KR19990064104A - Treatment of Helicobacter pylori infection - Google Patents

Abstract

The present invention is H. New therapies for the treatment of pylori infections and pharmaceutical compositions for use in the methods are provided.

Description

Treatment of Helicobacter pylori infection

H. The pylori is a Gram-negative bacterium closely associated with chronic active gastritis and gastrointestinal ulcer disease (Marshall et al., Medical Journal of Australia 142: 439-444 (1985); Buck, GE, Journal of Clinical Microbiology, 3: 1-12 (1990)). H. when cultured in vitro. The pylori exists in two distinct forms, cultivable helix balance and unculturable helix balance (Marshall et al., Microbios letters, 25: 83-88 (1984); Kung, JSL, and HO, B., Workshop on Gastroduodenal Pathology and Campylobacter pylori (Abstract p9), F. Megraud and H. Lamouliatte, edited by Bordeaux, Bordeaux, France (1988)). The helix balance bacteria do not survive for more than about 2 hours when exposed to air. Under unfavorable conditions, helix balance bacteria cause a shift to cobalt (Vijayakumari and Ho, Acta Gastro-enterologica Belgica, 56: 101 (1993)).

To date, only one example has been reported of successful in vitro transformation of cobalt to helix, but the experiment has proved to be non-repeatable (Mai et al., Gastorduodenal Pathology and Campylobacter pylori, p 28-). 33, edited by F. Megraud and H. Lamouliatte, Elsevier Science Fiblisher (1989). The formation of cobalt balances in vitro can be induced by antibiotics or by blocking the supply of nutrients (Nilius et al., Zbl. Bakt. 280: 259-272 (1993)), but information on cobalt balance and H. The study of cobalt balance was limited because there was no information on its role in the ecology of the pylori, nor was there any way to achieve such forms of coculture. So far, a balance H. The pylori has been regarded as a "dead" non-viable form, and its role in the ecology of Helicobacter pylori is unclear.

It has been found that the cobalt balance is viable and can lead to a transition to a spiral balance. One possible method for inducing conversion involves the administration of urease. WO 95/22987 describes H. The use of urease-based vaccines to treat pylori infections is disclosed. However, no general "chemical" non-immunological method based on urease that induces agglomerate conversion to helix balance is disclosed. Since the spiral balance of bacteria can be processed using antibiotics, these results are H. Offers the possibility of more effective treatment of pylori infection, and therefore H. Significantly reduces the recurrence of pylori mediated gastrointestinal diseases.

The present invention is Helicobacter. A method of treating Helicobacter pylori infection, a pharmaceutical composition for use in the method, H. The present invention relates to a method of making a pyrococci culture viable and inducing a conversion of a cocci balance into a spiral balance.

Thus, the first object of the present invention is H. H. A mammal, comprising administering to a mammal an agent capable of inducing the conversion of the pylori balance to a spiral balance. It provides a way to treat pylori infection.

It is preferred that the agent is not in the form of a vaccine, i.e. not administered to elicit any immune response.

In general, administration of the formulation will form part of a therapeutic regimen that includes administration of one or more antibiotics. Thus, in one aspect of the first object of the invention, the method further comprises administering to the mammal an effective amount of one or more antibiotics.

Administration of one or more antibiotics will be appropriate after administration of the formulation. Administration of the agent will allow the balance to be converted to a spiral balance prior to administration of the antibiotic (s).

Of course, antibiotics are known to induce a spiral balance to be converted back into a cobalt balance. Thus, several cycles of agent / antibiotic administration occur. Often needed to effectively remove pylori. Accordingly, a second object of the present invention is to have a mammal comprising two or more treatment cycles. A method of treating pylori infection is provided, wherein the treatment cycle comprises (i) and (ii):

(i) H. Administering to the mammal an agent capable of inducing the conversion of the pylori balance to the spiral balance;

(ii) administering to the mammal an effective amount of at least one antibiotic.

H after each treatment cycle. Much less amount of pylori will be left. The end point of treatment is commercially available H. This can be determined using diagnostic reagents for pylori.

In a preferred embodiment of the second object, the agent to be administered is H. It is a sufficient amount of urease that can induce the conversion of the pylori balance to the spiral balance. Herein, urease is a bacterium [eg, H. a. Individual subunits of one or more urease enzymes, or peptides derived from this subunit, as well as all urease forms of pylori or Protieus mirabitis urease] or non-bacteria (e.g., Jackbean urease) Include the form of. In one embodiment, only the C and D subunits of urease are administered.

If the agent administered is urease, the method of treatment may comprise administering urea to the mammal. Urea may be co-administered or administered separately.

H. Observing the characteristics of the life cycle of pylori organisms, it is normal to find a way to prevent the helix balance from converting to a balance. It can be seen that it is effective in enhancing the effect of the pylori treatment regimen. Therefore, the third object of the present invention is H. H. of mammals, comprising administering to a mammal an agent capable of preventing the helix balance of pylori from being converted to cobalt balance. It provides a way to treat pylori infection.

In addition, as an aspect of the third object, the present invention provides a preparation which is urease, optionally mixed with urea.

H. Another possible way to treat pylori infection is to "consume" the organism by allowing the organism to interconvert between helix balance and cobalt balance.

Thus, the fourth object of the present invention comprises one or more treatment cycles. To provide a method of treating pylori infection, each treatment cycle comprises the following steps (i) and (ii):

(i) H. Administering to the mammal an agent capable of inducing the conversion of the pylori balance to the spiral balance; And

(ii) administering to the mammal an agent capable of inducing a spiral balance of H. pylori to be converted into a cobalt balance.

In one aspect of the above object of the present invention, the present invention provides an agent capable of inducing a transition to helix equilibrium, and an agent capable of inducing conversion to a cobalt equilibrium is an "anti-urease", for example a urease. It is an antibody specific for an inhibitor or urease.

A fifth object of the present invention comprises administering to a mammal an agent capable of changing the pH in the stomach. It provides a way to treat pylori infection. In this embodiment, the lowering pH is present. Helix balance of pylori leads to increased urease production. Increased urease concentrations cause all of the existing balances to be converted to helix balances, thus making antibiotic treatment more effective. Thus, the method generally includes administering one or more antibiotics to the mammal. Methods of changing the pH may include administration of an edible acid or base.

In all the methods of treatment of the invention described above, the mammal is preferably a human.

The methods of the invention will generally use the formulation in the form of a pharmaceutical composition. Accordingly, a sixth object of the present invention is to provide a pharmaceutically acceptable carrier with one or more pharmaceutically acceptable carriers and / or excipients. It is to provide a pharmaceutical composition comprising an agent capable of inducing the conversion of the pylori balance to the spiral balance. It is also preferred that the pharmaceutical composition is not in the form of a vaccine.

In one aspect of this object of the invention, the formulation is a urease and optionally the pharmaceutical composition may also comprise urea.

The pharmaceutical compositions of the invention may be presented in unit dosage form containing a predetermined amount of agent per dose, for example urease (and optionally urea). This dosage unit may contain sufficient urease to convert 3 mg of urea for 30 minutes at 37 ° C., depending on the age, weight and symptoms of the patient.

The pharmaceutical compositions of the present invention may be applied for oral administration, and include discrete units such as capsules or tablets; Powder or granules; Aqueous or non-aqueous liquid solutions or suspensions; Edible foams or whips; Or a water-in-oil liquid emulsion or an oil-in-water liquid emulsion, or a conventional or new pharmaceutical form.

If urease and urea are administered together, the urease and urea are suitably administered in a form that prevents mixing of the two components.

The seventh object of the present invention is H. H in the manufacture of a medicament for the treatment of pylori infection. It is to provide a method of using an agent that can induce the conversion of the pylori balance to the spiral balance. In addition, as one aspect of this object, the formulation is a urease and the medicament will also optionally include urea. It is also preferred that the medicament is not a vaccine.

The results of the invention disclosed herein can be applied ex vivo. Accordingly, an eighth object of the present invention is to provide (a) and (b):

(a) H. H. in the culture medium, comprising adding an agent to the culture medium that can induce conversion of the pylori balance to the spiral balance. Converting the pylori balance to the spiral balance; And

(b) H. H. in the culture medium, comprising adding an agent to the spiral culture medium that can induce a spiral balance of the pylori to be converted to a cobalt balance. A method of converting the spiral balance of pylori into a balance.

In the case of (a), the preparation is provided in the form of a culture medium in which the spiral balance is multiplied but from which the spiral balance is removed. Spiral balance is preferably grown in the medium for at least 3 days. In addition, the formulation is a urease optionally mixed with urea.

In the case of (b), the preferred agent is an "anti-urease", for example a urease inhibitor or an antibody specific for urease.

Thus, the ex vivo conversion method is such that the products of the cocci and helix bacteria sources of antigen are produced. This method is particularly useful for generating specific antigens for use in identification tests for two types of bacteria.

Preferred features of the respective objects of the present invention are equally applicable to the modifications necessary for the respective embodiments.

The invention will be described in detail in the following examples, which are not intended to limit the invention in any way.

Example 1

(a) Bacterial strains and H. Balanced Preparation of Pylori

Other wild-type strains were similarly frequently used, but local Helicobacter pylori strain V ₂ isolated from patients with non-ulcer dyspepsia was used. First, the strain was grown on chocolate blood agar (CBA) to check purity. Subsequently, using a plate culture as an inoculum of a 250 ml short flat round bottle containing 30 ml of BHIH (a brain heart extract enriched with 10% horse serum and 0.4% yeast extract), followed by 37 ° C. for 72 hours. Incubated with. This served as an inoculum or batch culture of a continuous culture device.

A 1.5 L fermenter containing 540 mL of BHIH was set up as described in Ho and Vijayakumari's Microbios, 76: 59-66 (1993). 3 days old H on the badge. The pylori culture was inoculated with a ratio of 1:10 (culture: medium). The carbon dioxide was fed twice daily and the speed of the stirrer was adjusted to about 35 rpm. Samples were withdrawn at different times to investigate urease activity, pH, viability, and microscopic examination for morphological changes.

The cells were observed daily, maintaining the culture for a period of up to 3 months under these conditions. If homogeneous / simultaneous cultures were observed, cells were recovered by centrifugation at 10,000 g for 40 minutes and then washed once. This pellet was used to produce the coccus antigen using the modified glycine method (Ho, B., and Jiang, B., European Journal of Gastroenterology and Hepatology, 7: 121-124 (1995)).

Alternatively, 1 l short round bottom bottles or 1 l Erlenmeyer flasks with fixed rigid rubber stoppers and side-arms and containing 270 ml of BHIH were used. A 7 mm diameter hole was formed to fix the disposable filter unit with a 0.22 μm filter (eg gel only) with a diameter of 50 mm. 3 days old in 270 ml BHIH. 30 ml of pylori culture was inoculated. Subsequently, carbon dioxide was supplied twice a day through a 0.22 μm filter.

The cultures were incubated for up to 9 weeks in a 37 ° C. shaking incubator (commercially available in New Bronswick) maintained at 90 rpm and then centrifuged at 10,000 g for 40 minutes to harvest cells.

The cocci thus obtained are stored at −80 ° C. in glycerol-BHIH for a period of up to 2 years. If necessary, the cocci were collected by centrifugation at 10,000 × g for 30 minutes and washed once with PBS pH 7.2.

Preparation of Induced Juicy (IB)

3 days old H. Pylori spiral bacteria were inoculated into BHIH in a 1.5 L fermenter or a 1 L shorted bottle cap or 1 L Erenmeyer flask as described above. After 3 days, the supernatant was harvested by centrifugation at 10,000 × g for 10 minutes. The used medium was filtered twice with a 0.2 μm membrane filter. The filtrate is then called induction gravy (IB). Two filters were transferred to CBA plates and incubated at 37 ° C. for up to one week in a humidified carbon dioxide incubator.

Induces cocci into spiral balance

H. Pylori cocci were inoculated in 30 ml supernatant (IB) and incubated at 37 ° C. in a 5% carbon dioxide incubator to obtain a final concentration of 10 ⁸ cocci per ml. Similar amounts of cocci were inoculated with 30 ml of fresh BHIH as a control. As a result, fresh BHIH was added to the cocci culture over time (24 hours).

Transmission electron microscope

At 24, 48, 72 and 96 time intervals, aliquots of each culture were recovered by centrifugation and washed twice with PBS. Cells were resuspended in PBS and stained negative for transmission electron microscopy.

Alternatively, the cells were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer for 2-3 hours or overnight at 4 ° C. and then washed twice with 0.1M cacodylate buffer. The cells were resuspended in distilled water and negatively stained as described above.

Several drops of cell suspension were placed on a carbon coated 400 mesh copper grid for 1 minute. Excess fluid was blotted and the grid was fixed by air drying. The grid was then stained with one drop of 1% phosphotungstic acid for 1 minute before bloting excess dye. After air drying, the grid was tested using a Philips CM120 transmission electron microscope.

Treatment with antibiotics

The cocci were further treated with 5 μg / ml amoxycillin for 48 hours in BHIH to induce or alternatively kill any present helix. After treatment, the cocci suspension was washed twice and resuspended in the initial volume of BHIH. Aliquots of 1 ml suspension were inoculated with fresh BHIH and then incubated.

Urea addition to IB and BHIH

Urea was added at 5 mM concentration to cocci in IB or BHIH. Culture samples were drained at different times for microscopic testing and passaged in CBA and BHIH.

result

None of the cocci preparations proliferated upon passage alone in BHIH or CBA. Similarly, H. None of the second filters used for filtration of the pylori culture showed any growth in CBA or BHIH alone. IB over 3 days positively tested for urease within 30 minutes.

Cocci appeared dense under a phase contrast microscope. The contrast staining took 30 minutes to observe the effect. After 24 hours of induction at IB, the cocci were lean and fetal spiral cells began to form. Some early days of development can be seen attached to the "parental" cocci shell. After 48 hours of induction, this "birth" process becomes more pronounced. Upon influx of BHIH, the newly formed helix transformed into mature cells and developed motility. Spiral bacteria were more active at 2 × BHIH. When urea was added to IB and BHIH, the proliferation was more pronounced, and the helix appeared in as fast as 24 hours.

Helices produced from regenerated aureus are fully viable and functional. Easily adhered to KATO III cells was found. This is a process that mimics the pathogenic invasion of a normal helix balance formed of KATO III cells.

discussion

These results are H. It shows that pylori's equilibrium can be induced to convert into a spiral balance. Therefore, some elements in the IB ("cause factor") should influence this conversion. It was also observed that the conversion / propagation of helix without urea was partial.

The "causal factor" initiates the growth of the helical equilibrium from within the thick polysaccharide layer of the equilibrium. Thus, it is clear that nutrition / cause factors can pass through this sheath. Previously obtained data show that urease subunits C and D are present in insufficient or reduced concentrations in the equilibrium. The data provided above show that urease is present in IB. Thus, it can be concluded that subunits C and D of this urease enzyme are indeed "causal factors".

Introduction of fresh BHIH medium provides the necessary nutrition for the whole to switch (because IB is present in depleted or consumed concentrations). Providing physiological concentrations of urea indicates that urea plays an important role in the metabolism of this organism.

Estimating the in vivo situation from these findings, it can be seen that after colonization of the stomach by the organism, the helices secreted from the excreta are converted to a globule for survival in vitro. Upon reintroduction into the mouth, it is present under microaerobic conditions of subgingival plaques. Thus, the presence of other urease producing bacteria or indeed other sources may lead to a transition to a spiral balance. Once converted, the bacteria form colonies in the oral cavity, from which they travel along the digestive tract to form colonies in the stomach.

Therefore, it is clear that it is not enough to remove the bacterial spiral balance by conventional antibiotics. Gastrointestinal diseases can be recurred simply by the conversion of any cocci present into helix. Therefore, it is essential to eliminate the bacteria's helix and agglomerates to prevent recurrence. To this end, any H. pneumoniae can be converted into a spiral balance, and all H. pylori treated with antibiotic treatment. It can get rid of pylori infection.

This can be achieved by the addition of urease, optionally with urea as an additional component, to induce conversion into a spiral balance.

Claims (36)

H. H. of mammals, comprising administering to a mammal an agent capable of inducing a conversion of the pylori balance to a spiral balance. How to treat pylori infection. The method of claim 1 wherein the agent is not in the form of a vaccine. The method of claim 1 or 2, further comprising administering to the mammal an effective amount of one or more antibiotics. (i) H. Administering to the mammal an agent capable of inducing the conversion of the pylori balance to the spiral balance; (ii) H. of the mammal, comprising at least two treatment cycles comprising administering to the mammal an effective amount of one or more antibiotics. How to treat pylori infection. H. H. of mammals, comprising administering to a mammal an agent capable of preventing the conversion of the helix balance of pylori to cobalt balance. How to treat pylori infection. The method according to any one of claims 1 to 5, wherein the agent is urease. The method of claim 6, wherein the urease is a non-bacterial urease. 8. The method of claim 7, wherein the nonbacterial urease is Jackbean urease. The method of claim 6, wherein only the C and D subunits of urease are administered. The method of claim 6, wherein the urea is also administered to the mammal. (i) H. Administering to the mammal an agent capable of inducing the conversion of the pylori balance to the spiral balance; And (ii) H. of mammal, comprising at least one treatment cycle comprising administering to the mammal an agent that can induce a spiral balance of H. pylori to be converted into acupuncture. How to treat pylori infection. 12. The method of claim 11, wherein the agent of step (i) is urease 13. A method according to claim 12, modified by one or more of the features of any one or more of claims 7-9. The method of claim 12 or 13, wherein the urea is administered with urease. The method of claim 12, wherein the agent of step (ii) is “anti-urease”. The method of claim 15, wherein the “anti-urease” is a urease inhibitor. H. of mammals, including administering to a mammal an agent capable of changing the pH of the stomach. How to treat pylori infection. 18. The method of any one of claims 1 to 17, wherein the mammal is a human body. H. A pharmaceutical composition comprising an agent capable of inducing a conversion of a pylori balance to a spiral balance together with one or more pharmaceutically acceptable carriers and / or excipients. The pharmaceutical composition of claim 19, wherein the agent is urease. The pharmaceutical composition of claim 19 or 20, wherein the agent is not a vaccine. 22. A pharmaceutical composition according to any one of claims 19 to 21, modified by one or more of the features of any of claims 7-9. 23. The pharmaceutical composition of any one of claims 20 to 22, further comprising urea. H. H in the manufacture of a medicament for the treatment of pylori infection. A method of using an agent capable of inducing a conversion of a pylori balance to a spiral balance. The method of claim 24, wherein the agent is urease. The method of claim 24 or 25, wherein the agent is not a vaccine. 26. The method of claim 25, wherein the method of use is modified by at least one of the features of any of claims 7-9. 28. The method of any one of claims 25-27, wherein the medicament further comprises urease. H. H. in culture, comprising the addition of an agent to the culture of cocci that can induce the conversion of the pylori equilibrium into a spiral equilibrium. How to convert pylori's equilibrium to spiral equilibrium. The method of claim 29, wherein the formulation is H. A method provided in the form of a culture medium which propagates the spiral balance of the pylori, thereby eliminating the spiral balance. 31. The method of claim 30, wherein the spiral balance is propagated in the broth for at least 3 days. 32. The method of any one of claims 29-31, wherein the agent is urease. 33. The method of claim 32, modified by one or more of the features of any of claims 7-9. H. H. in the culture medium, comprising adding to the helix culture medium an agent capable of inducing the conversion of the pylori's helix balance to a balance. A method of converting the spiral balance of pylori into a balance. The method of claim 34, wherein the agent is an “anti-urease”. 36. The method of claim 35, wherein the "anti-urease" is a urease inhibitor.