Method of producing lipid nanoparticles for drug delivery

Patent metadata

Field	Value
Publication number	US-9579338-B2
Application number	US-201213669078-A
Country	US
Kind code	B2
Filing date	Nov 5, 2012
Priority date	Nov 4, 2011
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

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 lipid nanoparticle encapsulating a RNA molecule, comprising the steps of
(a) transferring to a mixing container a first aqueous buffer, pH 3.5-6.5, comprising the RNA molecule at 0.08 to 0.8 mg/ml;
(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 at a constant rate for 1-100 minutes while stirring the aqueous solution until a mixture comprising 25-45% (v:v) organic solvent is reached;
(c) diluting the organic solvent in the mixture to less than 10% (v:v); and
(d) removing the organic solvent from the diluted mixture by diafiltration against a second aqueous buffer, pH 6.5-8;
wherein step (b) is performed batchwise, and wherein the mixture comprises lipid nanoparticles having a RNA:lipid ratio 0.06 to 0.16 (w:w), and a RNA:lipid charge ratio of 1:2.5 to 1:1.

2. The method of claim of 1 , wherein the second aqueous buffer 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 lipid nanoparticle encapsulating the RNA molecule.

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

6. The method of claim 1 , wherein 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 lipids further comprise a targeting lipid.

10. The method of claim 1 , wherein the first aqueous solution and the second aqueous buffer are at 25-55° C.

11. The method of claim 1 , wherein the first aqueous solution comprises citrate.

12. The method of claim 5 , wherein the ethanol solution is added to the aqueous solution by injection to an air-water interface.

13. The method of claim 5 , wherein the ethanol solution is added to the aqueous solution by a submerged injection.

14. A pharmaceutical formulation comprising a lipid nanoparticle encapsulating a RNA molecule produced by a process of claim 1 wherein the nanoparticle does not require mechanical processing to obtain a polydispersity index of less than 0.2.

15. The pharmaceutical formulation of claim 14 , wherein 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 targeting lipid.

19. The pharmaceutical formulation of claim 14 , wherein the process further comprises lyophilization of the liposome-encapsulated RNA molecule.

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

21. The pharmaceutical formulation of claim 14 , 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 lipid nanoparticle encapsulating the RNA molecule is 50-100 nm in size.

23. The pharmaceutical formulation of claim 14 , wherein the lipid nanoparticle encapsulating the RNA molecule has a polydispersity index less than 0.2.

Assignees

• Nitto Denko Corp

Inventors

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

Classifications

• A61K47/26

  Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin · CPC title

• A61K31/7105 Primary

  Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links · CPC title

• A61K31/713

  Double-stranded nucleic acids or oligonucleotides · 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

• A61K9/10

  Dispersions; Emulsions · CPC title