Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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UREASE FOR THE TREATMENT OF HELICOBACTER PYLORI INFECTIONS
The present invention relates to methods of ~reating
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
A~stralia, 142: 439-444 (1985); Buc~, G.E., ~ournal of
clinical Microbiology, 3:1-12 (1990)). In in vitro
culture, H. pylori exists in two distinct morphological
forms, the culturable spiral form and the non-culturable
coccoid form (Marshall et al, ~icrobios letters, 25:83-88
(1984); Kung, J.S.L., and HO, B., Workshop on
Gastroduodenal Pathology and Campylo~acter pylori
(abstract P9), edited by F. Megraud and H. Lamouliatte,
Bordeaux, France (1988)). The spiral ~orm 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 vitro 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 vitro could be induced by
antibiotics or by deprivation of nutrients (Nilius et al,
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Zbl. Bakt. 280: 259-272 (1993), studies of the coccoid
form have been hampered due to the lack of inrormation
regarding this form, and its role in the life cycle of H.
py70ri, 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
A~m;n;stration of urease. WO 95/22987 discloses the use
of urease-based vaccines for the treatment of H. pyl ori
infections. However, there is no disclosure of a general
~chemical~ non-imm~nological 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 pro~ide the possibility
of a more effective treatment of H.pylori infection, such
that reoccurrence of H. pyl ori 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 ~mm~l,
which comprises administering to the m~mm~l an agent
capable of inducing conversion of the coccoid form of
H. pyl ori to the spiral form.
Preferably, the agent is not in the form of a vaccine,
i.e. it is not a~m; n; ~tered to elicit any immune
response.
In general, ~m; n; stration of the agent, will form part
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of a treatment regimen involving administration of one or
more antibiotics. Thus, in one embodiment of this aspect
or the invention, the method further comprises the step
of administering to the m~mm~l 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 a~m; n; stration of the
antibiotic (8) .
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 m~mm~l, which
comprises at least two treatment cycles, each treatment
cycle comprising:
m; n; stering to the m~mm~l an agent capable of
inducing conversion of the coccoid form of
H. pylori to the spiral form;
(ii) ~m;n;stering to the m~mm~l an effective amount
of at least one antibiotic.
After each treatment cycle a smaller and smaller number
of H. pyl ori will remain. The treatment endpoint can be
determined by the use of diagnostic tests for H. pyl ori
which are commercially available.
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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
Protie~s 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 ~m; n; qtered.
When the agent administered is urease, the method can
also comprise the administration of urea to the m~mm~ 1,
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 m~mm~l, which comprises
a~mi ni stering to the m~mm~ 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.
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Thus, in a fourth aspect, the present invention provides
a method of treating H. pylori infection, in a m~mm~l,
which comprises one or more treatment cycles, each
treatment cycle comprising:
s
(ï) administering to the m~mm~l an agent capable of
inducing the coccoid form of H.pylori to convert
to the spiral form; and
(ii) administering to the m~mm~l an agent capable of
inducing the spiral form of H. pyl ori 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 m~mm~l,which
comprises ~m;n;stering to the m~mm~l an agent capable of
changing the pH in the stomach. In this embo~;m~nt the
lowering of the pH induces the spiral form of H. pyl ori
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 ~min;stering at least one
antibiotic to the m~mm~l Methods of changing the pH can
include ~min;stration of edible acids or bases.
In all the methods of the invention described herein the
m~m~l is preferably a human.
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The methods of the present invention will generally
employ the agent in the ~orm 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. pyl ori 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 iB urease and optionally the pharmaceutical
formulation will also comprise urea.
The pharmaceutical formulations of the invention may be
presented in unit dose forms cont~;n;ng 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, dep~n~; ng 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
li~uid emulsions or water-in-oil liquid emulsions, or any
conventional or non-conventional pharmaceutical form.
Suitably, the urease and urea, if ~m;n;stered together,
will be administered in a form which prevents mixing of
the two components.
In a seventh aspect the present invention provides the
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.
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 ~accine.
The results described herein also find application in
vitro. 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
con~ert to the spiral form; and
(b) a method of converting the spiral form of
H.pylori in culture to the coccoid form which
comprises ~;ng 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 pro~ided in the form of culture
medium in which the spiral form has been growing ~ut from
which the spiral form has been ~e,no~ed. 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.
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Thus, the in vi tro conversion methods allow for t~e
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.
Pre~erred features of each aspect of the invention are as
for each other aspect mutatis mutandis.
The invention will now be described with re~erence to the
following example which not be construed as in any way
limiting the invention.
~X~MPLE 1
(a) Bacterial strain and preparat~on of the coccoid ~orm
of R. pyl or~ .
A local H. pyl ori 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 2x3Oml of 3 day
old H.~ylori cul ture, giving a ratio of 1:10
(inoculum:medium). Carbon dioxide was supplied twice
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daily and the impeller was adjusted to 35rpm. Samples
~ were withdrawn at time intervals and checked for urease
activity, pH, viability, and microscopic ~x~m; n~tion for
morphoiogical 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
lO,OOOg 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 Gastroenterology and Hepatology, 7:121-124 (1995).
Alternatively, a lL Schott round-bottomed bottle or lL
Erlenmeyer flask with a side-arm and fitted with a tight
fitting rubber bung, contAin;ng 270ml BHIH was used. A
7mm diameter hole was bored so as to accomodate the
fitting of a disposable filter unit cont~;n;ng a 0.22~m
filter having a diameter of SOmm (e.g. Gelman). Each
27Oml of BHIH was inoculated with 3Oml of 3 day old
~.pylori culture. Carbon dioxide was supplied twice daily
via the 0.22~m filter.
2S 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 lO,OOOg 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 lO,OOOxg for
3Omin, and washed once with PBS (pH 7.2).
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Preparation o~ Induction Broth(IB)
Three day old H. pyl ori spirals were inoculated into BHIH
in a 1.5B fermentor or a lL Schott screw capped bottle or
a lL Erlenmeyer flask as described above. After 3 days,
the supernatant was collected by centrifugation at
lO,OOOxg for lOmin. The spent medium was filtered twice
through 0.2~m 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 form
H. pyl o~i coccoids were inoculated into 3Oml of
supernatant (IB) and incubated at 37~C in a 5~ carbon
dioxide incubator to give a final concentration of 108
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).
Tr~ ~i ssion 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 st~; n; ng for transmission
electron microscopy.
Alternatively, the cells were fixed in 2~ glutaraldehyde
in O.lM cacodylate buffer (pH 7.0) for 2-3hrs or
overnight at 4~C and then washed in two changes of O.lM
cacodylate buffer. The cells were resuspended in
distilled water and processed for negative st~;n;ng as
above.
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.
11
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 ~xAm;ned using a Philips CM120
transmission electron microscope.
Treatment with antibiotic
Coccoids were further treated with ~g/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 lml of the suspension was inoculated into
fresh BHIH and incubated accordingly.
Addition o~ Urea to IB and 2HIH
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 ~m; nAtion and were
subcultured on CBA and in BHIH.
RES~TS
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 3Omin. for counter staining to ta~e
effect. After 24h induction in IB, the coccoids became
loose, and began to extrude the ~oetal spiral cells. Some
-
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12
nascent spirals could be seen attached to the "maternal"
coccoid shells. At 48h of induction, this "birthing"
process became more pronounced. Upon introduction of
BHI~, 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.
DTSCUSSION
These reults indicate that it is possible to induce
conversion of the coccoid form of ~. pyl ~ri 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 o~ the spirals was
partial without the addition of urea.
The "inducer" initiated the ~oetal ou~growth 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 i9 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
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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
organlsm .
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
pla~ues. 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
there~ore essential to eradicate both the spiral and
coccoid forms of the bacterium to prevent such
reoccurrence. To do that, any coccoids present must be
2S driven to convert into the spiral form such that the
antibiotic treatment will eradicate all N. pyl ori
infection.
This can be achieved by inducing conversion to the spiral
form by the addition of urease, optionally with urea as
an additional component.
.
