Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

AT Austria GB United Kingdom MR Mauritania

AU Australia GE Georgia MW Malawi

BB Barbados GN Guinea NE Niger

BE Belgium GR Greece NL Netherlands

BF Burkina Faso HU Hungary NO Norway

BG Bulgaria IE Ireland NZ New Zealand

BJ Benin IT Italy PL Poland

BR Brazil JP Japan PT Portugal

BY Belarus KE Kenya RO Romania

CA Canada KG Kyrgystan RU Russian Federation

CF Central African Republic KP Democratic People's Republic SD Sudan

CG Congo of Korea SE Sweden

CH Switzerland KR Republic of Korea SI Slovenia

CI Cδte d'lvoire KZ Kazakhstan SK Slovakia

CM Cameroon LI Liechtenstein SN Senegal

CN China LK Sri Lanka TO Chad

CS Czechoslovakia LU Luxembourg TG Togo

CZ Czech Republic LV Latvia TJ Tajikistan

DE Germany MC Monaco TT Trinidad and Tobago

DK Denmark MD Republic of Moldova UA Ukraine

ES Spain MG Madagascar US United States of America

FI Finland ML Mali UZ Uzbekistan

FR France MN Mongolia VN Viet Nam

GA Gabon

THE USE OF AN ANTI-HELICOBACTER SUBSTANCE.

The present invention relates to an anti Helicobacter pylori substance, and more specifically, the use of said substance to treat gastric and duodenal ulcers and gastritis type B, and to prevent gastric cancer.

The Gastric Ulcer disease has been recognized and treated for more than 100 years. Still, in spite of ever more potent and effective drugs the ethiology and cause has only been known for 10 years and recognized for less than 5 years (Refs: 5, 8, 9, 10, 11) .

In 1982, two Australian scientists set out to culture a spiral bacteria from the gastric mucosa of patients with gastritis. Partly by a mistake they eventually were successful (Refs: 9, 10, 11) and could later confirm the presence of this bacteria in patients with duodenal ulcer (DU) . This bacteria was subsequent¬ ly named Helicobacter pylori (H.p.) .

Today H.p. has been found in all human races and on all conti¬ nents and its prevalence in the developing countries can be as high as 90% (Ref: 4), whereas in the western world the prevalence can roughly be estimated to 20% at 20 years of age and 50% at 50 years. H.p. specifically resides in the gastric mucosa where it will stay under the mucus, in close vicinity or attached to the epithelial cells (Ref: 2) . The evolution has provided H.p. with excellent tools to survive in this hostile environment, like the ability to produce NH ₃ to protect against the acid, and the ability to transform to a highly resistant coccoid form, whenever danger is imminent (such as treatment with antibiotics or combi¬ nations with Bismuth) . The bacteria will preferably reside in the antral mucosa, but is also found in the corpus. Whenever present, an inflammation, so called Gastritis type B (type II) , affects the gastric mucosa (Refs: 2,11). In many cases this is of an unsymptomatic nature, but can be severe enough to be disabling in normal life. The presence of H.p. is very closely correlated with duodenal ulcer (DU) and highly correlated also to gastric ulcer

CONFIRMATION COPY

2 (Ref: 5) . Evidence are accumulating indicating an increased risk for gastric cancer if subjects are infected with H.p. already during childhood.

The hypothesis regarding the duodenal ulcer, which is the most common type, is that H.p. most probably does not initiate the ulcer, but that following a stress or medically induced ulcer in H.p. positive patients, the recurrence of the disease will depend on the formation of gastric metaplasia in the ulcer, followed by a permanent invasion of H.p. Overwhelming evidence today show that following a successful eradication of H.p., there is no relapse in DU (Refs: 1, 3, 5, 7, 12) .

Drug therapy: The present drug therapies against the ulcer disease ranges from antacids over H2-receptor antagonists to H ⁺ - pump blockers, however, none of these will affect the bacteria in spite of successful ulcer healing within 4-8 weeks (Ref: 1) . In contrast, the old traditional gastro-intestinal therapy based on Bismuth salts has been rejuvenated, based on reports of a sometimes amazing ability to heal ulcers and prevent recurrence. The effectiveness of Bismuth salts seems to be due to the fact that H.p. for reasons yet unexplained is very sensitive to Bismuth (Refs: 3, 12) . This was the first clue to the present knowledge that eradication of H.p. will prevent ulcer recurrence.

Anti-H.p. Therapy: Initially it was quite "obvious" that an infection of this kind should be treated with antibiotics. However, the lack of effect surprised everybody. H.p. was shown to be untouched by most monotherapies and had a fantastic ability to develop resistance, especially to metronidazole (Ref:

13) . Only, with combinations of broad spectrum antibiotics •together with Bismuth subcitrate (DeNol ®) , could long term (>1 year) eradication be achieved in 60-80% of the patients (depend¬ ing on compliance) , but the cost in terms of side effects and alteration of the natural gastrointestinal flora was very high (Refs: 3, 5, 7, 12) . Partial success (50%) has been reported following the use of Amoxicillin together with Omeprazole (Ref: 6) .

3 The problems within prior art regarding therapy for the eradication of H.p. are:

1. The drugs according to prior art are not able to reach the bacteria sitting under the mucus layer.

2. Drug resistance is often induced

3. Side effects exist at unsatisfactory frequencies.

4. Plural daily dosing is required, often in high doses. The present invention solves these problems by using low molecular sulfated carbohydrates or polyhydroxy alcohols to whic an aliphatic saturated or unsaturated hydrocarbon chain is linke through an ester or ether bond for the manufacture of an anti- adhesive substance (composition) , which when administered orally to a mammal can eliminate H. ylori from the gastric mucosa via a antiadhesive mechanism involving transformation of the spiral form of H. pylori to the coccoid form which is then shedded together with mucosal components.

The theory underlying the present invention is:

H. pylori exists both as an active, spiral shaped mobile organism and as the spore like coccoid form. When H. pylori is colonizing the human stomach it occurs as the mobile form which also can reproduce itself. The spiral shaped H. pylori may transform itself into the coccoid form. What initiates such a transformation is not known. A perturbation from the surrounding mucosa could be a reason for the bacterium to transform as a way to survive. Transformation into the coccoid form can also be a way for the bacterium to spread. The immobile coccoid form does not have adhesivity to the epithelial cells and along with the production of new mucosa the coccoid form is passively pushed towards the surface of the mucosa where it is shedded.

Outside the human body H.pylori exists as the coccoid form. When the bacterium arrives at the human stomach it has to transform to the spiral shaped form in order to penetrate the mucosa layer and adhere to the epithelial cells of the stomach. When the coccoid form of H. pylori arrives to the stomach the

4 bacterium must recognize human mucosa. The recognition includes adherence to the mucosa and a start signal to transformation. The coccoid form and the mobile form of H. pylori have receptors on their surfaces which interacts with specific structures of the gastric mucosa. The coccoid form need this interaction to start the transformation and the spiral shaped form needs a continuous mucosa signal to stay in the mobile form. If the mucosa signal is blocked or distorted the transformation to the coccoid form is initiated and then the coccoid form is passively shedded from the mucosa.

It is known that each tissue have a characteristic composition of sulfated mucopolysaccarides differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. Human gastric mucosa has its specific composition of sulfated mucopolysaccarides and this may be the tissue signal which H. pylori recognizes.

A compound which shall block or interfere with such a tissue signal must carry a structure that is able to effectively compete with H. pylori target structures in the gastric mucosa or cause distortions in the gastric mucosal layer. Penetration of the mucosal layer is a first prerequisite for distortion. In order to do this the compound must bear resemblance to the mucopolysaccarides of human mucosa. Thus a compound intended to disturb the tissue signal for H. pylori must be a fully or partially sulfated carbohydrate or polyhydroxy alcohol with a capability to penetrate the mucosa layer and act at the epithelial layer where H. pylori is adhered. A sulfated carbohydrate or a polyhydroxy alcohol with the ability to penetrate the gastric mucosa might be a suitably sized molecule and it must also have a lipophile character.

The experimental results now provided support that low molecular weight sulphated carbohydrates or polyhydroxy alcohols to which an aliphatic saturated or unsaturated hydrocarbon chain is linked through an ester or ether bond at a hydroxy group bear

5 enough resemblance to the mucopolysaccharides of human mucosa to cause the sufficient distortion of the mucosal layer, i.e. preferably at an alcoholic hydroxy group. The hydrocarbon chain may be an aliphatic saturated or unsaturated, preferably C ₆ -C ₁₈ , fatty acid or fatty alcohol. The hydrocarbon chain may be straight, branched or cyclic, and preferably carries no substituents comprising heteroatoms such as oxygen or nitrogen, i.e. in the preferred case there are only hydrogen and carbon in the chain. Both primary or secondary hydroxy groups in the carbohydrate or the polyhydroxy alcohol may be involved in ether or ester bond formation indicating a preference for alcoholic hydroxy groups compared to other hydroxy groups, such as glycosidic hydroxy groups. Hydroxy groups that are not demanded for ester or ether bonds are fully or partially sulfated.

Examples of polyhydroxy alcohols are: sugar alcohols such as glycol, glycerol, inositol, sorbitol and xylitol.

Examples of carbohydrates are mono-, di- and oligo saccharides and sugar alcohols.

Example of monosaccharides are: glyceraldehyde, glucose, mannose, galactose, xylose and ribose.

Examples of di-saccharides: sucrose, maltose and lactose.

By low molecular weight carbohydrates in connection with the invention is intended carbohydrates comprising up to 10, preferably up 1 or 2 monosaccharide units.

The compounds according to the invention possess antiadhesive properties due to their ability to distort the structure of the gastric mucosal layer, and have been shown to drastically lower the number of H.p. associated with gastric epithelium as studied both in vitro and in vivo . These compounds are designed to be strictly locally (topically) active.

The treatment method utilizing the present inventive concept means that a therapeutically active dose of the above-mentioned anti-H. pylori active compound/substance is administered orally to a mammal, preferably a human patient. Often the dosing schedule involves repeated administrations such as 1, 2, 3 etc

times a day under at least a week. The dosage given at each administration occasion varies and may depend on body weight, age, sex, administration occasions per day etc. Normally the dosage is with in the range of 0.1 - 100 mg/kg body and day, such as 1 - 25 mg/kg body weight and day.

The active principle may be formulated in those types of compositions that normally are used for oral administration, i.e. tablets, pellets, dragees, solutions, emulsions, capsules etc.

Formulation of the active anti-Helicobacter compound of the invention into the above-mentioned types of compositions may be performed in the conventional manner by incorporating it into the appropriate vehicle that may be in the solid or liquid form.

MATERIAL AND METHODS :

CHEMICAL

Example 1. SULFATED MIXTURE OF SUCROSE MONO DECANOYL ESTERS,

NA SALT

R = -S0 ₃ Na

Preparation of a mixture 6-, 1'- and 6'-sucrose decanoyl esters.

Sucrose (41 g, 119.8 mmol) was dissolved in anhydrous dimethyl formamide (200 ml) . The solution was placed in a three necked round-bottomed flask with a magnetic stirring bar. The reaction vessel was placed in an oil bath at 92-96°C. When the sucrose wa dissolved (7.46 g, 40 mmol) methyl decanoate and (0.56 g, 40 mmol) K ₂ C0 ₃ were added. The pressure above the dimethylformamide solution was lowered to =70 mm Hg and the stirred reaction mixture was maintained at 92°C for 8 hrs during which time methanol and a portion of the dimethylformamide distilled. After 8 hrs the solvent was distilled off in vacuo. The residue a whit solid was extracted with n-butanol at 92°C (4x200 ml) . The combined extracts were evaporated to dryness under reduced pressure and dried in vacuo. The composition of the preparation was analyzed by thin-layer chromatography on silica gel with ethyl acetate:methanol:water (85:15:5) . The preparation gave two large spots due to monoester isomers and several smaller spots with higher Rf. values which was attributable to di- and

7 polyesters. The yield of crude sucrose esters was 23.3 g of very hygroscopic flakes .

Purification of sucrose mono decanoate.

Sucrose mono decanoate isomers were isolated and purified from the crude sucrose decanoate mixture by column chromatography (70x30 mm) with silica gel 60 (Merck 40-63 μ ) . The column was eluted by suction with ethyl acetate:methanol (75:10, v/v) . The crude sucrose ester was dissolved in methanol (250 ml) , the solution was added to silica gel (50 g) and the mixture was evaporated to dryness in vacuo. The dry mixture was ground and 1 of it was applied on the top of the column packed with dry silic gel 60. Elution of the column was made by suction and the eluate was collected in fractions. The composition of each fraction was analyzed by thin-layer chromatography. Fractions containing spot due to sucrose mono ester isomers were pooled, evaporated to dryness, dissolved in water and lyophilized. In a typical • run 23.3 g of crude sucrose ester gave 6.1 g of sucrose mono ester mixture. HPLC analysis of the isolated monoester mixture of a Supersphere Si 60, 4 μm (250x4 mm) column (Merck) eluted with 95 ethyl acetate:3% methanol:2% water showed that the isolated sucrose mono decanoated predominantly was a mixture of 6-0- sucrose decanoate (47.2%), l'-O-sucrose decanoate (7.7%) and 6'- 0-sucrose decanoate (21.3%) .

Sulfatation of sucrose mono decanoate

Sucrose mono decanoate (3 g, 6 mmol) was added to a solution o pyridine-S0 ₃ (8.665 g, 54 mmol) in pyridine (20 ml) . The mixture was stirred at 50°C for 2 hrs. After cooling the pyridine was evaporated and the residual pyridium salt was dissolved in water (100 ml) . An aqueous solution of Ba(OH) ₂ was added to adjust the pH to 8.4. The precipitate of BaS0 ₄ was removed by centrifuga- tion. The supernatant was concentrated under reduced pressure to 50 ml and passed through a column of Amberlite IR-120 (Na ^* ) (35 ml) . The pH of the eluate was adjusted to 7.0 and concentrated t 10 ml. The material was separated by Superdex 20 column

8 chromatography in water. The eluate was monitored by refractive index and UV-absorbance at 210 nm. Eluted material was collecte in two fractions and lyophilized.

Fraction I. 0.71 g (0.58 mmol) . Fraction II. 6.35 g (5.24 mmol) .

Anal. calc. for C ₂₂ H ₃₃ S ₇ θ33Na ₇ fraction II: C 21.8%; H 2.7%; S 18.5%; 0 43.6%. Found: C 21.1%; H 3.4%; S 17.0%; O 46.0%

Example 2: SULFATED 6-0-SUCROSE DECANOATE

6-0-Sucrose decanoate.

6-sucrose decanoate was prepared from a crude sucrose decanoat mixture by column chromatography (25x500 mm) with silica gel 60, 40-63 μm (Merck) . The column was eluted with ethyl acetate:methanol:water 81000:25:5) and the eluate was monitored by a refractive index monitor. The eluted peaks of sucrose mono decanoate isomers were collected in three fractions . The fractions were concentrated with reduced pressure to dryness, dissolved in water and lyophilized. HPLC analysis of the fractions showed that only the 6-0-sucrose decanoate isomer was eluted as a single peak. The l'-O-sucrose isomer and the 6'-0- sucrose isomer did not separate from each other.

Yield of 6-0-sucrose decanoate: 2.6 g (34.2%) . The identity wa confirmed by ^-NMR-spectral data and the purity was estimated t 75% (mole) .

Sulfatation of 6-0-sucrose decanoate.

6-0-sucrose decanoate (1.5 g, 3.0 mmol) was added to a solutio of pyridine-SO _j (4.3 g, 27.0 mmol) in pyridine (10 ml) . The mixture was stirred at 50°C for 2 hrs. After cooling the pyridin

9 was evaporated and the residual pyridium salt was dissolved in water (100 ml). An aqueous solution Ba(OH) ₂ was added to adjust the pH to 8.3. The precipitate of BaS0 ₄ was removed by centrifu- gation. The supernatant was concentrated with reduced pressure t 10 ml and passed through a column of Amberlite IR-120(Na") (35 ml). The pH of the eluate was adjusted to 7.0 and concentrated t 10 ml. The material was separated by Superdex 20 column chromatography in water. The eluate was monitored by refractive index and UV-absorbance at 210 nm. The eluted material was collected in two fractions and lyophilized.

Fraction I: 1.24 g (1.02 mmol) (33.3%). Fraction II: 1.39 g (1.14 mmol) (46.3%)

Anal. calc. for C ₂₂ H ₃₃ S ₇ 0 ₃₃ Na ₇ : C 21.8; H 2.7; S 18.5; O 43.6. Found: C 21.0; H 3.5; S 15.8; 0 45.2.

Example 3. SULFATED 6'-0-SUCROSE DECANOATE

6'-0-Sucrose decanoate.

2,1' :4,6-Di-0-isopropylidensucrose.

A solution of 2,1' :4,6-di-0-isopropylidene sucrose tetra- acetate 1 g (1.69 mmol) in methanol (25 ml) was treated with 10- 15 g sodium. The reaction mixture was stirred at 23°C for 24 hrs. The solution was neutralized by carefully bubbling C0 ₂ and concentrated to dryness. The residue was dissolved in dichloromethane, filtrated and concentrated to dryness. Analysis by thin layer chromatography (silica gel; ethyl acetate: ethanol:water 85:15:5) showed that the reaction was complete. Yield: 0.71 g (1.68 mmol) (99.4%). Mp: 161-163°C.

10 6* -O-dβcanoyl-2,1' :4,6-di-0-isopropylidensucrose.

2, 1 ' : 4, 6-di-0-isopropylidene sucrose 0.71 g (1.68 mmol) was dissolved in 3 equiv. of anhydrous pyridine and dichloromethane 1:15. The solution was cooled to -15°C (ice/ethanol) . 1.1 equiv. of decanoyl chloride (0.360 ml) was added during 50 minutes with stirring and after another 20 minutes the reaction was stopped b addition of 5 drops of methanol. Analysis by thin layer chromatography (silica gel; ethylacetate:methanol: ater 85:15:5) showed one major spot and two small spots. The reaction mixture was treated with 2xNaHC0 ₃ and water. The organic solution was dried with MgS0 ₄ and evaporated to dryness . The residue was dissolved in ethyl acetate (1 g/10 ml) and separated by column chromatography (25x500 mm) with silica gel 60, 40-63 μm (Merck) and eluted with ethyl acetate. Three peaks were eluted. The majo peak was collected, evaporated to dryness, dissolved in water an lyophilized. Analysis by thin layer chromatography showed one spot. Yield: 0.41 g (45%) .

6'-0-Sucrose decanoate.

6 ' -0-decanoyl-2, 1 ' :4, 6-di-0-isopropylidene sucrose 2.35 g (4.12 mmol) was treated with aqueous 60% acetic acid (30 ml) for 30 mi at 50°C. The reaction mixture was concentrated to dryness, dissolved in water and lyophilized. The identity was confirmed b ^X H-NMR spectral data and the purity was estimated to 78% (mole) . Yield: 1.31 g (63.5%) .

Sulfatation of 6'-0-sucrose decanoate.

6'-0-sucrose decanoate 1.31 g (2.62 mmol) was added to a solution of 3.78 g (23.5 mmol) pyridine S0 ₃ in pyridine (10 ml) . The mixture was stirred at 50°C for 2 hrs. After cooling the pyridine was evaporated and the residual pyridium salt was dissolved in water (100 ml) . An aqueous solution of Ba(OH) ₂ was added to adjust the pH to 8.4. The precipitate of BaS0 ₄ was removed by centrifugation. The supernatant was concentrated with reduced pressure to 10 ml and passed through a column of Amberlite IR-120 (Na ⁺ ) (35 ml) . The pH of the eluate was adjusted

11 to 7.0 and concentrated to 10 ml. The material was separated by Superdex 20 column chromatography in water. Eluted material was collected in two fractions and lyophilized.

Fraction I: 2.0 g (1.65 mmol), (62.9%). Fraction II: 0.33 g (0.27 mmol) , (10.3%) .

Anal. calc. for C ₂₂ H ₃₃ S ₇ 0 ₃₃ Na ₇ : C 21.8; H 2.7; S 18.5; 0 43.6. Found: C 23.4; H 3.6; S 16.1; 0 46.2.

Example 4. SULFATED 6-0-GLUCOSE DECANOATE

6-0-glucosθ decanoate.

D(+)Glucose (3.6 g, 20 mmol), and N-decanoyl thiazolidine-2- thione (2.74 g, 10 mmol) were dissolved in 60 ml anhydrous pyridine. Sodium hydride (80% dispersion in mineral oil, 0.2 g, mmol) and dimethylamino-pyridine were added at room temperature. The reaction mixture was stirred for 2 hrs at 23°C and during that time the solution was decolorized (to pale yellow) . Acetic acid 1 ml was added and then the pyridine was removed at reduced pressure at 40°C. To the residue 0.1 M phosphate buffer pH 7.0

(30 ml) and ethyl acetate:n-butanol 4:1 (30 ml) were added and the reaction mixture distributed between the two phases. The aqueous phase was extracted twice (2x20 ml) with ethyl acetate:n butanol (4:1) . The combined organic layers were washed with wate (3x10 ml), dried with MgS0 ₄ and evaporated to dryness. Yield: 4.95 g. The material was purified by column chromatography (25x7 mm) on silica gel 60, 40-63 μm (Merck) . The column was eluted with dichloromethane:acetone 1:1. Yield: 1.75 g, (52.3%) a mixture of the α and β ano er of 6-0-glucose decanoate. The identity was confirmed by ^" -H-NMR spectral data on material recrystallized from acetone.

12

Sulfatation of 6-0-glucose decanoate.

6-0-Glucose decanoate (α and β anomers) was added to a solutio of pyridine-S0 ₃ (3.8 g, 23.9 mmol) in pyridine (10 ml) . The mixture was stirred at 50°C for 2 hrs.The pyridine was evaporate and the residual pyridium salt was dissolved in water (100 ml) . An aqueous solution of Ba(0H) ₂ was added to adjust the pH to 8.4 The precipitate of BaS0 ₄ was removed by centrifugation. The supernatant was concentrated to 10 ml and passed through a colum of Amberlite IR-120 (Na ⁺ ) (35 ml) . The pH of the eluate was adjusted to 7.0, concentrated to 10 ml and separated by Superdex 20 column chromatography in water. The eluted material was lyophilized. Yield: 0.72 g, (44.9.)

Anal. calc. for: C ₁₆ H ₂₆ S ₄ 0 ₁₉ Na ₄ : C 25.9; H 3.5; S 17.3; 0 40.9.

Found: C 23.3; H 3.8; S 15.3; O 41.6.

BIOLOGICAL EXPERIMENTS:

We have developed new biological tools in terms of an in vivo pig model (Refs: 16,17) and a unique in vi tro model where biopsies of pig and human mucosa can be cultured and infected with H.p. and studied for 72 hours (Refs: 14,15).

In vivo models:

PIG: Three strains of pigs were used.

1. Barrier born pigs of the Swedish native breed,

2. Yokotan "Micropigs" (specific pathogen free; SPF) , and 3. Gδttingen "Minipigs" (specific pathogen tested) .

When the experiment started, the animals were at least 7 weeks old. Pigs were inoculated as previously described (Refs 16,17) . In brief, the pigs were given omeprazole (to inhibit acid secre¬ tion) for 3 days prior to inoculation with 5 ml H. pylori suspen sion (10 ⁹ CFU/ml) . The inoculation was repeated 3 times during one week. Three to 14 weeks after the inocculation, presence of bacteria in the gastric mucosa was detected with culture from biopsies and by ¹³ C-Urea breath test. The anti-H. pylori effects of title compounds were always determined by ¹³ C-Urea breath tes

13 The anti-H. pylori effects of the title compounds were always detemined by the ¹³ C-Urea breath test. In general all pigs were found to be H. pylori positive by ¹³ C-Urea breath test 4 weeks after the inoculation.

^u C-Urea breath test: H. pylori has a uniquely potent urease enzyme. By giving pigs 2 mg/kg ¹³ C-Urea in a water solution, the amount of ¹³ C0 ₂ resulting from urease cleavage of urea into C0 ₂ and NH ₃ can be determined and correlated to the presence of H. pylori (Ref 17). Pigs were allowed to breath into a special mask before and at different time intervals after administration of ¹³ C-Urea. Samples of the exhaled air was subjected to mass-spectrometry, determining the ratio ¹³ C/ ¹² C . An increase in this ratio of 5 ppm above base-line was considered as a positive sign of H. pylori presence.

MICE: Athymic nude Balb/c mice can, as reported, successfully be infected with H. pylori (ref 18). The mice were kept under aseptic conditions. At 6 weeks of age they were inoculated under anaesthesia with 100 μl H. pylori suspension (5xl0 ⁸ /ml). Animals were sacrificed and the glandular stomach taken for H. pylori culture from 3 weeks after the inoculation. In positively infected animals the infection had stabilized after 4 weeks. Rinsed glandular stomachs were homogenized in 1 ml sterile NaCl. 100 μl of the suspension was spread out directly or after

1 4 dilution onto blood agar plates containing H. pylori selective supplement according to Dent and Nalidixic acid 20 μg/ml. Colonies were counted after a minimum of 4 days.

In vitro:

Biopsy culture: 2 mm wide biopsies were punched of from stripped antral gastric mucosa of pigs. The biopsies were subsequently cultured in tissue culture dishes on a steel grid support, in capillary contact with a special culture medium as previously reported (Refs 14,15). Biopsies could be maintained with full viability during at least 72 h. After 6 h in culture the biopsies were each inoculated with 10 ⁶ H. pylori. The amount of bacteria associated with the biopsies at different time points was deter¬ mined after vigorous rinsing of the biopsies, followed by bacte¬ rial count from homogenized biopsies and from the rinsing water. When the title compounds were tested they were added to the medium after 48 h culture and their effects evaluated during the following 24 h.

Urease activity: Bacteria in suspension were incubated at 37°C for 30 minutes in a medium containing 2% urea and 0.001% phenol red and the change in absorbance recorded at 559 nm. The effect on the urease activity was determined in the absence and presence of different compounds.

Bacterial culture in medium: H. pylori were incubated in 1.5 ml CMRL 1066 medium according to Autrup, without antibiotics. The title compound was added to different concentrations and the amount of living bacteria was determined after 24 h incubation at 37 ^β C.

RESULTS

The results will be described below in association with the accompanying drawings, in which

15

Fig. 1 is a graph showing ¹³ C urea breath test in pigs before and after treatment according to the invention;

Fig. 2 is a graph showing ¹³ C urea breath test in pigs before and after treatment using another dose than in Fig. 1;

Fig. 3 is a graph showing a comparison of treatment according to prior art and treatment according to the present invention;

Fig. 4 is a bar chart showing a comparison of treatment according to prior art and treatment according to the present invention;

Fig. 5 shows four bar charts of ¹³ C urea breath tests on different pigs;

Fig. 6 shows four graphs of ¹³ C urea breath tests on different pigs;

Fig. 7 is a bar chart of an in vivo mice test; and

Fig. 8 is a bar chart of an in vitro test of a biopsy cul¬ ture.

IN VIVO Pigs:

Effect on ¹³ C-Urea breath test in regular pigs when the mix of compounds according to Example 1 was given to barrier born pigs at a dose of 320 mg/day in water for 3 days. The Δ ppm at 60 min decreased from 39 to 7 in one and from 31 to 8 in another pig (fig 1). Administration of the mix at 2 x 250 mg during 5 days resulted in a decrease from 28 to 6 Δ ppm (fig. 2). Comparison between title compound 320 mg/day in water for 3 days with a suspension of Bismuth (DeNol) 2 x 480 mg/day for 3 days showed a much better effect of the mix (fig.3).

16 Treatment of regular pigs with 320 mg/day for 3 days showed a similar effect as triple therapy (Bismuth 2 x 480 mg; Pivampi- cillin 2 x 700 mg; Metronidazole 2 x 1200 mg) for 14 days, when studied immediately at the end of treatment. The duration of the H. pylori suppression was however shorter with the mix (Fig. 4).

The mix at a dose of 50 mg/day in water or 100 mg/day in capsules for 5 days given to Yokotan micropigs and to Gttttingen miniplgs showed significant inhibitory effects on the ¹³ C-Urea breath test. Fig. 5 shows the entire time-response pattern, where all animals are H. pylori positive 4 weeks after the inoculation. The mix was given as a solution (50 mg) during week 7 and as capsules (100 mg) during week 11, as indicated by the arrows. Fig 6 shows the response in the 4 different pigs before and after the 5 days treatment with 100 mg mix in capsules.

Mice:

Mice were treated with different daily doses of the mix according to Example 1 administered in the drinking water for 7 days. Control animals were given water only. All control animals were positive as determined by culture. A dose-dependent eradication of cultivable H. pylori was seen and at 1 mg/day 50% of the animals were culture negative (see Fig.7)

IN VITRO

Biopsy culture:

The mix according to Example 1 added to the biopsy incubation medium for 24 h, showed that the number of bacteria associated with the biopsies dramatically decreased and that the remaining H. pylori were loosely associated with the tissue, since they easily could be washed off (Fig.8). This is a direct proof that the compounds according to the invention have anti-adhesive properties.

17

Incubation of H. pylori in medium:

Incubating the bacteria with similar concentrations of the compounds as were effective in the biopsy culture did not affect the viability of the H. pylori. Thus the compounds do not have bacteriostatic or bacteriocidal effects.

Effect on Urease activity:

The compounds according to the invention in concentrations up to 0.417 mg/ml did not affect urease activity. The well known urease inhibitor Acetohydroxamic acid (AHA) showed a total inhibition at 0.013 mg/ml.

From the above it appears that the following effects and advantages are achieved by the present invention:

* The compounds according, to the invention are effective in suppressing the H. pylori infection in pigs in vivo, as measured by Urea breath test.

* Mice treated with the compounds cleared their H. pylori infec¬ tion in a dose-dependent way.

* Adhesion to cultured pig gastric biopsies decreased dramati¬ cally in the presence of compounds according to the invention.

* The compounds do not posses bacteriocidal or bacteriostatic properties.

* The compounds do not inhibit Urease activity.

18 REFERENCE LIST

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2. Thornsen, L.L., Gavin. B.J. and Tasman-Jones, C. : Relation of Helicobacter pylori to human gastric mucosa in chronic gastritis of the antrum. GUT, 31: 1230-1236, 1990.

3. Borody, T.J. et al.: ffelicoJ acfcer pylori eradication with doxycycline-metronidazole-bismuth subcitrate triple therapy. Scand. J. Gastroenterol. , 27: 281-284, 1992.

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1 9

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