Method of producing lipid nanoparticles for drug delivery

Patent metadata

Field	Value
Publication number	US-10155945-B2
Application number	US-201715430387-A
Country	US
Kind code	B2
Filing date	Feb 10, 2017
Priority date	Nov 4, 2011
Publication date	Dec 18, 2018
Grant date	Dec 18, 2018

Abstract

What is described is a method for preparing a liposome that efficiently encapsulates a negatively charged therapeutic polymer, e.g., siRNA. The process involves preparing a lipid mixture comprising a cationic lipid in a water miscible organic solvent, such as ethanol, at a concentration of 2.3 mg/ml, and adding this solution to the polymer dissolved in water to a final concentration of 35% ethanol in water. The final charge ratio of drug:lipid is 1:2.5. The resulting nanoparticles have a mean size of 50 to 150 nm.

First claim

What is claimed:

1. A method for preparing a population of lipid nanoparticles encapsulating a nucleic acid molecule, comprising the steps of
(a) transferring to a mixing container an aqueous solution comprising a buffer and the nucleic acid molecule;
(b) injecting a lipid solution comprising a cationic lipid, a helper lipid, a sterol, and a PEG lipid dissolved in a water-miscible organic solvent into the aqueous solution in the mixing container while stirring the aqueous solution;
(c) diluting the organic solvent in the mixing container by adding an aqueous buffer to prevent aggregation of the lipid nanoparticles in a diluted mixture; and
(d) removing the organic solvent from the diluted mixture to produce the population of lipid nanoparticles having a polydispersity index of less than 0.2 and an average size of 50 to 150 nm;

wherein step (b) is performed batchwise.

2. The method of claim of 1 , wherein the aqueous solution further comprises a polysaccharide.

3. The method of claim 2 , wherein the polysaccharide consists of sucrose, trehalose, mannitol, sorbitol, xylitol, lactose, maltose, or inulin.

4. The method of claim of 1 , further comprising the step of lyophilizing the population of lipid nanoparticles.

5. The method of claim 1 , wherein the water-miscible organic solvent is ethanol.

6. The method of claim 1 , wherein the population of lipid nanoparticles has a nucleic acid:lipid ratio 0.06 to 0.16 (w:w), and a nucleic acid:lipid charge ratio of 1:2.5 to 1:1, and the cationic lipid is 40 to 60 mole percent of the lipids.

7. The method of claim 1 , wherein the cationic lipid is selected from the group consisting of

8. The method of claim 1 , wherein the cationic lipid is selected from an ionizable cationic lipid or permanently charged cationic lipid.

9. The method of claim 1 , wherein the lipid solution further comprises a compound selected from
a compound of the formula (A)
L-X—R  (A)

wherein
lipid (L) is selected from the group consisting of DSPE, DOPE, and DC;
linker (X) is selected from the group consisting of nothing, PEG550, PEG2000, PEG-glutamate (-Glu), Glu, C6, glycine, and GluNH, N1,N19-bis(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-4,7,10,13,16-pentaoxanonadecane-1,19-diamide; and
retinoid (R) is selected from the group consisting of tretinoin, adapalene, retinol, 4-hydroxy(phenyl)retinamide (4-HPR), retinoic acid, 9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,2,6-trimethylcyclohexyl)nonanoic acid, and any partially or fully saturated retinoid;

a compound of the formula (B)
R—X—R  (B)

wherein
linker (X) is N1,N19-bis(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-4,7,10,13,16-pentaoxanonadecane-1,19-diamide or N1,N19-bis(16,20-diamino-15-oxo-4,7,10-trioxa-14-azaicosyl)-4,7,10,13,16-pentaoxanonadecane-1,19-diamide; and
retinoid (R) is selected from the group consisting of tretinoin, adapalene, retinol, 4-hydroxy(phenyl)retinamide (4-HPR), and retinoic acid, 9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,2,6-trimethylcyclohexyl)nonanoic acid, and any partially or fully saturated retinoid.

10. The method of claim 1 , wherein the population of lipid nanoparticles is prepared at 25-55° C.

11. The method of claim 1 , wherein the aqueous solution comprises citrate, pH 3.5-6.5.

12. The method of claim 1 , wherein the lipid solution is added to the aqueous solution by injection to an air-water interface.

13. The method of claim 1 , wherein the lipid solution is added to the aqueous solution by a submerged injection.

14. A pharmaceutical formulation comprising the population of lipid nanoparticles produced by the method of claim 1 .

15. The pharmaceutical formulation of claim 14 , wherein the population of lipid nanoparticles has a nucleic acid:lipid ratio 0.06 to 0.16 (w:w), and a nucleic acid:lipid charge ratio of 1:2.5 to 1:1, and the cationic lipid is 40 to 60 mole percent of the lipids.

16. The pharmaceutical formulation of claim 14 , wherein the cationic lipid is selected from the group consisting of

17. The pharmaceutical formulation of claim 14 , wherein the cationic lipid is selected from an ionizable cationic lipid or permanently charged cationic lipid.

18. The pharmaceutical formulation of claim 14 , wherein the lipids further comprise a compound selected from a compound of the formula (A)
L-X—R  (A)

wherein
lipid (L) is selected from the group consisting of DSPE, DOPE, and DC;
linker (X) is selected from the group consisting of nothing, PEG550, PEG2000, PEG-glutamate (-Glu), Glu, C6, glycine, and GluNH, N1,N19-bis(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-4,7,10,13,16-pentaoxanonadecane-1,19-diamide; and
retinoid (R) is selected from the group consisting of tretinoin, adapalene, retinol, 4-hydroxy(phenyl)retinamide (4-HPR), retinoic acid, 9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,2,6-trimethylcyclohexyl)nonanoic acid, and any partially or fully saturated retinoid;

a compound of the formula (B)
R—X—R  (B)

wherein
linker (X) is N1,N19-bis(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-4,7,10,13,16-pentaoxanonadecane-1,19-diamide or N1,N19-bis(16,20-diamino-15-oxo-4,7,10-trioxa-14-azaicosyl)-4,7,10,13,16-pentaoxanonadecane-1,19-diamide; and
retinoid (R) is selected from the group consisting of tretinoin, adapalene, retinol, 4-hydroxy(phenyl)retinamide (4-HPR), and retinoic acid, 9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nonanoic acid, 3,7-dimethyl-9-(2,2,6-trimethylcyclohexyl)nonanoic acid, and any partially or fully saturated retinoid.

19. The pharmaceutical formulation of claim 14 , wherein the process further comprises lyophilization of the population of lipid nanoparticles.

20. The pharmaceutical formulation of claim 14 , further comprising a polysaccharide.

21. The pharmaceutical formulation of claim 20 , wherein the polysaccharide consists of sucrose, trehalose, mannitol, sorbitol, xylitol, lactose, maltose, or inulin.

22. The pharmaceutical formulation of claim 14 , wherein the mean particle diameter of the population of lipid nanoparticles is 50-100 nm.

23. The pharmaceutical formulation of claim 14 , wherein the population of lipid nanoparticles has a polydispersity index less than 0.15.

Assignees

• Nitto Denko Corp

Inventors

• Knopov Victor
• Witte Richard P
• Karmali Priya
• Lee Robin
• Webb David
• Akopian Violetta

Classifications

• A61K9/0019

  Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00) · CPC title

• A61K47/12

  Carboxylic acids; Salts or anhydrides thereof · CPC title

• A61K9/1272

  comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543}) · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• A61K9/1277

  Preparation processes; Proliposomes · CPC title