Granted Patent B2

US 10,369,226 · App. 14/677,801 · Granted Aug 6, 2019

Abstract

Claims (98)

1. A compound of the formula:

or a salt thereof, wherein

R′ is absent, hydrogen, or C 1 -C 4 alkyl;

with respect to R 1 and R 2 ,

(i) R 1 and R 2 are each, independently, optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R 10 ;

(ii) R 1 and R 2 , together with the nitrogen atom to which they are attached, form an optionally substituted heterocylic ring; or

(iii) one of R 1 and R 2 is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10 member heterocyclic ring or heteroaryl with (a) the adjacent nitrogen atom and (b) the (R) a group adjacent to the nitrogen atom;

each occurrence of R is, independently, —(CR 3 R 4 )—;

each occurrence of R 3 and R 4 are, independently H, halogen, OH, alkyl, alkoxy, —NH 2 , R 10 , alkylamino, or dialkylamino;

each occurrence of R 10 is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits, (iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) the compound of formula (IIIA) has at most two R 10 groups;

the dashed line to Q is absent or a bond;

when the dashed line to Q is absent then Q is absent or is —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R 4 )—, —N(R 5 )C(O)—, —S—S—, —OC(O)O—, —O—N═C(R 5 )—, —C(R 5 )═N—O—, —OC(O)N(R 5 )—, —N(R 5 )C(O)N(R 5 )—, —N(R 5 )C(O)O—, —C(O)S—, —C(S)O— or —C(R 5 )═N—O—C(O)—; or

when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms;

each occurrence of R 5 is, independently, H or alkyl;

M 1 and M 2 are each, independently, a biodegradable group selected from —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R 5 )═N—, —N═C(R 5 )—, —C(R 5 )═N—O—, —O—N═C(R 5 )—, —C(O)(NR 5 )—, —N(R 5 )C(O)—, —C(S)(NR 5 )—, —N(R 5 )C(O)—, —N(R 5 )C(O)N(R 5 )—, —OC(O)O—, —OSi(R 5 ) 2 O—, —C(O)(CR 3 R 4 )C(O)O—, —OC(O)(CR 3 R 4 )C(O)—, or

wherein R 11 is a C 2 -C 8 alkyl or alkenyl;

each occurrence of R z is, independently, C 1 -C 8 alkyl;

a is 1, 2, 3, 4, 5 or 6;

b is 0, 1, 2, or 3;

L 1 and L 2 are each, independently, C 1 -C 5 alkylene or C 2 -C 5 alkenylene;

X and Y are each, independently, alkylene or alkenylene; and

Z 1 and Z 2 are each, independently, C 8 -C 14 alkyl or C 8 -C 14 alkenyl; wherein the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z 1 or Z 2 , and with the proviso that the terminus of at least one of Z 1 and Z 2 is separated from the group M 1 or M 2 by at last 8 carbon atoms.

2. The compound of claim 1 , wherein L 1 and L 2 are each —(CH 2 ) 2 —.

3. The compound of claim 1 , wherein L 1 and L 2 are each —(CH 2 ) 3 —.

4. The compound of claim 1 , wherein M 1 and M 2 are —C(O)O— (where the carbonyl group in M 1 and M 2 is bound to the variable X, and the oxygen atom in M 1 and M 2 is bound to the variable L′ and L 2 ).

5. The compound of claim 1 , wherein the R′R 1 R 2 N—(R) a -Q-(R) b — group is selected from (CH 3 ) 2 N—(CH 2 ) 3 —C(O)O—, (CH 3 ) 2 N—(CH 2 ) 2 —NH—C(O)O—, (CH 3 ) 2 N—(CH 2 ) 2 —OC(O)—NH—, and (CH 3 ) 2 N—(CH 2 ) 3 —C(CH 3 )═N—O—.

6. The compound of claim 1 , wherein Z 1 , Z 2 , and each R z are C 3 -C 8 alkyl.

7. The compound of claim 6 , wherein each R z is, independently, methyl, ethyl, isopropyl, n-butyl, n-pentyl or n-hexyl.

8. A cationic lipid or a salt thereof having:

(i) a central carbon atom,

ii) a nitrogen containing head group directly bound to the central carbon atom, and

(iii) two hydrophobic tails directly bound to the central carbon atom,

wherein

each hydrophobic tail is of the formula —R e -M-R f where R e is a C 4 -C 14 alkyl or alkenyl,

M is a biodegradable group selected from —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R 5 )═N—, —N═C(R 5 )—, —C(R 5 )═N—O—, —O—N═C(R 5 )—, —C(O)(NR 5 )—, —N(R 5 )C(O)—, —C(S)(NR 5 )—, —N(R 5 )C(O)—, —N(R 5 )C(O)N(R 5 )—, —OC(O)O—, —OSi(R 5 ) 2 O—, —C(O)(CR 3 R 4 )C(O)O—, —OC(O)(CR 3 R 4 )C(O)—, or

wherein

each occurrence of R 3 and R 4 are, independently H, halogen, OH, alkyl, alkoxy, —NH 2 , R 10 , alkylamino, or dialkylamino;

each occurrence of R 5 is, independently, H or C 1 -C 4 alkyl;

R 11 is a C 2 -C 8 alkyl or alkenyl, and

R f is a branched alkyl or alkenyl, such that (i) the total length of each tail from the first carbon atom after the central carbon atom to a terminus of the tail is at most 20 atoms, and (ii) the group —R e -M-R f has at least 20 carbon atoms, wherein

the alkyl or alkenyl group in R e is optionally substituted with one or two fluorine atoms at the alpha position to the M 1 or M 2 group; and

the alkenyl group in R f is optionally substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of R f .

9. The cationic lipid of claim 8 , wherein the cationic lipid is in the form of a pharmaceutically acceptable salt.

10. A lipid particle comprising a neutral lipid, a lipid capable of reducing aggregation, and a cationic lipid of claim 8 .

11. The lipid particle of claim 10 , wherein the neutral lipid is selected from DSPC, DPPC, POPC, DOPE, or SM; the lipid capable of reducing aggregation is a PEG lipid; and the lipid particle further comprises a sterol.

12. The lipid particle of claim 10 , wherein the cationic lipid is present in a mole percentage of about 20% and about 60%; the neutral lipid is present in a mole percentage of about 5% to about 25%; the sterol is present in a mole percentage of about 25% to about 55%; and the PEG lipid is PEG-DMA, PEG-DMG, or a combination thereof, and is present in a mole percentage of about 0.5% to about 15%.

13. The lipid particle of claim 10 , wherein the lipid capable of reducing aggregation is PEG-DMG.

14. The lipid particle of claim 13 , wherein the lipid particle comprises about 50 mole % of the cationic lipid, about 10% DSPC, about 38.5% cholesterol, and about 1.5% PEG-DMG (based on 100% of the lipid components in the lipid particle).

15. The lipid particle of claim 10 , further comprising an active agent.

16. The lipid particle of claim 15 , wherein the active agent is a nucleic acid selected from a plasmid, an immunostimulatory oligonucleotide, an siRNA, an antisense oligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme.

17. A pharmaceutical composition comprising a lipid particle of claim 10 and a pharmaceutically acceptable carrier.

18. A method of modulating the expression of a target gene in a cell, comprising providing to the cell a lipid particle of claim 10 .

19. The method of claim 18 , wherein the active agent is a nucleic acid is an siRNA.

20. A method of treating a disease or disorder characterized by the overexpression of a polypeptide in a subject, comprising providing to the subject in need thereof the pharmaceutical composition of claim 17 , wherein the active agent is a nucleic acid selected from the group consisting of an siRNA, a microRNA, and an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense oligonucleotide includes a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.

21. A method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising providing to the subject in need thereof the pharmaceutical composition of claim 17 , wherein the active agent is a plasmid that encodes the polypeptide or a functional variant or fragment thereof.

22. A method of inducing an immune response in a subject, comprising providing to the subject in need thereof the pharmaceutical composition of claim 17 , wherein the active agent is an immunostimulatory oligonucleotide.

23. A compound of the formula:

or a salt thereof, wherein

R′ is absent, hydrogen, or C 1 -C 4 alkyl;

with respect to R 1 and R 2 ,

(i) R 1 and R 2 are each, independently, optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R 10 ;

(ii) R 1 and R 2 , together with the nitrogen atom to which they are attached, form an optionally substituted heterocylic ring; or

(iii) one of R 1 and R 2 is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10 member heterocyclic ring or heteroaryl with (a) the adjacent nitrogen atom and (b) the (R) a group adjacent to the nitrogen atom;

each occurrence of R is, independently, —(CR 3 R 4 )—;

each occurrence of R 3 and R 4 are, independently H, halogen, OH, alkyl, alkoxy, —NH 2 , R 10 , alkylamino, or dialkylamino;

each occurrence of R 10 is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits, (iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) the compound of formula (IIIA) has at most two R 10 groups;

the dashed line to Q is absent or a bond;

when the dashed line to Q is absent then Q is absent or is —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R 4 )—, —N(R 5 )C(O)—, —S—S—, —OC(O)O—, —O—N═C(R 5 )—, —C(R 5 )═N—O—, —OC(O)N(R 5 )—, —N(R 5 )C(O)N(R 5 )—, —N(R 5 )C(O)O—, —C(O)S—, —C(S)O— or —C(R 5 )═N—O—C(O)—; or

when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms;

each occurrence of R 5 is, independently, H or alkyl;

M 1 and M 2 are each, independently, a biodegradable group selected from —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R 5 )═N—, —N═C(R 5 )—, —C(R 5 )═N—O—, —O—N═C(R 5 )—, —C(O)(NR 5 )—, —N(R 5 )C(O)—, —C(S)(NR 5 )—, —N(R 5 )C(O)—, —N(R 5 )C(O)N(R 5 )—, —OC(O)O—, —OSi(R 5 ) 2 O—, —C(O)(CR 3 R 4 )C(O)O—, —OC(O)(CR 3 R 4 )C(O)—, or

wherein R 11 is a C 2 -C 8 alkyl or alkenyl;

each occurrence of R z is, independently, C 1 -C 8 alkyl;

a is 1, 2, 3, 4, 5 or 6;

b is 0, 1, 2, or 3;

L 1 and L 2 are each, independently, straight chain C 1 -C 5 alkylene or C 2 -C 5 alkenylene;

X and Y are each, independently, alkylene or alkenylene; and

Z 1 and Z 2 are each, independently, C 1 -C 8 alkyl or C 2 -C 8 alkenyl;

wherein the compound is not one selected from:

24. The compound of claim 23 , wherein L 1 and L 2 are each —(CH 2 ) 2 —.

25. The compound of claim 23 , wherein L 1 and L 2 are each —(CH 2 ) 3 —.

26. The compound of claim 23 , wherein M 1 and M 2 are —C(O)O— (where the carbonyl group in M 1 and M 2 is bound to the variable X, and the oxygen atom in M 1 and M 2 is bound to the variable L 1 and L 2 ).

27. The compound of claim 23 , wherein the R′R 1 R 2 N—(R) a -Q-(R) b — group is selected from (CH 3 ) 2 N—(CH 2 ) 3 —C(O)O—, (CH 3 ) 2 N—(CH 2 ) 2 —NH—C(O)O—, (CH 3 ) 2 N—(CH 2 ) 2 —OC(O)—NH—, and (CH 3 ) 2 N—(CH 2 ) 3 —C(CH 3 )═N—O—.

28. The compound of claim 23 , wherein the compound is in the form of a cationic lipid.

29. The compound of claim 23 , wherein the compound is in the form of a pharmaceutically acceptable salt.

30. A lipid particle comprising a neutral lipid, a lipid capable of reducing aggregation, and a cationic lipid of claim 28 .

31. The lipid particle of claim 30 , wherein the neutral lipid is selected from DSPC, DPPC, POPC, DOPE, or SM; the lipid capable of reducing aggregation is a PEG lipid; and the lipid particle further comprises a sterol.

32. The lipid particle of claim 30 , wherein the cationic lipid is present in a mole percentage of about 20% and about 60%; the neutral lipid is present in a mole percentage of about 5% to about 25%; the sterol is present in a mole percentage of about 25% to about 55%; and the PEG lipid is PEG-DMA, PEG-DMG, or a combination thereof, and is present in a mole percentage of about 0.5% to about 15%.

33. The lipid particle of claim 30 , wherein the lipid capable of reducing aggregation is PEG-DMG.

34. The lipid particle of claim 33 , wherein the lipid particle comprises about 50 mole % of the cationic lipid, about 10% DSPC, about 38.5% cholesterol, and about 1.5% PEG-DMG (based on 100% of the lipid components in the lipid particle).

35. The lipid particle of claim 30 , further comprising an active agent.

36. The lipid particle of claim 35 , wherein the active agent is a nucleic acid selected from a plasmid, an immunostimulatory oligonucleotide, an siRNA, an antisense oligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme.

37. A pharmaceutical composition comprising a lipid particle of claim 30 and a pharmaceutically acceptable carrier.

38. A method of modulating the expression of a target gene in a cell, comprising providing to the cell a lipid particle of claim 30 .

39. The method of claim 38 , wherein the active agent is a nucleic acid is an siRNA.

40. A method of treating a disease or disorder characterized by the overexpression of a polypeptide in a subject, comprising providing to the subject in need thereof the pharmaceutical composition of claim 37 , wherein the active agent is a nucleic acid selected from the group consisting of an siRNA, a microRNA, and an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense oligonucleotide includes a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.

41. A method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising providing to the subject in need thereof the pharmaceutical composition of claim 37 , wherein the active agent is a plasmid that encodes the polypeptide or a functional variant or fragment thereof.

42. A method of inducing an immune response in a subject, comprising providing to the subject in need thereof the pharmaceutical composition of claim 37 , wherein the active agent is an immunostimulatory oligonucleotide.

Assignments (5)

Cited By (9)

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