Sphingoid polyalklamine conjugates, isomers and uses thereof

Abstract

The present application discloses methods for stimulating or enhancing an immune response of a subject to protect against an infection caused by an agent selected from Hepatitis B Virus (HBV), avian influenza virus, the bacterium Bacillus anthracis or the bacterium Streptococcus pneumoniae , the method comprising administering to said subject a combination of sphingoid-polyalkylamine conjugate and a biologically active molecule, the combination being effective to provide said stimulation or enhancement of the immune response. Also disclosed are vaccines comprising a combination of the sphingoid-polyalkylamine conjugate and a biologically active molecule for stimulating or enhancing an immune response of a subject to protect against an infection caused by such an agent. Preferred conjugates are N-palmitoyl D-erythro sphingosyl-1-carbamoyl spermine (C-1 CCS), N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine (C-3 CCS) and mixtures thereof. Also disclosed are uses of C-3 CCS for various applications.

1 . A method for stimulating or enhancing an immune response of a subject to provide protection against an infection, the method comprising administering to said subject a combination of sphingoid-polyalkylamine conjugate and a biologically active molecule, the combination being effective to provide said stimulation or enhancement of the immune response, wherein said infection is caused by an agent selected from the group consisting of hepatitis B virus (HBV), avian influenza virus (AIV), the bacterium  Bacillus anthracis  and the bacterium  Streptococcus pneumoniae.    
   
   
       2 . The method of  claim 1 , wherein said sphingoid-polyalkylamine conjugate comprises a sphingoid backbone carrying, via a carbamoyl linkage, at least one polyalkylamine chain.  
   
   
       3 . The method of  claim 1 , wherein said biologically active molecule is associated with said sphingoid-polyalkylamine conjugate.  
   
   
       4 . The method  claim 1 , wherein said biologically active molecule is selected from an antigenic protein, antigenic peptide, antigenic polypeptide, carbohydrate, or antigenic glyco-protein.  
   
   
       5 . The method  claim 1 , wherein: 
 (i) when said infection is caused by HBV, said biologically active molecule is an Hepatitis B antigen;    (ii) when said infection is caused by avian influenza virus, said biologically active molecule is an AIV antigen;    (iii) when said infection is caused by the bacterium  Bacillus anthracis , said biologically active molecule is an antigen of said bacterium; or    (iv) when said infection is caused by the bacterium  Streptococcus pneumoniae  said biologically active molecule is an antigen of said bacterium.    
   
   
       6 . The method of  claim 5 , wherein: 
 (i) said HBV antigen is an HBsAg particle;    (ii) said AIV antigen is an inactivated purified whole avian influenza virus;    (iii) said bacterium antigen is the protective antigen (PA) moiety of anthrax toxin; or    (iv) said  Streptococcus pneumoniae  antigen comprises a polysaccharide or protein conjugate of an antigen selected from pneumococcal surface protein A (PspA), pneumococcal adherence and virulence factor A (PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding protein A (CbpA), pneumococcal surface adhesion A (PsaA).    
   
   
       7 . The method of  claim 1 , further comprising administering to said subject an immunostimulating agent.  
   
   
       8 . The method of  claim 1 , wherein said sphingoid-polyalkylamine conjugate forms a lipid assembly.  
   
   
       9 . The method of  claim 1 , wherein said sphingoid is ceramide.  
   
   
       10 . The method of  claim 1 , wherein said polyalkylamine is selected from spermine, spermidine, a polyamine analog or a combination of same thereof.  
   
   
       11 . The method of  claim 1 , wherein said sphingoid-polyalkylamine conjugate is N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (CCS).  
   
   
       12 . The method of  claim 1 , wherein said sphingoid-polyalkylamine conjugate has the following formula (I):  
     
       
         
         
             
             
         
       
       wherein  
       R 1  represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R 5 ;  
       R 2  and R 5  represent, independently, a branched or linear C 10 -C 24  alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups;  
       R 3  and R 4  are independently a hydrogen or group —C(O)—NR 6 R 7 , R 6  and R 7  being the same or different for R 3  and R 4  and represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; provided that R 3  and R 4  are not simultaneously a hydrogen; or  
       R 3  and R 4  form together with the oxygen atoms to which they are bound a heterocyclic ring comprising —C(O)—NR 9 —[R 8 —NR 9 ] m —C(O)—, R 8  represents a saturated or unsaturated C 1 -C 4  alkyl and R 9  represents a hydrogen or a polyalkylamine of the formula —[R 8 —NR 9 ] n —, wherein said R 9  or each alkylamine unit R 8 NR 9  may be the same or different in said polyalkylamine; and  
       n and m, represent independently an integer from 1 to 10;  
       W represents a group selected from —CH═CH—, —CH 2 —CH(OH)— or —CH 2 —CH 2 —.  
     
   
   
       13 . The method of  claim 12 , wherein R 1  represents a —C(O)R 5  group; R 5  represents a C 12 -C 18  linear or branched alkyl or alkenyl; W represents —CH═CH—; R 2  represents a C 12 -C 18  linear or branched alkyl or alkenyl; R 3  and R 4  represent, independently, a hydrogen or a group C(O)—NR 6 R 7 , wherein said R 3  and R 4  are not simultaneously a hydrogen, and R 6  and R 7  represent, independently, a hydrogen or a polyalkylamine having the general formula (II):  
     
       
         
         
             
             
         
       
       wherein  
       R 8  represent a C 1 -C 4  alkyl;  
       R 9  represents a hydrogen or a polyalkylamine branch of formula (II), said R 8  and R 9  may be the same or different for each alkylamine unit, —R 8 NR 9 —, in the polyalkylamine of formula (II); and  
       n represents an integer from 3 to 6.  
     
   
   
       14 . The method of  claim 13 , wherein R 3  or R 4  is a hydrogen atom.  
   
   
       15 . The method of  claim 13 , wherein both R 3  and R 4  represent the same or a different polyalkylamine.  
   
   
       16 . The method of  claim 13 , wherein R 1  represents a C(O)R 5  group; R 5  represents a C 12 -C 18  linear or branched alkyl or alkenyl; W represents —CH═CH—; R 2  represents a C 12 -C 18  linear or branched alkyl or alkenyl; R 3  and R 4  form together with the oxygen atoms to which they are bonded a heterocyclic ring comprising —C(O)—[NH—R 8 ] n —NH—C(O)—, 
 wherein    R 8  represents a C 1 -C 4  alkyl, wherein for each alkylamine unit having the formula —NH—R 8 —, said R 8  may be the same or different; and n represents an integer from 3 to 6.    
   
   
       17 . The method of  claim 13 , wherein said R 8  is a C 3 -C 4  alkyl.  
   
   
       18 . The method of  claim 1 , comprising intranasal or parenteral administration of said combination of sphingoid-polyalkylamine conjugate and said biologically active molecule.  
   
   
       19 . The method of  claim 1 , comprising intranasal or intramuscular administration of a combination of said sphingoid-polyalkylamine conjugate with said biologically active molecule, wherein: 
 (i) when said infection is caused by HBV, said biologically active molecule is an HBV antigen;    (ii) when said infection is caused by avian influenza virus, said biologically active molecule is an AIV antigen;    (iii) when said infection is caused by the bacterium  Bacillus anthracis , said biologically active molecule is an antigen of said bacterium; or    (iv) when said infection is caused by the bacterium  Streptococcus pneumoniae  said biologically active molecule is an antigen of said bacterium.    
   
   
       20 . The method of  claim 19 , wherein: 
 (i) said HBV antigen is an HBV surface particle (HBsAg);    (ii) said AIV antigen is an inactivated purified whole avian influenza virus;    (iii) said bacterium antigen is the protective antigen (PA) moiety of anthrax toxin; or    (iv) said  Streptococcus pneumoniae  antigen is a polysaccharide or protein conjugate of an antigen selected from pneumococcal surface protein A (PspA), pneumococcal adherence and virulence factor A (PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding protein A (CbpA), pneumococcal surface adhesion A (PsaA).    
   
   
       21 . The method of  claim 13 , comprising intranasal or intramuscular administration of said N-palmitoyl D-erythro sphingosyl carbamoyl-spermine together with said biologically active molecule.  
   
   
       22 . A vaccine comprising a combination of a sphingoid-polyalkylamine conjugate and an amount of a biologically active molecule, the amount of said biologically active molecule, when combined with said sphingoid-polyalkylamine conjugate, being effective to stimulate or enhance an immune response of a subject to provide protection against an infection caused by an agent selected from the group consisting of HBV, AIV, the bacterium  Bacillus anthracis  and the bacterium  Streptococcus pneumoniae.    
   
   
       23 . The vaccine of  claim 22 , further comprising an immunostimulating agent.  
   
   
       24 . The vaccine of  claim 22 , wherein said sphingoid-polyalkylamine conjugate comprises a sphingoid backbone carrying, via a carbamoyl linkage at lest one polyalkylamine chain.  
   
   
       25 . The vaccine of  claim 22 , wherein said sphingoid backbone is selected from ceramide, dihydroceramide, phytoceramide, dihydrophytoceramide, ceramine, dihydroceramine, phytoceramine, dihydrophytoceramine.  
   
   
       26 . The vaccine of  claim 22 , wherein said sphingoid is ceramide and said polyalkylamine chain is selected from spermine, spermidine or a polyalkylamine analog of spermine or spermidine.  
   
   
       27 . The vaccine of  claim 22 , wherein said sphingoid-polyalkylamine conjugate comprises N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (CCS).  
   
   
       28 . The vaccine of  claim 22 , wherein: 
 (i) when said infection is caused by HBV, said biologically active molecule is an HBsAg particle;    (ii) when said infection is caused by avian influenza virus, said biologically active molecule is an inactivated purified whole avian influenza virus;    (iii) when said infection is caused by the bacterium  Bacillus anthracis , said biologically active molecule is a protective antigen (PA) moiety of anthrax toxin or    (iv) said  Streptococcus pneumoniae  antigen is a polysaccharide or protein conjugate of an antigen selected from pneumococcal surface protein A (PspA), pneumococcal adherence and virulence factor A (PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding protein A (CbpA), pneumococcal surface adhesion A (PsaA).    
   
   
       29 . A vaccine comprising a combination of N-palmitoyl D-erythro sphingosyl carbamoyl-spermine (CCS), being a single isomer of CCS or mixture of CCS isomers, with a biologically active molecule selected from the group consisting of HBsAg particle; an inactivated purified whole avian influenza virus comprising haemagglutinin (H5) antigen; the protective antigen (PA) moiety of anthrax toxin and pneumococcal surface protein A (PspA), or pneumococcal surface adhesion A (PsaA).  
   
   
       30 . The vaccine of  claim 22 , wherein said sphingoid-polyalkylamine conjugate has the following formula (I):  
     
       
         
         
             
             
         
       
       wherein  
       R 1  represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R 5 ;  
       R 2  and R 5  represent, independently, a branched or linear C 10 -C 24  alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups;  
       R 3  and R 4  are independently a hydrogen or a group —C(O)—NR 6 R 7 , R 6  and R 7  being the same or different for R 3  and R 4  and represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; wherein said R 4  and R 3  are not simultaneously a hydrogen; or  
       R 3  and R 4  form together with the oxygen atoms to which they are bound a heterocyclic ring comprising —C(O)—NR 9 —[R 8 —NR 9 ] m —C(O)—, R 8  represents a saturated or unsaturated C 1 -C 4  alkyl and R 9  represents a hydrogen or a polyalkylamine of the formula —[R 8 —NR 9 ],-, wherein said R 9  or each alkylamine unit R 8 NR 9  may be the same or different in said polyalkylamine; and  
       n and m, represent independently an integer from 1 to 10;  
       W represents a group selected from —CH═CH—, —CH 2 —CH(OH)— or —CH 2 —CH 2 —.  
     
   
   
       31 . A complex comprising a sphingoid-polyalkylamine conjugate and a biologically active molecule, the complex being capable of enhancing or stimulating an immune response of a subject to provide protection against an infection caused by an agent selected from HBV, AIV, the bacterium  Bacillus anthracis  or the bacterium  Streptococcus pneumoniae.    
   
   
       32 . The complex of  claim 31 , comprising N-palmitoyl D-erythro sphingosyl carbamoyl spermine (CCS) associated with said biologically active molecule.  
   
   
       33 . A method for the treatment or prevention of a disease or disorder comprising administering to a subject in need of the treatment a composition comprising a biologically active molecule and a sphingoid-polyalkylamine conjugate having the general formula (I′):  
     
       
         
         
             
             
         
       
       wherein  
       R 1  represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R 5 ;  
       R 2  and R 5  represent, independently, a branched or linear C 10 -C 24  alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups;  
       R 3  is a group —C(O)—NR 6 R 7 , R 6  and R 7  represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium;  
       W represents a group selected from —CH═CH—, —CH 2 —CH(OH)— or —CH 2 —CH 2 —.  
     
   
   
       34 . The method of  claim 33 , for stimulating or enhancing an immune response of a subject to provide protection against an infection.  
   
   
       35 . The method of  claim 33 , wherein said sphingoid-polyalkylamine conjugate is N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine.  
   
   
       36 . The method of  claim 33 , wherein said composition comprises a mixture of a first sphingoid-polyalkylamine conjugate of said formula (I′) and a second sphingoid-polyalkylamine conjugate of the following formula (I):  
     
       
         
         
             
             
         
       
       wherein  
       R 1  represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R 5 ;  
       R 2  and R 5  represent, independently, a branched or linear C 10 -C 24  alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups;  
       R 3  and R 4  are independently a group —C(O)—NR 6 R 7 , R 6  and R 7  being the same or different for R 3  and R 4  and represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; or R 3  is a hydrogen; or  
       R 3  and R 4  form together with the oxygen atoms to which they are bound a heterocyclic ring comprising —C(O)—NR 9 —[R 8 —NR 9 ] m —C(O)—, R 8  represents a saturated or unsaturated C 1 -C 4  alkyl and R 9  represents a hydrogen or a polyalkylamine of the formula —[R 8 —NR 9 ] n —, wherein said R 9  or each alkylamine unit R 8 NR 9  may be the same or different in said polyalkylamine; and  
       n and m represent independently an integer from 1 to 10;  
       W represents a group selected from —CH═CH—, —CH 2 —CH(OH)— or —CH 2 —CH 2 —.  
     
   
   
       37 . The method of  claim 36 , wherein said mixture comprises a first sphingoid-polyalkylamine conjugate being N-palmitoyl-D-erythro-sphingosyl-3-carbamoyl spermine and a second sphingoid-polyalkylamine conjugate being N-palmitoyl-D-erythro-sphingosyl-1-carbamoyl spermine.