Process for formulating an anionic agent
Formulations comprising anionic agents such as nucleic acids within a lipid-containing particle methods of formulating a lipid-containing particle comprising an anionic agent such as a nucleic acid, methods for preparing a lipid-containing particle comprising an anionic agent such as a nucleic acid, methods for therapeutic delivery of an anionic agent to a patient in need thereof, where the anionic agent is formulated in a lipid-containing particle as described herein.
1. A method of producing a particle comprising a first lipid or sterol, a second lipid or sterol and an anionic agent comprising:
(a) combining said second lipid or sterol with an alcohol to form a second lipid or sterol solution in said alcohol, wherein said alcohol is free of said first lipid or sterol;
(b) adding said second lipid or sterol solution in said alcohol of step (a) to said first lipid or sterol, thereby causing a concentration of said alcohol in said first lipid or sterol to increase from 0% to up to no more than 40% (v/v) as more of said second lipid or sterol solution in said alcohol of step (a) is added to said first lipid or sterol to form a solution of the second lipid or sterol and the first lipid or sterol, wherein the solubility of said first lipid or sterol in said alcohol in the presence of said second lipid or sterol is higher than the solubility of said first lipid or sterol in said alcohol in the absence of said second lipid or sterol; and
(c) combining the solution of the second lipid or sterol and the first lipid or sterol of step (b) with a complex-anionic agent comprising (i) a modified lipid which prevents particle aggregation during lipid-anionic agent particle formation, (ii) a cationic lipid, in an amount sufficient for a complex to form, and (iii) an anionic agent,
thereby producing a particle comprising a first lipid or sterol, a second lipid or sterol and an anionic agent.
2. The method of claim 1 , wherein said solution of the second lipid or sterol and the first lipid or sterol comprises at least one lipid selected from the group consisting of a neutral lipid, a sterol, a cationic lipid and a modified lipid which prevents particle aggregation during lipid-anionic agent particle formation.
3. The method of claim 1 , wherein said particle possesses a property selected from the group consisting of improved size and/or PDI, improved efficacy in a subject administered said particle and improved tolerability in a subject administered said particle, as compared to an appropriate control particle formed by an appropriate control process that comprises exposing said first lipid or sterol to said alcohol before said second lipid or sterol is exposed to said alcohol.
4. The method of claim 1 , wherein said anionic agent is a nucleic acid.
5. The method of claim 4 , wherein said nucleic acid is selected from the group consisting of an antisense oligonucleotide and a double-stranded nucleic acid.
6. The method of claim 5 , wherein said double-stranded nucleic acid is selected from the group consisting of a small hairpin RNA (shRNA) and a siRNA.
7. The method of claim 6 , wherein said double-stranded nucleic acid is a substrate for human Dicer.
8. The method of claim 1 , wherein said first lipid or sterol is a sterol selected from the group consisting of cholesterol, cholestanone, cholestenone, coprostanol, 3β-[-(N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol) and bis-guanidium-tren-cholesterol (BGTC).
9. The method of claim 1 , wherein said alcohol is ethanol.
10. A method of producing a particle comprising a first lipid or sterol, a second lipid or sterol and an anionic agent comprising:
(a) combining in an acidic aqueous solution (i) a modified lipid which prevents particle aggregation during lipid-anionic agent particle formation and (ii) a cationic lipid, in an amount sufficient for a complex to form;
(b) combining the complex of step (a) with an anionic agent;
(c) combining a neutral aqueous solution with the complex-anionic agent of step (b) to form a complex-anionic agent aqueous suspension;
(d) combining said second lipid or sterol with an alcohol to form a second lipid or sterol solution in said alcohol, wherein said alcohol is free of said first lipid or sterol;
(e) adding said second lipid or sterol solution in said alcohol to said first lipid or sterol, thereby causing a concentration of said alcohol in said first lipid or sterol to increase from 0% to up to no more than 40% (v/v) as more of said second lipid or sterol solution in said alcohol of step (d) is added to said first lipid or sterol to form a solution of the second lipid or sterol and the first lipid or sterol; and
(f) combining the solution of the second lipid or sterol and the first lipid or sterol of step (e) with the complex-anionic agent aqueous solution of step (c),
thereby producing a particle comprising a first lipid or sterol, a second lipid or sterol and an anionic agent.
11. The method of claim 10 , wherein the solubility of said first lipid or sterol in said alcohol in the presence of said second lipid or sterol in step (e) is higher than the solubility of said first lipid or sterol in said alcohol in the absence of said second lipid or sterol.
12. The method of claim 10 , wherein step (f) is performed by adding the solution of the second lipid or sterol and the first lipid or sterol of step (d) to the complex-anionic agent aqueous suspension of step (c) or in-line mixing of the solution of the second lipid or sterol and the first lipid or sterol of step (d) and the complex-anionic agent aqueous solution of step (c).
13. The method of claim 10 , wherein said first lipid or sterol is a sterol selected from the group consisting of cholesterol, cholestanone, cholestenone, coprostanol, 3β-[-(N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol) and bis-guanidium-tren-cholesterol (BGTC).
14. The method of claim 10 , wherein said alcohol is ethanol.
15. The method of claim 10 , wherein said acidic aqueous solution of step (a) comprises HCl.
16. The method of claim 10 , wherein said modified lipid is a polyethylene glycol (PEG)-, PEG-ceramide, or ganglioside-modified lipid.
17. The method of claim 10 , wherein the cationic lipid is one or more of DODMA, DOTMA, or a cationic lipid selected from the group consisting of L-1 to L-49:
L-1
L-2
L-3
L-4
L-5
L-6
L-7
L-8
L-9
L-10
L-11
L-12
L-13
L-14
L-15
L-16
L-17
L-18
L-19
L-20
L-21
L-22
L-23
L-24
L-25
L-26
L-27
L-28
L-29
L-30
L-31
L-32
L-33
L-34
L-35
L-36
L-37
L-38
L-39
L-40
L-41
L-42
L-43
L-44
L-45
L-46
L-47
L-48
L-49
18. The method of claim 10 , wherein the cationic lipid has the formula:
wherein each R 1 and R 2 is, independently, optionally substituted C 11-24 alkyl, optionally substituted C 11-24 alkenyl, optionally substituted C 11-24 alkynyl, optionally substituted C 11-24 heteroalkyl, optionally substituted C 11-24 heteroalkenyl, or optionally substituted C 11-24 heteroalkynyl; each n1 and n2 is, independently, an integer from 0 to 2; and R 5 is selected from the group consisting of H, optionally substituted C 1-6 alkyl, and optionally substituted heterocyclyl.
19. The method of claim 10 , wherein the cationic lipid has the formula:
wherein each R 1 and R 2 is, independently, optionally substituted C 11-24 alkyl, optionally substituted C 11-24 alkenyl, optionally substituted C 11-24 alkynyl, optionally substituted C 11-24 heteroalkyl, optionally substituted C 11-24 heteroalkenyl, or optionally substituted C 11-24 heteroalkynyl; R 3 is H or optionally substituted C 1-6 alkyl; L 1 is optionally substituted C 1-6 alkylene; and each R 5 and R 6 is, independently, H or optionally substituted C 1-6 alkyl, or where R 5 and R 6 combine to form an optionally substituted C 3-7 heterocyclyl.
20. The method of claim 10 , wherein the neutral aqueous solution of step (c) is water.