claim 7, comprising oral administration of said composition.

9. A method according to claim 7 or 8 for the treatment or prophylaxis of gastroduodenal disorders, such as gastric ulcer.

10. A protein with an apparent molecular weight of not more than about 120 kDa (on SDS-PAGE) and active subfragments thereof down to a molecular weight of about 20 kDa, said protein and subfragments specifically binding to heparan sulphate and being producable from H. pylori

strains 25 and 1139 as a surface protein antigen thereof.

11. A protein according to claim 10, wherein the binding strength ( K_d) to heparan sulphate at a pH of about 4 is more than about 10 ^-7M.

12. A protein according to claim 10 or 11, wherein the binding strength of a surface protein originating from H. pylori strains 25 and 1139 to heparan sulphate is about 9x10^-9M.

13. A mono- or polyclonal antibody directed against the protein of claim 10, 11 or 12.

14. A process for detecting the presence of H. pylori in a specimen, comprising contacting said specimen with the antibody of claim 13, and recording the presence or absence of interaction upon contact.

15. A composition for medicinal use comprising as active constituent a negatively charged polysaccharide selected from the group comprising: heparin, heparan sulphate and dextran sulphate immobilized to an insoluble carrier by covalent binding thereto, said carrier being selected from the group comprising: agar, algin, alginic acid, carrageenan, chitin, chitosan, insoluble carriers , coated by chitin or chitosan, collagen, gelatin, guar gum, locust bean gum, and xanthan gum, optionally together with a pharmaceutically acceptable carrier or excipient.

16. A composition according to claim 15, wherein said polysaccharide is heparin or heparan sulphate, and wherein said carrier is chitin or chitosan or an insoluble carrier coated by chitin or chitosan.

17. A reagent kit for binding, separation and identification of a protein or subfragment thereof according to any of claims 10 to 12.

18. A protein specifically binding to heparan sulphate and prepared by a process comprising the following steps:

a) preparing a cell extract by suspending H. pylori strain 25 in a buffer solution of an acid pH;

b) removing the cell debris from the suspension resulting from step a) above by centrifugation;

c) applying the supernatant resulting from step b) above onto a heparin-Sepharose column;

d) eluting said column using a solution of a pH above neutral;

e) collecting the fractions having the ability to inhibit binding of H.pylori to heparan sulphate; and

f) recovering the proteins having the inhibiting capacity from the fractions resulting from step e) above.

19. A protein according to claim 18, the approximate molecular weight of which is 120, 72 or 35.

20. A vaccine for use in creating resistance to infections caused by or associated with H.pylori, comprising as an active constituent a protein according to any of claims 10, 11, 12, 18 and 19 in an immunologically active amount in combination with a pharmaceutically acceptable carrier or diluent.