Gram positive bacteria preparations for the treatment of diseases comprising an immune dysregulation

Abstract

Compositions comprising components prepared from Gram positive bacteria such as Gram positive facultative intra-cellular bacteria, for example mycobacteria, including extended freeze-dried killed Gram positive bacteria, their method of preparation and their use in humans and animals, for the prevention and/or the treatment of disorders comprising an immune dysregulation such as cancer, autoimmune diseases, allergy and tuberculosis.

1 ) A bacterial preparation, characterized in that: 
 it contains killed Gram positive bacteria, obtainable by a process which does not denature the structure of the molecules from the bacteria cells, and    it is able to induce, in vivo, a modulation of the immune response against an antigen.    
     
     
         2 ) The bacterial preparation according to  claim 1 , characterized in that said Gram positive bacteria are chosen from Gram positive facultative intracellular bacteria.  
     
     
         3 ) The bacterial preparation according to  claim 2 , characterized in that said Gram positive facultative intracellular bacteria are selected from the group consisting of  Listeria  sp.,  Corynobacterium  sp., and Actinomycetes comprising  Mycobacteria  sp.,  Nocardia  sp. and  Rhodococcus  sp.  
     
     
         4 ) The bacterial preparation according to  claim 3 , characterized in that said  mycobacteria  is  Mycobacteria bovis,  more preferably  Mycobacteria bovis  BCG.  
     
     
         5 ) The bacterial preparation according to anyone of  claims 1  to  4 , characterized in that it contains extended-freeze-dried killed bacteria obtainable by an extended freeze-drying process.  
     
     
         6 ) The bacterial preparation according to  claim 4 , characterized in that it contains extended freeze-dried killed  mycobacteria.    
     
     
         7 ) The bacterial preparation according to  claim 5  or  claim 6 , characterized in that it contains less than 1.5% of residual water, preferably less than 1%, more preferably less than 0.5%.  
     
     
         8 ) The bacterial preparation according to anyone of  claims 5  to  7 , characterised in that it is prepared by an extended freeze-drying process, comprising at least the steps of: 
 (i) harvesting a culture of live bacteria cells,    (ii) washing the bacteria cells in water or in an aqueous solution of a salt such as borate,    (iii) freezing the bacteria cells in water or in an aqueous solution of a salt such as borate,    (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.    
     
     
         9 ) The bacterial preparation according to anyone of  claims 5  to  7 , characterized in that it is prepared by an extended freeze-drying process comprising the steps (i), (iii), (iv) and (v) as defined in  claim 8 .  
     
     
         10 ) The bacterial preparation according to claims  8  or  9 , characterized in that it is prepared by performing step (iv) at a drying chamber pressure of about 0.02 mBar to 0.2 mBar; more preferably of about 0.06 mBar to 0.1 mBar.  
     
     
         11 ) Fractions of bacterial preparation according to  claims 1  to  10 , selected in the group consisting of: 
 a fraction A consisting of an organic solvent extract of said killed bacterial preparation,    a fraction B consisting of a glycosidase-treated extract of said killed bacterial preparation,    a fraction C consisting of a DNase and/or a RNase-digested extract of said killed bacterial preparation,    a fraction D, consisting of a protease-treated extract of said killed bacterial preparation,    a fraction E, consisting of a extract of said killed bacterial preparation successively treated by an organic solvent, a glycosidase, a DNase and/or a RNase, and finally a protease.    
     
     
         12 ) Fractions according to  claim 11 , characterized in that: 
 said fraction A is obtained by extraction with a methanol/chloroform mixture,    said fraction B is obtained by digestion with lysozyme,    said fraction C is obtained by digestion with DNase I and/or RNase A, and    said fraction D is obtained by digestion with subtilisine.    
     
     
         13 ) A pharmaceutical composition, characterized in that it comprises an effective amount of a bacterial preparation according to anyone of  claims 1  to  10  and/or at least one fraction thereof, according to  claim 11  or  claim 12 , a pharmaceutically acceptable carrier and/or an additive, and/or an immunostimulant, and/or an adjuvant and/or an immunomodulator distinct from the bacterial preparation according to  claim 1 .  
     
     
         14 ) Use of the bacterial preparation according to anyone of  claims 1  to  10  or fraction(s) thereof according to claims  11  or  12  for the preparation of a medicament for the prevention and/or the treatment of diseases comprising an immune dysregulation.  
     
     
         15 ) Use according to  claim 14 , wherein said diseases comprising an immune dysregulation are selected from the group consisting of cancer, autoimmune diseases and allergy.  
     
     
         16 ) Use according to  claim 15 , wherein said autoimmune diseases are selected from the group consisting of multiple sclerosis, rhumatoïd arthritis, Crohn disease and diabetes mellitus,  
     
     
         17 ) Use according to  claim 15 , wherein said allergy is selected from the group consisting of asthma, allergic rhinitis, conjonctivis and atopic dermatitis.  
     
     
         18 ) Use according to anyone of  claims 14  to  17 , wherein said medicament is intended to be administered orally, sublingually, parenterally and preferably subcutaneously or intranasally.  
     
     
         19 ) A method for the preparation of the extended freeze-dried killed bacterial preparation as defined in anyone of  claims 5  to  10 , characterized in that it comprises at least the steps of: 
 (i) harvesting a culture of live bacteria cells,    (ii) washing the bacteria cells in water or in an aqueous solution of a salt,    (iii) freezing the bacteria cells in water or in an aqueous solution of a 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.    
     
     
         20 ) A method for the preparation of the extended freeze-dried killed bacterial preparation as defined in anyone of  claims 5  to  10 , characterized in that it comprises the steps (i), (iii), (iv) and (v) as defined in  claim 19 .  
     
     
         21 ) The method of claims  19  or  20 , characterised in that step (iv) is performed at a drying chamber pressure of about 0.02 mBar to 0.2 mBar, more preferably 0.06 mBar to 0.1 mBar.  
     
     
         22 ) Kit for the treatment of a disease comprising an immune dysregulation, characterized in that it comprises at least a pharmaceutical composition according to  claim 13 , means of administration of said pharmaceutical composition and/or means for insuring compliance with the prescribed treatment.  
     
     
         23 ) Products containing a bacterial preparation according to anyone of  claims 1  to  10  or fractions thereof according to  claim 11  or  claim 12  and a product selected from the group consisting of anticancer, anti-diabetes and immunomodulatory drugs as a combined preparation for simultaneous, separate or sequential use in the prevention and/or the treatment of diseases comprising an immune dysregulation.  
     
     
         24 ) Products according to  claim 23 , characterized in that said product is an anti-histaminic drug or an anti-inflammatory drug.  
     
     
         25 ) Use of a bacterial preparation according to anyone of  claims 1  to  10  or fractions thereof according to  claim 11  or  claim 12  for the preparation of a medicament for the treatment of asthma to be administered by the oral, parenteral, sublingual or intranasal route, in a dose range which is equivalent in mice to 1 μg to 10 000 μg, preferably 10 μg to 1 000 μg, most preferably 10 μg to 100 μg, at two months intervals, starting from at least two weeks before the usual period of allergen exposure.  
     
     
         26 ) Use of a bacterial preparation according to anyone of  claims 1  to  10  or fractions thereof according to  claim 11  or  claim 12  for the preparation of a medicament for the prevention of asthma in human baby, to be administered by the oral, parenteral, sublingual or intranasal route, in a dose range which is equivalent in mice to 1 μg to 10 000 μg, preferably 10 μg to 1 000 μg, most preferably 10 μg to 100 μg.