Note: Descriptions are shown in the official language in which they were submitted.
CA 02629057 2008-04-14
1
CONTROL OF DISEASES ASSOCIATED WITH DECREASE OF T-
REGULATORY CELLS WITH A PREPARATION OF EXTENDED FREEZE-
DRIED KILLED BACTERIA
Field of the invention
This invention relates to the use of a preparation of extended freeze-dried
(EFD)
killed Gram positive bacteria such as Gram positive facultative intracellular
bacteria, for example mycobacteria, for the treatment of T-regulatory cells
(Tregs)-associated diseases, such as multiple sclerosis (MS) and experimental
autoimmune encephalomyelitis (EAE).
Description of the prior art
Multiple sclerosis (commonly abbreviated as "MS") is a chronic neurological
disorder and is often incapacitating. Its clinical manifestations are linked
to a
demyelination of the nerve fibres of the white matter of the central nervous
system (brain, spinal cord and optical nerve) which is secondary to an
inflammation of autoimmune origin. Multiple sclerosis is a chronic disease,
which
is progressively incapacitating and which evolves in episodes. This syndrome,
(known as multiple sclerosis or MS) leads to a loss of motor or sensory
functions,
which are partially recovered after episodes, but unfortunately often
progressive.
The anti-inflammatory treatments tend to reduce the term or the intensity of
the
episodes, but are still very insufficient.
The patients are most often young adults (20 to 40 years old) and women, the
disease being observed at all ages. About 80,000 people are affected in France
(prevalence), with about 2,000 new cases a year (incidence).
The baseline treatments attempt to modify immune responses. The monoclonal
antibody, natalizumab, directed against the alpha chain of leucocyte integrins
inhibits the circulation of cells. Another baseline treatment is the anti-CD20
antibody, rituximab, an anti lymphocyte B antibody. Interferon 1-beta is also
CA 02629057 2008-04-14
2
administered by subcutaneous or intramuscular injection with the objective of
"modifying" the immune responses. A new product, FTY720, is undergoing phase
3 clinical trials. By sequestering lymphocytes in the sites of production, it
is
immunosuppressive and induces important lymphopenia which is apparently well
tolerated. Notable secondary effects have been reported: "A first undesired
effect
is the observation of a "reversible posterior leucoencephalitis" in a patient
treated
with FTY720. This syndrome is observed in cases of arterial hypertension or in
treatments with immunosuppressive agents. It is characterized by an edema of
the posterior portion of the brain and is well seen in an MRI. It is
associated to
confusion, visual disorders and headaches. While reversible if its cause is
rapidly
removed, it can otherwise evolve towards a definitive toxic edema. On one
hand,
FTY720 can cause arterial hypertension, especially at high doses. On the other
hand, it provokes an immunosuppressive effect. Finally, a case of a macular
(part
of the retina) edema was observed in a patient treated with FTY720 for a renal
transplant." (Comments from a newsletter of the Charcot Foundation).
Experimental autoimmune encephalomyelitis, sometimes Experimental
Allergic Encephalomyelitis (EAE) is an animal model of brain inflammation. It
is
an inflammatory demyelinating disease of the central nervous system (CNS). It
is
mostly used with rodents and is widely studied as an animal model of the human
CNS demyelinating diseases, including the diseases multiple sclerosis and
acute-
disseminated-encephalomyelitis. EAE can be induced by inoculation with whole
CNS tissue, purified myelin basic protein (MBP) or myelin proteolipid protein
(PLP), together with adjuvants. It may also be induced by the passive transfer
of
T cells specifically reactive to these myelin antigens. EAE may have either an
acute or a chronic relapsing course. Acute EAE closely resembles the human
disease acute disseminated encephalomyelitis, while chronic relapsing EAE
resembles multiple sclerosis. EAE is also the prototype for T-cell-mediated
autoimmune disease in general.
The role of T regulatory cells (Treg) in the control of autoagressive immune
responses is the subject of intense investigations. A review article, entitled
:
CA 02629057 2008-04-14
3
"Foxp3+ regulatory Tcells in the control of experimental CNS autoimmune
disease" summarizes our knowledge on the role of these cells to decrease
inflammatory reactions during multiple sclerosis (MS) and during experimental
autoimmune encephalomyelitis (EAE). According to these views the Tregs are
less abundant in tissues during relapsing periods, while Tregs were
accumulated
in the CNS during the recovery phases of EAE (O'Connor R.A. and Anderson
S.M. (2008) Journal of Neuroimmunology 193: 1-11)
Furthermore two recent papers report that the role of pertussis toxin (PTx),
injected with encephalitogen peptide (MOG35_55) and 48h later to enhance the
severity of EAE, is to reduce the number and function of Tregs (Chen X. et al.
(2006) Eur. J. lmmunol. 36 : 671-680 ; Chen X. et al. (2006) Eur. J. Immunol.
36:
671-680).
Summary of the invention
An object of the present invention is to provide a treatment for T-regulatory
cells (Tregs)-associated diseases, and more particularly for CNS inflammatory
diseases such as multiple sclerosis (MS) or experimental autoimmune
encephalomyelitis (EAE).
Accordingly, the present invention provides the use of a Gram positive
bacteria preparation for the prevention and treatment of a Tregs-associated
disease, the preparation being characterized in that the Gram positive
bacteria
are killed by extended freeze-drying (EFD).
The present invention still provides a method for preventing or treating a
Tregs-associated disease, the method comprising the step of administering to a
patient an effective amount of an extended freeze-dried (EFD) killed Gram
positive bacteria preparation, thereby stimulating the production of Tregs.
The present invention further provides a preparation of Mycobacterium
bovis BCG killed by extended freeze drying for treating a Tregs-associated
disease. This preparation is preferably obtained by a process comprising the
following steps:
CA 02629057 2008-04-14
4
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 present invention also provides a product comprising a preparation as
claimed in anyone of claims 19 to 24 and at least a drug selected from the
group
consisting of anti-inflammatory and immunoregulatory drugs, as a combined
preparation for simultaneous, separate or sequential use in the prevention
andlor
treatment of a CNS inflammatory disease such as multiple sclerosis (MS) or
experimental autoimmune encephalomyelitis (EAE).
Brief description of the drawinas
Figure 1 illustrates a marked increase of dendritric cells expressing ICOS-L
after
EFD treatment.
Figure 2 illustrates a selective view of the dendritric cells expressing ICOS-
L of
Figure 1 after EFD treatment.
Figure 3 is a Western blot showing the marked expression of the Tregs specific
marker Foxp-3 in EFD treated mice.
Figure 4 is a panel of 4 Western blots showing that EFD treatment promotes the
differentiation or multiplication of naive Tcelis toward Tregs expressing
Foxp3
and increases Tbet expression, whereas it reduces RORyand GATA3 expression
in a mouse model of Crohn disease.
CA 02629057 2008-04-14
Figure 5 illustrates that EFD treatment induces the maturation of Tregs.
Figure 6 illustrates that EFD protects from excessive inflammation in asthma
mouse model.
Figure 7 illustrates prevention of EAE by EFD treatment.
5 Figure 8 illustrates prevention of EAE by EFD treatment.
Detailed description of the invention
The present invention relates to the use of a killed Gram positive bacteria
preparation in a pharmaceutical composition for the prevention and/or
treatment
of a Tregs-associated disease.
Use of the Gram positive bacteria preparation
One embodiment of the invention relates to the use of a Gram positive
bacteria preparation for the prevention and treatment of a Tregs-associated
disease, particularly a CNS inflammatory disease, and more particularly
multiple
sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE). The
preparation is characterized in that the Gram positive bacteria are killed by
Extended Freeze-Drying (EFD). Preferably the preparation contains more than
50 %, or 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 a Tregs-associated disease, particularly a CNS inflammatory disease and
more particularly MS or EAE.
The Gram positive bacteria preparation may be a Gram positive facultative
intracellular bacterium. 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.
CA 02629057 2008-04-14
6
The bacterial preparation may contain Gram positive facultative
intracellular bacteria chosen from the group consisting of Listeria sp.,
Corynobacterium sp. and Actinomycetes comprising Mycobacteria sp., and even
more preferably, e.g. Mycobacterium bovis BCG, Nocardia sp. and Rhodococcus
sp.
The Gram positive bacteria preparation according to the invention is
preferably a preparation of Mycobacterium bovis BCG killed by extended freeze
drying.
The expression "extended freeze-dried Mycobacterium bovis BCG
preparation" "EFD preparation" or "EFD" as used in the context of the present
invention may refer to killed Mycobacterium bovis BCG bacteria prepared
according to the method as described in the International PCT Application WO
03/049752, said method comprising the steps of : (i) harvesting a culture of
live
bacteria cells, (ii) washing the bacteria cells in water or in aqueous
solution of a
salt, (iii) freezing the bacteria cells in water or in an aqueous solution of
salt, (iv)
killing the frozen bacteria cells by drying them in a lyophiliser, 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 (v) collecting the extended freeze-
dried killed bacteria cells.
The expression "EFD" as used in the context of the present invention
refers in other words to killed Mycobacterium bovis BCG bacteria for instance
where the structure of the molecules and in particular the structure of the
proteins
is preserved. An example of preserved protein is Apa which has the same
migrating characteristics in a SDS-PAGE gel as the protein extracted from
living
mycobacteria (Laqueyrerie et al., Infect. lmmun., 1995; 63; 4003-4010).
A fraction of EFD preparation as described above is covered by the
expression "EFD" or "EFD preparation" of the invention. This fraction is
selected
in the group consisting of : a fraction consisting of an organic solvent
extract of
said EFD preparation, a fraction consisting of a glycosidase-treated extract
of
said EFD preparation, a fraction consisting of a DNase and/or a RNase-digested
extract of said EFD preparation, a fraction consisting of a protease-treated
extract
CA 02629057 2008-04-14
7
of said EFD preparation, a fraction consisting of said EFD preparation
successively treated by an organic solvent, a glycosidase, a DNase and/or a
RNase, and finally a protease, and a fraction consisting of said EFD
preparation
treated by a protease (as subtilisine for example) and a DNase.
The EFD 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.
As used herein, the fraction of this extended-freeze-dried killed bacteria
refers to a fraction selected from 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.
Pharmaceutical composition
According to an embodiment of the invention, it is provided a Gram
positive bacteria preparation, such a preparation for the prevention and/or
treatment of a Tregs-associated disease. Such preparation may be 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
CA 02629057 2008-04-14
8
e) collecting the extended freeze-dried bacteria cells.
According to another advantageous embodiment of the invention, said
preparation is included in a pharmaceutical composition additionally
comprising
an acceptable carrier and/or an additive and/or an immunostimulant and/or an
adjuvant.
The composition of the present invention may be 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
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.
The composition may additionally contain a pharmaceutically acceptable
carrier and/or 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.
For parenteral administration, such as subcutaneous injection, the carrier
may comprise 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
CA 02629057 2008-04-14
9
pharmaceutical compositions of this invention. Suitable biodegradable
microspheres are disclosed for example in US patents 4,897,268 and 5,075,109.
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, Bordetelia
pertussis toxin. Suitable adjuvants are commercially available as well, for
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.
Method of treatment
According to an embodiment of the present invention, the EFD killed
Gram positive bacteria preparation is used for the prevention and treatment of
a
Tregs-associated disease such as MS or EAE. The method of the invention
comprises the step of administering to a patient an effective amount of the
EFD
killed Gram positive bacteria preparation, to stimulate the production of
Tregs,
such as CD4+ CD25+ Foxp-3+ T cells.
As mentioned above, the disease may be MS or EAE.
The amount of Gram positive bacteria preparation present in the
compositions of the present invention may be 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 stimulating the production of Tregs 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 be
administered
will vary according to factors such as the type of disease being treated, the
mode
of administration, as well as the other ingredients in the composition. The
CA 02629057 2008-04-14
composition may be composed of from about 10 pg to about 10 mg of EFD killed
Gram positive bacteria preparation.
For instance, during an oral administration of the composition of the
invention, host to be treated could be subjected to a 1 dose schedule of from
5 about 10 g to about 10 mg of EFD killed 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
be treated could be subjected to a 1 dose schedule of from about 10 g to
about
10 10 mg of EFD killed Gram positive bacteria preparation per month or every 6
months.
Example 1: EFD treatment promotes in vitro the Treg differentiation from a
naive T lymphocyte population.
Piasmacytoid dendritic cells (pDCs) expressing ICOS-L (Ito et al. 2007),
collected
from draining lymph nodes of a sub-cutaneous site of EFD injection are able to
promote in vitro the Treg differentiation from a nalve T lymphocyte
population.
a) Cells were collected from draining lymph nodes 4 days after subcutaneous
injection of lOOpg of heat-killed BCG (HK-BCG), live BCG, or EFD, or PBS in
naive mice.
Total number of ceils was in the same range (1 to 2 X 107 cells per lymph
node).
They were analysed for their membrane markers using adequate monoclonal
antibodies using FACS analysis.
A slight, (not significant) increase of dendritic cells (CD11 c+ Class2+
cells)
number was observed after EFD treatment (Fig. 1). A significant increase
number
of plasmacytoid dendritic cells (CD11c+B220+ cells) was observed after EFD
treatment.
CA 02629057 2008-04-14
11
The number of cells bearing the ICOS-L marker, considered to be a specific
marker for plasmacytoid dendritic cells able to promote differentiation and,
or
proliferation of Treg cells, was 10 fold increased after EFD (Fig. 2).
b) When these CD11c+ B220+ cells were sorted and cultivated 72h in vitro in
presence of naive Tcells the Foxp-3 transcription factor, considered to be
specific of Tregs, was highly expressed only in Tcells cultivated in presence
of
CD11 c+B220+ cells sorted from draining lymph nodes of EFD treated mice
(Fig. 3). The CD11c+B220+ cells collected from BCG injected mice induced also
the Foxp-3, it has to be noted that they were cultivated at the same number
than
those collected after EFD. In vivo they were at least 5 fold less numerous
after
BCG than after EFD as indicated previously.
Example 2: EFD treatment induces Treas in a Crohn mouse model.
Rag2-/- mice develop inflammatory bowel disease after intravenous transfer of
naive CD4+CD45h'9h Tcells (Fiona POWRIE).
Naive cells were collected from 4 spleens of C57B1/6 mice. They were sorted by
FACS Aria in order to select only CD4+CD45h'gh cells. 300 000 of these sorted
cells were transferred intravenously 10 Rag2 -/- mice, 5 of them received at
the
same time 100Ng of EFD subcutaneously at the base of the tail.
The spleens were collected 40 days later. The expression of transcription
factors
(Tbet, Foxp-3, GATA-3 and RORyt) was evaluated in spleen extracts.
EFD treatment promoted the differentiation and/or multiplication of naive
Tcells
toward Tregs expressing Foxp-3 and increased Tbet expression (Th1 cellular
immune response) whereas GATA-3 and RORyt were decreased (Th2 and Th17
cellular immune response) (Fig. 4).
CA 02629057 2008-04-14
12
Example 3: EFD treatment induces Treas in normal mice.
Mice were subcutaneaously injected with lOOpg of heat-killed BCG, live BCG or
EFD. Spleens were harvested 28 days later. Foxp-3 expression was higher in
cell
extracts from EFD than HK or live BCG injected mice (Fig. 5).
Example 4: EFD treatment induces Treas in an asthma mouse model.
In a murine model of asthma, BP2 mice were sensitized with ovalbumin (OVA),
injected subcutaneously two times and challenged intranasally 42 days later to
induce an airway hyperreactivity. Mice treated with EFD 8 days after
sensitisation
were protected as shown by their decreased sensitivity to methacholine (Penh
values) (Fig. 6).
The crucial role of Tregs was demonstrated by lost of the EFD protective
effect
after injection of a anti-CD25 monoclonal antibody (BD Biosciences, Ref.
553864,
J Exp Med (2000), Vol. 192, pp. 303-309). Contrary to this anti-CD25 mAb, an
isotype without any affinity for CD25 shows no result on EFD protective
effect,
demonstrating that EFD acts via Tregs immune response.
Example 5: EFD treatment protects mice aaainst paralysis observed in EAE.
EAE Induction protocol (Cassan et al. and Chen et al.):
C57B1/6 mice were injected at day 0 with 200 pg of MOG peptide (Invitrogen
Life Technologies, Carlsbad, CA, per mouse) emulsified in CFA (complete
Freund's adjuvant) supplement with killed Mycobacterium tuberculosis
Bacillus (total 300 pg per mouse) in equal volumes. Injection : subcutaneous ,
total volume 200 ui
Pertusis toxin: 200 ng per mouse (total) in 0.2 mi PBS at 0 and 48 hours, i.v
injection
CA 02629057 2008-04-14
13
Treatment protocol:
Twenty five mice received EFD 100 pg /100 pi per mouse in PBS by
subcutaneous injection at day 0 (induction), 32 mice received only PBS.
Results:
No mice among the 25 EFD treated mice developed paralysis during the period of
observation while 20 mice among the 32 which received PBS developed
paralysis. The difference between the two groups is highly significant using a
Chi2 test (Fig. 7).
Paralytic symptoms were recorded daily between days 12 and 28 after EAE
induction. Using a clinical scale,
0 no paralysis
0.5 weakness or partial tail paralysis
1 complete tail paralysis
2 complete tail paralysis, and uni/bilateral hind limb weakness
3 unilateral hind limb paralysis
4 bilateral hind limb paralysis
5 forelimb paralysis, motionless
a mean of paralytic symptoms was established daily.
The difference between EFD treated mice, without symptoms and control mice
was highly significant (Fig. 8). It can be concluded that EFD treatment
prevents
symptoms of EAE from appearing.
