Composition comprising a carrier and a purified mycobacterial lipid cell-wall component and its use in the prevention, treatment and diagnosis of disease

Abstract

A composition including a purified lipid cell-wall component or analog or derivative thereof and a suitable pharmaceutical carrier, medium, excipient or adjuvant is described. The composition is useful in prophylactic and therapeutic methods of treating a microbial infection in a subject, typically a mycobacterial infection such as tuberculosis, and immune disorders, inflammatory conditions and allergies in a subject, typically autoimmune diseases. It is also useful in diagnostic methods. The purified lipid cell-wall component is typically a purified mycolic acid or a mixture of purified mycolic acids form a bacterium which produces mycolic acids. The bacterium if from Mycobacterium, Corynbacterium, Nocardia or Rhodococcus.

1 . A composition comprising a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof and a pharmaceutically acceptable carrier or adjuvant, wherein the pharmaceutically acceptable carrier is suitable for direct administration to the lungs of a subject.  
     
     
         2 . A composition according to  claim 1 , wherein the purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids is from  Mycobacterium tuberculosis.    
     
     
         3 . A composition according to  claim 1 , wherein the pharmaceutically acceptable carrier is a vaporisable liquid.  
     
     
         4 . A composition according to  claim 3 , wherein the vaporisable liquid is a saline solution.  
     
     
         5 . A composition comprising a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof and a pharmaceutically acceptable carrier or adjuvant, wherein the composition comprises a tolerogenic amount of the purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof.  
     
     
         6 . A composition according to  claim 5 , wherein the purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids is from  Mycobacterium tuberculosis.    
     
     
         7 . A prophylactic pharmaceutical composition for an immune disorder, comprising a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof and a pharmaceutically acceptable carrier or adjuvant.  
     
     
         8 . A vaccine according to  claim 7 , wherein the immune disorder is arthritis.  
     
     
         9 . A method of decreasing the pathogenic effects of a mycobacterial infection in the lungs of a subject by administering to the subject a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof to stimulate the innate immunity of the subject.  
     
     
         10 . A method according to  claim 9 , which is a prophylactic method or a therapeutic method.  
     
     
         11 . A method according to  claim 10 , which is an immunoregulatory method.  
     
     
         12 . A method according to  claim 10  or  11 , which is a prophylactic method which enhances resistance or reduces susceptibility to a mycobacterial infection in a subject.  
     
     
         13 . A method according to  claim 12 , wherein the prophylactic method promotes a pro-inflammatory response in the lungs of the subject.  
     
     
         14 . A method according to  claim 10  or  11 , wherein the prophylactic method or the therapeutic method modulates or manipulates the humoral immune system or cellular immune system or both in a subject.  
     
     
         15 . A method according to  claim 12  or  13 , wherein the prophylactic method stimulates the expression of IL12 IFN-γ in the lungs of the subject.  
     
     
         16 . A method according to  claim 10 , wherein the therapeutic method stimulates the expression of TGF-β in the lungs of the subject.  
     
     
         17 . A method according to  claim 10 , wherein the mycobacterial infection is tuberculosis.  
     
     
         18 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof for use in a method of decreasing the pathogenic effects of a mycobacterial infection in the lungs of a subject by stimulating the innate immunity of the subject.  
     
     
         19 . Use of a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof in a method of making a medicament for use in a method of decreasing the pathogenic effects of a mycobacterial infection in the lungs of a subject by stimulating the innate immunity of the subject.  
     
     
         20 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids according to  claim 18  or  19 , wherein the method of treatment is a prophylactic method or a therapeutic method.  
     
     
         21 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids according to  claim 20 , wherein the method of treatment is an immunoregulatory method.  
     
     
         22 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof according to  claim 18  or  19 , wherein the method of treatment is a prophylactic method which enhances resistance or reduces susceptibility to a mycobacterial infection in a subject.  
     
     
         23 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids according to  claim 22 , wherein the prophylactic method promotes a pro-inflammatory response in the lungs of the subject.  
     
     
         24 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids according to  claim 20  or  21 , wherein the prophylactic method or the therapeutic method modulates or manipulates the humoral immune system or cellular immune system or both in a subject.  
     
     
         25 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids according to  claim 22 , wherein the mycobacterial infection is tuberculosis.  
     
     
         26 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids according to  claim 20 , wherein the method of treatment is a prophylactic method which stimulates the expression of IL12 and IFN-γ in the lungs of the subject.  
     
     
         27 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids according to  claim 20 , wherein the method of treatment is a therapeutic method which stimulates the expression of TGF-β in the lungs of the subject.  
     
     
         28 . A method of diagnosing an immune disorder in a subject comprising the steps of: 
 contacting a sample from the subject with a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof; and    detecting any immunological reaction between the purified mycobacterial mycolic acid or mixture of purified mycolic acids and the sample.    
     
     
         29 . A method according to  claim 28 , wherein the step of detecting any reaction between the purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof and the sample comprises detecting the binding of an antibody present in the sample to the purified mycobacterial mycolic acid component.  
     
     
         30 . A method of treatment of an immune disorder in a subject comprising the step of administering to the subject a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof.  
     
     
         31 . A method according to  claim 30 , which is a prophylactic method or a therapeutic method.  
     
     
         32 . A method according to  claim 31 , which is an immunoregulatory method.  
     
     
         33 . A method according to  claim 31  or  32 , which is a prophylactic method which enhances resistance or reduces susceptibility to an immune disorder in a subject.  
     
     
         34 . A method according to  claim 33 , wherein the prophylactic method suppresses inflammation of the joints in the subject.  
     
     
         35 . A method according to  claim 31  or  32 , wherein the prophylactic method or the therapeutic method modulates or manipulates the humoral immune system or cellular immune system or both in a subject.  
     
     
         36 . A method according to any one of  claims 30  to  35 , wherein the immune disorder is an inflammatory condition or allergy.  
     
     
         37 . A method according to  claim 36 , wherein the inflammatory condition is an autoimmune disease.  
     
     
         38 . A method according to  claim 37 , wherein the autoimmune disease is arthritis.  
     
     
         39 . A purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof for use in a method of treatment of an immune disorder in a subject.  
     
     
         40 . Use of a purified mycobacterial mycolic acid or mixture of purified mycobacterial mycolic acids or ester form thereof or synthetic form thereof in a method of making a medicament for use in the treatment of an immune disorder in a subject.  
     
     
         41 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to  claim 39  or  40 , wherein the method of treatment is a prophylactic method or a therapeutic method.  
     
     
         42 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to  claim 41 , wherein the method of treatment is an immunoregulatory method.  
     
     
         43 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to  claim 41  or  42 , wherein the method of treatment is a prophylactic method which enhances resistance or reduces susceptibility to an immune disorder in a subject.  
     
     
         44 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to  claim 43 , wherein the prophylactic method suppresses inflammation of the joints in the subject.  
     
     
         45 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to  claim 41  or  42 , wherein the prophylactic method or the therapeutic method modulates or manipulates the humoral immune system or cellular immune system or both in a subject.  
     
     
         46 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to any one of  claims 39  to  45 , wherein the immune disorder is an inflammatory condition or allergy.  
     
     
         47 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to  claim 46 , wherein the inflammatory condition is an autoimmune disease.  
     
     
         48 . A purified mycobacterial mycolic acid, mixture, ester or synthetic form according to  claim 47 , wherein the autoimmune disease is arthritis.  
     
     
         49 . A method of separating and purifying a specific microbial cell-wall component of a lipid or carbohydrate nature or a derivative or analog thereof from an extracted mixture of the cell-wall component or derivative or analog thereof and contaminants or from a synthetic mixture of the cell-wall component or derivative or analog thereof and contaminants, comprising the steps of: 
 dissolving the extracted mixture or synthetic mixture in a bi-phasic solvent containing sodium chloride to form a solution;    allowing the solution to separate to from an upper phase and a lower phase;    subjecting the phases to countercurrent distribution/separation comprising a required number of cycles to separate the microbial cell-wall component or analog or derivative thereof in the upper phase or the lower phase; and    removing the separated microbial cell-wall component or derivative or analog thereof from the upper or lower phase.    
     
     
         50 . A method according to  claim 49 , which also comprises the additional pre-purification steps of: 
 dissolving the extracted mixture of cell-wall component or derivative or analog thereof and contaminants or the synthetic mixture of the cell-wall component or derivative or analog thereof and contaminants in a first solvent without sodium chloride;    adding thereto a second solvent without sodium chloride;    mixing and allowing the solution to separate to form a first upper phase (second solvent) and first lower phase (first solvent); and    removing the first upper phase and/or the first lower phase for further processing.    
     
     
         51 . A method according to  claim 49  or  50 , wherein the lower phase containing the extracted mixture is removed and subjected to countercurrent distribution/separation comprising a required number of cycles to separate the microbial cell-wall component or analog or derivative thereof in a second upper phase or lower phase and the separated microbial cell-wall component or derivative or analog thereof is removed from the second upper or lower phase.  
     
     
         52 . A method according to any one of  claims 49  to  51 , which also comprises the additional post-purification steps of: 
 dissolving the extracted microbial cell-wall component or derivative or analog thereof in a suitable solvent; and  
 adding a precipitant to the solution to precipitate out the dissolved further purified microbial cell-wall component or derivative or analog thereof.  
 
     
     
         53 . A method according to  claim 52 , wherein the solvent is chloroform.  
     
     
         54 . A method according to  claim 52  or  53 , wherein the precipitant is acetone.  
     
     
         55 . A method according to any one of  claims 49  to  54 , which also comprises the steps of: 
 saponifying a microbial culture prior to preparing therefrom an extracted mixture of a cell-wall component or derivative or analog thereof on which to perform the method; and  
 resaponifying the separate and purified microbial cell-wall component or derivative or analog thereof.  
 
     
     
         56 . Detection means for detecting the presence of antibodies to a purified mycolic acid or mixture of purified mycolic acids comprising a solid phase and an unconjugated purified mycolic acid or a mixture of unconjugated purified mycolic acids in a methyester form or in a freshly resaponified form associated therewith.  
     
     
         57 . Detection means according to  claim 56 , wherein the solid phase is an ELISA plate.  
     
     
         58 . A prophylactic pharmaceutical composition according to  claim 7 , wherein the immune disorder is an autoimmune disorder.