Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CONTROL OF INTESTINAL INFLAMMATORY SYNDROMES
WITH A PREPARATION OF KILLED OR NON INFECTIOUS BACTERIA
FIELD OF THE INVENTION
This invention relates to the use of a preparation of killed or non
infectious Gram positive bacteria such as Gram positive facultative
intracellular
bacteria, for example mycobacteria, for the treatment of intestinal
inflammatory
syndromes.
DESCRIPTION OF THE PRIOR ART
With an increase in incidence, Crohn's disease is a disease found in
children, adolescents and adults which can be serious, and requires
specialized
long term attention. Ulcerative colitis is also an Inflammatory Bowel Disease
(IBD)
which is disabling, of unknown etiology, and which affects young subjects. The
cumulative prevalence of these two diseases is 10 to 200 per 100 000
inhabitants
depending on the country. Recent dietary modifications and bacterial flora
imbalances have been implicated in the origin of these afflictions, without
precise
results being reported. For Crohn's disease, bacteria of the genus
Mycobacterium
avium sub. sp. paratuberculosis have been proposed to be at the origin of the
disease in part because of its analogy with Johne's disease, a disease
affecting
cattle which appears a few years after the initial infection. More recently,
E. coli
adhesins, flagellin or fimbriae, have been implicated in the pathology of the
disease.
Idiopathic inflammatory bowel disease (IBD) includes a collection of
disorders of the gastrointestinal tract of unknown aetiology, characterized by
intestinal inflammation and a chronic relapsing course associated with local
and
systemic complications.
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The aetiology of IBD remains unclear, but it is well established that the
lesions and symptoms are associated with over-production of pro-inflammatory
cytokines. IBD comprises two entities, ulcerative colitis (UC) and Crohn's
disease
(CD) and an intermediate variant of these diseases, indeterminate colitis
which
shows overlapping features of the two major forms.
The immune response is implicated in these inflammatory bowel
diseases. The clinical table, by successive advances, and with extended
periods
of remission, has evoked the participation of autoimmune mechanisms; however,
are these causes or consequences of the diseases? The active treatments
brought about particularly by anti-TNF-a, underline that immunological
mechanisms in the broad sense are implicated in these diseases, once again
without providing any precise indication of the initial mechanism.
The different murine experimental models partially reproduce human
diseases. It has been shown that flora participates to the disease. Mice
maintained in a germ free breeding environment do not develop inflammatory
syndrome by contrast to the mice in which the flora is conventional. Some of
these inflammatory bowel disease (IBD) models in the rat or in the mouse
involve
a local sensitization by phenolic derivatives (TNBS) deposited locally in the
colon
or by the ingestion of dextran sodium sulphate (DSS) given in the drinking
water
for a few days. Regulatory T lymphocytes have been described to play a role in
IBD models.
Intestinal inflammatory syndromes are chronic pathologies which are
disabling and which may be mortal since they bring about intestinal stenoses
and
repetition of necessary surgical interventions. The medical treatment for
these
inflammatory syndromes is essentially based on the use of powerful anti-
inflammatory agents, corticoids, antimitotics, and more recently anti-TNF
agents.
Any treatment bringing about a decrease in the doses of anti-inflammatory
agents
to be administered or that may be substituted to these anti-inflammatory
agents
are important.
In the course of analyzing mechanisms that may control experimental
asthma in mice, it was discovered that CD4+ CD25+ regulatory cells are
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produced. The IL-10 that is secreted in turn insures an important part of the
anti-
inflammatory activity. The important decrease in the number of inflammatory
cells
present after the administration of an allergen in the lungs of the animals
that
were treated with Mycobacteria bovis BCG killed by Extended Freeze Drying
(EFD) led the inventors to determine the eventual activity of EFD in other
syndromes where immunoallergic or autoimmune phenomena have been
described. In models reproducing rheumatoid arthritis, the participation of
CD4+ T
lymphocytes was also reported, the presence or transfers of CD4+CD25+ T
lymphocytes can stop the disease.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a treatment for intestinal
inflammatory syndromes such as inflammatory bowel disease (IBD).
Another object of the present invention is to provide a treatment for IBD
which does not require or decrease the dose of anti-inflammatory agents
required. Accordingly, the present invention provides the use of a Gram
positive
bacteria preparation for the prevention and treatment of intestinal
inflammatory
syndromes, the preparation being characterized in that the Gram positive
bacteria
are killed or non infectious, and contains more than 50 %, and preferentially
more
than 90%, of the bacterial protein components which are in a native structure.
The present invention further provides a method for preventing or treating
a disease caused by a Thl/Th2 imbalance, the method comprising the steps of :
a. providing a killed or non infectious Gram positive bacteria
preparation or a portion thereof retaining the capacity to inhibit
intestinal inflammatory syndromes, and
b. administering an effective amount of the killed Gram positive
bacteria preparation or the portion thereof to a patient affected by
the disease.
The present invention still provides a method for preventing or treating an
intestinal inflammatory syndrome, the method comprising the step of
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administering to a patient an effective amount of a Gram positive bacteria
preparation, thereby stimulating the production of leukocyte regulatory cells.
The present invention further provides a composition for the treatment
and/or prevention of intestinal inflammatory bowel diseases containing a Gram
positive bacteria composition prepared by the following steps
a) harvesting a culture of live bacteria cells,
b) washing the bacteria cells in water or in an aqueous solution of a
salt such as borate,
c) freezing the bacteria cells in water or in an aqueous solution of a
salt such as borate,
d) killing the frozen bacteria cells by drying them in a freeze-dryer, for
a time sufficient to remove at least 98.5 % of the water, preferably
at least 99% of the water, more preferably at least 99.5% of the
water, and
e) collecting the extended freeze-dried bacteria cells.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates changes in body weight and anus inflammation after
addition
of 2.5% DSS in drinking water during 7 days (C57B1/6 male mice).
Figure 2 illustrates a difference in length of colon between mice receiving
DSS in
their drinking water and control (no DSS feeding) mice. The experimental
model:
C57BI/6 male mice received 2.5% of dextran sodium sulphate (DSS) in their
drinking water during 7 days. Colon was collected on day 10 after the
beginning
of DSS feeding.
Figure 3 illustrates the difference in length of the colon between EFD treated
and
untreated mice. Mice received 2.5% DSS in their drinking water during 7 days.
A
group of mice were EFD treated: 100 pg given subcutaneously 21 days before
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DSS feeding; or 1 mg per os at days 21, 20, 17 and 16 before DSS feeding.
Colon was collected on day 10 after the beginning of DSS feeding.
Figure 4 illustrates the difference in length of colon between the control
group (no
EFD treatment and no DSS feeding), the EFD treated mice (DSS feeding) and
5 untreated mice (DSS feeding). The colons of EFD treated mice were similar to
those of control mice. They were measured between caecum and anus.
Figure 5 illustrates prevention of inflammatory bowel disease by EFD
treatment.
The colons of EFD treated mice were similar to those of control mice (no EFD
and no DSS) (*** means p<0.001 with ANOVA statistical test). They were
measured at day 8 or 10 i.e. 1 or 3 days after the end of DSS feeding.
Figure 6 illustrates prevention of inflammatory bowel disease by EFD
treatment.
EFD treated mice did not develop a serious diarrhea or no diarrhea whereas the
controls (DSS treated) did.
Figure 7 illustrates a histological cut of the colon of a control (no DSS
feeding, no
EFD treatment) mice.
Figure 8 is an enlarged view of a portion of the picture in figure 7.
Figure 9 illustrates a histological cut of the colon of a mouse feeded with
DSS.
Figure 10 illustrates an enlarged view of a portion of the picture in figure
9.
Figure 11 illustrates the histological cut of the colon of a mouse treated
with EFD
before DSS feeding.
Figure 12 illustrates en enlarged view of a portion of the picture in figure
11.
Figure 13a illustrates a histological cut of the colon of a control mouse.
Figure 13b illustrates a histological cut of a colon of a mouse feeded with
DSS.
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Figure 13c illustrates a histological cut of a colon of a mouse treated with
EFD
before DSS.
Figures 14a, b and c are enlarged views of figures 13a, b and c respectively.
Figure 15 illustrates the preliminary experiment with TNBS. C57BI/6 male mice
received or not TNBS (1 mg in 100 pl of ethyl alcohol at 50%) twice, locally
in
colon at day -5 and 0. Their stools were observed during 10 days. At day 10,
the
mice were weighted. A group received EFD 100 pg subcutaneously 5 days
before the first TNBS delivery (i.e. at day -10). A second group received PBS.
The control group did not receive TNBS neither EFD. Weights in grams are
reported on the figure (y-axis).
Figure 16 illustrates the effects of EFD on DSS induced IBD in mice of
protocol 2
during the 10 days of observation : a) on weight and b) on anus inflammation
and
stools. According to protocol 2, C57BI/6 male mice received or not 2.5% of
dextran sodium sulfate (DSS) in their drinking water during 5 days. A group
received EFD lOOpg subcutaneously 21 days before DSS. A group received
PBS. The last group, control, did not drink DSS. This treatment protocol is
the
same for figure 16 and following figures 17 to 24. On x-axis day 0 corresponds
to
the first day of DSS feeding. The pre-treatment with EFD decreases IBD
symptoms.
Figure 17 illustrates the effects of EFD pre-treatment on DSS feeded mice of
protocol 2, as assessed by colon and caecum length. Colon and caecum lengths
were measured at day 10 after the beginning of DSS fedding. Important
inflammatory reaction due to DSS feeding conducted to a thickening of
intestinal
walls and a decrease in colon length of PBS treated mice, the colon weight
being
unchanged. The colons and caecums of EFD treated mice were similar to those
of confrol mice (*** means p<0.001 with ANOVA statistical test).
Figure 18 illustrates the effects observed at day 10 post the beginning of DSS
feeding on mesenteric lymph node cell number. Mesenteric lymph nodes were
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collected, dissociated, and their cell contents were determined. An increased
number of cells (x 2.5) was observed. This increase was marginal when mice had
been previously EFD treated (*** means p<0.001 with ANOVA statistical test).
Figure 19 illustrates EFD effects on cytokines and lymphokines present in
colonic
tissues (a). Samples of colonic tissues were collected, weighed, dissociated
in
presence of protease inhibitors at day 10 post the beginning of DSS feeding.
Their contents in different cytokines and lymphokines were determined. For IL-
12p40 and RANTES no statistically significant differences were observed
between PBS and EFD treated mice.
Figure 20 illustrates EFD effects on cytokines and lymphokines present in
colonic
tissues (b). For IL-1p, TNF-a and MIP-la, all implicated in inflammatory
processes, statistically significant differences were observed between PBS and
EFD treated mice, thus EFD treatment prevented the inflammation induced by
DSS feeding.
Figure 21 illustrates EFD effects on cytokines and lymphokines present in
colonic
tissues (c). IL-17, produced by activated T-cells, stimulates different cell
lineages
to produce inflammatory and hematopoietic cytokines. Less activated T-cells
were present (according to IFN-y measured quantity) and less IL-17 production
was observed after EFD treatment.
Figures 22 illustrates EFD effects on cytokines and lymphokines present in
colonic tissues (d). KC, murin equivalent of IL-8, IL-6 and IL-la are
"inflammatory" cytokines or implicated in the NFKB signalling pathway. Their
production was decreased after EFD treatment.
Figure 23 illustrates EFD effects on cytokines and lymphokines present in
colonic
tissues (e). Hematopoeitic cytokines, IL-3, GM-CSF and G-CSF, were less
produced after EFD treatment as suggested by preceding results showing a
decreased IL-17 production.
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Figure 24 illustrates the fact that the increase in GATA-3 protein level
observed
in the spleen of mice drinking DSS supplemented water is prevented by EFD
treatment. T-bet protein was highly produced in spleen 30 days after EFD
treatment. Two samples, a and b, from DSS feeded mice were processed.
Figure 25 illustrates the difference in body weight between EFD treated and
untreated mice receiving dextran sodium sulphate (DSS) three times in their
drinking water some days after EFD treatments. This model explored EFD
preventive treatments in chronic IBD. Mice (C57B1/6 male mice) received 1.5%
DSS in their drinking water during 7 days, ordinary tap water during 8 days,
1.5%
DSS in their drinking water during 5 days, tap water during 10 days, 1.5% DSS
during 5 days and tap water thereafter. A group of mice were EFD treated: 100
pg given subcutaneously 21 days before the first day of DSS feeding; or 1 mg
per
os at days 23, 22 and 21 before the first DSS feeding. They did not receive
more
EFD treatment. The weight of individual mouse was checked each day, 5 days a
week. The experiment ended at day 52.
Figure 26 illustrates the colitis score performed on mice included in the
preventive assay (figure 25). Scores were checked according to:
- 0: no modification of feces
- 1: anus Inflammation
- 2: anus inflammation plus soft stool
- 3: anus inflammation plus diarrhoea
- 4: anus inflammation plus bloody diarrhoea
Figure 27 up illustrates the difference in length of colon, measured between
caecum and anus, at day 52 on mice included in the preventive assay (figure
25).
The colons of EFD treated mice were similar to those of naive mice never fed
with DSS (** means p<0.01 and *** means p<0.001 with ANOVA statistical test ).
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Figure 27 down illustrates the number of cells found in the mesenteric lymph
nodes collected at day 52 on mice included in the preventive assay (figure
25).
The number of cells were decreased in the lymph nodes of EFD treated mice
compared to those of PBS treated mice (*** means p<0.001 with ANOVA
statistical test).
Figure 28 illustrated the spleen weight and number of spleen cells at day 52
on
mice included in the preventive assay (figure 25). The differences were not
statistically different.
Figure 29 illustrated the spontaneous release of some inflammatory cytokines
by
spleen cells collected at day 52 from mice included in the preventive assay
(figure
25). 2x105 cells were incubated in 0.2 ml of tissue culture medium during 96h
at
37 C, the supernatant were collected and the concentrations in IFN-y, IL-6 and
IL-17 were determined using the Multiplex BioRad assay. The differences
observed after PBS and EFD treatments were extremely significant, whatever the
cytokine, but much marked for IL-17.
Figure 30 up illustrated the concentration of the transcription factor NFKB.
NFKB
was measured using a kit sell by Active Motif and used according to
manufacturer
protocol. This transcription factor was measured as optical density on 5pg of
nuclear extracts of spleen cells collected at day 52 from mice included in the
preventive assay (figure 25). The differences observed after PBS and EFD
treatments were highly significant (** means p<0.01 with ANOVA statistical
test)
or extremely significant (*** means p<0.001 with ANOVA statistical test).
Figure 30 down illustrated the concentration of the transcription factor
PPARy. PPARy was measured using a kit sell by Active Motif and used according
to manufacturer protocol. This transcription factor was measured as optical
density on 5pg of nuclear extract of spleen cells collected at day 52 from
mice
included in the preventive assay (figure 25). The differences observed after
PBS
and EFD treatments were highly (** means p<0.01 with ANOVA statistical test)
or
extremely significant (*** means p<0.001 with ANOVA statistical test).
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Figure 31 illustrated the difference in body weight between EFD treated and
untreated mice which have received dextran sodium sulphate (DSS) during 5
days in their drinking water. This model explored EFD curative treatment in
acute
IBD. Mice (C57BI/6 male mice) received 2.5% DSS in their drinking water during
5 5 days, and tap water thereafter. A group of mice were EFD treated: 100 pg
given
subcutaneously 24 hours after the last day of DSS feeding i.e. at day 6; or 1
mg
per os at days 6, 7, and 8 after the 1st day of DSS feeding. They did not
receive
more EFD treatment. The weight of individual mouse was checked each day, 5
days a week. The experiment ended at day 34. After EFD treatment, a slight,
10 significant, curative effect was observed on day 13 and 15 (* p<0.05) with
a faster
recovery of weight loss.
Figure 32 illustrates the colitis score performed on mice included in the
curative
assay (figure 31). Scores were checked according to previous description
(figure26). The clinical symptoms decreased faster in EFD treated mice.
Figure 33 up illustrates the difference in length of colon, measured between
caecum and anus, at day 34 on mice included in the curative assay (figure 31).
The colons of EFD treated mice were similar to those of naive mice never fed
with DSS (*** means p<0.001 with statistical ANOVA test).
Figure 33 down illustrates the number of cells found in the mesenteric lymph
nodes collected at day 34 on mice included in the curative assay (figure 31).
The
number of cells were decreased in the lymph nodes of EFD treated mice
compared to those of PBS treated mice, the difference were statistically
significant.
Figure 34 illustrated the spleen weight and number of spleen cells at day 34
on
mice included in the curative assay (figure 31). The differences were not
statistically significant.
Figure 35 illustrated the spontaneous release of some inflammatory cytokines
by
spleen cells collected at day 34 from mice included in the curative assay
(figure
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31). 2x105 cells were incubated in 0.2 ml of tissue culture medium during 96h
at
37 C, the supernatant were collected and the concentrations in IFN-y, IL-6 and
IL-17 were determined using the Multiplex BioRad assay. The differences
observed after PBS and EFD treatments were extremely significant (p<0.001) for
IL-17, not significant for IFN-y and IL-6.
Figure 36 up illustrated the concentration of NFKB measured as optical density
on 5pg of nuclear extracts of spleen cells collected at day 34 from mice
included
in the curative assay (figure 31). The differences observed after PBS and EFD
treatments were extremely significant (*** means p<0.001 with statistical
ANOVA
test).
Figure 36 down illustrated the concentration of PPAR7 measured as optical
density on 5pg of nuclear extract of spleen cells collected at day 34 from
mice
included in the curative assay (figure 31). The differences observed after PBS
and EFD treatments were extremely significant (*** means p<0.001 with
statistical ANOVA test).
Figure 37 illustrates the difference in body weight between EFD treated and
untreated mice receiving dextran sodium sulphate (DSS) three times in their
drinking water some days and EFD treatments after the first series of DSS.
This
model explored EFD curative and preventive treatments in chronic IBD. Mice
(C57B1/6 male mice) received 1.5% DSS in their drinking water during 7 days,
ordinary tap water during 8 days, 1.5% DSS in their drinking water during 5
days,
tap water during 10 days, 1.5% DSS during 5 days and tap water thereafter. A
group of mice were EFD treated: 100 pg given subcutaneously 9 days after the
first DSS feeding; or 1 mg per os at days 9, 10 and 11 after the first DSS
feeding.
They did not receive more EFD treatment. The weight of individual mouse was
checked each day, 5 days a week. The experiment ended at day 52. After EFD
treatment, a slight, significant, curative effect was observed on days 13, 15
with a
faster and stable recovery of weight loss.
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Figure 38 illustrates the colitis score performed on mice included in the
curative
and preventive assay (figure 37). Scores were checked according to previous
description (figure26). The clinical symptoms decreased in subcutaneously EFD
treated mice.
Figure 39 up illustrates the difference in length of colon, measured between
caecum and anus, at day 52 on mice included in the curative and preventive
assay (figure 37). The colons of EFD treated mice were grossly similar to
those of
PBS mice.
Figure 39 down illustrates the number of cells found in the mesenteric lymph
nodes collected at day 52 on mice included in the curative and preventive
assay
(figure 37). The number of cells were decreased only in the lymph nodes of EFD
treated subcutaneously (*** means p<0.001 with statistical ANOVA test).
Figure 40 illustrated the spleen weight and number of spleen cells at day 52
on
mice included in the curative and preventive assay (figure 37). The spleen
weight
and spleen cell number were decreased only in mice which have been
subcutaneously EFD treated (* means p<0.05 and *** means p<0.001 with
statistical ANOVA test).
Figure 41 illustrated the spontaneous release of some inflammatory cytokines
by
spleen cells collected at day 52 from mice included in the preventive and
curative
assay (figure 37). 2x105 cells were incubated in 0.2 ml of tissue culture
medium
during 96h at 37 C, the supernatant were collected and the concentrations in
IFN-y, IL-6 and IL-17 were determined using the Multiplex BioRad assay. The
differences observed after PBS and EFD treatments were extremely significant
(p
< 0.001) whatever the cytokine, but much marked for IL-17.
Figure 42 up illustrated the concentration of NFKB measured as optical density
on 5pg of nuclear extracts of spleen cells collected at day 52 from mice
included
in the curative and preventive assay (figure 37). The differences observed
after
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PBS and EFD treatments were extremely significant (*** means p<0.001 with
statistical ANOVA test).
Figure 42 down illustrated the concentration of PPARy measured as optical
density on 5pg of nuclear extract of spleen cells collected at day 52 from
mice
included in the curative and preventive assay (figure 37). The differences
observed after PBS and EFD treatments were extremely significant (*** means
p<0.001 with statistical ANOVA test).
Figure 43 illustrates the difference in body weight between EFD treated and
untreated mice receiving dextran sodium sulphate (DSS) three times in their
drinking water and EFD treatments after the last series of DSS. This model
explored EFD curative treatments in chronic IBD. Mice (C57B116 male mice)
received 1.5% DSS in their drinking water during 7 days, ordinary tap water
during 8 days, 1.5% DSS in their drinking water during 5 days, tap water
during
10 days, 1.5% DSS during 5 days and tap water thereafter. A group of mice were
EFD treated: 100 pg given subcutaneously 7 days after the last DSS feeding; or
1
mg per os at days 42, 43 and 45 after the first DSS feeding. The weight of
individual mouse was checked each day, 5 days a week. The experiment ended
at day 54. After subcutaneous EFD treatment, a slight, curative effect was
observed on day 54 with a recovery of weight loss.
Figure 44 illustrates the colitis score performed on mice included in the
curative
assay of chronic IBD (figure 43). Scores were checked according to previous
description (figure 26). The clinical symptoms decreased in subcutaneously EFD
treated mice.
Figure 45 up illustrates the difference in length of colon, measured between
caecum and anus, at day 54 on mice included in the curative assay of chronic
IBD (figure 43). The colons of EFD treated mice were grossly similar to those
of
PBS mice (* means p<0.05 with statistical ANOVA test).
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Figure 45 down illustrates the number of cells found in the mesenteric lymph
nodes collected at day 54 on mice included in the curative assay (figure 43).
The
number of cells were decreased only in the lymph nodes of EFD treated
subcutaneously (*** means p<0.001 with statistical ANOVA test).
Figure 46 illustrates the spleen weight and number of spleen cells at day 54
on
mice included in the curative assay of chronic IBD (figure 43). The spleen
weight
and spleen cell number were decreased only in mice which have been
subcutaneously EFD treated (* means p<0.05 and ** means p<0.01 with
statistical ANOVA test).
Figure 47 illustrates the spontaneous release of some inflammatory cytokines
by
spleen cells collected at day 54 from mice included in the curative assay of
chronic IBD (figure 43). 2x105 cells were incubated in 0.2 ml of tissue
culture
medium during 96h at 37 C, the supernatant were collected and the
concentrations in IFN-y, IL-6 and IL-17 were determined using the Multiplex
BioRad assay. The differences observed after PBS and EFD treatments were
extremely significant (p < 0.001) whatever the cytokine, but much marked for
IL-
17.
Figure 48 up illustrates the concentration of NFKB measured as optical density
on 5pg of nuclear extracts of spleen cells collected at day 54 from mice
included
in the curative assay of chronic IBD (figure 43). The differences observed
after
PBS and EFD treatments were significant or extremely significant (* means
p<0.05 and *** means p<0.001 with statistical ANOVA test).
Figure 48 down illustrates the concentration of PPARy measured as optical
density on 5pg of nuclear extract of spleen cells collected at day 54 from
mice
included in the curative assay of chronic (figure 43). The differences
observed
after PBS and EFD treatments were very or extremely significant (** means
p<0.01 and *** means p<0.001 with statistical ANOVA test).
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
5
The expression "killed or non infectious Gram positive bacteria
preparation" as used in the context of the present invention refers to a
preparation of killed or non infectious Gram positive bacteria as described in
W003049752. This Gram positive bacteria preparation contains killed or non
10 infectious Gram positive bacteria, obtainable by a process which does not
denature the structure of the molecules contained therein and particularly the
proteins contained therein. Advantageously, this Gram positive bacteria
preparation contains extended-freeze-dried killed bacteria and less than 1.5 %
of
residual water, preferably less than 1%, more preferably less than 0.5 %.
These
15 extended-freeze-dried killed bacteria are prepared by harvesting a culture
of live
bacteria cells, washing the bacteria cells in water or in an aqueous solution
of a
salt such as borate, freezing the bacteria cells in water or in an aqueous
solution
of salt such as borate, killing the frozen bacteria cells by drying them in a
freeze-
dryer, for a time sufficient to remove at least 98.5% of the water, preferably
at
least 99% of the water, more preferably at least 99.5% of the water, and
collecting the extended-freeze-dried killed bacteria cells.
A fraction of this extended-freeze-dried killed bacteria preparation is
covered by the expression "Gram positive bacteria preparation" of the
invention.
This Fraction is selected in the group consisting of : a fraction consisting
of an
organic solvent extract of said killed bacterial preparation, a fraction
consisting of
a glycosidase-treated extract of said killed bacterial preparation, a fraction
consisting of a DNAse and/or RNase-digested extract of said killed bacterial
preparation and a fraction consisting of said killed bacterial preparation
successively treated by an organic solvent, a glycosidase, a DNase and/or
RNase, and finally a protease.
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The expression "intestinal inflammatory syndromes" as used in the context
of the present invention relates to any inflammatory bowel diseases including
the
two entities Crohn's disease and ulcerative colitis and an intermediate
variant of
these diseases, indeterminate colitis which shows overlapping features of the
two
diseases previously enumerated.
Use of the Gram positive bacteria preparation
The invention also relates to the use of a Gram positive bacteria
preparation for the prevention and treatment of intestinal inflammatory
syndromes, the preparation being characterized in that the Gram positive
bacteria
are killed or non infectious, and containing more than 50 %, and
preferentially
more than 90 %, of the bacterial protein components which are in a native
structure. The Gram positive bacteria preparation of the invention may also be
useful for the preparation of a medicament for the prevention and/or treatment
of
intestinal inflammatory syndromes.
According to a preferred embodiment of the present invention, the Gram
positive bacteria preparation is a Gram positive facultative intracellular
bacteria.
Gram positive facultative intracellular bacteria means Gram positive bacteria
with
a capacity of growing in synthetic medium in vitro as well as of infecting
eucaryotic cells from a mammalian or non-mammalian host, in vivo and
multiplying in those cells, for example, macrophages.
According to another preferred embodiment, the bacterial preparation
contains Gram positive facultative intracellular bacteria chosen from the
group
consisting of Listeria sp., Corynobacterium sp. and Actinomycetes comprising
Mycobacteria sp., Nocardia sp. and Rhodococcus sp.
More preferably, the bacterial preparation contains Mycobacterium bovis
and even more preferably, Mycobacterium bovis BCG.
The instant invention also relates to the use of the killed or non infectious
bacteria preparation or fractions thereof for the preparation of a medicament
for
the prevention and/or treatment of a disease comprising an immune
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dysregulation such as a Th1/Th2 imbalance. According to a preferred
embodiment of the invention, the disease is Crohn's disease or ulcerative
colitis.
The killed bacteria preparation or fractions thereof may be associated with
a pharmaceutically acceptable carrier, and/or an immunostimulant, and/or an
adjuvant and/or any conventional additives as defined herein below. The Gram
positive bacteria preparation and/or the medicament of the invention may be
administered by the oral, sublingual, parenteral or intranasal route.
Pharmaceutical compositions
As mentioned herein above, the present invention relates to the use of a
killed or non infectious Gram positive bacteria preparation for the
preparation of a
pharmaceutical composition for the prevention and/or treatment of intestinal
inflammatory disorders.
According to an embodiment of the invention, such a composition is
obtained by:
a) harvesting a culture of live bacteria cells,
b) washing the bacteria cells in water or in an aqueous solution of a
salt such as borate,
c) freezing the bacteria cells in water or in an aqueous solution of a
salt such as borate,
d) killing the frozen bacteria cells by drying them in a freeze-dryer, for
a time sufficient to remove at least 98.5 % of the water, preferably
at least 99 % of the water, more preferably at least 99.5 % of the
water, and
e) collecting the extended freeze-dried bacteria cells.
The composition of the present invention is preferably in a form suitable for
oral administration. For example, the composition may be in the form of
tablets,
ordinary capsules, gelatine capsules or syrup for oral administration. These
gelatine capsules, ordinary capsules and tablet forms can contain excipients
CA 02571886 2006-12-21
18
conventionally used in pharmaceutical formulations such as adjuvants or
binders
like starches, gums and gelatine, adjuvants like calcium phosphate,
disintegrating
agents like corn starch or algenic acids, a lubricant like magnesium stearate,
sweeteners or flavourings. Solutions or suspensions can be prepared in aqueous
or non-aqueous media by the addition of pharmacologically compatible solvents.
These include glycols, polyglycols, propylene glycols, polyglycol ether, DMSO
and ethanol.
According to another preferred embodiment, the composition of the
present invention is preferably in a form suitable for parenteral
administration,
such as subcutaneous injection. For parenteral administration, such as
subcutaneous injection, the carrier preferably comprises water, saline buffer,
lactose, glutamate, a fat or a wax. For oral administration, any of the above
carriers or a solid carrier such as mannitol, lactose, starch, magnesium
stearate,
sodium saccharine, talcum, cellulose, glucose, sucrose and magnesium
carbonate may be employed. Biodegradable microspheres (e.g. polylactic
galactide) may also be employed as carriers for the pharmaceutical
compositions
of this invention. Suitable biodegradable microspheres are disclosed for
example
in US patents 4,897,268 and 5,075,109.
The compositions of the invention may additionally contain an additive
and/or an immunostimulant and/or an adjuvant such as a liposome containing the
bacteria cells or fractions thereof according to the present invention. The
additives used for preparing the pharmaceutical composition of the present
invention may be chosen among anti-aggregating agents, antioxidants, dyes,
flavour enhancers, or smoothing, assembling or isolating agents, and in
general
among any excipient conventionally used in the pharmaceutical industry.
Any of the variety of adjuvants may be employed in the compositions of the
present invention to enhance the immune response. Most adjuvants contain a
substance designed to protect the antigen from rapid catabolism or to create
controlled inflammatory reactions, such as aluminium hydroxide or mineral oil,
and a non-specific stimulator of immune response such as lipid A, Bordetella
pertussis toxin. Suitable adjuvants are commercially available as well, for
CA 02571886 2006-12-21
19
example, Freund's incomplete adjuvant and Freund's complete adjuvant which
cannot be used for injection in human. Other suitable adjuvants which can be
used in human include aluminium hydroxide, biodegradable microspheres,
monophospheryl A and Quil A.
Methods of prevention or treatment
According to an embodiment of the present invention, the killed or non
infectious Gram positive bacteria preparation is used for the prevention and
treatment of intestinal inflammatory disorders selected from the group
consisting
of Crohn's disease and ulcerative colitis. The method of the invention
comprises
the step of administering to a patient an effective amount of the killed or
non
infectious Gram positive bacteria preparation, to stimulate the production of
leukocytic regulatory cells, such as CD4+, CD25+, T cells, B cells and/or
dendritic
cells.
According to another embodiment of the present invention, there is
provided a method for preventing or treating a disease caused by a Thl/Th2
imbalance. The method comprises the steps of a) providing a killed or non
infectious Gram positive bacteria preparation or a portion thereof retaining
the
capacity of inhibiting intestinal inflammatory syndromes or a pharmaceutical
composition of the invention and b) administering an effective amount of the
Gram positive bacteria preparation to a patient affected by the disease.
As mentioned above, the intestinal inflammatory syndrome may be
Crohn's disease or ulcerative colitis.
The amount of Gram positive bacteria preparation present in the
compositions of the present invention is preferably a therapeutically
effective
amount. A therapeutically effective amount of Gram positive bacteria
preparation
is that amount necessary so that the Gram positive bacteria preparation
performs
its role of inhibiting intestinal inflammatory syndrome without causing,
overly
negative effects in the host to which the composition is administered. The
exact
amount of Gram positive bacteria preparation to be used and the composition to
CA 02571886 2006-12-21
be administered will vary according to factors such as the type of intestinal
inflammatory syndrome being treated, the mode of administration, as well as
the
other ingredients in the composition. Preferably, the composition is composed
of
from about 10 pg to about 10 mg and more preferably from about 100 g to about
5 1 mg, of killed or non infectious Gram positive bacteria preparation. By
"about", it
is meant that the value of said quantity (pg or mg) of killed or non
infectious Gram
positive bacteria preparation can vary within a certain range depending on the
margin of error of the method used to evaluate such quantity.
For instance, during an oral administration of the composition of the
10 invention, host to be treated could be subjected to a 1 dose schedule of
from
about 10 g to about 10 mg of killed or non infectious Gram positive bacteria
preparation per day during 3 consecutive days. The treatment may be repeated
once one week later.
For parenteral administration, such as subcutaneous injection, the host to
15 be treated could be subjected to a 1 dose schedule of from about 10 g to
about
10 mg and more preferably from about 100 g to about 1 mg, of killed or non
infectious Gram positive bacteria preparation per month or every 6 months.
20 Method of preparation of the Gram positive bacterial preparation
For instance, the Gram positive bacteria of the present invention may be
killed by "soft methods" which do not denature the molecules from the bacteria
cells and thus are able to stimulate leukocyte regulatory cells (CD4+, CD25+,
T
cells and/or B cells and/or dendritic cells) in vivo when they are
administered to
subjects suffering from immune dysregulation. Thus, the Gram positive bacteria
preparation according to the present invention may consist of heat-killed
preparations of Mycobacteria. According to a preferred embodiment of the
present invention, the Gram positive bacteria are killed by lyophilization.
These
processes which are denominated soft processes include with no limitation the
use of physical means which disrupt the bacteria cell membranes while
CA 02571886 2006-12-21
21
preserving the structure of its macromolecular components. These processes
include with no limitation: extended freeze-drying, grinding in the presence
of
silica or zirconium beads, use of a so-called "French press", sonification and
gamma-rays irradiation. Other processes which may be used for obtaining the
killed bacteria preparation as defined above are known to those of ordinary
skill in
the art.
A process which does not denature the structure of the molecules from the
bacteria cells means a process which results in no extensive denaturation of a
configuration of the molecules. Preferably, such process preserves the three
dimensional structure of the micromolecules from the bacteria cells such as
proteins, polysaccharides and lipids.
EXAMPLES
General information
To test the capacity of EFD to decrease inflammatory reactions in IBD, two
mouse models of IBD were explored :
- IBD secondary to TNBS local sensitisation
- IBD secondary to DSS feeding.
IBD secondary to TNBS local sensitisation
TNBS (2,4,6-trinitrobenzene sulfonic acid) is a chemical containing a phenol
derivative which creates a "pure" T-lymphocyte dependent sensitization. The
sensitization is performed by local delivery into the colon of a TNBS solution
through a small tube inserted in the anal canal. Some days later the local
delivery
of the same chemical creates local inflammatory lesions with ulcerative
aspects
due to the local T-lymphocyte response.
The advantage of this model is to explore T-lymphocyte dependent
inflammatory reactions.
The disadvantages of this modei are the following :
CA 02571886 2006-12-21
22
- each mouse has to be anesthetised twice, to be carefully handle to
introduce a tiny tube into the anus
- TNBS is a potent sensitizer for human beings. The risks to sensitize
laboratory workers are high, knowing that equivalent phenol derivatives are
frequent in our environment. It has to be handle with high precocious.
IBD secondary to DSS feeding
DSS (Dextran sodium sulphate) is a chemical which creates local intestinal
lesions in mice when ingested. Its delivery via drinking water is easy. It is
a less
potent sensitizer than TNBS, with fewer risks for the laboratory workers. The
mechanisms of lesions are partly understood, the absence of DSS-induced
lesions in germ-free mice excludes a direct, single, immunological mechanism.
The necessity for the presence of intestinal bacteria being also observed in
the
Crohn's disease in human, DSS model was proposed by some researchers to be
closer than TNBS model to human diseases.
Example I. Preventive model of DSS induced IBD (short term effect)
Protocols
Protocol 1
C57BI/6 mice, 6 to 7 weeks old, were distributed in 4 groups of 10 mice :
= Mice from Group 1 did not receive anything; they composed the control
group.
= Mice from Group 2 received 100p1 of isotonic saline solution
subcutaneously. Their drinking water was replaced 21 days later by a
2.5% DSS solution in water for 7 days.
= Mice from Group 3 were fed 1 mg of EFD on days 1 and 2, and again were
fed 1 mg EFD on days 8 and 9. Their drinking water was replaced by the
DSS solution on day 21 for 7 days.
CA 02571886 2006-12-21
23
= Mice from Group 4 received lOOpg EFD in lOOpI saline solution
subcutaneously at the base of the tail on day 1. Their drinking water was
replaced by the DSS solution on day 21 for 7 days.
Day -21 : EFD lOOpg s.c.
or 1 mg per os x 4(days -21,-20, -14 and 13)-
DSS 2.5% Day 8 Day 10
0
All mice were killed at day 8 (5 mice) or day 10 (5 mice) to analyse early
pathological modifications: colon length, histological findings.
Protocol 2
This protocol is similar to protocol 1. C57BI/6 male mice (per group of 10
mice)
received or not 2.5% of dextran sodium sulfate (DSS) in their drinking water
during 5 days. This protocol was slightly lighter than the protocol 1, DSS
being
given during 5 days instead of 7 days. A group received EFD lOOpg
subcutaneously 21 days before DSS. A group received PBS instead of EFD. The
last group, control, receive neither DSS nor EFD.
Day -21 : EFD lOOpg s.c.
DSS 2.5% Day 10
44~
0
Daily weighing and observation of the animals were made during the 8 or 10
days
after the beginning of DSS ingestion. A clinical score summarizing the
intensity of
the observed signs was established:
= 0, normal mouse
0 1, inflammation of the anus
CA 02571886 2006-12-21
24
= 2, inflammation of the anus and soft stools
= 3, inflammation of the anus and diarrhea
= 4, inflammation of the anus and bloody diarrhea
On the 8th day after the beginning of DSS ingestion, 4 animals of each group
were sacrificed, their colon was collected and measured (length between the
anus and the caecum), placed in Tissue-Tek O.C.T. (Sakura Finetek) and frozen
for histological examination. The surviving mice were sacrificed on day 10.
Samples on colonic tissues were collected on day 10 in order to determine
their
content in different cytokines/lymphokines using a Multiplex kit (BioRad).
Results
1. Weight curves and clinical appearance of mice
The mice not treated with EFD receiving the dextran sodium sulphate
solution as drinking water, Group 2, lose weight rapidly (Figure 1 left side).
The
vivaciousness of the animals is very reduced on the 6th or 7th day of this
ingestion; they do not recuperate at all or do not recuperate well 24 or 72
hours
after stopping DSS ingestion. 3 mice died on day 8.
The mice treated with EFD, per os or subcutaneously, from Groups 3 and
4 appear sick, but in a less important manner; they remain vivacious and
appear
susceptible to recuperation. There is no mortality among those groups.
The group of mice treated with EFD according to protocol 2 showed a similar
weight curve as the group of control mice (Figure 16a), demonstrating that the
pretreatment with EFD decreases IBD symptoms.
CA 02571886 2006-12-21
2. Clinical scores
The mice that were not treated with EFD present an elevated clinical
score (inflammation and diarrhea) after 4 or 5 days of ingestion of DSS. This
5 score becomes increasingly elevated after 7 to 8 days (Figure 1 right side).
The mice treated with EFD present identical clinical scores when
compared to each other, scores less elevated than those observed for the non-
treated animals on days 4 and 5 after the beginning of DSS ingestion. This
score
regresses, and is then normalized. The mice having been treated with EFD do
10 not present inflammation of the anus, nor diarrhea, on days 7 and 8 (Figure
6).
Mice having been treated with EFD according to protocol 2 showed clinical
scores similar to the ones of control mice (Figure 16b).
3. Inflammation of colon
a) Macroscopic examination
The animals were sacrificed 8 or 10 days after the beginning of DSS
ingestion, either 1 or 3 days after stopping such ingestion. No difference was
observed depending on the date of autopsy. The colon of the control animals
contained many feces which became increasingly harder with their migration
towards the anus. The average length of the colon is 6.7 cm (Figure 5). The
length of the colon of the animals having received DSS is reduced to
approximately 3.8 cm (Figures 2 and 5). The stools are soft and the colon very
often hemorrhagic in DSS treated mice.
The colon of animals having been treated with EFD contained feces that
were more or less hard, of identical appearance to those of the control
animals
(Figure 4). The average length of the colon is of 5.8 cm (Figure 5). No
significant
difference was observed in the effect of EFD treatments according to the mode
of
administration (Figure 5).
CA 02571886 2006-12-21
26
The mice having been treated with EFD in accordance with the protocol 2
showed colons and caecum of lengths similar to those of control mice never fed
with DSS (Figure 17).
b) Microscopic examination
The microscopic examination of the lesions after the administration of
DSS shows intense inflammatory responses at the level of the intestinal
mucosa,
an oedema of the sub-mucosa, important at the level of the insertion of the
mesentery, associated to a thickening of the intestinal wall. The intestinal
villi are
dissociated and partially unstructured (Figures 9 and 10 versus Figures 7 and
8
for control, Figures 13 and 14).
In the animals that were treated with EFD before the ingestion of DSS,
the lesions are much less important (Figures 11 and 12 versus Figures 7 and 8
for control). The intestinal villi have a morphology close to that observed in
the
control animals (Figures 13 and 14).
C) Inflammatory regulators present in colonic tissues and spleen
Dosage of IL-12p40, IL-12p70, RANTES, lL-1 beta, TNF alpha, MIP-1
alpha, IL-17, IFN gamma, IL-10, KC, IL-6, IL-1 alpha, IL-3, GM-CSF, and G-CSF.
All cytokines and lymphokines were measured using Bioplex method according to
manufacturer recommendations (Bio-Rad).
Dosage of T-bet and GATA-3 proteins in spleen.
Total proteins extracted from the spleen cells were resolved on 7.5% SDS-PAGE,
then protein bands transferred to nitrocellulose sheets were probed with
polyclonal rabbit anti-mouse FOXP3 IgG kindly provided by E. Schmitt and C.
Richter (Institute of Immunology, Mainz, Germany) with mouse monoclonal anti-
T-bet, mouse monoclonal anti-GATA-3 (Santa Cruz Biotechnology, Santa Cruz,
CA) or R-actin mouse monoclonal Ab (Ac-15 Abcam, Cambridge, UK). As
CA 02571886 2006-12-21
27
secondary Ab, we used HRP-labeled polyclonal goat anti rabbit (Dako
Cytomation, Denmark). The immune complex was revealed by enhanced
chemiluminescence detection system (Amersham, France).
Inflammatory cytokines and lymphokines are present in high
concentration in the colonic samples collected from mice having DSS in their
drinking water. These molecules are the origin or the consequence of the
important pathological changes observed in the mice. In the animals that were
treated subcutaneously with EFD before the ingestion of DSS, the
concentrations
of these molecules were in the range observed for control animals receiving
tap
water without DSS (see Figures 19 - 24).
d) Mesenteric lymph nodes cell number
An increase of the size of mesenteric lymph nodes is commonly
associated with colonic disease and more particularly with inflammatory bowel
disease.
At day 10 post beginning of DSS feeding, mesenteric lymph nodes of
mice from protocol 2 were collected, crushed on cell-strainers (Falcon). The
dissociated cells were washed in AIM V medium (Gibco) supplemented with 5%
FCS, centrifuged and the pelleted cells resuspended in 0.5 or 1 ml of medium.
Cells were diluted 10 fold in trypan blue (0.1% in PBS) and counted under
microscope in a Malassez cell. An increased number of cells (x 2.5) is
observed
for mice PBS-treated and feeded with DSS in comparison with mice control. EFD
preventive treatment permits to significantly reduce this number to the value
of
control mice (Figure 18).
Example II. Preventive model of DSS induced chronic IBD (long term effect)
This model explored EFD preventive treatments in chronic IBD.
CA 02571886 2006-12-21
28
Protocol
C57B1/6 mice, 6 to 7 weeks old, were distributed in 4 groups of 10 mice.
Mice (C57B1/6 male mice) received 1.5% DSS in their drinking water during 7
days, ordinary tap water during 8 days, 1.5% DSS in their drinking water
during 5
days, tap water during 10 days, 1.5% DSS during 5 days and tap water
thereafter. A group of mice were EFD treated: 100 pg given subcutaneously 21
days before the first day of DSS feeding; or 1 mg per os at days 23, 22 and 21
before the first DSS feeding. They did not receive more EFD treatment. The
experiment ended at day 52.
Cl:+,'-21. EFD 100Eag s-.c.
ar1 rrig pEro5;,3
DSS 1.5'.:-b C?SS 1 .573 E?J'S 1 uGa;1 5 2
I i ilr T ~,r
!!l111
oT '~ 16 ** 30
Results
Weight curves
The mice treated with EFD, per os or subcutaneously, appear to lose
weight and gain weight again in a similar manner than mice not treated with
EFD
and feeded with DSS but at the end of the experiment (day 50) after the three
series of DSS ingestion, the preventive treatment permits to mice to have a
weight very close to the one of mice control (Figure 25).
Clinical scores
According to Figure 26, at the end of the experiment (day 50), mice not
treated with EFD present a clinical score arising continuously after the end
of
DSS feeding. The EFD pretreatment permits mice to have a normalized clinical
score at the end of experiment, when the mice not treated with EFD see their
clinical scores continuously arise even after the three IBD simulations.
CA 02571886 2006-12-21
29
Inflammation of colon
At the end of experiment (day 52), the colons of EFD treated mice were
similar to those of naive mice never fed with DSS (Figure 27 up).
Lymph node and spleen
At the end of experiment (day 52), the number of cells in the lymph nodes
of EFD treated mice were decreased compared to those of PBS treated mice and
can be expected to revert progressively to the cell number of control mice
never
fed with DSS (Figure 27 down).
As illustrated in Figure 28, the difference in spleen weight and number of
spleen cells at day 52 on mice included in this preventive assay are not
statistically different.
Inflammatory regulators release from spleen
The spontaneous release of some inflammatory cytokines by spleen cells
collected at the end of the experiment (day 52) was studied. The differences
observed after PBS and EFD pretreatments were extremely significant whatever
the cytokine (IFN gamma, IL-6 and IL-17) but much marked for IL-17. EFD
pretreatment permits after chronic IBD to keep levels of cytokines release
similar
to the ones of naive mice never fed with DSS (Figure 29).
The nuclear factor-kappaB (NF-KB) is known to be activated in
inflammatory bowel diseases and up-regulate inflammatory cytokines. As
illustrated in Figure 30 up, the high concentration of NF-KB provoked by
recurrent DSS treatments of mice is significantly decreased by EFD
pretreatment.
The peroxisome proliferator-activated receptor-y (PPAR-y), a member of
the nuclear receptor superfamily of ligand-dependent transcription factors
predominantly expressed in adipose tissue, adrenal gland and spleen is
involved
in the regulation of numerous inflammatory responses and in particularly of
intestinal inflammation. It has been proposed as a key inhibitor of colitis
though
attenuation of nuclear factor kappa B (NF-KB) activity. Levels of PPAR-y in
spleen
CA 02571886 2006-12-21
are decreased during chronic inflammation as illustrated by Figure 30 down.
The
preventive treatment of mice with EFD permits to increase the level of PPAR-y
in
chronic IBD model at and beyond the level of naive mice (without chronic IBD).
5
Example III. Curative model of DSS induced IBD (long term effect)
This model explored EFD curative treatment in acute IBD.
10 Protocol
C57B1/6 mice, 6 to 7 weeks old, were distributed in 4 groups of 10 mice.
Mice (C57BI/6 male mice) received 2.5% DSS in their drinking water during 5
days, and tap water thereafter. A group of mice were EFD treated: 100 pg given
subcutaneously 24 hours after the last day of DSS feeding (i.e at day 6); or 1
mg
15 per os at days 6, 7, and 8 after the 1 St day of DSS feeding. They did not
receive
more EFD treatment. The experiment ended at day 34.
C+-ay 6. EFD 100pg s.c.
or 1 mg I?er- c-s x 3
C. IS .~,; ~
~ ~, . ,.~
TI,111 T
0
20 Results
Weight curves
In this model of curative treatment in acute DSS-induced IBD, the weight of
individual mouse was checked each day, 5 days a week from the first day of DSS
ingestion and during 35 days after. After treatment with EFD, a slight,
significant,
25 curative effect was observed on days 13 and 15 with a faster recovery of
weight
loss (Figure 31).
CA 02571886 2006-12-21
, ' .
31
Clinical scores
Scores were checked according to previous description (see Example I). The
clinical symptoms decreased faster in EFD treated mice than in PBS treated
mice
(Figure 32).
Inflammation of colon
At the end of experiment (day 34), the colons of EFD treated mice were similar
to
those of naive mice never fed with DSS (Figure 33 up).
Lymph node and spleen
At the end of experiment (day 34), the number of cells in the lymph nodes of
EFD
treated mice were decreased compared to those of PBS treated mice, the
difference being statistically different (Figure 33 down). As illustrated in
Figure
34, the differences in spleen weight and number of spleen cells at day 34 are
not
statistically significant.
Inflammatory regulators release from spleen
The spontaneous release of some inflammatory cytokines by spleen cells
collected at the end of the experiment (day 34) was studied. The differences
observed between PBS and EFD treatments were extremely significant for IL-17,
not for IFN-y and IL-6 (Figure 35).
As illustrated in Figure 36 up, a high concentration of NF-KB is maintained in
spleens of PBS treated mice a month later after the end of DSS ingestion. At
the
same time, EFD treatment permits to have NF-KB concentrations significantly
lower than without EFD treatment and similar to the ones of naive mice never
fed
with DSS.
At the end of experiment, levels of PPAR-y observed in spleens of PBS and EFD
treated mice are extremely different as illustrated by Figure 36 down.
CA 02571886 2006-12-21
32
Example IV. Curative and preventive model of DSS induced chronic IBD
(long term effect) (figures 37- 42)
This model explored EFD curative and preventive treatments in chronic IBD.
Protocol
C57B1/6 mice, 6 to 7 weeks old, were distributed in 4 groups of 10 mice.
Mice (C57BI/6 male mice) received 1.5% DSS in their drinking water during 7
days, ordinary tap water during 8 days, 1.5% DSS in their drinking water
during 5
days, tap water during 10 days, 1.5% DSS during 5 days and tap water
thereafter. A group of mice were EFD treated: 100 pg given subcutaneously 9
days after the first DSS feeding; or 1 mg per os at days 9, 10 and 11 after
the first
DSS feeding. They did not receive more EFD treatment. The experiment ended
at day 52.
Day 9: EFD 10Opg S.C.
:cir 1 rng per os
D ~ G..~~.~.~ U ' ~ L~~~ _, EZ
~' DSS 1.~, .~~ ~ ~1.~,~~~ ~ ~'' ~ ~
'If
0 16 30
Results
Weight curves
In this model of curative/preventive treatment in chronic DSS-induced IBD, the
weight of individual mouse was checked each day, 5 days a week from the first
day of DSS ingestion and during 52 days after. After treatment with EFD, a
slight,
significant, curative effect was observed on days 13 and 15 with a faster and
stable recovery of weight loss (Figure 37).
CA 02571886 2006-12-21
33
Clinical scores
Score were checked according to previous description (see Example I). The
clinical symptoms decreased in subcutaneously EFD treated mice compared to in
PBS treated mice (Figure 38).
Inflammation of colon
At the end of experiment (day 52), the colons of EFD treated mice were grossly
similar to those of PBS treated mice (Figure 39 up).
Lymph node and spleen
At the end of experiment (day 52), the number of cells were decreased only in
the
lymph nodes of subcutaneously EFD treated mice, the difference with PBS
treated mice being extremely significant (Figure 39 down). Similarly, spleen
weight and spleen cell number were decreased only in mice which have been
subcutaneously EFD treated (Figure 40).
Inflammatory regulators release from spleen
The spontaneous release of some inflammatory cytokines by spleen cells
collected at the end of the experiment (day 52) was studied. The differences
observed between PBS and EFD treatments were extremely significant, whatever
the cytokine, but much marked for IL-17 (Figure 41).
As illustrated in Figure 42 up, after chronic IBD high concentration of NF-KB
were
observed in spleens of PBS treated mice. NF-KB concentrations in spleen of EFD
treated mice were lower than the ones in spleen of PBS treated mice, the
differences being extremely significant.
After chronic 1BD, levels of PPAR-y observed in spleens of PBS and EFD treated
mice were very different, the differences being extremely significant (Figure
42
down).
CA 02571886 2006-12-21
34
Example V. Curative model of DSS induced chronic IBD (short term effect)
(figures 43-48)
This model explored EFD curative treatments in chronic IBD.
Protocol
C57B1/6 mice, 6 to 7 weeks old, were distributed in 4 groups of 10 mice.
Mice (C57B1/6 male mice) received 1.5% DSS in their drinking water during 7
days, ordinary tap water during 8 days, 1.5% DSS in their drinking water
during 5
days, tap water during 10 days, 1.5% DSS during 5 days and tap water
thereafter. A group of mice were EFD treated: 100 pg given subcutaneously 7
days after the last DSS feeding; or 1 mg per os at days 42, 43 and 45 after
the
first DSS feeding. The experiment ended at day 54.
Day 42: EFD 100{.ig s.c.
or 1 mg per os x 3
DSS 1.5% DSS 1.5% DSS 1.5% Day 54
S1..r :i +I v V" 41% "}}TI ,'~~f S 1,
0 Tj
Results
Weight curves
In this model of curative treatment in chronic DSS-induced IBD, the weight of
individual mouse was checked each day, 5 days a week from the first day of DSS
ingestion and during 54 days after. After subcutaneous EFD treatment, a
slight,
curative effect was observed on day 54 with a recovery of weight loss (Figure
43).
Clinical scores
Score were checked according to previous description (see Example I). After
only
subcutaneous EFD treatment, the clinical symptoms decreased and get close to
the normality (Figure 44).
CA 02571886 2006-12-21
Inflammation of colon
At the end of experiment (day 54), the colons of EFD treated mice were grossly
similar to those of PBS treated mice (Figure 45 up).
5
Lymph node and spleen
At the end of experiment (day 54), the number of cells were decreased only in
the
lymph nodes of subcutaneously EFD treated mice, the difference observed
between EFD treated mice and PBS treated mice being extremely significant
10 (Figure 45 down). Similarly, spleen weight and spleen cell number were
decreased only in mice which have been subcutaneously EFD treated (Figure
46).
Inflammatory regulators release from spleen
The spontaneous release of some inflammatory cytokines by spleen cells
15 collected at the end of the experiment (day 54) was studied. The
differences
observed between PBS and EFD treatments were extremely significant, whatever
the cytokine, but much marked for IL-17 (Figure 47).
As illustrated in Figure 48 up, after chronic IBD NF-KB concentrations in
spleen
of EFD treated mice were lower than the ones in spleen of PBS treated mice,
the
20 differences being very (subcutaneous treatment) or extremely (per os
treatment)
significant.
After chronic IBD, concentrations of PPAR-y observed in spleens of PBS and
EFD treated mice showed an extremely significant difference (Figure 48 down).
EFD treatment, whatever the mode of administration, permits to have a PPAR-y
25 concentration similar or lightly higher to the one of naive mice.
CA 02571886 2006-12-21
36
Example VI. Preventive model of TNBS induced IBD (short term effect)
Protocol
C57BI/6 male mice received or not TNBS (1 mg of 2,4,6-trinitrobenzene sulfonic
acid (TNBS) in 100 pl of ethyl alcohol at 50% twice, locally in colon at day -
5 and
0. Their stools were observed during 10 days. At day 10, the mice were
weighted.
A group of mice received EFD 100 pg subcutaneously 5 days before the first
TNBS delivery (i.e. at day -10). A second group received PBS. The control
group
did not receive TNBS neither EFD.
Day - 10: EFD lOOpg s.c.
TNBS 10g/I Day 10
W W W
-5 0
Results
Weighing of the animals was made at day 0 and at day 10 after the beginning of
TNBS treatment. Mice which have been received TNBS lost approximately 2
grams, when naive mice not treated with TNBS gained more than 1 gram and
mice which have received EFD before TNBS had the same weight than at the
beginning of experiment (Figure 15).
CA 02571886 2006-12-21
37
Conclusion of Examples
DSS models
Preventive models : EFD was given (subcutaneously or per os) 21 days before
DSS feeding in an acute model (DSS feeding during 5 or 7 days - Example I)
and in a chronic model (DSS feeding during 5 days, three times separated by 5
days- Example II). The protective effect was observed on clinical symptoms
(body weight, aspect of stools), histopathological findings,
cytokine/lymphokine
concentrations in colonic tissues and spleen, levels of transcriptional
factors
NFKB and PPARy in spleen.
Curative acute model : EFD was given (subcutaneously or per os) 24 hours
after feeding with DSS during 5 days (Example III), In the EFD treated group
the
recovery of body weight was faster, the symptoms were less marked, the
biological markers were closer to normal values than in the PBS treated group
of
mice at the end of experiment, one month later.
Curative and preventive chronic model : EFD was given (subcutaneously or
per os) 48 hours after feeding with DSS during 7 days, 2 series of 5 days of
DSS
feeding were delivered before the end of experiment at day 52 (Example IV). In
the EFD treated group the biological markers were closer to normal values than
in the PBS treated group of mice, the effect on clinical findings was present
but
less marked.
Curative chronic model : EFD was given (subcutaneously or per os) 48 hours
after a series of DSS feedings (DSS during 7 days and 2 series of 5 days of
DSS
feeding) (Example V). At the end of experiment at day 54, in the EFD treated
group the biological markers were closer to normal values than in the PBS
treated group of mice.
CA 02571886 2006-12-21
38
TNBS model
The experiment performed with TNBS included few mice with limited
explorations. The decrease in inflammatory reactions observed after EFD
treatment indicated that EFD is able to decrease pure T-lymphocyte dependent
immune responses.
All these results indicate that EFD has potent anti-inflammatory activities in
different DSS induced IBD models. As a preventive treatment, it acts on
clinical
symptoms and biological parameters. As a curative treatment, its acts
essentially
on biological parameters. The effects on clinical symptoms were present but
less
marked in these drastic models of chronic IBD, knowing that the delay between
the treatment and the end of experiments (when measures are done) was short,
12 to 19 days.
The EFD efficacy was also observed in a TNBS induced IBD model (Example VI)
demonstrating its anti-inflammatory potencies in pure T-lymphocyte dependent
acquired immune responses.
In light of these examples, one skilled in the art may appreciate that the
inventors
have confirmed that the Gram positive bacteria preparation according to the
present invention is useful for the prevention and treatment of intestinal
inflammatory syndromes. Indeed, many cytokines (namely IFN gamma, IL-6 and
IL-17) as well as two transcription factors (NFkappaB and PPARgamma) which
are involved in the inflammatory response, have been assayed by the inventors.
The results shown in the Example Section demonstrate that the Gram positive
bacteria preparation according to the present invention has the original
property
of acting not only on the Th1 signalisation pathway to which TNF-alpha, IL-12,
IFN-gamma and T-bet are associated with, but also on the Th2 signalisation
pathway to which IL-4, IL-13 and GATA-3 are associated with, involving and on
a
CA 02571886 2006-12-21
39
new signalisation pathway to which IL-17 and PPAR-gamma seem to be
associated with (Young Y. Current Gastroenterolo Rep 2006 Dec; 8 (6) : 470-7).
