PROCESSES FOR PREPARING LIPID NANOPARTICLE COMPOSITIONS FOR THE DELIVERY OF PAYLOAD MOLECULES TO AIRWAY EPITHELIUM
Provided are lipid nanoparticle compositions, and processes for their preparation, which are useful for the delivery of therapeutic or prophylactic agents to airway epithelium in patients.
1 . A process of preparing a filled lipid nanoparticle composition comprising:
(a) mixing a lipid solution comprising:
(i) an ionizable lipid,
(ii) a phospholipid,
(iii) a structural lipid, and
(iv) a PEG-lipid,
with an aqueous buffer solution having a pH of about 4.5 or less, resulting in an empty lipid nanoparticle composition; and
(b) combining the empty lipid nanoparticle composition with payload to form a filled lipid nanoparticle composition, wherein the payload is for delivery to epithelium cells; and
(c) adding a cationic agent to the filled lipid nanoparticle composition.
2 . The process of claim 1 , wherein the payload comprises a nucleic acid.
3 . The process of claim 1 , wherein the payload comprises a nucleic acid which is mRNA.
4 . The process of claim 2 or 3 , wherein the nucleic acid is provided as a nucleic acid solution comprising (i) the nucleic acid and (ii) a buffer capable of maintaining acidic pH.
5 . The process of claim 4 , wherein the nucleic acid solution has a pH of about 3 to about 6.
6 . The process of claim 4 , wherein the nucleic acid solution has a pH of about 5.
7 . The process of any one of claims 4 to 6 , wherein the nucleic acid solution has a buffer concentration of about 5 mM to about 140 mM.
8 . The process of any one of claims 4 to 7 , wherein the nucleic acid is present in the nucleic acid solution at a concentration of about 0.05 to about 5.0 mg/mL.
9 . The process of any one of claims 1 to 8 , wherein the combining of step (b) is carried out at a pH of about 5 to about 6.
10 . The process of any one of claims 1 to 9 , wherein the encapsulation efficiency of step (b) is 90% or greater.
11 . The process of any one of claims 1 to 10 , wherein the cationic agent is a cationic lipid.
12 . The process of claim 11 , wherein the cationic lipid is a sterol amine comprising a sterol-based hydrophobic moiety and a hydrophilic moiety.
13 . The process of claim 12 , wherein the hydrophilic moiety of the sterol amine comprises an amine group comprising one to four primary, secondary, or tertiary amines or mixtures thereof.
14 . The process of claim 12 , wherein the hydrophilic moiety of the sterol amine comprises one or two terminal primary amines.
15 . The process of claim 12 , wherein the hydrophilic moiety of the sterol amine comprises one or two terminal primary amines and one internal secondary amine.
16 . The process of claim 12 , wherein the hydrophilic moiety of the sterol amine comprises one or two tertiary amines.
17 . The process of any one of claims 13 to 16 , wherein at least one amine group has a pKa value of greater than about 8.
18 . The process of any one of claims 12 to 17 , wherein the sterol amine is a compound of Formula (A1):
A-L-B (A1)
or a salt thereof, wherein: A is an amine group, L is an optional linker, and B is a sterol.
19 . The process of any one of claims 12 to 17 , wherein the sterol amine is a compound of Formula A2a:
or a salt thereof, wherein:
is a single or double bond
R 1 is C 1-14 alkyl or C 1-14 alkenyl;
L a is absent, —O—, —S—S—, —OC(═O), —C(═O)N—, —OC(═O)N—, CH 2 —NH—C(O)—, —C(O)O—, —OC(O)—CH 2 —CH 2 —C(═O)N—, —S—S—CH 2 , —SS—CH 2 —CH 2 —C(O)N—, or a group of formula (a):
Y 1 is C 1-10 alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), or C 1-6 alkyl-(5 to 6 membered heteroaryl)
wherein the alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), and C 1-6 alkyl-(5 to 6 membered heteroaryl) comprises one to five primary, secondary, or tertiary amines or combination thereof
wherein the alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), and C 1-6 alkyl-(5 to 6 membered heteroaryl) are each optionally substituted with 1, 2, 3, or 4 substituents selected from C 1-6 alkyl, halo, OH, O(C 1-6 alkyl), C 1-6 alkyl-OH, NH 2 , NH(C 1-6 alkyl), N(C 1-6 alkyl) 2 , 3 to 8 membered heterocycloalkyl (optionally substituted with C 1-14 alkyl comprising one to five primary, secondary, or tertiary amines or combination thereof), 5 to 6 membered heteroaryl, NH (3 to 8 membered heterocycloalkyl), and NH (5 to 6 membered heteroaryl); and
n=1 or 2.
20 . The process of any one of claims 12 to 17 , wherein the sterol amine is a compound of Formula A3a:
or a salt thereof, wherein:
is a single or double bond;
R 2 is H or C 1-6 alkyl;
L a is absent, —O—, —S—S—, —OC(═O), —C(═O)N—, —OC(═O)N—, CH 2 —NH—C(O)—, —C(O)O—, —OC(O)—CH 2 —CH 2 —C(═O)N—, —S—S—CH 2 , —SS—CH 2 —CH 2 —C(O)N—, or a group of formula (a):
Y 1 is C 1-10 alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), or C 1-6 alkyl-(5 to 6 membered heteroaryl),
wherein the alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), and C 1-6 alkyl-(5 to 6 membered heteroaryl) comprises one to five primary, secondary, or tertiary amines or combination thereof,
wherein the alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), and C 1-6 alkyl-(5 to 6 membered heteroaryl) are each optionally substituted with 1, 2, 3, or 4 substituents selected from C 1-6 alkyl, halo, OH, O(C 1-6 alkyl), C 1-6 alkyl-OH, NH 2 , NH(C 1-6 alkyl), N(C 1-6 alkyl) 2 , 3 to 8 membered heterocycloalkyl (optionally substituted with C 1-14 alkyl comprising one to five primary, secondary, or tertiary amines or combination thereof), 5 to 6 membered heteroaryl, NH (3 to 8 membered heterocycloalkyl), and NH (5 to 6 membered heteroaryl); and
n=1 or 2.
21 . The process of any one of claims 12 to 17 , wherein the sterol amine is a compound of Formula A4:
or a salt thereof, wherein:
Z 1 is OH or C 3-6 alkyl;
L is absent, —O—, —S—S—, —OC(═O), —C(═O)N—, —OC(═O)N—, CH 2 —NH—C(O)—, —C(O)O—, —OC(O)—CH 2 —CH 2 —C(═O)N—, —S—S—CH 2 , or —SS—CH 2 —CH 2 —C(O)N—;
Y 1 is C 1-10 alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), or C 1-6 alkyl-(5 to 6 membered heteroaryl),
wherein the alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), and C 1-6 alkyl-(5 to 6 membered heteroaryl) comprises one to five primary, secondary, or tertiary amines or combination thereof,
wherein the alkyl, 3 to 8 membered heterocycloalkyl, 5 to 6 membered heteroaryl, C 1-6 alkyl-(3 to 8 membered heterocycloalkyl), and C 1-6 alkyl-(5 to 6 membered heteroaryl) are each optionally substituted with 1, 2, 3, or 4 substituents selected from C 1-4 alkyl, halo, OH, O(C 1-4 alkyl), C 1-4 alkyl-OH, NH 2 , NH(C 1-6 alkyl), N(C 1-4 alkyl) 2 , 3 to 8 membered heterocycloalkyl (optionally substituted with C 1-14 alkyl comprising one to five primary, secondary, or tertiary amines or combination thereof), 5 to 6 membered heteroaryl, NH (3 to 8 membered heterocycloalkyl), and NH (5 to 6 membered heteroaryl); and
n=1 or 2.
22 . The process of any one of claims 19 to 21 , wherein Y 1 is selected from:
23 . The process of any one of claims 12 to 17 , wherein the sterol amine is a compound of Formula A5:
or a salt thereof, wherein:
Z 2 is OH or isopropyl; and
L 3 is —CH 2 —NH—C(O)—, —C(O)NH—, or —C(O)O—.
24 . The process of any one of claims 12 to 17 , wherein the sterol amine is a compound selected from:
Sterol amine
no.
Structure
SA1
SA2
SA3
SA4
SA5
SA6
SA7
SA8
SA9
SA10
SA11
SA12
SA13
SA14
SA15
SA16
SA17
SA18
SA19
SA20
SA21
SA22
SA23
SA24
SA25
SA26
SA27
SA28
SA29
SA30
SA31
SA32
SA33
SA34
SA35
SA36
SA37
SA38
SA39
SA40
SA41
SA42
SA43
or a salt thereof.
25 . The process of any one of claims 12 to 17 , wherein the sterol amine is SA3:
or a salt thereof.
26 . The process of any one of claims 1 to 25 , wherein the cationic agent is provided in a cationic agent solution comprising the cationic agent and a buffer.
27 . The process of claim 26 , wherein the cationic agent solution has a pH of about 7 to about 8.
28 . The process of claim 26 or 27 , wherein the buffer concentration of the cationic agent solution is about 5 mM to about 100 mM.
29 . The process of any one of claims 26 to 28 , wherein the buffer of the cationic agent solution comprises Tris.
30 . The process of any one of claims 26 to 29 , wherein the cationic agent concentration of the cationic agent solution is about 0.1 to about 50 mg/mL.
31 . The process of any one of claims 1 to 30 , further comprising adding a surface-acting agent to the filled lipid nanoparticle.
32 . The process of claim 31 , wherein the surface-acting agent is a PEG lipid.
33 . The process of claim 31 , wherein the surface-acting agent is provided together with the cationic agent.
34 . The process of any one of claims 31 to 33 , wherein the surface-acting agent is a PEG-lipid having a concentration of about 0.1 to about 50 mg/mL.
35 . The process of any one of claims 1 to 34 further comprising one or more additional steps selected from:
diluting the composition with a dilution buffer;
adjusting the pH of the composition;
filtering the composition;
concentrating the composition;
exchanging buffer of the composition; and
adding an osmolality modifier to the composition.
36 . The process of claim 35 , wherein the one or more additional steps is adding an osmolality modifier to the composition.
37 . The process of claim 36 , wherein the osmolality modifier is sodium chloride.
38 . The process of claim 36 or 37 , wherein the osmolality modifier is provided as a salt solution comprising an inorganic salt and a buffer.
39 . The process of claim 38 , wherein the inorganic salt in the salt solution has a concentration of about 100 to about 750 mM.
40 . The process of claim 38 or 39 , wherein the salt solution has a pH of about 7 to about 8.
41 . The process of any one of claims 1 to 40 , wherein the aqueous buffer solution in step (a) has a pH of about 3.5 to about 4.5.
42 . The process of any one of claims 1 to 41 , wherein the aqueous buffer solution of step (a) has a buffer concentration greater than about 30 mM.
43 . The process of any one of claims 1 to 42 , wherein the aqueous buffer solution of step (a) has an ionic strength of about 15 mM or less.
44 . The process of any one of claims 1 to 42 , wherein the aqueous buffer solution of step (a) has an ionic strength of about 0.1 mM to about 15 mM.
45 . The process of any one of claims 1 to 44 , wherein the aqueous buffer solution of step (a) comprises an acetate buffer, a citrate buffer, a phosphate buffer, a tris buffer, or a mixture thereof.
46 . The process of any one of claims 1 to 45 , wherein the empty lipid nanoparticle composition of step (a) is characterized by a zeta potential of about 35 mV or more.
47 . The process of any one of claims 1 to 45 , wherein the empty lipid nanoparticle composition of step (a) is characterized as having a zeta potential which is substantially at maximum.
48 . The process of any one of claims 1 to 47 , wherein the lipid solution has a lipid concentration of about 5 to about 100 mg/mL.
49 . The process of any one of claims 1 to 48 , wherein the mixing of step (a) is carried out in a multi-inlet vortex mixer.
50 . The process of any one of claims 1 to 49 , wherein the empty lipid nanoparticles of the empty lipid nanoparticle composition have an average diameter of about 30 nm or less.
51 . The process of any one of claims 1 to 49 , wherein the lipid nanoparticles of the empty lipid nanoparticle composition are substantially free of payload.
52 . The process of any one of claims 1 to 51 , wherein the empty lipid nanoparticles of the empty lipid nanoparticle composition are stable.
53 . The process of claim 52 , wherein the average diameter of the empty lipid nanoparticles of the empty lipid nanoparticle composition increases less than about 150% over 25 hours.
54 . The process of claim 52 , wherein the average diameter of the lipid nanoparticles of the empty lipid nanoparticle composition remains below 50 nm over 25 hours.
55 . The process of any one of claims 1 to 54 , wherein step (a) further comprises one or more additional steps selected from:
diluting the composition with a dilution buffer;
adjusting the pH of the composition to a pH of about 5 to about 6;
filtering the composition;
concentrating the composition;
exchanging buffer of the composition; and
adding cryoprotectant to the composition.
56 . The process of claim 55 , wherein the one or more additional steps is adjusting the pH of the empty lipid nanoparticle composition to a pH of about 5 to about 6.
57 . The process of claim 55 , wherein the one or more additional steps is adding cryoprotectant to the empty lipid nanoparticle composition.
58 . The process of claim 57 , wherein the cryoprotectant is sucrose.
59 . The process of claim 55 , which includes the steps of:
adjusting the pH of the composition to a pH of about 5; and
adding cryoprotectant to the composition.
60 . The process of any one of claims 1 to 59 , wherein the ionizable lipid comprises a compound of Formula (I):
or an N-oxide or a salt thereof, wherein:
R 1 is:
wherein
denotes a point of attachment;
R aα , R aβ , R aγ , and R aδ are each independently selected from H, C 2-12 alkyl, and C 2-12 alkenyl;
R 2 and R 3 are each independently selected from C 1-14 alkyl and C 2-14 alkenyl;
R 4 is selected from —(CH 2 ) n OH and
wherein n is selected from 1, 2, 3, 4, and 5;
wherein
denotes a point of attachment,
wherein R 10 is N(R) 2 ;
wherein each R is independently selected from C 1-6 alkyl, C 2-3 alkenyl, and H;
wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
each R 5 is independently selected from C 1-3 alkyl, C 2-3 alkenyl, and H;
each R 6 is independently selected from C 1-3 alkyl, C 2-3 alkenyl, and H;
M and M′ are each independently selected from —C(O)O— and —OC(O)—;
R′ is C 1-12 alkyl or C 2-12 alkenyl;
l is selected from 1, 2, 3, 4, and 5; and
m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.
61 . The process of claim 60 , wherein the ionizable lipid has the structure:
or an N-oxide or a salt thereof.
62 . The process of any one of claims 1 to 61 , wherein the phospholipid is selected from:
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),
1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC),
1-hexadecyl-sn-glycero-3-phosphocholine (C 1-6 Lyso PC),
1,2-dilinolenoyl-sn-glycero-3-phosphocholine,
1,2-diarachidonoyl-sn-glycero-3-phosphocholine,
1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,
1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,
1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.
63 . The process of any one of claims 1 to 62 , wherein the structural lipid is selected from: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, a hopanoid, a phytosterol, a steroid, or a mixture thereof.
64 . The process of any one of claims 1 to 63 , wherein the PEG-lipid is selected from: a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
65 . The process of any one of claims 1 to 64 , wherein the lipid solution, empty lipid nanoparticle composition, or filled lipid nanoparticle composition comprises about 30 mol % to about 60 mol % of ionizable lipid with respect to total lipids.
66 . The process of any one of claims 1 to 65 , wherein the lipid solution, empty lipid nanoparticle composition, or filled lipid nanoparticle composition comprises about 5 mol % to about 15 mol % of phospholipid with respect to total lipids.
67 . The process of any one of claims 1 to 66 , wherein the lipid solution, empty lipid nanoparticle composition, or filled lipid nanoparticle composition comprises about 30 mol % to about 50 mol % of structural lipid with respect to total lipids.
68 . The process of any one of claims 1 to 67 , wherein the lipid solution, empty lipid nanoparticle composition, or filled lipid nanoparticle composition comprises about 0.1 mol % to about 2 mol % of PEG-lipid with respect to total lipids.
69 . The process of any one of claims 1 to 68 , wherein the lipid solution, empty lipid nanoparticle composition, or filled lipid nanoparticle composition comprises:
about 40 mol % to about 50 mol % of ionizable lipid;
about 10 mol % to about 12 mol % of phospholipid;
about 37 mol % to about 42 mol % of structural lipid; and
about 0.25 mol % to about 0.75 mol % of PEG-lipid; each with respect to total lipids.
70 . The process of any one of claims 1 to 69 , wherein a weight ratio of the cationic agent to payload is about 1:1 to about 4:1.
71 . A lipid nanoparticle composition prepared by the process of any one of claims 1 to 70 .
72 . The lipid nanoparticle composition of claim 71 , comprising the following components:
(i) an ionizable lipid,
(ii) a phospholipid,
(iii) a structural lipid,
(iv) a PEG-lipid,
(v) a cationic agent, and
(vi) a payload;
wherein the lipid nanoparticle composition has a pH of about 4.5 to about 8;
73 . The lipid nanoparticle composition of claim 72 having a pH of about 7 to about 8.
74 . The lipid nanoparticle composition of claim 72 or 73 , wherein the concentration of payload is about 0.1 to about 10 mg/mL.
75 . The lipid nanoparticle composition of any one of claims 72 to 74 , further comprising about 0.1% to about 10% w/v sucrose.
76 . The lipid nanoparticle composition of any one of claims 72 to 75 , further comprising about 5 mM to about 150 mM NaCl.
77 . The lipid nanoparticle composition of any one of claims 72 to 76 , further comprising about 5 mM to about 100 mM buffer.
78 . The lipid nanoparticle composition of any one of claims 72 to 76 , wherein the buffer comprises an acetate buffer and a Tris buffer.
79 . The lipid nanoparticle composition of any one of claims 72 to 78 , which is frozen or lyophilized.
80 . A pharmaceutical composition, comprising the lipid nanoparticle composition of any one of claims 71 to 79 and at least one pharmaceutically acceptable excipient.
81 . A method of delivering a payload into a cell comprising contacting the cell with a lipid nanoparticle composition of any one of claims 71 to 80 .
82 . The method of claim 81 , wherein the cell is an airway epithelium cell.
83 . A method of treating or preventing a disease in a patient comprising administering to the patient a lipid nanoparticle composition of any one of claims 71 to 80 comprising administering the lipid nanoparticle composition to the patient.
84 . The method of claim 83 , wherein the disease is associated with dysfunction of the airway epithelium.
85 . The method of any one of claims 82 to 84 , wherein the lipid nanoparticle composition is administered by intranasal, intrabronchiol, or pulmonary administration.