OIL MIXTURES / SURFACTANT FOR SELF-EMULSIFICATION

专利摘要:
Oil-in-water emulsions with small droplet sizes can be formed without requiring either microfluidization or heating to cause phase inversion, but rather by a simple mixing of a premixed composition of oil and a component surfactant comprising at least one surfactant component with aqueous material. The BHL value of the surfactant component can be chosen to provide a composition which, when mixed with an excess volume of aqueous material, spontaneously forms an oil-in-water emulsion with submicron oil droplets having a diameter <250 nm , suitable for sterilization by filtration. Droplet diameters <40 nm can also be obtained.
公开号:BE1022714B1
申请号:E2015/5157
申请日:2015-03-17
公开日:2016-08-23
发明作者:Luis Brito；Stephanie Kay Dodd；Derek O'hagan；Manmohan Singh
申请人:Glaxosmithkline Biologicals S.A.；
IPC主号:

专利说明:

OIL MIXTURES / SURFACTANT FOR SELF-EMULSIFICATION
This application claims the priorities of European Patent Application No. 14160391.0, filed March 17, 2014, and European Patent Application No. 14167083.6, filed May 5, 2014, the entirety of which is incorporated herein. by reference for all objects.
Technical area
This invention relates to improved methods of making oil-in-water emulsions having small particle sizes of oil droplets, for example, which are useful as vaccine adjuvants.
BACKGROUND OF THE INVENTION The vaccine adjuvant known as "MF5 9" [1-3] is a submicron oil-in-water emulsion of squalene, polysorbate 80 (also known as Tween 80), and sorbitan trioleate (also known as Span 85). It may also include citrate ions, for example, 10 mM sodium citrate buffer. The composition of the volume emulsion can be about 5% squalene, about 0.5% Tween 80 and about 0.5% Span 85. The adjuvant and its production are described in more detail in the references. 4 (Chapter 10), 5 (Chapter 12) and 6 (Chapter 19). As described in reference 7, it is manufactured commercially as a dispersant of Span 85 in squalene, dispersing Tween 80 in an aqueous phase (citrate buffer), and then mixing these two phases to form a coarse emulsion. which is then microfluidized. The emulsion is prepared at double concentration and is diluted 1/1 (by volume) with the vaccine in question. The emulsion adjuvant known as "AS03" [8] is prepared by mixing an oily mixture (consisting of squalene and α-tocopherol) with an aqueous phase (Tween 80 and buffer), followed by microfluidization (9) is also prepared at double concentration The emulsion adjuvant known as "AF03" is prepared by cooling an emulsion water in the preheated oil until it crosses its temperature. phase reversal of the emulsion, at which point it thermoretrically converts to an oil-in-water emulsion [10] The "AF03" emulsion comprises squalene, sorbitan oleate, ether polyoxyethylene cetostearyl and mannitol Mannitol, cetostearyl ether and phosphate buffer are mixed in one container to form an aqueous phase, while the sorbitan ester and squalene are mixed in another container to form an oily component The ph The aqueous solution is added to the oily component and the mixture is then heated to ~ 60 ° C and cooled to provide the final emulsion. The emulsion is initially prepared with a composition of 32.5% squalene, 4.8% sorbitan oleate, 6.2% polyoxyethylene cetostearyl ether and 6% mannitol, which is at least 4x final concentration.
As has been demonstrated above, the prior art methods known for producing emulsions suitable for use as adjuvants require either vigorous mechanical methods (such as homogenization and microfluidization) or relatively high temperatures (for example, in a phase inversion temperature method) to obtain the small sizes of oil droplets required for the activity of the adjuvant. The use of these methods is associated with several disadvantages, for example, high manufacturing costs. Summary of the invention
Therefore, it is an object of the present invention to provide other improved and (for example, simpler) methods for the production of submicron oil-in-water emulsions. In particular, an object of the present invention is to provide methods that are suitable for use on a commercial scale and that do not require the use of methods involving vigorous mechanical processing or significantly elevated temperatures.
The inventors have found that oil-in-water emulsions with small droplet sizes can be formed without requiring either microfluidization or heating to cause phase inversion, but rather by simply mixing a premixed oil composition. and surfactant with an aqueous material. In general, if the oil / surfactant composition contains a larger volume of surfactant than oil then, when mixing with an excess volume of aqueous material, the system spontaneously forms an oil-in-water emulsion with droplets. submicron oil (and even with droplets having a diameter <250 nm, suitable for sterilization by filtration). These emulsions exhibit adjuvant activity even if they have different compositions from known adjuvants.
In a first aspect, the present invention provides a pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm (e.g. between 40 and 250 nm, between 90 and 240 nm, for example from 100 to 220 nm), said composition comprising at least one biocompatible, metabolizable oil (such as squalene); and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 2.5 to 9.0, and wherein the oil / surfactant composition is substantially free of aqueous components. This oil / surfactant composition contrasts with the sorbitan squalene / trioleate composition that is used to make MF59 (see Figure 1 of Ref. 11) in which the surfactant component has a BHL of less than 2.
In a second aspect, the present invention provides an oil-in-water emulsion comprising the oil / surfactant composition according to the first aspect of the invention in combination with an aqueous phase, wherein said emulsion has an average particle diameter of oil less than 250 nm. In general, when compared to microfluidized emulsions having equivalent oil droplet sizes and distributions, such emulsions will have slightly higher surfactant / oil weight ratios, for example, greater than 1: 4.3. for an emulsion similar to MF59, and more than 1 / 4.7 for an emulsion similar to AS03.
In a third aspect, the present invention provides a method of forming an oil-in-water emulsion comprising at least one biocompatible, metabolizable oil; and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 2.5 to 9.0 and wherein the emulsion has an average particle diameter of oil less than 250 nm; said method comprising: providing an oil / surfactant composition according to the first aspect of the invention; providing an aqueous phase; combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition; and the light mixture of the diluted composition to form an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm.
In a fourth aspect, the present invention provides a pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of not more than 40. nm, said composition comprising at least one biocompatible, metabolizable oil; and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 9.0 to 14.0 and wherein the oil / surfactant composition is substantially free of aqueous components.
In a fifth aspect, the present invention provides an oil-in-water emulsion comprising the oil / surfactant composition according to the fourth aspect of the invention in combination with an aqueous phase, wherein said emulsion has an average particle diameter of oil which is not greater than 40 nm.
In a sixth aspect, the present invention provides a method of forming an oil-in-water emulsion comprising at least one biocompatible, metabolizable oil; and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 9.0 to 14.0 and wherein the emulsion has an average oil particle diameter of not greater than 40 nm; said method comprising: providing an oil / surfactant composition according to the fourth aspect of the invention; providing an aqueous phase; combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition; and the light mixture of the diluted composition to form an oil-in-water emulsion having an average particle diameter of oil which is not greater than 40 nm. The invention also provides oil-in-water emulsions of the invention for use as additives.
In another aspect, the present invention provides an immunogenic composition comprising an oil-in-water emulsion according to the present invention, and an antigen component.
In one aspect, the present invention provides a process for preparing an immunogenic composition, said method comprising mixing an oil-in-water emulsion according to the present invention with an antigen component.
In another aspect, the present invention provides a kit comprising: an oil / surfactant composition according to the present invention; an aqueous phase; and optionally instructions for combining the oil / surfactant composition and the aqueous phase.
According to embodiments of the invention, the oil / surfactant composition and / or the aqueous phase may comprise an antigen component.
In another aspect, the invention provides a method of preparing a kit comprising the steps of: providing an oil / surfactant composition according to the present invention; and conditioning the composition in a kit as a kit component together with an aqueous phase; and optionally conditioning an antigen component in the kit as a kit component together with the oil / surfactant composition and the aqueous phase.
The present invention also provides a kit comprising: an oil-in-water emulsion according to the present invention; and an antigen component.
The present invention further provides a method of preparing a kit comprising the steps of: providing an oil-in-water emulsion according to the present invention; and packaging the emulsion in a kit as a kit component together with a separate antigen component.
The present invention also provides a dry material (e.g., a lyophilizate) which, when reconstituted with an aqueous phase, provides an oil-in-water emulsion according to the present invention. As used herein, "dry material" and "dried material" mean a material that is substantially free of water or substantially free of an aqueous phase. The invention also proposes a process for preparing a dried emulsion, comprising: (i) obtaining an oil-in-water emulsion of the invention; and (ii) drying the emulsion to provide the dried emulsion. This dried material can be reconstituted in an emulsion of the invention by combining it with a suitable aqueous carrier. Suitable drying techniques are described below.
The present invention also provides a kit for preparing an oil-in-water emulsion according to the present invention, wherein the kit comprises: an oil-in-water emulsion according to the present invention in dried form; and an aqueous phase.
Embodiments of the present invention are set forth in the following numbered clauses: 1. A pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm, said composition comprising (i) at least one biocompatible, metabolizable oil; and (ii) a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 2.5 to 9.0, and wherein the oil / surfactant composition is substantially free of aqueous components. 2. The oil / surfactant composition according to clause 1, wherein the surfactant component has a BHL value of 3.3 to 8.4. 3. The oil / surfactant composition according to Clause 1 or Clause 2, wherein the volume ratio of the oil / surfactant component is 1/1 to 1/10. 4. The oil / surfactant composition of any of Clauses 1 to 3, wherein the surfactant component comprises at least two surfactants. The oil / surfactant composition of any one of Clauses 1 to 4, wherein the surfactant component comprises a first surfactant having a BHL value of less than 10 and a second surfactant having a BHL value of at least The oil / surfactant composition of any of Clauses 1 to 5, wherein the surfactant component comprises a first surfactant having a BHL value of 1 to 4 and a second surfactant having a BHL value of The oil / surfactant composition of any one of Clauses 1 to 6, wherein the oil / surfactant composition comprises squalene, sorbitan trioleate and polysorbate 80. oil / surfactant according to clause 7, wherein the combined% by volume of sorbitan trioleate and polysorbate 80 is 50 to 90% of the total volume of the oil / surfactant composition. 9. The oil / surfactant composition according to Clause 7 or Clause 8, wherein the combined% by volume of squalene and polysorbate 80 is 20 to 70% of the total volume of the oil / surfactant composition. The oil / surfactant composition of any one of clauses 7 to 9, wherein the combined volume percent of squalene and sorbitan trioleate is from 60 to 90 percent of the total volume of the oil composition. surfactant. 11. The oil / surfactant composition according to clause 7, wherein the combined% by volume of sorbitan trioleate and polysorbate 80 is from 50 to 90% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition. 12. The oil / surfactant composition according to clause 7 or clause 11, wherein the combined volume percent of squalene and polysorbate 80 is 20 to 70% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition. The oil / surfactant composition according to any of Clauses 7, 11 or 12, wherein the combined% by volume of squalene and sorbitan trioleate is from 60 to 90% of the total volume of squalene, trioleate sorbitan and polysorbate 80 in the oil / surfactant composition. 14. The oil / surfactant composition according to any one of the clauses 1 to 13, chosen from:
15. An oil-in-water emulsion comprising the oil / surfactant composition according to any one of Clauses 1 to 14 in combination with an aqueous phase, wherein said emulsion has an average particle diameter of oil of less than 250 nm. 16. The oil-in-water emulsion according to clause 15, wherein the emulsion has an average oil particle diameter of 100 to 220 nm. 17. A method of forming an oil-in-water emulsion comprising (i) at least one biocompatible, metabolizable oil; and (ii) a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 2.5 to 9.0 and the emulsion has an average oil particle diameter of less than 250 nm; said method comprising: a) providing an oil / surfactant composition according to any one of clauses 1 to 14; (b) the supply of an aqueous phase; c) combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition; and d) lightly mixing the diluted composition to form an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm. 18. The method according to clause 17, wherein the emulsion has an average oil particle diameter of 100 to 220 nm. 19. The process according to any one of clauses 17 to 18, wherein the process is carried out in the absence of mechanical agitation. 20. The process according to any one of clauses 17 to 19, wherein the light mixture of the diluted composition comprises inverting the mixture by hand or by agitation or by a mechanical device. 21. The process of any one of clauses 17 to 20, wherein the step of combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition comprises: (i) the mixing equal volumes of the oil / surfactant composition and the aqueous phase; and (ii) diluting the mixture of the oil / surfactant composition and the aqueous phase with another volume of aqueous phase to form a diluted composition. 22. The process of any of Clauses 17 to 21, wherein the step of combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition comprises adding the oil / surfactant composition in the aqueous phase. 23. The process of any of Clauses 17 to 22, wherein the oil / surfactant composition is added to an excess volume of the aqueous phase at a temperature below 55 ° C. 24. The process of any one of clauses 17 to 23, wherein the process takes place at a temperature of about 25 ° C. 25. The process of any of Clauses 17 to 24, wherein the ratio of the aqueous phase to the oil / surfactant composition in the diluted composition is from 10/1 to 50/1 by volume, preferably / 1 to 40/1 by volume. 26. The process of any of Clauses 17 to 25, wherein said method further comprises the step of subjecting the oil-in-water emulsion to filtration sterilization. 27. The process according to any one of clauses 17 to 26, wherein the aqueous phase is substantially free of surfactant (s) and / or oil (s). 28. The oil-in-water emulsion according to Clause 15 or Clause 16, for use as an adjuvant. 29. An immunogenic composition comprising an oil-in-water emulsion according to Clause 15 or Clause 16, and an antigenic component. 30. A process for preparing an immunogenic composition comprising mixing an oil-in-water emulsion according to clause 15 or clause 16 with an antigen component. 31. A kit comprising: (i) an oil / surfactant composition according to any one of clauses 1 to 14; (ii) an aqueous phase; and optionally (iii) instructions for combining the oil / surfactant composition and the aqueous phase. 32. The kit according to clause 31, wherein the oil / surfactant composition further comprises an antigenic component. 33. The kit according to clause 31 or 32, wherein the aqueous phase further comprises an antigen component. 34. A process for preparing a kit comprising the following steps: (i) providing an oil / surfactant composition according to any one of clauses 1 to 14; and (ii) conditioning the composition in a kit as a kit component together with an aqueous phase. 35. A method of preparing a kit according to clause 34, further comprising: (iii) conditioning an antigen component in the kit as a kit component together with the oil / surfactant composition and the phase aqueous. 36. A kit comprising: (i) an oil-in-water emulsion in accordance with clause 15 or clause 16; and (ii) an antigen component. 37. A method of preparing a kit comprising the following steps: (i) providing an oil-in-water emulsion according to clause 15 or clause 16; and (ii) conditioning the emulsion in a kit as a kit component together with a separate antigen component. 38. A dried material which, when reconstituted with an aqueous phase, provides an oil-in-water emulsion according to clause 15 or clause 16. 39. The dried material according to clause 38, further comprising an antigenic component in dried form. 40. The dried material according to clause 38 or clause 39, further comprising one or more freeze-drying stabilizers. 41. A kit for the preparation of an oil-in-water emulsion in accordance with clause 15 or clause 16, in which the kit comprises: (i) an oil-in-water emulsion in accordance with clause 15 or clause 16 in the form dried; and (ii) an aqueous phase. 42. The kit according to clause 41, wherein the oil-in-water emulsion in dried form is combined with an antigen component in dried form. 43. A pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of not more than 40 nm, said composition comprising (i) ) at least one biocompatible, metabolizable oil; and (ii) a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 9.0 to 14.0 and wherein the oil / surfactant composition is substantially free of aqueous components. 44. The oil / surfactant composition according to clause 43, wherein the surfactant component has a BHL value of 9.3 to 13.5. 45. The oil / surfactant composition according to Clause 43 or Clause 44, wherein the volume ratio of the oil / surfactant component is 1/1 to 1/10. 46. The oil / surfactant composition according to any one of clauses 43 to 45, wherein the surfactant component comprises at least two surfactants. 47. The oil / surfactant composition according to any one of clauses 43 to 46, wherein the surfactant component comprises a first surfactant having a BHL value of less than 10 and a second surfactant having a BHL value of at least The oil / surfactant composition of any one of clauses 43 to 47, wherein the surfactant component comprises a first surfactant having a BHL value of 1 to 4 and a second surfactant having a BHL value. from 13 to 17. 49. The oil / surfactant composition according to any one of clauses 43 to 48, wherein the oil / surfactant composition comprises squalene, sorbitan trioleate and polysorbate 80. The composition of oil / surfactant according to clause 49, wherein the combined% by volume of sorbitan trioleate and polysorbate 80 is 60 to 90% of the total volume of the oil / surfactant composition. 51. The oil / surfactant composition according to Clause 49 or Clause 50, wherein the combined% by volume of squalene and polysorbate 80 is 70 to 90% of the total volume of the oil / surfactant composition. 52. The oil / surfactant composition of any of Clauses 49 to 51, wherein the combined% by volume of squalene and sorbitan trioleate is 20 to 60% of the total volume of the oil composition. surfactant. 53. The oil / surfactant composition according to clause 49, wherein the combined% by volume of sorbitan trioleate and polysorbate 80 is 60 to 90% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition. 54. The oil / surfactant composition according to Clause 49 or Clause 53, wherein the combined% by volume of squalene and polysorbate 80 is 70 to 90% of the total volume of squalene, sorbitan trioleate and polysorbate. 80 in the oil / surfactant composition. 55. The oil / surfactant composition according to any of Clauses 49, 53 or 54, wherein the combined% by volume of squalene and sorbitan trioleate is from 20 to 60% of the total volume of squalene, trioleate of sorbitan and polysorbate 80 in the oil / surfactant composition. 56. The oil / surfactant composition according to any one of clauses 43 to 55, chosen from:
57. An oil-in-water emulsion comprising the oil / surfactant composition according to any one of clauses 43 to 56 in combination with an aqueous phase, wherein said emulsion has an average particle diameter of oil which is not not greater than 40 nm. 58. The oil-in-water emulsion according to clause 57, wherein the emulsion has an average particle diameter of oil which is not greater than 30 nm. A method of forming an oil-in-water emulsion comprising (i) at least one biocompatible, metabolizable oil; and (ii) a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 9.0 to 14.0 and wherein the emulsion has a mean particle diameter of oil not greater than 40 nm; said method comprising: a) providing an oil / surfactant composition according to any one of clauses 43 to 56, b) providing an aqueous phase; c) combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition; and d) mixing the diluted composition lightly to form an oil-in-water emulsion having an average particle diameter of oil of not more than 40 nm. 60. The process according to clause 59, wherein the emulsion has an average particle diameter of oil which is not greater than 30 nm. 61. The process according to clause 59 or clause 60, where the process is carried out in the absence of mechanical agitation. 62. The process of any one of clauses 59 to 61, wherein the light blend of the diluted composition comprises inverting the mixture by hand or by agitation or by mechanical means. 63. The process of any one of clauses 59 to 62, wherein the step of combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition comprises: (i) the mixing equal volumes of the oil / surfactant composition and the aqueous phase; and (ii) diluting the mixture of the oil / surfactant composition and the aqueous phase with another volume of aqueous phase to form a diluted composition. The process of any one of clauses 59 to 63, wherein the step of combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition comprises adding the oil / surfactant composition in the aqueous phase. 65. The process of any one of clauses 59 to 64, wherein the oil / surfactant composition is added to an excess volume of the aqueous phase at a temperature below 55 ° C. 66. The process according to any one of clauses 59 to 65, wherein the process takes place at a temperature of about 25 ° C. 67. The process according to any one of clauses 59 to 66, wherein the ratio of the aqueous phase to the oil / surfactant composition in the diluted composition is from 10/1 to 50/1 by volume, preferably from 20/1 to 40/1 by volume. 68. The process of any one of clauses 59 to 67, wherein said method further comprises the step of subjecting the oil-in-water emulsion to filtration sterilization. 69. The process according to any one of clauses 59 to 68, wherein the aqueous phase is substantially free of surfactant (s) and / or oil (s).
The oil / surfactant composition
According to the invention, the processes for preparing emulsions make use of an oil / surfactant composition.
This composition is a mixture which comprises at least one oil and a surfactant component comprising at least one surfactant, examples of which are presented in more detail below. The oil / oils and the surfactant (s) are ideally miscible with each other in the composition. The composition may be an oil / surfactant dispersion. If the phases of the oil and the surfactant are completely miscible with each other, the composition will be an oil / surfactant solution.
Because the emulsions of the invention are intended for pharmaceutical use, the oil / oils and surfactant (s) in the composition will be generally metabolizable (biodegradable) and biocompatible. If only one of these two components is metabolizable and biocompatible, it should be oil / oils.
The composition may comprise one or more components in addition to the oil (s) and surfactant (s), but in some embodiments the oil / oils and the surfactant (s) constitute substantially all of the composition. the composition. When other components are included, they may form up to 15% of the composition (by weight), preferably 1 to 15% of the composition (by weight), more preferably 5 to 10%. For example, in some embodiments, the composition comprises one or more pharmacologically active agents, which will usually be lipophilic. Such lipophilic agents include, but are not limited to, vitamins (e.g., vitamins A, D, E and K), carotenoids (e.g., β-carotene), fatty acids (e.g. arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid), pyrimidines (for example, 5-hydroxy-4,6-dimethyl-2- (6-phenylhexyl) aminopyrimidine), ansamitocins, receptor antagonists angiotensin II (eg, candesartan cilexetil), and immunopotentiators (eg, muramyl-dipeptides). Typical lipophilic agents have a positive logP value (partition coefficient measured in 1-octanol and water) at pH 7.4 and 37 ° C, for example, they may have a logP ^ 1,> 2, h 3,> 4,> 5,> 6, h 7, etc.
In some embodiments, the composition may comprise a cholesterol and / or a phospholipid. Suitable classes of phospholipids include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, phosphatidylglycerols, and the like.
In embodiments of the invention, the oil / surfactant composition may comprise an antigen component.
In a first aspect, the present invention provides a pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm, said composition comprising at least one biocompatible, metabolizable oil; and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 2.5 to 9.0, and wherein the oil / surfactant composition is substantially free of aqueous components.
In a fourth aspect, the present invention provides a pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of not more than 40. nm, said composition comprising at least one biocompatible, metabolizable oil; and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 9.0 to 14.0 and wherein the oil / surfactant composition is substantially free of aqueous components.
The volume ratio of the oil / surfactant component in the oil / surfactant composition of the present invention is preferably from 1: 1 to 1: 10.
Preferably, the oil / surfactant composition of the present invention comprises an oil, a surfactant having a BHL value of from 13 to 17, and a surfactant having a BHL value of from 1 to 5. More preferably, the composition The oil / surfactant of the present invention consists essentially of an oil, a surfactant having a BHL value of 13 to 17, and a surfactant having a BHL value of from 1 to 5. In certain embodiments, In that embodiment, the oil / surfactant composition consists of an oil, a surfactant having a BHL value of 13 to 17, and a surfactant having a BHL value of 1 to 5.
According to embodiments of the first aspect of the present invention wherein the oil / surfactant composition comprises an oil, a surfactant having a BHL value of 13 to 17, and a surfactant having a BHL value of 1 to 5: the combined% by volume of the surfactant having a BHL value of 1 to 5 and the surfactant having a BHL value of 13 to 17 in the oil / surfactant composition may be 50 to 90%, preferably 50 to 70% the total volume of the oil / surfactant composition; and / or the combined% by volume of the oil and surfactant having a BHL value of 13 to 17 may be 20 to 70%, preferably 60 to 70% of the total volume of the oil / surfactant composition; and / or the combined volume% of the oil and surfactant having a BHL value of 1 to 5 may be 60 to 90%, preferably 70 to 80% of the total volume of the oil / surfactant composition.
Preferably, the oil / surfactant compositions according to embodiments of the first aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the first aspect of the invention satisfy all three of these criteria.
According to embodiments of the first aspect of the present invention wherein the oil / surfactant composition comprises an oil, a surfactant having a BHL value of 13 to 17, and a surfactant having a BHL value of 1 to 5: the combined% by volume of the surfactant having a BHL value of 1 to 5 and the surfactant having a BHL value of 13 to 17 in the oil / surfactant composition may be 50 to 90%, preferably 50 to 70% total volume of oil, surfactant having a BHL value of 1 to 5 and surfactant having a BHL value of 13 to 17 in the oil / surfactant composition; and / or the combined volume% of the oil and surfactant having a BHL value of 13 to 17 may be 20 to 70%, preferably 60 to 70% of the total volume of oil, surfactant having a value BHL of 1 to 5 and surfactant having a BHL value of 13 to 17 in the oil / surfactant composition; and / or the combined% by volume of the oil and surfactant having a BHL value of 1 to 5 may be 60 to 90%, preferably 70 to 80% of the total volume of oil, surfactant having a value of BHL of 1 to 5 and surfactant having a BHL value of 13 to 17 in the oil / surfactant composition.
Preferably, the oil / surfactant compositions according to embodiments of the first aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the first aspect of the invention satisfy all three of these criteria.
In a particular embodiment, the oil / surfactant composition of the present invention comprises squalene, sorbitan trioleate and polysorbate 80. In another embodiment, the oil / surfactant composition of the present invention comprises essentially, squalene, sorbitan trioleate and polysorbate 80. In embodiments of the present invention, the oil / surfactant composition is composed of squalene, sorbitan trioleate and polysorbate 80.
According to embodiments of the first aspect of the present invention wherein the oil / surfactant composition comprises squalene, sorbitan trioleate and polysorbate 80: the combined% by volume of sorbitan trioleate and polysorbate 80 in the composition oil / surfactant may be 50 to 90%, preferably 50 to 70% of the total volume of the oil / surfactant composition; and / or the combined% by volume of squalene and polysorbate 80 may be from 20 to 70%, preferably 60 to 70% of the total volume of the oil / surfactant composition; and / or the combined volume% of squalene and sorbitan trioleate may be from 60 to 90%, preferably from 70 to 80% of the total volume of the oil / surfactant composition.
Preferably, the oil / surfactant compositions according to embodiments of the first aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the first aspect of the invention satisfy all three of these criteria.
According to embodiments of the first aspect of the present invention in which the oil / surfactant composition comprises squalene, sorbitan trioleate and polysorbate 80: the combined% by volume of sorbitan trioleate and polysorbate 80 may be 50 to 90%, preferably 50 to 70%, of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition; and / or the combined volume% of squalene and polysorbate 80 may be 20 to 70%, preferably 60 to 70%, of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition ; and / or the combined% by volume of squalene and sorbitan trioleate may be 60 to 90%, preferably 70 to 80% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil composition. surfactant.
Preferably, the oil / surfactant compositions according to embodiments of the first aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the first aspect of the invention satisfy all three of these criteria.
According to some embodiments of the first aspect of the present invention, the oil / surfactant composition is selected from:
According to embodiments of the fourth aspect of the present invention wherein the oil / surfactant composition comprises an oil, a surfactant having a BHL value of 13 to 17, and a surfactant having a BHL value of 1 to 5: the combined volume% of the surfactant having a BHL value of 1 to 5 and the surfactant having a BHL value of 13 to 17 in the oil / surfactant composition may be 60 to 90% of the total volume of the composition of the oil / surfactant: and / or the combined% by volume of the oil and surfactant having a BHL value of 13 to 17 in the oil / surfactant composition may be 70 to 90% of the total volume of the composition oil / surfactant; and / or the combined% by volume of the oil and surfactant having a BHL value of 1 to 5 in the oil / surfactant composition may be 20 to 60% of the total volume of the oil / surfactant composition .
Preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the invention satisfy all three of these criteria.
According to embodiments of the fourth aspect of the present invention wherein the oil / surfactant composition comprises an oil, a surfactant having a BHL value of 13 to 17, and a surfactant having a BHL value of 1 to 5: the combined% by volume of the surfactant having a BHL value of from 1 to 5 and the surfactant having a BHL value of from 13 to 17 may be from 60 to 90% of the total volume of oil, surfactant having a BHL value of 1-5 and surfactant having a BHL value of 13-17 in the oil / surfactant composition; and / or the combined volume% of the oil and surfactant having a BHL value of 13 to 17 may be 70 to 90% of the total oil volume, surfactant having a BHL value of 1 to 5 and surfactant having a BHL value of 13 to 17 in the oil / surfactant composition; and / or the combined volume% of the oil and surfactant having a BHL value of 1 to 5 may be 20 to 60% of the total oil volume, surfactant having a BHL value of 1 to 5 and surfactant having a BHL value of 13 to 17 in the oil / surfactant composition.
Preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the invention satisfy all three of these criteria.
According to embodiments of the fourth aspect of the present invention in which the oil / surfactant composition comprises squalene, sorbitan trioleate and polysorbate 80: the combined volume% of sorbitan trioleate and polysorbate 80 may be 60 to 90% of the total volume of the oil / surfactant composition; and / or the combined volume% of squalene and polysorbate 80 is 70 to 90% of the total volume of the oil / surfactant composition; and / or the combined volume% of squalene and sorbitan trioleate is from 20 to 60% of the total volume of the oil / surfactant composition.
Preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the invention satisfy all three of these criteria.
According to embodiments of the fourth aspect of the present invention wherein the oil / surfactant compositions comprise squalene, sorbitan trioleate and polysorbate 80: the combined% by volume of sorbitan trioleate and polysorbate 80 is 60% by weight. at 90% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition; and / or the combined volume% of squalene and polysorbate 80 is 70 to 90% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition; and / or the combined volume% of squalene and sorbitan trioleate is from 20 to 60% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition.
Preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the present invention satisfy at least two of these three criteria. More preferably, the oil / surfactant compositions according to embodiments of the fourth aspect of the invention satisfy all three of these criteria.
According to some embodiments of the fourth aspect of the present invention, the oil / surfactant composition is selected from:
In embodiments of the invention, the oil / surfactant composition is substantially free of aqueous components.
Oily component (s)
The composition comprises one or more oils. Suitable oil / oils include those from, for example, an animal (such as a fish) or plant source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify nut oils. Jojoba oil can be used, for example, obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like can be also used. The 6- to 10-carbon fatty acid esters of glycerol and 1,2-propanediol, while not naturally occurring in seed oils, can be prepared by hydrolysis, separation and esterification of appropriate materials by starting from nut oils and seeds. Fats and oils from mammalian milks are metabolizable and so can be used. The procedures for separation, purification, saponification and other means necessary to obtain pure animal source oils are well known in the state of the art.
Most fish contain metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify many of the fish oils that can be used herein. A number of branched chain oils are synthesized by biochemistry in 5-carbon isoprene units, and are generally referred to as terpenoids. A preferred oil for use according to the invention is squalene, which is a branched unsaturated terpenoid ([(CH 3) 2 C [= CHCH 2 CH 2 C (CH 3)] 2 = chch 2 -] 2; c 30 H 50; 2,6,10,15 , 19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, CAS RN 7683-649). Squalane, the squalene-saturated analogue, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or can be obtained by methods known from the state of the art. Other useful oils are tocopherols, particularly in combination with squalene. When the oily phase of an emulsion comprises a tocopherol, any of the α, β, γ, δ, ε or ξ tocopherols may be used, but α-tocopherols are preferred. D-α-tocopherol and DL-α-tocopherol can both be used. A preferred α-tocopherol is DL-α-tocopherol. An oily combination comprising squalene and a tocopherol (e.g., DL-α-tocopherol) may be used.
The composition of the present invention may comprise a combination of oils, for example, squalene and at least one other oil. When the composition comprises more than one oil, these may be present in various ratios, for example between 1/5 and 5/1 by volume, for example between 1/2 and 2/1, as at equal volumes.
Within the composition, the total quantity of oil ideally does not constitute more than 50% (by volume). As shown in the examples, a higher oil content tends not to promote spontaneous emulsification. In a particular embodiment, the preferred amounts of oil in the composition are between 10 and 50%, more preferably between 25 and 50%. An oil content in the composition of about 20%, or 30% or 40% (by volume) is particularly useful.
Within the composition, the ratio of the total amount of oil to the total amount of surfactant ideally is not more than 1/1. In a particular embodiment, the surfactant is present in a volume in excess of the oil. As shown in the examples, an excess oil content tends not to promote spontaneous emulsification. In a particular embodiment, an oil / surfactant volume ratio is 1/1 to 1/10. The oil / oils in the compositions and emulsions of the invention will generally be metabolizable (biodegradable) and biocompatible.
The surfactant component (s)
The composition comprises a surfactant component comprising one or more surfactants. In a particular embodiment, the surfactant component comprises at least two surfactants. In another embodiment, the surfactant component consists of two surfactants. The surfactant component in the compositions and emulsions of the invention will generally be metabolizable (biodegradable) and biocompatible.
Surfactants can be classified by their "BHL" (Griffin hydrophilic / lipophilic balance), where a BHL in the range of 1 to 10 generally means that the surfactant is more soluble in oil than in water, and BHL in the range of 10 to 20 means that the surfactant is more soluble in water than in oil. The BHL values are readily available for the surfactants of interest, for example, polysorbate 80 ("Tween 80") has a BHL of 15.0 and sorbitan trioleate ("Span 85") has a BHL of 1, 8.
When two or more surfactants are mixed, the resulting BHL of the mixture is easily calculated by the weighted average, for example, a mixture of 70/30% by weight of polysorbate 80 and sorbitan trioleate with a BHL of (15.0%). x 0.70) + (1.8 x 0.30) i.e. 11.04.
According to the first, second and third aspects of the present invention, the surfactant component comprises at least one surfactant and the surfactant component has a BHL value of from 2.5 to 9.0, preferably from 3.3 to 8.4. For example, the surfactant component may have a BHL value of 3.5 to 7.7, 4.0 to 7.5, 4.7 to 7.1 or 5.1 to 6.2.
According to the fourth, fifth and sixth aspects of the present invention, the surfactant component comprises at least one surfactant and the surfactant component has a BHL value of 9.0 to 14.0, preferably 9.3 to 13.5. For example, the surfactant component can have a BHL value of 10.1 to 11.2. In some embodiments, it may even be possible to prepare an emulsion of the invention using a surfactant component with a BHL greater than 14.0, for example, using polysorbate (s) alone. Thus, for example, an AS03-like emulsion may be prepared by applying the methods of the invention to the known non-aqueous components of this emulsion (ie, squalene, α-tocopherol, and polysorbate 80), but with surfactant / oil weight ratio slightly higher than that in AS03. This can be obtained by increasing the proportion of surfactant (polysorbate 80) relative to the oil and / or by decreasing the proportion of oil (squalene and / or α-tocopherol) relative to the surfactant.
When the surfactant component comprises more than one surfactant, then at least one of them will generally have a BHL of at least 10 (for example, in the range of 12 to 16, or 13 to 17) and at least one of them has a BHL less than 10 (for example, in the range of 1 to 9, or 1 to 4). For example, the surfactant component of the composition may comprise polysorbate 80 and sorbitan trioleate. In embodiments of the present invention, the surfactant component comprises a first surfactant having a BHL value of 1 to 5, preferably 1 to 4 and a second surfactant having a BHL value of 13 to 17.
As shown in the examples of the application, the oil / surfactant compositions comprising a surfactant component having a BHL value in the range of 2.5 to 9.0 tend to form oil-in-water emulsions having average particle diameter of oil less than 250 nm, for example, in the range of 100 to 220 nm. The oil / surfactant compositions comprising a surfactant component having a BHL value in the range of 9.0 to 14.0 tend to form emulsions having an even smaller average particle diameter of oil than is not greater at 40 nm, more preferably not more than 30 nm. The invention can be used with various surfactants, including ionic, nonionic and zwitterionic surfactants. Nonionic surfactants are preferred. The invention can thus utilize surfactants including, but not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tween or polysorbates), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the trade name DOWFAX ™, as linear block copolymers of EO / PO; octoxynols, which may vary in the number of ethoxy (oxy-1,2-ethanediyl) repeating groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; 1 '(octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidyl choline (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known as Span), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Many examples of pharmaceutically acceptable surfactants are known from the state of the art for use in the composition and thus in the final emulsion.
In a particular embodiment, the surfactants to be included in the composition are polysorbate 80 (polyoxyethylene sorbitan monooleate) and sorbitan trioleate. If the composition comprises a single surfactant, then this is a surfactant having a BHL value of 13 to 17, preferably polysorbate 80 (BHL value of 15). If the composition comprises two surfactants, a mixture of polysorbate 80 and sorbitan trioleate is preferred. In general terms, the compositions comprising from 20 to 80% (by volume) of sorbitan trioleate have good ability to form an emulsion, while compositions with only 10% (by volume) of sorbitan trioleate behave less well. Similarly, the compositions comprising from 20 to 80% (by volume) of polysorbate 80 have a good ability to form an emulsion, while the compositions with only 10% (by volume) of polysorbate 80 in general have behaved less well. , although their behavior can be improved by ensuring a volume in excess of at least 4 times of sorbitan trioleate.
Another useful surfactant mixture (as observed in "AFO3") is sorbitan oleate and polyoxyethylene cetostearyl ether. Sorbitan monooleate has a BHL of 4.3 while polyoxyethylene cetostearyl ether has a BHL of 13.5.
Within the composition, the total amount of surfactant is ideally at least 50% (by volume). As shown in the examples, a lower surfactant content does not tend to promote spontaneous emulsification. In a particular embodiment, the amounts of surfactant in the composition are between 50 and 90%. In another embodiment, the amounts of surfactant in the composition are between 60 and 90%. A surfactant content in the composition of about 60%, 70%, 80% or 90% (by volume) is particularly useful.
When an oil / surfactant composition comprises a surfactant having a BHL greater than 8, then the concentration of this surfactant is preferably at least 4000 x greater than its critical micelle concentration (CMC), for example, at least 5000 x greater , 6000 x upper, 8000 x upper, 10000 x upper, 12000 x upper, 15000 x upper, or even at least 20000 x greater than the CMC. . The ratio of the total amount of surfactant to the total amount of oil is ideally at least 1/1. In a particular embodiment, the surfactant is present in a volume in excess of the oil. As shown in the examples, an excess oil content does not tend to promote spontaneous emulsification. In a particular embodiment, a volume ratio of surfactant / oil is 1/1 to 10/1. Oil / surfactant mixtures containing squalene
In a particularly preferred embodiment, the oil for use of which the invention is squalene (which will usually be used as the only oily component, but which may be used optionally in combination with another oil such as a-tocopherol ). In combination with a surfactant component comprising or consisting of polysorbate 80 (such as a mixture of polysorbate 80 and sorbitan trioleate), squalene provides useful compositions.
The surfactant component in these compositions may have a BHL value of 10 to 18 (for example, 13 to 17, or in particular embodiments of 14 to 16), or a high BHL surfactant may be mixed with a surfactant lower BHL to provide a surfactant component with a BHL value of 6 to 11 or 2.5 to 9.0 (for example, 3.3 to 8.4). The surfactant component may comprise a first surfactant having a BHL value of less than 10 and a second surfactant having a BHL value of at least 10. For example, it may comprise a first surfactant having a BHL value of 1 to 4 ( for example, sorbitan trioleate) and a second surfactant having a BHL value of 13 to 17 (e.g., polysorbate 80).
Ideally, these oil / surfactant compositions have an excess volume of squalene relative to the surfactant component. Thus, for example, they may comprise (by volume): 60 to 80% squalene, with the remainder being either polysorbate 80 or a mixture of polysorbate 80 and sorbitan trioleate. For example, the amount of squalene may be 65 to 75%, 68 to 72% squalene, 69 to 71% squalene, or 70% squalene by volume.
Ideally, these compositions consist essentially of only these two or three components, i.e., squalene, polysorbate 80 and, optionally, sorbitan trioleate. For example, 95% by volume of the composition consists of (a) squalene and polysorbate 80 or (b) squalene, polysorbate 80, and sorbitan trioleate. A composition consisting of squalene and polysorbate 80 may be used, ideally with an excess volume of squalene.
The combined% by volume of squalene and polysorbate 80 in these compositions may be 70 to 90% of the total volume of the oil / surfactant composition. When the composition comprises squalene, sorbitan trioleate and polysorbate 80, the combined% by volume of squalene and polysorbate 80 can be from 70 to 90% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition.
In more general terms, the combined% by volume of the oil and a surfactant having a BHL value of 13 to 17 may be 70 to 90% of the total oil volume, with a surfactant having a BHL value of 1-5 and surfactant having a BHL value of 13-17 in the oil / surfactant composition. In place of polysorbate 80, the composition may use another nonionic surfactant having a BHL in the range of 14 to 16, but polysorbate 80 is preferred.
The aqueous phase
According to the invention, the processes for preparing the emulsions make use of an aqueous phase. This aqueous phase can be. clear water (for example, water for injectables) or it may include other components, for example, solutes. For example, in particular embodiments, it preferably comprises salts, which can be used to influence tone and / or control pH. For example, the salts may form a pH buffer, for example, citrate or phosphate salts, such as sodium salts. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. In preferred embodiments, the aqueous phase is free of buffers. In other embodiments of the invention, a buffered aqueous phase is preferred, and the buffers will generally be in the range of 1 to 20 mM.
The aqueous phase can be buffered appropriately. Any physiologically acceptable buffer may be used herein, such as water, citrate buffers, phosphate buffers, acetate buffers, Tris buffers, bicarbonate buffers, carbonate buffers, succinate buffer, or the like. The pH of the aqueous phase will preferably be from 6.0 to 8.0, preferably from about 6.2 to about 6.8. In an exemplary embodiment, the buffer is 10 mM citrate buffer with a pH of 6.5. The aqueous phase may include Pickering agents such as mannitol to reduce surface tension.
The aqueous phase may comprise solutes to influence tone and / or osmolality. Tonicity can be chosen to be isotonic with human tissues. To control tonicity, the emulsion may comprise a physiological salt, such as a sodium salt. Sodium chloride (NaCl), for example, can be used at about 0.9% (w / v) (physiological saline). Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, magnesium chloride, calcium chloride, and the like. Nonionic tonicity agents may also be used to control tonicity. Monosaccharides classified as aldoses such as glucose, mannose, arabinose, and ribose, as well as those classified into ketoses such as fructose, sorbose, and xylulose can be used as nonionic tonicity agents in the present invention. . Disaccharides such as sucrose, maltose, trehalose, and lactose may also be used. In addition, alditols (acyclic polyhydroxy alcohols, also known as sugar alcohols) such as glycerol, mannitol, xylitol, and sorbitol are nonionic tonicity agents useful in the present invention. Nonionic tonicity modifiers may be present at a concentration of from about 0.1% to about 10% or about 1% to about 10% of the aqueous phase depending on the agent that is used.
The aqueous phase ideally has a pH between 5.5 and 8.5, for example, between 6.0 and 8.0, or between 6.5 and 7.5. This pH range maintains compatibility with normal physiological conditions and, in some cases, may be required to ensure the stability of certain emulsion components.
In particular embodiments, the aqueous phase is substantially free of oil (s). Thus, when mixing with the oil / surfactant composition to form an emulsion, substantially all of the oil in the emulsion will have to come from the composition. In another embodiment, the aqueous phase is substantially free of surfactant (s). Thus, when mixing with the oil / surfactant composition to form an emulsion, substantially all of the surfactant in the emulsion will have to come from the composition. In another embodiment, the aqueous phase is substantially free of both oil (s) and surfactant (s).
In embodiments of the invention, the aqueous phase may comprise an antigen component. Mixing Unlike MF59 and AS03, the emulsions of the invention can be prepared without requiring the use of homogenizers or microfluidizers. Unlike AF03, the emulsions of the invention can be prepared without requiring heating to> 50 ° C. Instead, mixing the oil / surfactant composition with the aqueous phase can lead to the spontaneous formation of a submicron emulsion even with only slight agitation / mixing (e.g., simple manual inversion).
The present invention provides a method of forming an oil-in-water emulsion according to the invention, said method comprising: providing an oil / surfactant composition according to the invention; providing an aqueous phase; combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition; and the light mixture of the diluted composition to form an oil-in-water emulsion. The step of forming a diluted composition can take place by simply mixing the oil / surfactant composition with the aqueous phase. Preferably, formation of a diluted composition is achieved by adding the oil / surfactant composition to the aqueous phase. The step of combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition may sometimes comprise two distinct steps: (i) mixing equal volumes of the oil / surfactant composition and the aqueous phase; and (ii) diluting the mixture of the oil / surfactant composition and the aqueous phase with another volume of aqueous phase to form a diluted composition. In a particular embodiment, steps (i) and (ii) are each obtained by adding the oil / surfactant in the aqueous phase.
Mixing can be accomplished without the need for any shearing pressure, without using a rotor / stator mix, at normal pressures, and without components flowing through a pump. The process can be carried out in the absence of mechanical agitation.
The mixture of the composition and the aqueous phase may be stirred / mixed slightly to form an oil-in-water emulsion. The light mixture is provided by means other than homogenization, microfiltration, microfluidization, sonication (or other high shear or high energy processes) or a phase inversion temperature process in which the temperature of the the emulsion is increased until the emulsion reverses. Suitably, the light agitation may include inverting the mixture by hand, or it may comprise agitation, or it may comprise mixing by syringe passage, or it may comprise any similar method. Overall, the mixture is obtained by applying a controlled minimum dispersion force. The step of combining the oil / surfactant composition and the aqueous phase of a process of the invention can take place below 55 ° C, for example, anywhere in the range of 5 to 50 ° C, for example between 10 and 20 ° C, between 20 and 30 ° C, between 30 and 50 ° C, or between 40 and 50 ° C. The process can conveniently take place at room temperature, i.e. about 20 to 25 ° C. The composition and / or the aqueous phase are preferably equilibrated to the desired temperature before being mixed. For example, both components can be equilibrated at 40 ° C and then mixed. After mixing, the mixture can be maintained at a temperature below 55 ° C while the emulsion is formed. In a particular embodiment, the oil / surfactant composition and / or the aqueous phase are heated prior to mixing and maintained at the desired temperature (below 55 ° C) until mixing of the two components is complete and then the temperature is reduced.
The oil / surfactant composition is ideally mixed with an excess volume of the aqueous phase to ensure that an oil-in-water emulsion is formed (rather than a water-in-oil emulsion). In a particular embodiment, the aqueous phase is substantially free of surfactant (s) and / or oil (s). The process preferably uses the aqueous phase in a volume in excess of at least 5 times, for example, between 5 times and 50 times of greater volume. In a particular embodiment, the ratio of the aqueous phase to the oil / surfactant composition is from 10/1 to 50/1 (by volume). In another embodiment, the ratio of the aqueous phase to the oil / surfactant composition is from 20/1 to 40/1 (by volume).
The process of the invention can be used on a laboratory or bench scale or on an industrial scale. Thus, the composition and / or the aqueous phase can have a volume in the range of 10 to 100 ml, in the range of 100 to 1000 ml, in the range of 1 to 10 1, or even in the range of 10 to 100 1.
The methods of the invention may further include the step of subjecting the oil-in-water emulsion to filtration sterilization. Sterilization by filtration may take place at any suitable stage, for example, when placing the emulsion in containers (the filling stage), or before drying (which can be done aseptically, to maintain a sterile emulsion during and after drying).
In general, a method of the invention may involve the following steps: preparing an emulsion without the use of microfluidization; sterilization by filtration; and conditioning the sterilized emulsion, optionally after combining with an immunogen. The emulsion has adjuvant activity, and the conditioned material is suitable for injection, to elicit an immune response. The emulsion oil in water
In a second aspect, the present invention provides an oil-in-water emulsion comprising: a pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm, said composition comprising at least one biocompatible, metabolizable oil; and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of from 2.5 to 9.0, and wherein the oil / surfactant composition is substantially free of aqueous components; in combination with an aqueous phase; wherein said emulsion has an average particle diameter of oil of less than 250 nm.
When an emulsion is described as having an average particle diameter of oil less than 250 nm, this average is ideally located within the range of 100 to 220 nm, for example between 120 and 200 nm or 150 to 200 nm.
In a fifth aspect, the present invention provides an oil-in-water emulsion comprising: a pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of an oil which is not greater than 40 nm, said composition comprising at least one biocompatible, metabolizable oil; and a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of 9.0 to 14.0, and wherein the oil / surfactant composition is substantially free of aqueous components; in combination with an aqueous phase; wherein said emulsion has an average particle diameter of oil which is not greater than 40 nm.
In a particular embodiment, the average oil particle diameter of the oil-in-water emulsions according to the fifth aspect of the present invention is not more than 30 nm, more preferably not more than 20 nm.
The average diameter of the oil particles in an emulsion can be determined in various ways, for example, using dynamic light scattering and / or optical single particle detection techniques, using apparatus such as Accusizer ™ and Nicomp ™ instruments available from Partiele Sizing Systems (Santa Barbara, USA), Malvern Instruments Zetasizer ™ instruments (UK), or Horiba Partiele Size Distribution Analyzer instruments (Kyoto, Japan). See also reference 12. In a particular embodiment, dynamic light scattering (DLS) is the process by which the diameters of the oil particles are determined. In a particular embodiment, the method for defining the average oil particle diameter is a Z-average, i.e. the average hydrodynamic size weighted by the intensity of the collection as a whole of droplets measured by DLS. The mean Z is derived from the cumulant analysis of the measured correlation curve, where the size of a single particle (droplet diameter) is assumed and a simple exponential fit is applied to the autocorrelation function. Thus, the references herein to an average diameter should be taken as an intensity weighted average, and ideally the Z average.
In a particular embodiment, the droplets within the emulsions of the invention have a polydispersity index of less than 0.4. Polydispersity is a measure of the width of the particle size distribution, and is traditionally expressed as a polydispersity index (IPd). A polydispersity index greater than 0.7 indicates that the sample has a very large size distribution and a reported value of 0 means that size variation is absent, although values below 0.05 are rarely observed. In a particular embodiment, it is preferable for the oil droplets within an emulsion of the invention to be relatively uniform in size. Thus, in particular embodiments, the oil droplets in the emulsions have an IPd of less than 0.35, for example, less than 0.3, 0.275, 0.25, 0.225, 0.2, 0.175, 0. , 15, 0.125, or even less than 0.1. IPd values are easily provided by the same instrumentation that measures the diameter.
Downstream processing
The oil-in-water emulsions of the present invention can be filtered. This filtration eliminates all large droplets of oil from the emulsion. Although few in number, these oil droplets may be large in volume and may act as nucleation sites for aggregation, leading to degradation of the emulsion during storage. In addition, this filtration step can obtain sterilization by filtration.
The particular filtration membrane suitable for filtration sterilization depends on the fluidity characteristics of the oil-in-water emulsion and the degree of filtration required. The characteristics of a filter may affect its suitability for the filtration of the emulsion. For example, its pore size and surface characteristics may be important, especially when filtering a squalene emulsion.
The pore size of the membranes used in the context of the invention should allow the passage of the desired droplets while retaining the undesirable droplets. For example, it will have to retain the droplets that have a size ^ 1 μιη while allowing the passage of droplets <200 nm. A filter of 0.2 μιη or 0.22 μιη is ideal, and can thus obtain sterilization by filtration. The emulsion may be prefiltered, for example, through a 0.45 μιη filter. Pre-filtration and filtration can be achieved in one step by the use of known double layer filters which include a first membrane layer with larger pores and a second membrane layer with smaller pores. Double layer filters are particularly useful in the context of the invention. The first layer ideally has a pore size> 0.3 μπι as between 0.3 and 2 μιτι or between 0.3 and 1 μιη, or between 0.4 and 0.8 μιη, or between 0.5 and 0, 7 μπι. A pore size of 0.75 μιη in the first layer is preferred. Thus, the first layer may have a pore size of 0.6 μπι or 0.45 μπι, for example. The second layer ideally has a pore size that is less than 75% of (and ideally less than half of) the pore size of the first layer, such as between 25 and 70% or between 25 and 49% of the size of the first layer. pore of the first layer, for example, between 30 and 45%, like 1/3 or 4/9, of the pore size of the first layer. Thus, the second layer may have a pore size <0.3 μπι, such as between 0.15 and 0.28 μπι or between 0.18 and 0.24 μπι, for example, a second pore size layer of 0 , 2 μπι or 0.22 μπι. In one example, the first membrane layer with larger pores provides a 0.45 μπι filter, while the second membrane layer with smaller pores provides a 0.22 μπι filter.
The filtration membrane and / or the prefiltration membrane may be asymmetrical. An asymmetric membrane is a membrane in which the pore size varies from one side of the membrane to the other, for example, in which the pore size is larger on the face of the inlet relative to the face of the membrane. the exit. One side of the asymmetric membrane may be termed a "coarse pore surface" while the other side of the asymmetric membrane may be termed a "fine pore surface". In a double layer filter, one or (ideally) both layers may be asymmetrical.
The filtration membrane may be porous or homogeneous. A homogeneous membrane is usually a dense film in the range of 10 to 200 μιη. A porous membrane has a porous structure. In one embodiment, the filtration membrane is porous. In a double layer filter, the two layers may be porous, the two layers may be homogeneous, or there may be a porous layer and a homogeneous layer. A preferred double-layer filter is a filter in which both layers are porous.
In one embodiment, the oil-in-water emulsions of the invention are prefiltered through an asymmetric hydrophilic porous membrane and then filtered through another asymmetric hydrophilic porous membrane having pores smaller than the prefiltration membrane. A double layer filter can be used.
The membrane (s) of the filters can be autoclaved before use to ensure sterility.
Filtration membranes are generally made of polymeric support materials such as PTFE (poly-tetrafluoroethylene), PES (polyethersulfone), PVP (polyvinylpyrrolidone), PVDF (polyvinylidene fluoride), nylons ( polyamides), PP (polypropylene), celluloses (including cellulose esters), PEEK (polyetheretherketone), nitrocellulose, etc. These have variable characteristics, with some carriers being inherently hydrophobic (eg, PTFE) and others being inherently hydrophilic (eg, cellulose acetates). However, these intrinsic characteristics can be modified by treating the surface of the membrane.
For example, it is known to prepare hydrophilized or hydrophobized membranes by treating them with other materials (such as other polymers, graphite, silicone, etc.) to coat the surface of the membrane, for example, see section 2.1 of reference 13. In a double-layer filter, the two membranes may be made of different materials or (ideally) of the same material.
Details of the appropriate filtration techniques are available, for example, in reference 14.
During filtration, the emulsion can be maintained at a temperature of 40 ° C or lower, for example, 30 ° C or lower, to facilitate successful filtration sterilization. Some emulsions may not pass through a sterile filter when they are above 40 ° C.
It is advantageous to carry out the filtration step within 24 hours, for example within 18 hours, within 12 hours, within 6 hours, within 2 hours, within 30 minutes, following the production of the emulsion. because after this time, it may not be possible to pass the second emulsion through the sterile filter without clogging the filter, as discussed in reference 15.
The methods of the invention can be used on a large scale. Thus, a process may involve filtration of a volume greater than 1 liter, for example, 5 liters, 10 liters, 20 liters, 50 liters, 100 liters, 250 liters, etc.
In some embodiments, an emulsion that is prepared according to the invention may be microfluidized. Thus, for example, the invention can be used prior to microfluidization to reduce the degree of microfluidization that is required to achieve a desired result. Thus, if desired, microfluidization can be used but the overall shear forces imparted to the emulsion can be reduced.
Antigens
Although it is possible to administer only adjuvants in the oil-in-water emulsion to patients (for example, to provide an adjuvant effect for an antigen that has been administered separately to the patient), it is more usual to mix adjuvant with an antigen prior to administration, to form an immunogenic composition, for example, a vaccine. The mixture of the emulsion and the antigen can take place extemporaneously, at the time of use, or it can take place during the manufacture of the vaccine, before filling. The emulsions of the invention can be used in one situation or the other.
Various antigens can be used with oil-in-water emulsions, including, but not limited to: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and / or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens. The invention is particularly useful for vaccines against influenza virus, HIV, hookworms, hepatitis B virus, Herpes simplex virus (and other herpesviridae), rabies, respiratory syncytial virus, cytomegalovirus,
Staphylococcus aureus, Chlamydiae, Coronavirus SARS, Varicella zoster virus, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus, Human papillomavirus, Malaria, etc. For example :
Antigens of the influenza virus. These can take the form of a live virus or an inactivated virus. When an inactivated virus is used, the vaccine may comprise the whole virion, the fragmented virion, or purified surface antigens (including hemagglutinin and usually also including neuraminidase). Flu antigens may also be in the form of virosomes. The antigens may have any subtype of hemagglutinin, selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and / or H16. The vaccine may include one or more antigens of one or more (eg, 1, 2, 3, 4 or more) strains of influenza virus, including influenza A virus and / or influenza virus. influenza B, for example, a monovalent A / H5N1 or A / H1N1 vaccine, or a trivalent A / H1N1 + A / H3N2 + B vaccine. Vaccines may be for seasonal or pandemic use. The influenza virus may be a reassortant strain, and may have been obtained by reverse genetics techniques [eg, 16-20]. Thus the virus may comprise one or more RNA segments of A / PR / 8/34 virus (usually 6 segments of A / PR / 8/34, with the HA and N segments being of a vaccine strain, c. that is, a 6/2 reassigned strain). The viruses used as the source of the antigens can be developed either on eggs (for example, embryonated chicken eggs) or on cell culture. When a cell culture is used, the cell substrate will generally be a mammalian cell line, such as MDCK; CHO; 2 93T; BHK; Vero; MRC-5; PER.C6; WI-38; etc. Preferred mammalian cell lines for developing influenza viruses include: MDCK cells [21 - 24], derived from the Madin Darby dog kidney; Vero cells [25-27], derived from the kidney of the African green monkey; or PER.C6 cells [28], derived from human embryonic retinoblasts. When the virus has been grown on a mammalian cell line, then the composition will advantageously be free of egg protein (e.g. ovalbumin and ovomucoid) and chicken DNA, thereby reducing allergenicity. Unit doses of vaccine are generally standardized with reference to hemagglutinin (HA) content, usually measured by SRID. Existing vaccines generally contain about 15 μg of HA per strain, although lower doses may be used, particularly when an adjuvant is used. Fractional doses such as 1/2 (that is, 7.5 μρ of HA per strain), 1/4 and 1/8 were used [29,30], as were higher doses. (for example, doses of 3x or 9x [31,32]). Thus, the vaccines may comprise between 0.1 and 150 μρ of HA per influenza strain, preferably between 0.1 and 50, for example, 0.1 to 20 μl, 0.1 to 15 μq, 0.1 to 10 μρ , 0.1 to 7.5 μqr 0.5 to 5 μρ, etc. Particular doses include, for example, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc. by strain.
Human immunodeficiency virus, including HIV-1 and HIV-2. The antigen will generally be an antigen of the envelope.
Surface antigens of hepatitis B virus. This antigen is preferably obtained by recombinant DNA methods, for example, after expression in yeast Saccharomyces cerevisiae. Unlike native HBsAg, recombinant antigen expressed by yeast is unglycosylated. It may be in the form of substantially spherical particles (average diameter of about 20 nm), including a lipid matrix comprising phospholipids. Unlike native HBsAg particles, particles expressed by yeast may comprise phosphatidylinositol. The HBsAg can be any of the aywl, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq-, and adrq + subtypes. Hookworm, especially as observed in dogs (Ancylostoma caninum). This antigen may be recombinant Ac-MTP-1 (astacin metalloprotease) and / or aspartic hemoglobinase (Ac-APR-1), which may be expressed in a baculovirus / insect cell system in the form of a secreted protein [33,34].
Herpes simplex virus (HSV) antigen. A preferred HSV antigen for use of which the invention is the gD membrane glycoprotein. It is preferable to use gD from a strain of HSV-2 ('gD2' antigen). The composition can use a form of gD in which the C-terminal membrane anchoring region has been deleted [35], for example, a truncated gD comprising amino acids 1 to 306 of the natural protein with addition of asparagine and glutamine at the C-terminus. This form of the protein comprises the signal peptide which is cleaved to produce a mature protein of 283 amino acids. The deletion of the anchoring region makes it possible to prepare the protein in soluble form. The invention can also be used with other herpesviridae, such as varicella zoster virus (VZV), Epstein-Barr virus (EBV), or human cytomegalovirus (hCMV). An anti-hCMV composition may comprise a glycoprotein B antigen (gB) in some embodiments, or it may comprise one or more of the gH, gL and gO antigens.
Antigens of human papillomavirus (HPV). The preferred HPV antigens for use of which the invention are the capsid L1 proteins, which can assemble to form structures known as pseudoviral particles (PPV). VLPs can be produced by recombinant expression of L1 in yeast cells (e.g., in S. cerevisiae) or in insect cells (e.g., in Spodoptera cells, such as S. frugiperda, or in cells of Drosophila). For yeast cells, plasmid vectors may carry the L1 gene (s); for insect cells, baculovirus vectors may carry the L1 gene (s). More preferably, the composition comprises LV VLPs from both HPV-16 and HPV-18 strains. This bivalent combination has been shown to be highly effective [36]. In addition to the HPV-16 and HPV-18 strains, it is also possible to include LHVPs of the HPV-6 and HPV-11 strains. The use of oncogenic strains of HPV is also possible. A vaccine may comprise between 20 and 60 μg / ml (e.g., about 40 μg / ml) of L1 per HPV strain.
Antigens of anthrax. Anthrax is caused by Bacillus anthracis. Suitable antigens of B. anthracis include components A (lethal factor (LF) and edema factor (EF)), both of which can share a common component B known as the protective antigen (PA). The antigens can be optionally detoxified. Further details can be found in references [37 to 39].
Malaria antigens. A composition for protecting against malaria may include a portion of the circumsporozoite protein of P. falciparum from the pre-erythrocytic stage of the body. The C-terminal portion of this antigen can be expressed as a fusion protein with HBsAg, and this fusion protein can be coexpressed with HBsAg in yeast such that both proteins assemble to form a particle.
Rage. The rabies protection compositions will generally comprise an inactivated rabies virus virion, as observed in products such as RABIPUR, RABIVAC, and VERORAB.
Antigens of S. aureus. Various antigens of S. aureus are known. Suitable antigens include capsular saccharides (e.g. of type 5 and / or type 8 strain) and proteins (e.g., IsdB, Hla, etc.). Capsular saccharide antigens are ideally conjugated to a carrier protein.
Antigens of S. pneumoniae. Various antigens of S. pneumoniae are known. Suitable antigens include capsular saccharides (e.g., one or more of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and / or 23F) and proteins (e.g., pneumolysin, detoxified pneumolysin, PhtD (polyhistidine triad protein D), etc.). Capsular saccharide antigens are ideally conjugated to a carrier protein.
Meningococcal antigens. Neisseria meningitidis is a cause of bacterial meningitis. Suitable meningococcal antigens include conjugated capsular saccharides (particularly for serogroups A, C, W135, X and / or Y), recombinant proteins (e.g., factor H binding protein) and / or membrane vesicles. external.
Cancer antigens. Various tumor-specific antigens are known. The invention can be used with antigens that elicit a therapeutic immune response against lung cancer, melanoma, breast cancer, prostate cancer, and the like.
A solution of the antigen will normally be mixed with the emulsion, for example, in a 1/1 volume ratio. This mixture can either be done by a vaccine manufacturer before filling or can be done at the time of use by a health professional. However, as noted below, a formulation variant includes both the antigen and the emulsion in dried form in a single container to be reconstituted.
Uses of oil-in-water emulsions of the invention
According to one aspect of the present invention, the oil-in-water emulsions of the present invention are for use as an adjuvant, preferably a vaccine adjuvant. Suitably, said adjuvant is administered as part of a vaccine.
In another aspect, the present invention provides an immunogenic composition (e.g., a vaccine) comprising an oil-in-water emulsion according to the present invention, and an antigenic component. The invention further provides a process for preparing such immunogenic compositions, said method comprising mixing an oil-in-water emulsion according to the present invention with an antigen component.
The present invention also provides a kit comprising: an oil / surfactant composition according to the present invention; an aqueous phase; and optionally instructions for combining the oil / surfactant composition and the aqueous phase.
According to embodiments of the invention, the oil / surfactant composition and / or the aqueous phase may comprise an antigen component.
In another aspect, the present invention provides a method of preparing a kit comprising the steps of: providing an oil / surfactant composition according to the present invention; and conditioning the composition in a kit as a kit component together with an aqueous phase; and optionally conditioning an antigen component in the kit as a kit component together with the oil / surfactant composition and the aqueous phase.
The present invention also provides a kit comprising: an oil-in-water emulsion according to the present invention; and an antigen component.
The present invention further provides a method of preparing a kit comprising the steps of: providing an oil-in-water emulsion according to the present invention; and packaging the emulsion in a kit as a kit component together with a separate antigen component.
Although it is possible to administer only oil-in-water emulsion adjuvants to the patients (for example, to provide an adjuvant effect for an antigen that has been administered separately to the patient), it is more usual to mix the adjuvant with an antigen prior to administration, to form an immunogenic composition, for example, a vaccine. The mixture of the emulsion and the antigen can take place extemporaneously, at the time of use, or it can take place during the manufacture of the vaccine, before filling.
Overall, therefore, the invention can be used in the preparation of reconstituted vaccines or in the preparation of kits comprising an antigen and adjuvant ready to mix. When the mixing takes place during manufacture, then the bulk volumes of antigen and emulsion which are mixed will generally be greater than 1 liter, for example 5 liters, 10 liters, 20 liters, 50 liters, ^ 100 liters, ^ 250 liters, etc. When mixing takes place at the time of use, then the volumes that are mixed will generally be smaller than 1 milliliter, for example, <0.6 ml, 0.5 ml, 0.4 ml, ^ 0, 3 ml, <0.2 ml, etc. In both cases, it is usual to mix substantially equal volumes of the emulsion and the antigen solution, i.e., substantially 1/1 (e.g., between 1.1 / 1 and 1/1). 1.1, preferably between 1.05 / 1 is 1 / 1.05, and more preferably between 1.025 / 1 and 1 / 1.025). However, in some embodiments, excess emulsion or excess antigen may be used [40]. When an excess volume of a component is used, the excess will generally be at least 1.5 / 1, for example,> 2/1,> 2.5 / 1,> 3/1,> 4 / 1,> 5/1, etc.
When the antigen and adjuvant are presented as separate components within a kit, they are physically separated from each other within the kit, and this separation can be achieved in a variety of ways. For example, the components may be in separate containers, such as flasks. The contents of two vials can then be mixed when necessary, for example by taking the contents of one vial and adding it to the other vial, or by taking the contents of the two vials separately and mixing them together. in a third container.
In another arrangement, one of the kit components is in one syringe and the other is in a container such as a vial. The syringe can be used (for example, with a needle) to insert its contents into the vial for mixing, and the mixture can then be drawn into the syringe. The mixed contents of the syringe can then be administered to a patient, usually via a new sterile needle. Pouring a component into a syringe eliminates the need to use a separate syringe for administration to the patient.
In another preferred arrangement, the two kit components are held together but separately in the same syringe, for example, a dual chamber syringe, such as those described in references 41-48, etc. When the syringe is actuated (for example, during administration to a patient), then the contents of both chambers are mixed. This arrangement avoids the need for a separate mixing step at the time of use.
The contents of the various components of the kit will generally be in liquid form.
Vaccines are usually given by injection, particularly by intramuscular injection. The compositions of the invention are generally presented at the time of use as aqueous solutions or suspensions. In certain embodiments of the invention, the compositions are in aqueous form from the conditioning stage to the administration stage. In other embodiments, however, one or more components of the compositions may be packaged in dried form (e.g., lyophilized), and an adjuvant for effective delivery may be reconstituted where necessary. The emulsion can thus be distributed in the form of a freeze-dried cake. Drying can conveniently be obtained by lyophilization, but other techniques can also be used, for example, spray drying. Drying by lyophilization is preferred.
Accordingly, in one aspect, the present invention provides a dried material (e.g., a lyophilizate) which when reconstituted with an aqueous phase provides an oil-in-water emulsion according to the present invention. The dried material is preferably a lyophilisate. As used herein, "dry material" or "dried material" refers to a material that is substantially free of water or a material that is substantially free of aqueous phase. Ideally, an emulsion can be reconstituted in its original composition simply by adding water (for example, it loses no component with the exception of water during drying). The invention also provides a process for preparing said dried material, wherein said process comprises preparing an oil-in-water emulsion according to the invention and subjecting the emulsion to a drying process. Suitably, the emulsion is combined with one or more freeze-drying stabilizers. The emulsion may also be combined with at least one antigen component prior to drying, optionally in addition to one or more lyophilization stabilizers.
In some arrangements, a component (typically the emulsion component) is in dry form (e.g., in freeze-dried form), with the remaining components (usually, the antigen and / or an aqueous phase) being in liquid form. The two or more components may be mixed to reactivate the dry component and provide a liquid composition for administration to a patient. A dried component will usually be placed within a vial rather than a syringe.
A lyophilized component (e.g. emulsion) may include freeze-drying stabilizers. These stabilizers include substances such as sugar alcohols (eg, mannitol, etc.) or simple saccharides such as disaccharides and trisaccharides. The lyophilization stabilizers are preferably small saccharides such as disaccharides. They preferably comprise saccharide monomers selected from glucose, fructose and galactose, and disaccharides containing glucose and disaccharides containing fructose are particularly preferred. Examples of preferred disaccharides include sucrose (containing glucose and fructose), trehalose (containing two glucose monosaccharides) and maltulose (containing glucose and fructose), more preferably sucrose, such as lactose, sucrose or mannitol, and mixtures thereof, for example, lactose / sucrose mixtures, sucrose / mannitol mixtures, etc.
In a particular embodiment, a possible arrangement comprises a dried emulsion component in a vial and an antigen component and / or an aqueous phase in a pre-filled syringe.
The present invention also provides an arrangement comprising a dried emulsion of the present invention and a separate liquid antigen component.
Also provided by the present invention is a dried cake formed from the emulsion of the invention. The cake may be provided in combination with a separate aqueous phase. The arrangement may further comprise an antigen component which may be in liquid or dried form.
The present invention also provides a dried blend wherein the blend comprises the emulsion of the present invention in combination with an antigen component. In a particular embodiment, the mixture is a freeze-dried mixture.
An advantage of the oil-in-water emulsions of the invention and their manufacturing processes according to the invention is that when the oil-in-water emulsion is reconstituted with an aqueous phase following the drying of the emulsion, the resulting oil-in-water emulsion retains its original properties prior to drying (e.g., its average particle size of oil). The invention also provides a kit for preparing an oil-in-water emulsion of the invention, wherein the kit comprises an oil-in-water emulsion of the invention in dried form and an aqueous phase in liquid form. The kit may comprise two vials (one containing the dried emulsion and one containing the aqueous phase) or it may comprise a pre-filled syringe and a vial, for example, with the contents of the syringe (the aqueous phase) being used to reconstitute the vial contents (dried emulsion) before administration to a subject. In embodiments of the invention, the oil-in-water emulsion in dried form is combined with an antigen component which is also in dried form.
If vaccines contain components in addition to the emulsion and the antigen, then these other components may be included in one of the two kit components according to embodiments of the invention, or they may be part of the kit. a third component of the kit.
Suitable containers for the reconstituted vaccines of the invention, or for individual kit components, include disposable vials and syringes. These containers should be sterile.
When a composition / component is placed in a vial, the vial is preferably made of glass or plastic material. The vial is preferably sterilized prior to the addition of the composition therein. To avoid problems with latex-sensitive patients, the vials are preferably sealed with a latex-free cap, and the absence of latex throughout the packaging material is preferred. In one embodiment, a vial with a butyl rubber stopper. The vial may comprise a single dose of vaccine / component, or it may comprise more than one dose (a 'multidose' vial), for example, 10 doses. In one embodiment, a vial comprises 10 doses of 0.25 ml of emulsion. The preferred bottles are made of colorless glass.
A vial may have a cap (eg, a luer lock) adapted such that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial (for example, to reconstitute the material dried inside), and the contents of the vial can be taken again in the syringe. After removal of the syringe from the vial, a needle may then be attached and the composition may be administered to a patient. The cap is preferably housed inside a seal or lid so that the seal or lid must be removed before the cap can be accessed.
When a composition / component is packaged in a syringe, the syringe will normally have no needle attached, although a separate needle may be provided with the syringe for assembly and use. Safety needles are preferred. The 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may be supplied with peel-off labels on which the batch number, influenza season and expiry date of the contents can be printed to facilitate record keeping. The plunger in the syringe has a blocker to prevent the plunger from being accidentally removed during aspiration. The syringes may have a rubber latex cap and / or plunger. Disposable syringes contain a single dose of adjuvant or vaccine. The syringe will generally have a cap at its end for sealing the end prior to attachment of a needle, and the end cap is preferably made of butyl rubber. If the syringe and the needle are packaged separately, then the needle is preferably equipped with a butyl rubber cover. The emulsion can be diluted with buffer prior to packaging in a vial or syringe. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Dilution can reduce the concentration of the adjuvant components while maintaining their relative proportion, for example, to provide a "half-strength" adjuvant.
The containers can be labeled to indicate the volume of a half-dose, for example, to facilitate administration to children. For example, a syringe containing a dose of 0.5 ml may have a mark indicating a volume of 0.25 ml.
When a glass container (for example, a syringe or vial) is used, then it is preferable to use a container made of a borosilicate glass rather than a soda-lime glass.
Compositions made using the methods of the invention are pharmaceutically acceptable. They may include components in addition to the emulsion and any antigen.
The composition may comprise a preservative such as thiomersal or 2-phenoxyethanol. However, it is preferable that the adjuvant or the vaccine is substantially free of (i.e., less than 5 μg / ml) mercurial material, i.e., free of thiomersal [49, 50]. Vaccines and components containing no mercury are more preferred.
The pH of a composition will generally be from 5.0 to 8.1, and more generally from 6.0 to 8.0, for example, from 6.5 to 7.5. A method of the invention may therefore include a step of adjusting the pH of the adjuvant of the vaccine prior to conditioning.
The composition is preferably sterile. The composition is preferably non-pyrogenic, for example, containing <1 EU (endotoxin unit, a standard measurement) per dose, and preferably <0.1 EU per dose. The composition is preferably free of gluten.
The composition may comprise material for a single immunization, or it may comprise material for multiple immunizations (i.e., a multidose kit). The inclusion of a preservative is preferred in multi-dose arrangements.
The compositions can be administered in a variety of ways. The most preferred route of immunization is by intramuscular injection (eg, into the arm or leg), but other available routes include subcutaneous injection, intranasal [51-53], oral [54] , intradermal [55,56], transcutaneous, transdermal [57] etc.
Adjuvants or vaccines prepared according to the invention can be used to treat both children and adults. The patient may be less than 1 year old, 1 to 5 years old, 5 to 15 years old, 15 to 55 years old, or at least 55 years old. The patient may be an elderly person (for example, at age 50, preferably at age 65), a young person (for example, age 5), an inpatient, a health professional, a member of the armed services staff, and military, a pregnant woman, a chronic patient, an immunodeficient patient, and a traveler abroad. Vaccines are not appropriate only for these groups, however, and they can be used more generally in a population.
Adjuvants or vaccines of the invention may be administered to patients substantially at the same time (for example, during the same medical consultation or visit to a health professional) as other vaccines. Definitions
The term "comprising" includes "including" as well as "consisting of", for example, a composition "comprising" X may consist exclusively of X or it may comprise something else, for example, X + Y.
The term "substantially" does not exclude "completely", for example, a composition that is "substantially devoid" of Y may be completely devoid of Y. When necessary, the term "substantially" may be omitted from the definition. of the invention.
The term "about" in relation to a numerical value x is optional and means, for example, x ± 10%.
As used herein, the singular forms "one", "one", "the" and "the" include plural articles unless the context clearly indicates otherwise.
Unless otherwise indicated, a process comprising a step of mixing two or more components does not require any specific mixing order. Thus, the components can be mixed in any order. When there are three components, then two components can be combined with each other, and then the combination can be combined with the third component, etc.
Where animal (and particularly bovine) material is used in cell culture, it must be obtained from sources that are free of transmissible spongiform encephalopathies (TSE), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is best to grow the cells in the complete absence of animal materials.
Embodiments of the invention
The examples presented below are for illustrative purposes and are not intended to limit the scope of the invention. 1 / Formation of an oil-in-water emulsion and measurement of the average particle size
The oil / surfactant compositions comprising squalene, Span 85 and Tween 80 were prepared with the percentages of the compositions shown in Tables 1 and 2. These compositions were mixed at 37-40 ° C overnight. The following day, the oily compositions were mixed with aqueous material by adding the oil / surfactant composition to the aqueous material in a ratio of 1/10 (oil / surfactant to water) (v / v) at room temperature; it has been observed that oil-in-water emulsions have formed spontaneously. The average size of the oil particles of the various emulsions was measured using Zetasizer Nano ZS (Malvern Instruments) according to the manufacturer's instructions (to give a Z-mean and IPd), or a particle size measuring instrument. Wyatt DynaPro broadband (by the intensity). The results of this study are presented in Tables 1 and 2.
Table 1
Table 2
The oil / surfactant compositions comprising squalene, Span 85 and Tween 80 were prepared with the percentages of the compositions shown in Table 3 below. These compositions were combined with aqueous material by adding the oil / surfactant composition to the aqueous material in a ratio of 1/10 (oil / surfactant to water) (volume / volume) at room temperature and it was observed that the Oil-in-water emulsions did not form spontaneously or an emulsion formed but having an average particle size of 250 nm or more. The average oil particle size of various emulsions was measured using a Wyatt DynaPro high throughput particle size measuring instrument (by intensity) according to the manufacturer's instructions.
Table 3
As shown in Table 1, the oil-in-water emulsions according to the present invention can have an average particle size of oil of less than 200 nm. The oil-in-water emulsions according to some embodiments of the invention may have an average particle size of oil which is not greater than 40 nm as shown in Table 2. 2 / Formation of a oil-in-water emulsion and measurement of the average particle size 400 μl of squalene, 400 μl of Span 85 (sorbitan trioleate), and 200 μl of Tween 80 (polysorbate 80, polyoxyethylene sorbitan monooleate) were mixed together to form a 1 ml composition according to the present invention. After formation of the oil-containing and surfactant-containing composition, said composition was added to 19 ml of an aqueous phase comprising water to form a 20 ml mixture. The resulting mixture was then inverted gently by hand, resulting in the formation of an oil-in-water emulsion according to the present invention. The average size of the oil droplet particles in the oil-in-water emulsion was measured as 43.15 nm.
This experiment was repeated with various different combinations of squalene, Span 85 and Tween 80 according to the present invention. The results of these experiments are shown in Table 4 below. For reference, the corresponding data for the known adjuvant MF59 are also provided in Table 4. The average diameters of the oil particles of each emulsion were measured using a ZS Zetasizer Nano (Malvern Instruments, Worcestershire, UK). according to the manufacturer's instructions.
Table 4
As shown in Table 4, each of the emulsions tested had an average particle size of oil less than 175 nm. In particular, it has been found that the SEA19 emulsion has a squalene particle size of less than 20 nm.
Therefore, the present invention allows the manufacture of highly dispersed oil-in-water emulsions having an average particle diameter of oil of less than 250 nm. The present invention also allows the manufacture of highly dispersed oil-in-water emulsions with very small oil particles (squalene) having an average particle diameter of oil that is not greater than 40 nm. In addition, small particle sizes are obtained without the need for expensive processes such as microfluidization.
Table 4 also shows the relative percentages of each component in the emulsion compared to the corresponding amount of each component in the known MF59 vaccine adjuvant. For example, SEA composition 19 of the present invention contains only two-fifths of the amount of squalene present in MF59, twice as much Span 85, and four times as much Tween 80 as MF59, and has an average particle diameter which is more than eight times smaller than that in the MF59. In general, each of the emulsions tested in Example 2 contained less than or equal to half the amount of squalene present in the MF59 vaccine adjuvant. In addition, the emulsions of the present invention tested in Example 2 contain anywhere between two and four times the amount of each surfactant found in the MF59. 3 / In vivo analysis of oil-in-water emulsions for their potential use as adjuvants
In this experiment, the SEA19 oil-in-water emulsion of Example 2 was tested in vivo for its ability to amplify an immune response in a subject. Comparative experiments were also performed in which no adjuvant or known adjuvant MF59 was administered in place of the SEA19 emulsion. In addition, varying dose concentrations of the SEA19 emulsion were tested.
Each experiment listed in Table 5 was performed on two groups of mice, each group consisting of four mice. Each group was given 1 or 10 μg of influenza antigens (Brisbane strain) in combination with MF59, SEA19 adjuvant or no adjuvant according to the experiments presented in Table 5 below.
Table 5
As shown in Table 5, the SEA19 emulsion of the present invention was effective in inducing an immune response against the influenza antigen. Therefore, the emulsions of the present invention are suitable for use as an adjuvant, for example in vaccines. 4 / Variants of oils and surfactants
The composition of SEA20 as presented above was defined more precisely based on a volume ratio of 600/200/1400 (squalene / sorbitan trioleate / polysorbate 80), thus leading to 27.3% by volume of squalene, 9.1% sorbitan trioleate, and 63.6% polysorbate 80. When combined with a 19-fold excess of aqueous material (1.1 ml of mixture with 19 ml of 100 mM citrate buffer, pH 6.5), this mixture SEA20 gives an emulsion with droplets with a diameter of ~ 20 nm, with an IPd of ~ 0.1.
While the three components in SEA20 have an excess surfactant volume, the same components can be combined in different ratios with an excess volume of squalene. Unlike SEA20, with 70% by volume of squalene, this mixture provides an emulsion with droplets with a diameter of ~ 160 nm, also with an IPd of ~ 0.1. The oil and surfactants in these two emulsions have been varied. Three variants of oils were used (squalane, soybean oil, sunflower seed oil) and three variants of high BHL surfactants (polysorbate 20, 16.7 BHL, SDS, 40 BHL, or polyoxyethylene lauryl ether). (10), BHL of 17) in the same volumes (except for SDS, which was used at the same weight). These emulsion variants had the following characteristics:
Thus, none of the oil variants was a suitable substitute for squalene, and none of the surfactant component variants were useful either.
It should be understood that the invention has been described by way of example only and that modifications can be made while remaining within the scope and spirit of the invention. Embodiments within the description provide an illustration of the embodiments of the invention and should not be construed as limiting the scope of the invention. Those skilled in the art understand that many other embodiments are encompassed by the invention. References [1] WO 90/14837.
[2] Podda &amp; Del Giudice (2003) Expert Rev Vaccines 2: 197-203.
[3] Podda (2001) Vaccine 19: 2673-2680.
[4] Vaccine Design: The Subunit and Adjuvant Approach (eds.
Powell &amp; Newman) Plenum Press 1995 (ISBN 0-306-44867-X).
[5] Adjuvant Vaccine: Preparation Methods and Research
Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed O'Hagan.
[6] New Generation Vaccines (eds, Levine et al.). 3rd edition, 2004. ISBN 0-8247-4071-8.
[7] O'Hagan (2007) Expert Rev Vaccines 6 (5): 699-710.
[8] Boy et al. (2012) Expert Rev Vaccines 11: 349-66.
[9] WO 2006/100109.
[10] US 2007/0014805.
[11] Ott et al. (2000) Chapter 12 of Vaccine Adjuvants: Preparation Methods and Research Protocols (ed. 0'Hagan), Volume 42 of Methods in Molecular Medicine.
[12] Light Scattering from Polymer Solutions and Nanoparticle Dispersions (W. Schartl), 2007. ISBN: 978-3-540-71950-2.
[13] WO 90/04609.
[14] WO 2011/067669 [15] Lidgate et al (1992) Pharmaceutical Research 9 (7): 860-863.
[16] Hoffmann et al. (2002) Vaccine 20: 3165-3170.
[17] Subbarao et al. (2003) Virology 305: 192-200.
[18] Liu et al. (2003) Virology 314: 580-590.
[19] Ozaki et al. (2004) J. Virol. 78: 1851-1857.
[20] Webby et al. (2004) Lancet 363: 1099-1103.
[21] WO 97/37000.
[22] Brands et al. (1999) Dev Biol Stand 98: 93-100.
[23] Halperin et al. (2002) Vaccine 20: 1240-7.
[24] Tree et al. (2001) Vaccine 19: 3444-50.
[25] Kistner et al. (1998) Vaccine 16: 960-8.
[26] Kistner et al. (1999) Dev Biol Stand 98: 101-110.
[27] Brühl et al. (2000) Vaccine 19: 1149-58.
[28] Pau et al. (2001) Vaccine 19: 2716-21.
[29] WO 01/22992.
[30] Hehme et al. (2004) Virus Res. 103 (1-2): 163-71.
[31] Treanor et al. (1996) J Infect Dis 173: 1467-70.
[32] Keitel et al. (1996) Clin Diagn Lab Immunol 3: 507-10.
[33] Williamson et al. (2006) Infection and Immunity 74: 961 7.
[34] Loukas et al. (2005) PLoS Med 2 (10): e295.
[35] EP-A-0139417.
[36] Harper et al. (2004) Lancet 364 (9447): 1757-65.
[37] J Toxicol Clin Toxicol (2001) 39: 85-100.
[38] Demicheli et al. (1998) Vaccine 16: 880-884.
[39] Stepanov et al. (1996) J Biotechnol 44: 155-160.
[40] WO 2007/052155.
[41] WO 2005/089837.
[42] US 6,692,468.
[43] WO 00/07647.
[44] WO 99/17820.
[45] US 5,971,953.
[46] US 4,060,082.
[47] EP-A-0520618.
[48] WO 98/01174.
[49] Banzhoff (2000) Immunology Letters 71: 91-96.
[50] WO 02/097072.
[51] Greenbaum et al. (2004) Vaccine 22: 2566-77.
[52] Zurbriggen et al. (2003) Expert Rev Vaccines 2: 295-304.
[53] Piaseik (2003) Am Pharm Assoc (Wash DC). 43: 728-30.
[54] Mann et al. (2004) Vaccine 22: 2425-9.
[55] Halperin et al. (1979) Am J Public Health 69: 1247-50.
[56] Herbert et al. (1979) J. Infect Dis 140: 234-8.
[57] Chen et al. (2003) Vaccine 21: 2830-6.

权利要求:
Claims (14)
[1]
A pharmaceutically acceptable oil / surfactant composition for use in the preparation of an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm, said composition comprising (i) squalene; and (ii) a biocompatible, metabolizable surfactant component comprising at least one surfactant, wherein: (a) the surfactant component has a BHL value of 2.5 to 9.0; (b) the surfactant component has a BHL value of 3.3 to 8.4; (c) the surfactant component has a BHL value of 10 to 18; (d) the surfactant component has a BHL value of 13 to 17; (e) the surfactant component has a BHL value of 14 to 16; or (f) the surfactant component has a BHL value of 9 to 14, wherein the oil / surfactant composition is substantially free of aqueous components.
[2]
The oil / surfactant composition of claim 1, wherein the surfactant component comprises or consists of polysorbate 80.
[3]
The oil / surfactant composition according to claim 1, wherein: (a) the surfactant component has a BHL value of 2.5 to 9.0 and the oil-in-water emulsion has an average particle diameter oil between 40 and 250 nm; or (b) the surfactant component has a BHL value of 9.0 to 14.0 and the oil-in-water emulsion has an average oil particle diameter of not greater than 40 nm.
[4]
An oil / surfactant composition according to any one of the preceding claims, wherein: (a) the composition comprises an excess volume of squalene relative to the surfactant component; or (b) the composition comprises an excess volume of the surfactant component relative to squalene.
[5]
An oil / surfactant composition according to any one of the preceding claims, comprising (by volume) 60 to 80% squalene (eg, 65 to 75% squalene, or 70% squalene), with the remainder. being polysorbate 80 or a combination of polysorbate 80 and sorbitan trioleate.
[6]
An oil / surfactant composition according to any one of the preceding claims, wherein (a) the composition comprises squalene and polysorbate 80, and the combined volume percent of squalene and polysorbate 80 is 70 to 90 % of the total volume of the oil / surfactant composition; or (b) the composition comprises squalene, sorbitan trioleate and polysorbate 80, and the combined volume percent of squalene and polysorbate 80 is 70-90% of the total volume of squalene, sorbitan trioleate and polysorbate 80 in the oil / surfactant composition.
[7]
An oil-in-water emulsion comprising the oil / surfactant composition according to any one of the preceding claims in combination with an aqueous phase, wherein said emulsion has an average particle diameter of oil of less than 250 nm, preferably in the range of 20 to 200 nm.
[8]
8. A process for forming an oil-in-water emulsion comprising (i) squalene; and (ii) a biocompatible, metabolizable surfactant component comprising at least one surfactant; wherein the surfactant component has a BHL value of (a), (b), (c), (d), (e) or (f) as defined in claim 1, and wherein the emulsion has an average diameter oil particle less than 250 nm; said method comprising: a) providing an oil / surfactant composition according to any one of claims 1 to 6; (b) the supply of an aqueous phase; c) combining the oil / surfactant composition with an excess volume of the aqueous phase to form a diluted composition; and d) lightly mixing the diluted composition to form an oil-in-water emulsion having an average particle diameter of oil of less than 250 nm.
[9]
An oil-in-water emulsion according to claim 7 for use as an adjuvant.
[10]
An immunogenic composition comprising an oil-in-water emulsion according to claim 7, and an antigen component.
[11]
A process for preparing an immunogenic composition comprising mixing an oil-in-water emulsion according to claim 7 with an antigen component.
[12]
Kit comprising: (i) an oil / surfactant composition according to any one of claims 1 to 6; (ii) an aqueous phase; and optionally (iii) instructions for combining the oil / surfactant composition and the aqueous phase, or (i) an oil-in-water emulsion according to claim 7; and (ii) an antigen component.
[13]
13. A dried material which, when reconstituted with an aqueous phase, provides an oil-in-water emulsion according to claim 7.
[14]
The kit for preparing an oil-in-water emulsion of claim 7, wherein the kit comprises: (i) an oil-in-water emulsion according to claim 7 in dried form; and (ii) an aqueous phase.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US9700616B2|2017-07-11|Arranging interaction and back pressure chambers for microfluidization

---

US11141376B2|2021-10-12|Circulation of components during microfluidization and/or homogenization of emulsions

---

US20210077395A1|2021-03-18|Hydrophilic filtration during manufacture of vaccine adjuvants

---

JP5751678B2|2015-07-22|Configuration of interaction chamber and back pressure chamber for microfluidization

---

BE1022714A1|2016-08-23|OIL MIXTURES / SURFACTANT FOR SELF-EMULSIFICATION

---

US8678184B2|2014-03-25|Methods for producing vaccine adjuvants

---

US20180036237A1|2018-02-08|Oil/surfactant mixtures for self-emulsification

---

US8871816B2|2014-10-28|Methods for producing vaccine adjuvants

---

BE1022714B1|2016-08-23|OIL MIXTURES / SURFACTANT FOR SELF-EMULSIFICATION

---

AU2013213678B2|2016-06-23|Arranging interaction and back pressure chambers for microfluidization

---

WO2022003560A1|2022-01-06|Cold filtration of oil-in-water emulsion adjuvants

同族专利:

---

公开号 | 公开日

---

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

EP14160391.0|2014-03-17|

---