EP0862458A1

EP0862458A1 - Urease for the treatment of helicobacter pylori infections

Info

Publication number: EP0862458A1
Application number: EP96932735A
Authority: EP
Inventors: Bow National University Of Singapore Ho
Original assignee: Cortecs International Ltd; Chapman Paul William
Current assignee: Cortecs International Ltd; Chapman Paul William
Priority date: 1995-10-09
Filing date: 1996-10-08
Publication date: 1998-09-09
Also published as: BR9611033A; NO981594L; JPH11514998A; MX9802809A; AU7141096A; WO1997013527A1; NO981594D0; KR19990064104A; CN1201395A

Abstract

The invention provides novel therapies for the treatment of H. pylori infections, as well as pharmaceutical formulations for use in such methods.

Description

UREASE FOR THE TREATMENT OF HELICOBACTER PYLORI INFECTIONS

The present invention relates to methods of treating H. pylori infection, pharmaceutical formulations for use in such methods, methods of rendering coccoid cultures of H. pylori viable, as well as methods of inducing the coccoid form to convert to the spiral form.

H. pylori is a Gram negative bacteria that has been strongly implicated in chronic active gastritis and peptic ulcer disease (Marshall et al, Medical Journal of Australia, 142:439-444 (1985); Buck, G.E. , Journal of clinical Microbiology, 3:1-12 (1990)) . In in vi tro culture, H. pylori exists in two distinct morphological forms, the culturable spiral form and the non-culturable coccoid form (Marshall et al , Microbios letters, 25:83-88 (1984); Rung, J.S.L., and HO, B., Workshop on Gastroduodenal Pathology and Campylobacter pylori (abstract P9) , edited by F. Megraud and H. Lamouliatte, Bordeaux, France (1988)). The spiral form of the bacterium does not survive beyond about 2 hrs when exposed to air. Under unfavourable conditions, the spiral form undergoes differentiation into the coccoid form (Vijayakumari and Ho, Acta Gastro-enterologica Belgica , 56:101 (1993) ) .

To date, there has been only a single report of the successful in vi tro transformation of coccoids to spirals, which experiment has proved unrepeatable (Mai et al , Gastroduodenal Pathology and Campylobacter pylori , pp28-33, edited by F. Megraud and H. Lamouliatte, Elsevier Science Publishers (1989)). Although the formation of coccoids in vi tro could be induced by antibiotics or by deprivation of nutrients (Nilius et al , Zbl . Bakt . 280: 259-272 (1993), studies of the coccoid form have been hampered due to the lack of information regarding this form, and its role in the life cycle of H. pylori , as well as the lack of any method for obtaining a culture of this form. Up to now the coccoid form of H. pylori has been regarded effectively as a "dead", non¬ viable form whose role in the life-cycle of the bacterium is unclear.

It has now been found that the coccoid form is viable and can be induced to convert to the spiral form. One possible method for inducing the conversion involves the administration of urease. WO 95/22987 discloses the use of urease-based vaccines for the treatment of H. pylori infections. However, there is no disclosure of a general "chemical" non-immunological method based on urease driven conversion of the coccoid form to the spiral form. Since the spiral form of the bacterium can be treated using antibiotics these results provide the possibility of a more effective treatment of H. pylori infection, such that reoccurrence of H. pylori mediated gastric disease will itself be significantly reduced.

Thus, in a first aspect, the present invention provides a method of treating H. pylori infection, in a mammal, which comprises administering to the mammal an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form.

Preferably, the agent is not in the form of a vaccine, i.e. it is not administered to elicit any immune response.

In general, administration of the agent, will form part of a treatment regimen involving administration of one or more antibiotics. Thus, in one embodiment of this aspect of the invention, the method further comprises the step of administering to the mammal an effective amount of at least one antibiotic.

Suitably, the administration of the one or more antibiotics will take place after administration of the agent. This will allow for the coccoid form to convert to the spiral form prior to the administration of the antibiotic(s) .

Of course, antibiotics are known to induce the spiral form to revert back to the coccoid form. It will often be the case, therefore, that several cycles of agent/antibiotic administration will be required to ensure effecient eradication of H. pylori . In a second aspect, therefore, the present invention provides a method of treating H. pylori infection, in a mammal, which comprises at least two treatment cycles, each treatment cycle comprising:

(i) administering to the mammal an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form;

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

After each treatment cycle a smaller and smaller number of H. pylori will remain. The treatment endpoint can be determined by the use of diagnostic tests for H. pylori which are commercially available. In preferred embodiments of these aspects of the present invention the agent administered is an amount of urease sufficient to induce conversion of the coccoid form of H. pylori to the spiral form. In the context of the present invention, urease is intended to include all forms of urease, either bacterial (eg H. pylori or Protieus mirabi tis urease) or non-bacterial (eg Jackbean urease) , as well as one or more individual subunits of the urease enzyme, or indeed peptides derived from such subunits. In one embodiment, only the C and D subunits of the urease are administered.

When the agent administered is urease, the method can also comprise the administration of urea to the mammal. The urea can be co-administered or administered separately.

Given the nature of the life cycle of the H. pylori organism, it can be seen that methods of preventing conversion of the spiral form to the coccoid form will also be effective in increasing the effectiveness of normal H. pylori treatment regimes. Therefore, in a third aspect, the present invention provides a method of treating H. pylori infection, in a mammal, which comprises administering to the mammal an agent capable of preventing conversion of the spiral form of H. pylori to the coccoid form.

Again, in one embodiment of this aspect of the invention the agent will be urease, optionally together with urea.

Another potential method of treating H. pylori infection is to "exhaust" the organism by means of forcing the organism to switch between the spiral and coccoid forms. Thus, in a fourth aspect, the present invention provides a method of treating H. pylori infection, in a mammal, which comprises one or more treatment cycles, each treatment cycle comprising:

(i) administering to the mammal an agent capable of inducing the coccoid form of H. pylori to convert to the spiral form; and

(ii) administering to the mammal an agent capable of inducing the spiral form of H. pylori to convert to the coccoid form.

In one embodiment of this aspect of the invention the agent capable of inducing conversion to the spiral form is urease and the agent capable of inducing conversion to the coccoid form is "anti-urease", eg a urease inhibitor or antibody specific for urease.

In a fifth aspect the present invention provides a method of treating H. pylori infection, in a mammal,which comprises administering to the mammal an agent capable of changing the pH in the stomach. In this embodiment the lowering of the pH induces the spiral form of H. pylori present to increase urease production. This increased urease level induces conversion of any coccoid form present to the spiral form, thus making antibiotic treatment more effective. Thus, this method generally also includes the step of administering at least one antibiotic to the mammal. Methods of changing the pH can include administration of edible acids or bases.

In all the methods of the invention described herein the mammal is preferably a human. The methods of the present invention will generally employ the agent in the form of a pharmaceutical formulation. Thus, in a sixth aspect, the present invention provides a pharmaceutical formulation comprising an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form together with one or more pharmaceutically acceptable carriers and/or excipients. Again, preferably, the pharmaceutical formulation is not in the form of a vaccine.

In one embodiment of this aspect of the invention the agent is urease and optionally the pharmaceutical formulation will also comprise urea.

The pharmaceutical formulations of the invention may be presented in unit dose forms containing a predetermined amount of the agent, eg urease (and optionally urea) per dose. Such a unit may contain for example enough urease to convert 3 mg urea in 30 min at 37°C, depending on the age, weight and condition of the patient.

The pharmaceutical formulations of the invention will be adapted for oral administration and may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non¬ aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions, or any conventional or non-conventional pharmaceutical form.

Suitably, the urease and urea, if administered together, will be administered in a form which prevents mixing of the two components.

In a seventh aspect the present invention provides the use of an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form in the manufacture of a medicament for the treatment of H. pylori infection. Once again, in a one embodiment of this aspect the agent is urease and the medicament will optionally also include urea. Also, once again, preferably the medicament is not a vaccine.

The results described herein also find application in vi tro . Thus, in other aspects the present invention provides:

(a) a method of converting the coccoid form of H. pylori in culture to the spiral form which comprises adding to the coccoid culture an agent capable of inducing the coccoid form of H. pylori to convert to the spiral form; and

(b) a method of converting the spiral form of

H. pylori in culture to the coccoid form which comprises adding to the spiral culture an agent capable of inducing the spiral form of H. pylori to convert to the coccoid form.

For (a) the agent can be provided in the form of culture medium in which the spiral form has been growing but from which the spiral form has been removed. Preferably, the spiral form has been growing in the medium for at least 3 days. Alternatively, the agent is urease, optionally together with urea.

For (b) a preferred agent is "anti-urease", e.g. a urease inhibitor or an antibody specific for urease. Thus, the in vi tro conversion methods allow for the production of both coccoid and spiral sources of antigen to be produced. These will be particularly useful in producing specific antigens for use in diagnostic tests for the two forms of the bacterium.

Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis.

The invention will now be described with reference to the following example which not be construed as in any way limiting the invention.

EXAMPLE 1

(a) Bacterial strain and preparation of the coccoid foπn of H. pylori .

A local H. pylori strain V₂ isolated from a patient with non-ulcer dyspepsia was used, although other wild type strains can be used equally well. This strain was initially grown on chocolate blood agar (CBA) to check for purity. The plate culture was then used as inoculum for a 250ml Schott flat-bottomed round bottle containing 30ml BHIH (brain heart infusion supplemented with 10% horse serum and 0.4% yeast extract), and incubated at 37°C for 72h. This in turn serves as the inoculum for chemostat or batch cultures.

A 1.5L fermenter containing 540ml BHIH was set up as described in Ho and Vijayakumari {Microbios , 76:59-66

(1993) . The medium was inoculated with 2x30ml of 3 day old H. pylori cul ture, giving a ratio of 1:10

(inoculum:medium) . Carbon dioxide was supplied twice daily and the impeller was adjusted to 35rpm. Samples were withdrawn at time intervals and checked for urease activity, pH, viability, and microscopic examination for morphological changes.

The culture was maintained under these conditions for up to 3 months during which daily monitoring of the cells was continued. When homogenous/synchronous culture was observed, the cells were harvested by centrifugation at 10,000g for 40min. and washed once. The pellet was then used for preparing coccoid antigen by using the modified glycine method (Ho, B., and Jiang, B., European Journal of Gas tr center ology and Hepatology, 7:121-124 (1995) .

Alternatively, a IL Schott round-bottomed bottle or IL Erlenmeyer flask with a side-arm and fitted with a tight fitting rubber bung, containing 270ml BHIH was used. A 7mm diameter hole was bored so as to accomodate the fitting of a disposable filter unit containing a 0.22μm filter having a diameter of 50mm (e.g. Gelman) . Each 270ml of BHIH was inoculated with 30ml of 3 day old H. pylori culture. Carbon dioxide was supplied twice daily via the 0.22μm filter.

The culture was incubated in a 37°C shaker incubator (New Brunswick) maintained at 90rpm for up to nine weeks and the cells were subsequently harvested by centrifugation at 10,000g for 40min.

The coccoids thus obtained are stored at -80°C in glycercl-BHIH for up to two years. When required, the coccoids were collected by centrifugation at 10,000xg for 30min, and washed once with PBS (pH 7.2) . Preparation of Induction Broth(IB)

Three day old H. pylori spirals were inoculated into BHIH in a 1.5L fermentor or a IL Schott screw capped bottle or a IL Erlenmeyer flask as described above. After 3 days, the supernatant was collected by centrifugation at 10,000xg for lOmin. The spent medium was filtered twice through 0.2μm membrane filters. The filtrate was henceforth referred to as induction broth (IB) . The two filters were transferred to CBA plates and incubated at 37°C in a humidified carbon dioxide incubator for up to 1 week.

Induction of coccoids into Spiral foπn

H. pylori coccoids were inoculated into 30ml of supernatant (IB) and incubated at 37°C in a 5% carbon dioxide incubator to give a final concentration of 10^β coccoids per ml. Similar amounts of coccoids were inoculated into 30ml of fresh BHIH served as controls. Subsequently, fresh BHIH was added to the coccoid cultures over time (24hr) .

Transmission Electron Microscopy

At time intervals of 24, 48, 72 and 96 hours, an aliquot of each culture was harvested by centrifugation and washed twice in PBS. The cells were resuspended in PBS and processed by negative staining for transmission electron microscopy.

Alternatively, the cells were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.0) for 2-3hrs or overnight at 4°C and then washed in two changes of 0.IM cacodylate buffer. The cells were resuspended in distilled water and processed for negative staining as above. Drops of the cell suspension were placed on carbon coated 400 mesh copper grids for 1 minute. The excess fluid was blotted and the grids air dried to fix. the grids were then stained with a drop of 1% phosphotungstic acid for 1 minute before the excess stain was blotted. After air drying, the grids were examined using a Philips CM120 transmission electron microscope.

Treatment with antibiotic Coccoids were further treated with 5μg/ml amoxycillin for 48hrs in BHIH to ensure that any spiral present would be induced to the coccoid form or, alternatively, killed. Following treatment, the coccoid suspension was washed 2 times and resuspended in the original volume of BHIH. An aliquot of 1ml of the suspension was inoculated into fresh BHIH and incubated accordingly.

Addition of Urea to IB and BHIH

Urea was added at a concentration of 5mM to coccoids in IB or BHIH. Samples of the culture were withdrawn at time intervals for microscopic examination and were subcultured on CBA and in BHIH.

RESULTS None of the coccoid preparations grew on subculturing in BHIH or CBA alone. Similarly, none of the second filters that were used for filtering the H. pylori cultures showed any growth on CBA or in BHIH alone. IB of three days or more showed positive a test for urease within 30min.

The coccoids appeared dense under phase contrast microscopy. It took 30min. for counter staining to take effect. After 24h induction in IB, the coccoids became loose, and began to extrude the foetal spiral cells. Some nascent spirals could be seen attached to the "maternal" coccoid shells. At 48h of induction, this "birthing" process became more pronounced. Upon introduction of BHIH, the new spirals changed into mature cells and became motile. The spirals were more active in 2x BHIH. Upon addition of urea to the IB and BHIH, the growth was more evident, with spirals appearing as early as 24h.

Spirals generated from regenerated coccoids became fully viable and functional. They were found to attach easily to KATO III cells. This process mimic that of the pathogenic invasion of normal spiral forms with KATO III cells.

DISCUSSION

These reults indicate that it is possible to induce conversion of the coccoid form of H. pylori into the spiral form. Some factor (the "inducer") in the IB must therefore be influencing this conversion. In addition, it was observed that conversion/growth of the spirals was partial without the addition of urea.

The "inducer" initiated the foetal outgrowth of the spiral form from within the thick polysaccharide layer of the coccoid form. Thus, clearly, nutrients/inducers can pass through this coat. Data obtained previously has indicated that urease subunits C and D are lacking or are present at reduced levels within the coccoid form. The data given above indicates that urease is present in the IB. Thus, it can be concluded that subunits C and D of the urease enzyme are in fact the "inducer".

The introduction of freshBHIH medium provided the necessary nutrients to ensure full conversion (since the IB would have had deleted or even exhausted levels) . Provision of physiological levels of urea demeonstrates that urea plays a pivotal role in the metabolism of this organism.

Extrapolating these findings to the in vivo situation, it is proposed that after colonisation of the stomach by the organism, spirals excreted in the faeces convert to the coccoid form to ensure survival ex vivo . When reintroduced into the oral cavity, they remain there under the microaerophilic conditions of the subgingival plaques. The presence of other urease producing bacteria or indeed other sources, can thus induce conversion into spirals. Once so converted the bacteria can colonise the mouth, and from there can travel down the alimentary canal and colonise the stomach.

Clearly therefore, elimination of the spiral form of the bacterium by convential antibiotics is not enough. Gastric disease can simply reoccur by virtue of conversion of any coccoids present into sprirals. It is therefore essential to eradicate both the spiral and coccoid forms of the bacterium to prevent such reoccurrence. To do that, any coccoids present must be driven to convert into the spiral form such that the antibiotic treatment will eradicate all H. pylori infection.

This can be achieved by inducing conversion to the spiral form by the addition of urease, optionally with urea as an additional component.

Claims

CLAIMS :

1. A method of treating H. pylori infection, in a mammal, which comprises administering to the mammal an agent capable of inducing conversion of the coccoid form of H. pylori to the spiral form.

2. A method as claimed in claim 1, wherein the agent is not in the form of a vaccine.

3. A method as claimed in claim 1 or claim 2 which further comprises the step of administering to the mammal an effective amount of at least one antibiotic.

4. A method of treating H. pylori infection, in a mammal, which comprises at least two treatment cycles, each treatment cycle comprising:

(i) administering to the mammal an agent capable of inducing conversion of the coccoid form of

H. pylori to the spiral form;

5. A method of treating H. pylori infection, in a mammal, which comprises administering to the mammal an agent capable of preventing conversion of the spiral form of H. pylori to the coccoid form.

6. A method as claimed in any one of claims 1 to 5 wherein the agent is urease.

7. A method as claimed in claim 6 wherein the urease is a non-bacterial urease.

8. A method as claimed in claim 7 wherein the non- bacterial urease is Jackbean urease.

9. A method as claimed in any one of claims 6 to 8 wherein only the C and D subunits of the urease are administered.

10. A method as claimed in any one of claims 6 to 9 wherein urea is also administered to the mammal.

11. A method of treating H. pylori infection, in a mammal, which comprises one or more treatment cycles, each treatment cycle comprising:

12. A method as claimed in claim 11 wherein the agent in step (i) is urease.

13. A method as claimed in claim 12 modified by any one or more of the features of any one or more of claims 7 to 9.

14. A method as claimed in claim 12 or claim 13 wherein urea is administered with the urease.

15. A method as claimed in any one of claims 12 to 14 wherein the agent in step (ii) is "anti-urease" .

16. A method as claimed in claim 15 wherein the "anti- urease" is a urease inhibitor.

17. A method of treating H. pylori infection, in a mammal, hich comprises administering to the mammal an agent capable of changing the pH in the stomach.

18. A method as claimed in any one of claims 1 to 17 wherein the mammal is a human.

19. A pharmaceutical formulation comprising an agent capable of inducing the coccoid form of H. pylori to convert to the spiral form together with one or more pharmaceutically acceptable carriers and/or excipients.

20. A pharmaceutical formulation as claimed in claim 19 wherein the agent is urease.

21. A pharmaceutical formulation as claimed in claim 19 or claim 20 which is not a vaccine.

22. A pharmaceutical formulation as claimed in any one of claims 19 to 21 modified by any one or more of the features of any one of claims 7 to 9.

23. A pharmaceutical formulation as claimed in any one of claims 20 to 22 which also comprises urea.

24. The use of an agent capable of inducing the coccoid form of H. pylori to convert to the spiral form in the manufacture of a medicament for the treatment of H. pylori infection.

25. The use as claimed in claim 24 wherein the agent is urease.

26. The use as claimed in claim 24 or claim 25 wherein the agent is not a vaccine.

27. The use as claimed in claim 25 modified by any one or more of the features of any one or more of claims 7 to

9.

28. The use as claimed in any one of claims 25 to 27 wherein the medicament also comprises urea.

29. A method of converting the coccoid form of H. pylori in culture to the spiral form which comprises adding to the coccoid culture an agent capable of inducing the coccoid form of H. pylori to convert to the spiral form.

30. A method as claimed in claim 29 wherein the agent is provided in the form of culture medium in which the spiral form of H. pylori has been growing, but for which the spiral form has been removed.

31. A method as claimed in claim 30, wherein the spiral form has been growing in the broth for at least three days.

32. A method as claimed in any one of claims 29 to 31 wherein the agent is urease.

33. A method as claimed in claim 32 modified by any one or more of the features of any one of claims 7 to 9.

34. A method of converting the spiral form of H. pylori in culture to the coccoid form which comprises adding to the spiral culture an agent capable of inducing the spiral form of H. pylori to convert to the coccoid form.

35. A method as claimed in claim 34 wherein the agent is "anti-urease" .

36. A method as claimed in claim 35 wherein the "anti- urease" is a urease inhibitor.