CATIONIC OIL-IN-WATER EMULSIONS

• US 20140220083A1
• Filed: 07/06/2012
• Published: 08/07/2014
• Est. Priority Date: 07/06/2011
• Status: Active Grant

• Current Assignee: Glaxosmithkline Biologicals SA (GlaxoSmithKline PLC)
• Original Assignee: Glaxosmithkline Biologicals SA (GlaxoSmithKline PLC)
• Inventors: Brito, Luis, Chan, Michelle, Geall, Andrew, O'Hagan, Derek, Singh, Manmohan
• Granted Patent: US 9,636,410 B2
• Time in Patent Office: Days
• US Class Current: 424/400
• CPC Class Codes: A61K 2039/5256 expressing foreign proteins
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• CPC Class Codes: A61K 2039/55555 Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• CPC Class Codes: A61K 2039/55566 Emulsions, e.g. Freund's adjuvant, MF59
• CPC Class Codes: A61K 2039/575 humoral response
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/155 Paramyxoviridae, e.g. parainfluenza virus
• CPC Class Codes: A61K 39/245 Herpetoviridae, e.g. herpes simplex virus
• CPC Class Codes: A61K 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/06 Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
• CPC Class Codes: A61K 47/12 Carboxylic acids; Salts or anhydrides thereof
• CPC Class Codes: A61K 47/22 Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• CPC Class Codes: A61K 47/24 containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• CPC Class Codes: A61K 47/26 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 Class Codes: A61K 47/34 Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers A61K47/10 takes precedence
• CPC Class Codes: A61K 9/1075 Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers A61K9/0026 takes precedence
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2710/16134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2710/16171 : Demonstrated in vivo effect
• CPC Class Codes: C12N 2710/16734 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2710/16771 : Demonstrated in vivo effect
• CPC Class Codes: C12N 2760/18534 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2760/18571 : Demonstrated in vivo effect
• CPC Class Codes: C12N 2770/36134 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/36143 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2840/203 : having an IRES
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

This invention generally relates to cationic oil-in-water emulsions that contain high concentrations of cationic lipids and have a defined oil:lipid ratio. The cationic lipid can interact with the negatively charged molecule thereby anchoring the molecule to the emulsion particles. The cationic emulsions described herein are useful for delivering negatively charged molecules, such as nucleic acid molecules to cells, and for formulating nucleic acid-based vaccines.

Claims

1. An oil-in-water emulsion comprising particles that are dispersed in an aqueous continuous phase, wherein the average diameter of said particles is from about 80 nm to 180 nm;

the emulsion comprises an oil and a cationic lipid, and

wherein;

(i) the ratio of oil;

lipid (mole;

mole) is at least about 8;

1 (mole;

mole);

(ii) the concentration of cationic lipid in said emulsion is at least about 2.5 mM; and

(iii) the cationic lipid is not DC-Cholesterol.

2. The oil-in-water emulsion of claim 1, wherein the average diameter of said particles is from about 80 nm to about 150 nm or from about 80 nm to about 130 nm.

5. The oil-in-water emulsion of claim 1, wherein the ratio of oil:

lipid (mole;

mole) is from about 10;

1 (mole;

mole) to about 43;

1 (mole;

mole).

6. The oil-in-water emulsion of claim 1, wherein said oil-in-water emulsion comprises from about 0.2% to about 8% (w/v) oil.

7. The oil-in-water emulsion of claim 6, wherein said oil is squalene or squalane.

11. The oil-in-water emulsion of claim 1, wherein the cationic oil-in-water emulsion further comprises from about 0.01% to about 2.5% (v/v) surfactant.

12. The oil-in-water emulsion of claim 11, wherein said surfactant is SPAN85 (Sorbtian Trioleate), Tween 80 (polysorbate 80), or a combination thereof.

13. The oil-in-water emulsion of claim 12, wherein the cationic oil-in-water emulsion comprises about 0.5% (v/v) Tween 80 and about 0.5% (v/v) SPAN85.

15. The oil-in-water emulsion of claim 1, wherein the cationic lipid is selected from the group consisting of:

1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

18. The oil-in-water emulsion of claim 15, wherein the cationic lipid is DOTAP and the concentration of DOTAP in said emulsion is from about 2.58 mM (1.8 mg/mL) to about 7.16 mM (5 mg/mL).

19. A method for preparing the oil-in-water emulsion of claim 1, comprising:

(a) directly dissolving the cationic lipid in the oil to form an oil phase;

(b) providing an aqueous phase of the emulsion; and

(c) dispersing the oil phase in the aqueous phase by homogenization.

20. The method of claim 19, wherein step (a) further comprises heating the oil to a temperature between about 30°

C. to about 65°

C.

44. A method for preparing a composition comprising an RNA molecule complexed with a particle of a cationic oil-in-water emulsion, comprising:

(i) providing an oil-in-water emulsion of claim 1;

(ii) providing an aqueous solution comprising the RNA molecule; and

(iii) combining the oil-in-water emulsion of (i) and the aqueous solution of (ii), thereby preparing the composition.

45. The method of claim 44, wherein the cationic oil-in-water emulsion of (i) and RNA solution of (ii) are combined at about 1:

1 (v/v) ratio.

46. The method of claim 44, wherein the aqueous solution comprising the RNA molecule comprises a salt.

49. The method of claim 44, wherein the aqueous solution comprising the RNA molecule is a buffer.

52. The method of claim 44, wherein the aqueous solution comprising the RNA molecule comprises a nonionic tonicifying agent selected from a sugar or sugar alcohol.

54. The method of claim 52, wherein the nonionic tonicifying agent is selected from the group consisting of sucrose, trehalose, sorbitol, dextrose and combination thereof.

57. The method of claim 44, wherein the aqueous solution comprising the RNA molecule comprises from about 0.05% to about 20% (w/v) polymer.

60. The method of claim 57, wherein the aqueous solution comprises about 1% (w/v) Pluronic®

F127.

163. The oil-in-water emulsion or composition of claim 1 further comprising an antioxidant.

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21. A composition comprising an RNA molecule complexed with a particle of a cationic oil-in-water emulsion, wherein the particle comprises an oil that is in liquid phase at 25°

C., and a cationic lipid;

wherein;

(i) the ratio of oil;

lipid (mole;

mole) is at least about 8;

1 (mole;

mole);

(ii) the concentration of cationic lipid in said composition is at least about 1.25 mM; and

(iii) the cationic lipid is not DC-Cholesterol.

22. The composition of claim 21, wherein the average diameter of said particles is from about 80 nm to 180 nm or from about 80 nm to 150 nm or from about 80 nm to about 130 nm.

26. The composition of claim 21, wherein the N/P ratio of the composition is from about 4:

1 to about 20;

1 or from about 4;

1 to about 15;

1.

28. The composition of claim 21, wherein the ratio of oil:

lipid (mole;

mole) is from about 10;

1 (mole;

mole) to about 43;

1 (mole;

mole).

29. The composition of claim 21, wherein said oil-in-water emulsion comprises from about 0.1% to about 5% (w/v) oil.

30. The composition of claim 29, wherein said oil is squalene or squalane.

34. The composition of claim 21, wherein the cationic oil-in-water emulsion further comprises from about 0.005% to about 1.25% (v/v) surfactant.

35. The composition of claim 34, wherein said surfactant is SPAN85 (Sorbtian Trioleate), Tween 80 (polysorbate 80), or a combination thereof.

38. The composition of claim 21, wherein said cationic lipid is selected from the group consisting of:

1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

41. The composition of claim 21, wherein the cationic lipid is DOTAP and the concentration of DOTAP in said composition is from about 1.29 mM (0.9 mg/mL) to about 3.58 mM (2.5 mg/mL).

42. The composition of claim 21, wherein the RNA molecule is a self-replicating RNA molecule that encodes an antigen.

43. The composition of claim 42, wherein the self-replicating RNA is an alphavirus-derived RNA replicon.

80. The composition of claim 21, wherein the composition is buffered and has a pH of about 6.0 to about 8.0.

81. The composition of claim 21, wherein the composition further comprises an inorganic salt, and the concentration of inorganic salt is no greater than 30 mM.

82. The composition of claim 21, wherein the composition further comprises a nonionic tonicifying agent, and is isotonic.

96. A method of generating an immune response in a subject, comprising administering to a subject in need thereof the composition of claim 21.

97. The method of claim 96, wherein the total amount of the cationic lipid administered to the subject in a single administration is no more than about 30 mg.

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61. An oil-in-water emulsion comprising particles that are dispersed in an aqueous continuous phase, wherein the emulsion comprises an oil and a cationic lipid, the average diameter of said particles is from about 80 nm to 180 nm, the oil is present from 0.6% to 4% (w/v);

and the concentration of cationic lipid in said emulsion is at least about 1.25 mM.

63. The oil-in-water emulsion of claim 61, wherein the concentration of cationic lipid in said emulsion is at least about 2.5 mM.

64. The oil-in-water emulsion of claim 61, wherein the oil is squalene or squalane.

68. The oil-in-water emulsion of claim 61, wherein the cationic oil-in-water emulsion further comprises from about 0.01% to about 2.5% (v/v) surfactant.

69. The oil-in-water emulsion of claim 68, wherein said surfactant is SPAN85 (Sorbtian Trioleate), Tween 80 (polysorbate 80), or a combination thereof.

71. The oil-in-water emulsion of claim 61, wherein the cationic lipid is selected from the group consisting of:

1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

73. A composition comprising an RNA molecule complexed with a particle of the cationic oil-in-water emulsion of claim 61.

75. The composition of claim 73, wherein the N/P ratio of the composition is from about 4:

1 to about 20;

1 or from about 4;

1 to about 15;

1.

77. The composition of claim 73, wherein the RNA molecule is a self-replicating RNA molecule that encodes an antigen.

78. The composition of claim 77, wherein the self-replicating RNA is an alphavirus-derived RNA replicon.

84. A composition comprising an RNA molecule complexed with a particle of a cationic oil-in-water emulsion, wherein the composition is prepared using a process that comprises:

(i) providing an oil-in-water emulsion of claim 61;

(ii) providing an aqueous solution comprising the RNA molecule; and

(iii) combining the oil-in-water emulsion of (i) and of the aqueous solution (ii), thereby preparing the composition.

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