Cationic oil-in-water emulsions

• US 9,636,410 B2
• Filed: 07/06/2012
• Issued: 05/02/2017
• 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
• Primary Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US14/130,886
• Publication Number:
  US 20140220083A1
• Time in Patent Office: 1,761 Days
• Field of Search: 4241931, 42419611, 424400, 424450
• 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. A stable 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 150 nm, wherein the average diameter does not change by more than 10% when the emulsion is stored at 4°

C. for one month; and

wherein the emulsion comprises an oil and a cationic lipid, wherein the head group of the cationic lipid comprises a quaternary amine, 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 130 nm.

3. 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).

4. 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.

5. The oil-in-water emulsion of claim 4, wherein said oil is squalene or squalane.

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

7. The oil-in-water emulsion of claim 6, wherein said surfactant is SPAN85 (SorbitanTrioleate), Tween 80 (polysorbate 80), or a combination thereof.

8. The oil-in-water emulsion of claim 7, wherein the oil-in-water emulsion comprises about 0.5% (v/v) Tween 80 and about 0.5% (v/v) SPAN85.

9. 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).

10. The oil-in-water emulsion of claim 9, 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).

11. 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.

12. The method of claim 11, wherein step (a) further comprises heating the oil to a temperature between about 30°

C. to about 65°

C.

13. The oil-in-water emulsion of claim 1 further comprising an antioxidant.