Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7024—Esters of saccharides

Definitions

• 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.
• 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.
• an inflammation so called Gastritis type B (type II)
• Gastritis type B type II
• Refs gastric mucosa
• 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.
• 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) .
• 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:
• the drugs according to prior art are not able to reach the bacteria sitting under the mucus layer.
• 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.
• H. pylori exists both as an active, spiral shaped mobile organism and as the spore like coccoid form.
• H. pylori 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.
• H.pylori exists as the coccoid form.
• 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.
• 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.
• 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.
• 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 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.
• 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.
• polyhydroxy alcohols examples include sugar alcohols such as glycol, glycerol, inositol, sorbitol and xylitol.
• carbohydrates are mono-, di- and oligo saccharides and sugar alcohols.
• Example of monosaccharides are: glyceraldehyde, glucose, mannose, galactose, xylose and ribose.
• di-saccharides sucrose, maltose and lactose.
• 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.
• 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.
• 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.
• sucrose wa dissolved (7.46 g, 40 mmol) methyl decanoate and (0.56 g, 40 mmol) K 2 C0 3 were added.
• 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 ⁇ ) .
• 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.
• 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.
• 6-sucrose decanoate was prepared from a crude sucrose decanoat mixture by column chromatography (25x500 mm) with silica gel 60, 40-63 ⁇ m (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.
• 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.
• PIG Three strains of pigs were used.
• H. pylori has a uniquely potent urease enzyme.
• 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 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 ⁇ l 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 ⁇ l of the suspension was spread out directly or after
• 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 ⁇ C.
• 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.
• Fig. 8 is a bar chart of an in vitro test of a biopsy cul ⁇ ture.
• 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 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)
• the compounds according to the invention in concentrations up to 0.417 mg/ml did not affect urease activity.
• the compounds according, to the invention are effective in suppressing the H. pylori infection in pigs in vivo, as measured by Urea breath test.

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Citations (5)

• 1994
  • 1994-05-27 SE SE9401826A patent/SE9401826D0/sv unknown
• 1995
  • 1995-05-24 WO PCT/SE1995/000594 patent/WO1995032717A1/en active Application Filing
  • 1995-05-24 AU AU26340/95A patent/AU2634095A/en not_active Abandoned

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