CA2629057A1 - Control of diseases associated with decrease of t-regulatory cells with a preparation of extended freeze-dried killed bacteria - Google Patents
Control of diseases associated with decrease of t-regulatory cells with a preparation of extended freeze-dried killed bacteria Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
Abstract
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 diseases associated with a decrease of T-regulatory cells (Tregs), such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE).
Description
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 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 :
"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:
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 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 :
"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:
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.
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.
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.
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.
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.
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 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 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 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 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.
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 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 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 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 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.
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).
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).
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 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.
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 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.
Claims (25)
1. Use of a Gram positive bacteria preparation or a fraction thereof for the prevention and treatment of a T-regulatory cells (Tregs)-associated disease, the preparation being characterized in that the Gram positive bacteria are killed by extended freeze-drying (EFD).
2. Use according to claim 1, wherein said preparation contains more than 50 %, and preferentially more than 90%, of the bacterial protein components which are in a native structure.
3. Use according to claim 1 or 2, wherein said preparation contains less than 1% of residual water and preferably less than 0.5%.
4. Use according to anyone of claims 1 to 3, characterized in that the Gram positive bacteria is a Gram positive facultative intracellular bacterium.
5. Use according to anyone of claims 1 to 4, characterized in that the Gram positive facultative intracellular bacteria is Mycobacterium bovis BCG.
6. Use according to anyone of claims 1 to 5, wherein the Tregs are CD4+
CD25+ Foxp3+ T cells.
CD25+ Foxp3+ T cells.
7. Use according to anyone of claims 1 to 6, characterized in that the disease is a central nervous system (CNS) inflammatory disease.
8. Use according to anyone of claims 1 to 7, characterized in that the disease is multiple sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE).
9. A method for preventing or treating a T-regulatory cells (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.
10. The method according to claim 9, wherein said preparation contains more than 50 %, and preferentially more than 90%, of the bacterial protein components which are in a native structure.
11. The method according to claim 9 or 10, wherein said preparation contains less than 1% of residual water and preferably less than 0.5%.
12. The method according to anyone of claims 9 to 11, characterized in that the Tregs are CD4+ CD25+ Foxp3+ T cells.
13. The method according to anyone of claims 9 to 12, characterized in that the disease is multiple sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE).
14. The method according to anyone of claims 9 to 13, characterized in that the Gram positive bacteria cell is a Gram positive facultative intracellular bacterium.
15. The method according to anyone of claims 9 to 14, characterized in that the Gram positive facultative intracellular bacteria is Mycobacterium bovis BCG.
16. The method according to anyone of claims 9 to 15, characterized in that the Tregs are CD4+ CD25+ Foxp3+ T cells.
17. The method according to anyone of claims 9 to 16, characterized in that the disease is a central nervous system (CNS) inflammatory disease.
18. The method according to anyone of claims 9 to 17, characterized in that the disease is multiple sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE).
19. A preparation of Mycobacterium bovis BCG killed by extended freeze drying for treating a T-regulatory cells (Tregs)-associated disease.
20. The preparation according to claim 19, characterized in that the disease is a CNS inflammatory disease such as multiple sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE).
21. The preparation according to claim 19 or 20, wherein said preparation contains more than 50 %, and preferentially more than 90%, of the bacterial protein components which are in a native structure.
22. The preparation according to anyone of claims 19 to 21, wherein said preparation contains less than 1% of residual water and preferably less than 0.5%.
23. The preparation according to anyone of claims 19 to 22, which is composed of from about 10 µg to about 10 mg of Mycobacterium bovis BCG
killed by extended freeze drying.
killed by extended freeze drying.
24. The preparation according to anyone of claims 19 to 23, which is obtained by a process comprising 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.
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.
25. Product comprising a preparation as claimed in anyone of claims 19 to 24 and at least another product selected from the group consisting of anti-inflammatory and immunoregulatory drugs, as a combined preparation fro simultaneous, separate or sequential use in the prevention and/or treatment of a CNS inflammatory disease such as multiple sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE).
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002629057A CA2629057A1 (en) | 2008-04-14 | 2008-04-14 | Control of diseases associated with decrease of t-regulatory cells with a preparation of extended freeze-dried killed bacteria |
| PCT/IB2009/005498 WO2009127971A2 (en) | 2008-04-14 | 2009-04-14 | Control of diseases associated with decrease of t-regulatory cells with a preparation of extended freeze-dried killed bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002629057A CA2629057A1 (en) | 2008-04-14 | 2008-04-14 | Control of diseases associated with decrease of t-regulatory cells with a preparation of extended freeze-dried killed bacteria |
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| Publication Number | Publication Date |
|---|---|
| CA2629057A1 true CA2629057A1 (en) | 2009-10-14 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002629057A Abandoned CA2629057A1 (en) | 2008-04-14 | 2008-04-14 | Control of diseases associated with decrease of t-regulatory cells with a preparation of extended freeze-dried killed bacteria |
Country Status (2)
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| CA (1) | CA2629057A1 (en) |
| WO (1) | WO2009127971A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2316466B1 (en) * | 2001-12-11 | 2013-08-21 | Institut Pasteur | Gram positive bacteria preparations for the treatment of diseases comprising an immune dysregulation |
| CA2531261A1 (en) * | 2005-12-21 | 2007-06-21 | Institut Pasteur | Control of intestinal inflammatory syndromes with a preparation of killed or non infectious bacteria |
-
2008
- 2008-04-14 CA CA002629057A patent/CA2629057A1/en not_active Abandoned
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2009
- 2009-04-14 WO PCT/IB2009/005498 patent/WO2009127971A2/en active Application Filing
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| Publication number | Publication date |
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| WO2009127971A2 (en) | 2009-10-22 |
| WO2009127971A3 (en) | 2009-12-10 |
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