Lipids

Abstract

Bipolar lipids are described which are able to form complexes with polyanions. The lipids comprise a cationic head linked to a hydrophobic backbone and a hydrophilic tail and are capable of self assembly to form stable complexes in aqueous solutions. The lipids are of particular use for the delivery of bioactive substances such as nucleic acids to cells in vitro and especially in vivo.

1 . A bipolar lipid comprising a cationic head (1) a hydrophobic backbone (2) and a hydrophilic tail (3) in which: 
 (A) the cationic head comprises two or more cationic centres, each centre being covalently linked to one or more others by one or more carbon containing spacer groups;    (B) the hydrophobic backbone comprises one or more hydrocarbon chains; and    (C) the hydrophilic tail comprises one or more hydrophilic hydrocarbons each containing two or more atoms or groups capable of being solvated by water;    each of said components (1) to (3) being covalently linked head (1) to backbone (2) to tail (3) and arranged such that at least one hydrocarbon chain in the hydrophobic backbone (2) is covalently linked to a carbon atom of a spacer group in the cationic head (1) and each hydrophilic hydrocarbon in the hydrophilic tail (3) is covalently linked to a chain in the backbone (2) to achieve at least a ten atom spacing along the chain between the tail (3) and the head (1)    
     
     
         2 . A lipid according to  claim 1  wherein each cationic centre is an amino group.  
     
     
         3 . A lipid according to  claim 1  or  claim 2  wherein the number of cationic centres in the cationic head is from three to six.  
     
     
         4 . A lipid according to any one of  claim 1  to  claim 3  wherein each carbon containing spacer group is an optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or heteroaromatic group.  
     
     
         5 . A lipid according to any one of  claim 1  to  claim 4  wherein each hydrocarbon chain in the hydrophobic backbone is an optionally substituted straight or branched aliphatic or heteroaliphatic chain containing from ten to around one hundred chain-linked atoms.  
     
     
         6 . A lipid according to  claim 5  wherein the hydrophobic backbone has one or two hydrocarbon chains indirectly linked through a linker atom or group to a carbon atom in a spacer group connecting two cationic centres in the cationic head (1).  
     
     
         7 . A lipid according to any of  claim 1  to  claim 5  wherein each hydrophilic hydrocarbon in the hydrophilic tail (3) is attached to a hydrocarbon chain of the hydrocarbon backbone (2) at the terminal carbon atom of said chain distal to the chain carbon atom attached to the cationic head (1).  
     
     
         8 . A lipid according to any of the preceding claims which has the formula (1): 
       [R 1 ] m —(L 1 ) n —[—C(R 2 )(R 3 )(R 4 )]  (1) 
       wherein R 1  is a hydrocarbon chain optionally substituted by one or more hydrophilic hydrocarbons each containing two or more atoms or groups capable of being solvated by water, provided that at least one hydrocarbon chain is substituted by at least one hydrophilic hydrocarbon and each hydrophilic hydrocarbon is attached to the hydrocarbon chain to achieve at least a ten atom spacing along the chain between the hydrophilic hydrocarbon and the group —(L 1 ) n —[—C (R 2 )(R 3 )(R 4 )]; 
 m is an integer from 1 to 6;  
 L 1  is a linker atom or group;  
 n is zero or the integer 1;  
 —[—C(R 2 )(R 3 )(R 4 )] is a cationic head in which R 2  is a hydrogen atom or an optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, hetero-cycloaliphatic, aromatic or heteroaromatic group optionally containing one or more cationic centres, and R 3  and R 4  which may be the same or different is each an optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or heteroaromatic group containing one or more cationic centres, or R 3  and R 4  together with the carbon atom to which they are attached form a cycloaliphatic, heterocycloaliphatic, aromatic or heteroaromatic group containing two or more cationic centres;  
 and the salts, solvates and hydrates thereof.  
 
     
     
         9 . A lipid according to  claim 8  which has the formula (1a): 
       [R 7 ] p —(L 3 ) q —[R 6 ] m —(L 1 ) n —[—C(R 2 )(R 3 )(R 4 )]  (1a) wherein R 2 , R 3 , R 4 , L 1 , m and n are as defined for formula (1);    R 6  is a hydrocarbon chain;    L 3  is a linker atom or group;    R 7  is a hydrophilic hydrocarbon containing two or more atoms or groups capable of being solvated by water;    q is zero or an integer from one to six;    p is an integer from one to six;    and the salts, solvates and hydrates thereof, provided that each R 7  or L 3  group, when present, is attached to a group R 6  to achieve at least a ten atom spacing along R 6  between R 7  or L 3  and the group —(L 1 ) n —[C(R 2 )(R 3 )(R 4 )].    
     
     
         10 . A lipid according to  claim 9  wherein R 2  is a hydrogen atom and R 3  and R 4  is each a group Sp 1 [WSp 2 ] b WSp 3  or —Sp 1 [WSp 2 ] b WH in which Sp 1 , Sp 2  and Sp 3 , which may be the same or different, is each a spacer group, W is a cationic centre and b is zero or an integer from one to six.  
     
     
         11 . A lipid according to  claim 10  where Sp 1 , Sp 2  and Sp 3  is each an optionally substituted aliphatic, cycloaliphatic, aromatic or heteroaromatic group.  
     
     
         12 . A lipid according to  claim 11  wherein Sp 1 , Sp 2  and Sp 3  is each an optionally substituted C 1-6 alkylene chain.  
     
     
         13 . A lipid according to any one of  claim 9  to  claim 12  wherein W is a —NH— group.  
     
     
         14 . A lipid according to any one of  claim 9  to  claim 13  wherein b is an integer from 1 to 3.  
     
     
         15 . A lipid according to  claim 9  wherein the group —C(R 2 )(R 3 )(R 4 ) is a group —CH[Sp 1 NHSp 2 NH 2 ] 2 , —CH[Sp 1 NHSp 2 NHSp 2 NH 2 ] 2  or —CH[Sp 1 NHSp 2 NHSp 2 NHCH 3 ] 2  wherein Sp 1  is —CH 2 — and each Sp 2  is —(CH 2 ) 3 — or —(CH 2 ) 4 —.  
     
     
         16 . A lipid according to any one of  claim 9  to  claim 15  wherein n in —(L 1 ) n — is the integer 1.  
     
     
         17 . A lipid according to  claim 16  wherein L 1  is a group —X 1 Alk 2 — or —[X 1 ] 2 Alk 1 X 1 Alk 2 — in which X 1  is an —O— or —S— atom or a —C(O)—, —C(O)O—, —C(S)—, —S(O), —S(O) 2 — —N(R 5 )—, [where R 5  is a hydrogen atom, straight or branched alkyl group such as a methyl or ethyl group or an —Alk 1 X 1 — chain], —CON(R 5 )—, —OC(O)N(R 5 )—, —CSN(R 5 )—, —N(R 5 )CO—, N(R 5 )C(O)O—, —N(R 5 )CS—, —S(O)N(R 5 )—, —S(O) 2 N(R 5 )—, —N(R 5 )S(O)—, —N(R 5 )S(O) 2 —, —N(R 5 )CON(R 5 )—, or —N(R 5 )SO 2 N(R 5 )— group [where any of these groups contains two R 5  substituents these may be the same or different]; and Alk 1  and Alk 2  which may be the same or different is each an optionally substituted straight or branched C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene chain optionally interrupted or terminated by one or more, e.g. one, two or three, carbocyclic or heterocarbocyclic groups and/or heteroatoms or heteroatom containing groups X 1  as just defined.  
     
     
         18 . A lipid according to  claim 17  wherein X 1  is a —CONH— group, Alk 1  is a —CH 2 —CH< chain and Alk 2  is a —(CH 2 ) 4 —, —(CH 2 )5— or —(CH 2 ) 6 — chain.  
     
     
         19 . A lipid according to any one of  claim 9  to  claim 18  wherein m is an integer 1 or 2.  
     
     
         20 . A lipid according to any one of  claim 9  to  claim 19  wherein R 6  is an optionally substituted C 10-60 aliphatic chain.  
     
     
         21 . A lipid according to  claim 20  wherein R 6  is a linear, optionally substituted C 16-38 alkylene chain.  
     
     
         22 . A lipid according to any one of  claim 9  to  claim 21  wherein q is the integer 1 and p is the integer 1 or 2.  
     
     
         23 . A lipid according to any one of  claim 9  to  claim 22  wherein L 3  is an atom or group —X 1 —, —X 1 Alk 1 X 1 — or [X 1 Alk 1 ] 1 X 1 Alk 2 X 1  in which X 1  is an —O— or —S— atom or a —C(O)—, —C(O)O—, —C(S)—, —S(O), —S(O) 2 — —N(R 5 )—, [where R 5  is a hydrogen atom, straight or branched alkyl group such as a methyl or ethyl group or an —Alk 1 X 1 — chain], —CON(R 5 )—, —OC(O)N(R 5 )—, —CSN(R 5 )—, —N(R 5 )CO—, N(R 5 )C(O)O—, —N(R 5 )CS—, —S(O)N(R 5 )—, —S(O) 2 N(R 5 )—, —N(R 5 )S(O)—, —N(R 5 )S(O) 2 —, —N(R 5 )CON(R 5 )—, or —N(R 5 )SO 2 N(R 5 )— group [where any of these groups contains two R 5  substituents these may be the same or different]; and Alk 1  and Alk 2  which may be the same or different is each an optionally substituted straight or branched C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene chain optionally interrupted or terminated by one or more, e.g. one, two or three, carbocyclic or heterocarbocyclic groups and/or heteroatoms or heteroatom containing groups X 1  as just defined.  
     
     
         24 . A lipid according to  claim 23  wherein L 3  is a —NHCO—, —CONH—, —CONH(CH 2 ) 2 NHCO—, or —[CONH(CH 2 ) 2 —] 2 NCO(CH 2 ) 2 CONH group.  
     
     
         25 . A lipid according to any one of  claim 9  to  claim 24  wherein R 7  is a synthetic or naturally occurring polyol or a poly(alkylene oxide) or a derivative thereof.  
     
     
         26 . A lipid according to  claim 25  wherein R 7  is a poly(alkylene oxide) or a derivative thereof.  
     
     
         27 . A lipid according to  claim 26  wherein R 7  is a poly(ethylene oxide).  
     
     
         28 . A lipid according to any one of the Examples herein.  
     
     
         29 . A lipid complex comprising a bipolar lipid according to any one of the preceding claims in association with one or more bioactive substances.  
     
     
         30 . A complex according to  claim 29  wherein each bioactive substance is a bioactive protein, peptide, polysaccharide, nucleic acid, oligonucleotide or a derivative thereof, lipid, glycolipid, lipoprotein, lipopolysaccharide or viral, bacterial, protozoal, cellular or tissue fraction.  
     
     
         31 . A complex according to  claim 30  wherein the bioactive substance is a polyanion.  
     
     
         32 . A complex according to  claim 31  wherein the bioactive substance is a nucleic acid.  
     
     
         33 . A complex according to any one of  claims 29  to  32  containing two or more different bipolar lipids.  
     
     
         34 . A complex according to  claim 33  wherein one bipolar lipid has a hydrophilic tail formed by a poly(alkylene oxide) or a derivative thereof and each of the others has a hydrophilic tail formed by a synthetic or naturally occurring polyol.  
     
     
         35 . A complex according to  claim 34  wherein the poly(alkylene oxide) is poly(ethylene oxide).  
     
     
         36 . A composition comprising a complex according to any one of  claim 29  to  claim 35  and one or more other lipids.  
     
     
         37 . A composition according to  claim 36  wherein each other lipid is a neutral or cationic lipid.  
     
     
         38 . A composition comprising a complex according to any one of  claim 29  to  claim 35  and one or more pharmaceutically acceptable carriers, excipients or diluents.  
     
     
         39 . A method of preparing a lipid as claimed in any one of  claims 1  to  29 , comprising coupling: 
 (A) a cationic head comprising two or more cationic centres, each centre being covalently linked to one or more others by one or more carbon containing spacer groups;  
 (B) a hydrophobic backbone comprising one or more hydrocarbon chains; and  
 (C) a hydrophilic tail comprising one or more hydrophilic hydrocarbons each containing two or more atoms or groups capable of being solvated by water, wherein starting materials A), B) and C) contain one or more reactive functional groups suitable for facilitating coupling.  
 
     
     
         40 . A method of preparing a lipid as claimed in any one of  claims 1  to  29 , comprising the step of deprotecting a protected derivative of said lipid.  
     
     
         41 . A method of preparing a complex as claimed in any one of  claims 30  to  35  comprising mixing a lipid as claimed in any one of  claims 1  to  29  with a bioactive substance.  
     
     
         42 . Use of a complex as claimed in any one of  claims 30  to  35  for delivering a bioactive substance to cells in vitro.  
     
     
         43 . A composition as claimed in any one of  claims 36  to  38  for use in delivering a bioactive substance to cells in vivo.  
     
     
         44 . A composition as claimed in any one of  claims 36  to  38  for use as a medicament.  
     
     
         45 . Use of a composition as claimed in any one of  claims 36  to  38  for the preparation of a medicament for the delivery of a bioactive substance, preferably a therapeutic, diagnostic or immunomodulatory agent.  
     
     
         46 . Method of delivering a bioactive substance to a human or non-human animal wherein said bioactive substance, preferably a therapeutic, diagnostic or immunomodulatory agent, is administered in the form of a complex as defined in any one of  claims 30  to  35 .