METHODS FOR PREPARING VESICLES AND FORMULATIONS PRODUCED THEREFROM
The present disclosure provides methods for preparing vesicles. In one aspect, the methods involve providing a molten mixture of vesicle-forming lipids and adding the molten mixture of vesicle-forming lipids to an aqueous solution comprising an antigen such that antigen-containing vesicles are formed, wherein in the step of adding the molten mixture of vesicle forming lipids is at a temperature of less than 120° C. In another aspect, the methods involve providing a molten mixture of vesicle-forming lipids and adding an aqueous solution comprising an antigen to the molten mixture of vesicle-forming lipids such that antigen-containing vesicles are formed, wherein the resulting mixture is placed under temperature-controlled conditions of less than 60° C. In yet another aspect, the methods involve providing a solution of vesicle forming lipids and adding the solution of vesicle-forming lipids to an aqueous solution comprising an antigen by injection such that antigen-containing vesicles are formed.
1 . A method comprising:
providing a molten mixture of vesicle-forming lipids; and
adding the molten mixture of vesicle-forming lipids to an aqueous solution comprising an antigen such that antigen-containing vesicles are formed, wherein in the step of adding the molten mixture of vesicle-forming lipids is at a temperature of less than 120° C.
2 . A method comprising:
providing a molten mixture of vesicle-forming lipids; and
adding an aqueous solution comprising an antigen to the molten mixture of vesicle-forming lipids such that antigen-containing vesicles are formed, wherein the resulting mixture is placed under temperature-controlled conditions of less than 60° C.
3 . The method of claim 2 , wherein the molten mixture of vesicle-forming lipids is placed under temperature-controlled conditions of less than 60° C. before step of adding.
4 . The method of claim 2 , wherein the molten mixture of vesicle-forming lipids is not placed under temperature-controlled conditions of less than 60° C. before the step of adding.
5 . The method of claim 1 or 2 , wherein the aqueous solution comprising an antigen is at a temperature of less than about 50° C. in the step of adding.
6 . The method of claim 1 or 2 , wherein the aqueous solution comprising an antigen is at a temperature of less than about 40° C. during the step of adding.
7 . The method of claim 1 or 2 , wherein the aqueous solution comprising an antigen is at a temperature of less than about 30° C. during the step of adding.
8 . The method of claim 1 , wherein the aqueous solution comprising an antigen is temperature controlled during the step of adding.
9 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature that is no more than 50° C. above its melting point during the step of adding.
10 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature that is no more than 40° C. above its melting point during the step of adding.
11 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature that is no more than 30° C. above its melting point during the step of adding.
12 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature that is no more than 20° C. above its melting point during the step of adding.
13 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature that is no more than 10° C. above its melting point during the step of adding.
14 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature that is no more than 5° C. above its melting point during the step of adding.
15 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature of less than about 110° C. during the step of adding.
16 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature of less than about 100° C. during the step of adding.
17 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature of less than about 90° C. during the step of adding.
18 . The method of claim 1 or 2 , wherein the molten mixture of vesicle-forming lipids is at a temperature of less than about 80° C. during the step of adding.
19 . The method of any one of claims 1 - 18 , wherein the vesicle-forming lipids comprise a phospholipid.
20 . The method of any one of claims 1 - 18 , wherein the vesicle-forming lipids comprise a non-ionic surfactant.
21 . The method of claim 20 , wherein the non-ionic surfactant is a glycerol ester.
22 . The method of claim 20 , wherein the non-ionic surfactant is a glycol or glycol ether.
23 . The method of claim 20 , wherein the non-ionic surfactant is 1-monopalmitoyl glycerol.
24 . The method of claim 20 , wherein the non-ionic surfactant is 1-monocetyl glycerol ether or diglycolcetyl ether.
25 . The method of any one of claims 1 - 18 , wherein the molten mixture of vesicle-forming lipids further comprises a transport enhancer which facilitates the transport of lipids across mucosal membranes.
26 . The method of claim 25 , wherein the transport enhancer is a cholesterol derivative in which the C 23 carbon atom of the side chain carries a carboxylic acid.
27 . The method of claim 25 , wherein the transport enhancer is cholic acid, chenodeoxycholic acid or a salt thereof.
28 . The method of claim 25 , wherein the transport enhancer is glycocholic acid, taurocholic acid, deoxycholic acid, ursodeoxycholic acid, or a salt thereof.
29 . The method of claim 25 , wherein the transport enhancer is an acyloxylated amino acid or a salt thereof.
30 . The method of claim 25 , wherein the transport enhancer is an acylcarnitine containing a C 6-20 alkanoyl or alkenoyl moiety or a salt thereof.
31 . The method of any one of claims 1 - 18 , wherein the molten mixture of vesicle-forming lipids does not comprise a transport enhancer which facilitates the transport of lipids across mucosal membranes.
32 . The method of any one of claims 1 - 18 , wherein the molten mixture of vesicle-forming lipids further comprises an ionic surfactant.
33 . The method of claim 32 , wherein the ionic surfactant is an alkanoic acid or an alkenoic acid.
34 . The method of claim 32 , wherein the ionic surfactant is a phosphate.
35 . The method of claim 32 , wherein the ionic surfactant is dicetylphospate, phosphatidic acid or phosphatidyl serine.
36 . The method of claim 32 , wherein the ionic surfactant is a sulphate monoester.
37 . The method of claim 32 , wherein the ionic surfactant is cetylsulphate.
38 . The method of any one of claims 1 - 18 , wherein the molten mixture of vesicle-forming lipids further comprises a steroid.
39 . The method of claim 38 , wherein the steroid is cholesterol.
40 . The method of any one of claims 1 - 39 , wherein the aqueous antigen solution further comprises a lyoprotectant.
41 . The method of claim 40 , wherein the lyoprotectant is selected from the group consisting of sucrose, trehalose, polyethylene glycol (PEG), dimethyl-succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran.
42 . The method of claim 40 , wherein the lyoprotectant is sucrose.
43 . The method of any one of claims 1 - 18 , wherein the antigen is a virus.
44 . The method of claim 43 , wherein the virus is an attenuated virus.
45 . The method of claim 43 , wherein the virus is an inactivated virus.
46 . The method of any one of claims 43 - 45 , wherein the virus is an influenza virus.
47 . The method of any one of claims 43 - 45 , wherein the virus is a measles virus, a mumps virus, a rubella virus, a varicella virus or a combination thereof.
48 . The method of any one of claims 43 - 45 , wherein the virus is selected from the group consisting of rotavirus, herpes zoster virus, vaccinia virus, yellow fever virus, and combinations thereof.
49 . The method of any one of claims 1 - 18 , wherein the antigen is a polypeptide.
50 . The method of claim 49 , wherein the polypeptide is a viral polypeptide.
51 . The method of claim 50 , wherein the polypeptide is an influenza polypeptide.
52 . The method of any one of claims 1 - 18 , wherein the antigen is thermolabile.
53 . The method of any one of claims 1 - 18 , wherein the aqueous solution comprises a mixture of antigens.
54 . The method of claim 53 , wherein the aqueous solution comprises a mixture of polypeptides.
55 . The method of claim 54 , wherein the mixture of polypeptides comprises a mixture of polypeptides from the same virus.
56 . The method of any one of claims 1 - 18 , wherein the antigen is a polynucleotide.
57 . The method of any one of claims 1 - 18 , wherein the antigen is a polysaccharide.
58 . The method of any one of claims 1 - 18 , further comprising a step of adding an adjuvant after the antigen-containing vesicles are formed.
59 . The method of claim 58 , wherein the adjuvant is a TLR-3 or TLR-4 agonist.
60 . The method of any one of claims 1 - 18 , wherein the molten mixture of vesicle-forming lipids comprises an adjuvant.
61 . The method of claim 60 , wherein the adjuvant is a TLR-3 or TLR-4 agonist.
62 . The method of any one of claims 1 - 61 , further comprising a step of lyophilizing a formulation that comprises the antigen-containing vesicles.
63 . The method of claim 62 , further comprising a step of rehydrating the antigen-containing vesicles after they have been lyophilized.
64 . A method comprising:
providing a solution of vesicle-forming lipids in an organic solvent; and
adding the solution of vesicle-forming lipids to an aqueous solution comprising an antigen by injection such that antigen-containing vesicles are formed.
65 . The method of claim 64 , further comprising preparing the solution of vesicle-forming lipids in the organic solvent by dissolving vesicle-forming lipids in the organic solvent.
66 . The method of claim 65 , wherein the vesicle-forming lipids are dissolved in an organic solvent without any co-solvents.
67 . The method of claim 65 , wherein the vesicle-forming lipids are dissolved in an organic solvent with one or more co-solvents.
68 . The method of claim 65 , wherein the vesicle-forming lipids are dissolved in a water-free solvent system.
69 . The method of any one of claims 64 - 68 , wherein the organic solvent is a water-miscible solvent.
71 . The method of any one of claims 64 - 69 , wherein the organic solvent is a polar-protic organic solvent.
72 . The method of claim 71 , wherein the polar-protic organic solvent is an aliphatic alcohol having 2-5 carbon atoms.
73 . The method of claim 71 , wherein the polar-protic organic solvent is an aliphatic alcohol having 4 carbon atoms.
74 . The method of claim 71 , wherein the polar-protic organic solvent is tert-butanol.
75 . The method of claim 71 , wherein the polar-protic organic solvent is ethanol.
76 . The method of any one of claims 64 - 69 , wherein the organic solvent is diethyl ether.
77 . The method of any one of claims 64 - 76 , wherein the vesicle-forming lipids comprise a phospholipid.
78 . The method of any one of claims 64 - 77 , wherein the vesicle-forming lipids comprise a non-ionic surfactant.
79 . The method of claim 78 , wherein the non-ionic surfactant is a glycerol ester.
80 . The method of claim 78 , wherein the non-ionic surfactant is a glycol or glycol ether.
81 . The method of claim 78 , wherein the non-ionic surfactant is 1-monopalmitoyl glycerol.
82 . The method of claim 78 , wherein the non-ionic surfactant is 1-monocetyl glycerol ether or diglycolcetyl ether.
83 . The method of any one of claims 64 - 82 , wherein in the step of adding the solution of vesicle-forming lipids is at a temperature of less than 90° C.
84 . The method of any one of claims 64 - 82 , wherein in the step of adding the solution of vesicle-forming lipids is at a temperature of less than 70° C.
85 . The method of any one of claims 64 - 82 , wherein in the step of adding the solution of vesicle-forming lipids is at a temperature of 55° C. to 65° C.
86 . The method of any one of claims 64 - 82 , wherein the aqueous solution comprising an antigen is at a temperature of less than 50° C. in the step of adding.
87 . The method of any one of claims 64 - 82 , wherein the aqueous solution comprising an antigen is at a temperature of less than 40° C. during the step of adding.
88 . The method of any one of claims 64 - 82 , wherein the aqueous solution comprising an antigen is at a temperature of 30° C. to 35° C. during the step of adding.
89 . The method of claim 64 , wherein the aqueous solution comprising an antigen is temperature controlled during the step of adding.
90 . The method of any one of claims 64 - 89 , wherein the solution of vesicle-forming lipids further comprises a transport enhancer which facilitates the transport of lipids across mucosal membranes.
91 . The method of claim 90 , wherein the transport enhancer is a cholesterol derivative in which the C 23 carbon atom of the side chain carries a carboxylic acid.
92 . The method of claim 90 , wherein the transport enhancer is cholic acid, chenodeoxycholic acid or a salt thereof.
93 . The method of claim 90 , wherein the transport enhancer is glycocholic acid, taurocholic acid, deoxycholic acid, ursodeoxycholic acid, or a salt thereof.
94 . The method of claim 90 , wherein the transport enhancer is an acyloxylated amino acid or a salt thereof.
95 . The method of claim 90 , wherein the transport enhancer is an acylcarnitine containing a C 6-20 alkanoyl or alkenoyl moiety or a salt thereof.
96 . The method of any one of claims 64 - 95 , wherein the solution of vesicle-forming lipids does not comprise a transport enhancer which facilitates the transport of lipids across mucosal membranes.
97 . The method of any one of claims 64 - 96 , wherein the solution of vesicle-forming lipids further comprises an ionic surfactant.
98 . The method of claim 97 , wherein the ionic surfactant is an alkanoic acid or an alkenoic acid.
99 . The method of claim 97 , wherein the ionic surfactant is a phosphate.
100 . The method of claim 97 , wherein the ionic surfactant is dicetylphospate, phosphatidic acid or phosphatidyl serine.
101 . The method of claim 97 , wherein the ionic surfactant is a sulphate monoester.
102 . The method of claim 97 , wherein the ionic surfactant is cetylsulphate.
103 . The method of any one of claims 64 - 102 , wherein the solution of vesicle-forming lipids further comprises a steroid.
104 . The method of claim 103 , wherein the steroid is cholesterol.
105 . The method of any one of claims 64 - 104 , wherein the aqueous antigen solution further comprises a lyoprotectant.
106 . The method of claim 105 , wherein the lyoprotectant is selected from the group consisting of sucrose, trehalose, polyethylene glycol (PEG), dimethyl-succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran.
107 . The method of claim 105 , wherein the lyoprotectant is sucrose.
108 . The method of any one of claims 64 - 107 , wherein the antigen is a virus.
109 . The method of claim 108 , wherein the virus is an attenuated virus.
110 . The method of claim 108 , wherein the virus is an inactivated virus.
111 . The method of any one of claims 108 - 110 wherein the virus is an influenza virus.
112 . The method of any one of claims 108 - 110 , wherein the virus is a measles virus, a mumps virus, a rubella virus, a varicella virus or a combination thereof.
113 . The method of any one of claims 108 - 110 , wherein the virus is selected from the group consisting of rotavirus, herpes zoster virus, vaccinia virus, yellow fever virus, and combinations thereof.
114 . The method of any one of claims 64 - 107 , wherein the antigen is a polypeptide.
115 . The method of claim 114 , wherein the polypeptide is a viral polypeptide.
116 . The method of claim 115 , wherein the polypeptide is an influenza polypeptide.
117 . The method of any one of claims 64 - 116 , wherein the antigen is thermolabile.
118 . The method of any one of claims 64 - 116 , wherein the aqueous solution comprises a mixture of antigens.
119 . The method of claim 118 , wherein the aqueous solution comprises a mixture of polypeptides.
120 . The method of claim 119 , wherein the mixture of polypeptides comprises a mixture of polypeptides from the same virus.
121 . The method of any one of claims 64 - 107 , wherein the antigen is a polynucleotide.
122 . The method of any one of claims 64 - 107 , wherein the antigen is a polysaccharide.
123 . The method of any one of claims 64 - 122 , further comprising a step of adding an adjuvant after the antigen-containing vesicles are formed.
124 . The method of claim 123 , wherein the adjuvant is a TLR-3 or TLR-4 agonist.
125 . The method of any one of claims 64 - 122 , wherein the solution of vesicle-forming lipids comprises an adjuvant.
126 . The method of claim 125 , wherein the adjuvant is a TLR-3 or TLR-4 agonist.
127 . The method of any one of claims 64 - 126 , further comprising a step of lyophilizing a formulation that comprises the antigen-containing vesicles.
128 . The method of claim 127 , further comprising a step of rehydrating the antigen-containing vesicles after they have been lyophilized.
129 . A formulation comprising antigen-containing vesicles prepared according to the method of any one of claims 1 - 128 .
130 . A method comprising administering a formulation of claim 129 to a patient in need thereof.
131 . A kit comprising:
a first container that includes a lyophilized antigen-containing vesicle formulation that was prepared according to the method of claim 62 or claim 127 ; and
a second container that includes an aqueous solution such that, when the contents of the second container are mixed with the contents of the first container, the antigen-containing vesicles are rehydrated.
132 . The kit of claim 131 further comprising:
instructions for mixing the contents of the first and second containers in order to rehydrate the antigen-containing vesicles.