JPWO2020160397A5

JPWO2020160397A5 -

Info

Publication number: JPWO2020160397A5
Application number: JP2021544660A
Authority: JP
Publication date: 2023-02-07

Claims

A method of making a lipid nanoparticle (LNP) formulation, comprising:
mixing a lipid solution comprising ionizable lipids with an aqueous buffer solution comprising a first buffering agent, thereby forming a lipid nanoparticle solution comprising lipid nanoparticles; and mixing a nucleic acid solution comprising nucleic acids with said lipid nanoparticles. adding to a solution, thereby forming a lipid nanoparticle (LNP) formulation comprising said lipid nanoparticles associated with said nucleic acid.

2. The method of claim 1, wherein the lipid nanoparticles of the lipid nanoparticle solution are empty lipid nanoparticles.

The lipid nanoparticles of the lipid nanoparticle solution are less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, about 55 nm. 2. The method of claim 1 , having an average diameter of less than, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, or less than about 30 nm.

The lipid nanoparticles of the lipid nanoparticle solution have an average diameter of about 25 nm to about 125 nm, about 30 nm to about 110 nm, about 35 nm to about 100 nm, about 40 nm to about 90 nm, about 45 nm to about 80 nm, or about 50 nm to about 70 nm. 2. The method of claim 1 , having a uniform diameter .

The aqueous buffer solution is about 4.5 to about 6.5, about 4.6 to about 6.0, about 4.7 to about 5.75, about 4.8 to about 5.5, or about 4.5. 2. The method of claim 1 , having a pH in the range of 9 to about 5.25.

wherein the lipid solution has a pH ranging from about 7.0 to about 8.0, from about 7.1 to about 7.8, from about 7.2 to about 7.6, or from about 7.3 to about 7.5; 2. The method of claim 1, comprising:

The nucleic acid solution is about 4.5 to about 6.5, about 4.8 to about 6.25, about 4.8 to about 6.0, about 5.0 to about 5.8, or about 5.2 2. The method of claim 1 , having a pH in the range of to about 5.5.

The lipid nanoparticle solution is about 4.5 to about 6.25, about 4.6 to about 6.0, about 4.8 to about 5.8, about 5.0 to about 5.75, about 5.5. 2. The method of claim 1 , having a pH in the range of 0 to about 5.5.

The lipid nanoparticle formulation is about 4.5 to about 6.0, about 4.6 to about 5.8, about 4.8 to about 5.6, about 5.0 to about 5.5, or about 5 2. The method of claim 1 , having a pH in the range of .1 to about 5.4.

2. The method of claim 1, wherein the lipid nanoparticle solution or lipid nanoparticle formulation has a pH below the pKa of the ionizable lipid.

2. The method of claim 1 , wherein said nucleic acid is ribonucleic acid.

12. The method of claim 11 , wherein said nucleic acid is messenger RNA (mRNA).

2. The method of claim 1, wherein said lipid solution and/or said lipid nanoparticle solution further comprises a first organic solvent.

2. The method of claim 1, wherein said aqueous buffer solution, nucleic acid solution, and/or said lipid nanoparticle solution comprises a first aqueous buffer.

2. The method of claim 1, wherein said aqueous buffer solution, nucleic acid solution, and/or said lipid nanoparticle solution further comprises a second aqueous buffer, a second organic solvent, or both.

wherein the first aqueous buffer and the second aqueous buffer are at least one buffer selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, and Tris buffer; 16. The method of claim 15 .

17. The method of claim 16 , wherein the first organic solvent, the second organic solvent, or both are alcohols.

2. The method of claim 1 , wherein the lipid solution, the nucleic acid solution, or both further comprise a third organic solvent.

19. The method of claim 18 , wherein said third organic solvent is an alcohol.

2. The method of claim 1, further comprising processing said lipid nanoparticle formulation.

2. The method of claim 1 , wherein said lipid solution, said lipid nanoparticle (LNP) solution, and/or said lipid nanoparticle (LNP) formulation further comprises an encapsulating agent.

The encapsulant is
i) Formula (EA-I):

or a salt thereof, wherein
R ₂₀₁ and R ₂₀₂ are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, and (C═NH)N(R ₁₀₁ ) ₂ , wherein each R ₁₀₁ is independently selected from the group consisting of H, C ₁ -C ₆ alkyl, and C ₂ -C ₆ alkenyl;
R ₂₀₃ is selected from the group consisting of C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl;
R ₂₀₄ is H, C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C(O)(OC ₁ -C ₂₀ alkyl), C(O)(OC ₂ -C ₂₀ alkenyl), C(O) (NHC ₁ -C ₂₀ alkyl), and C(O) (NHC ₂ -C ₂₀ alkenyl);
n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
ii) Formula (EA-II):

or a salt thereof, wherein
X ₁₀₁ is a bond, NH, or O;
R ₁₀₁ and R ₁₀₂ are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, and C ₂ -C ₆ alkenyl;
R ₁₀₃ and R ₁₀₄ are each independently selected from the group consisting of C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl, and
n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; or
iii) ethyl lauroyl alginate or a salt thereof;
22. The method of claim 21 .

2. The method of claim 1, wherein the weight/weight ratio of said lipid nanoparticle (LNP) formulation to said nucleic acid ranges from about 5:1 to about 60:1.

2. The method of claim 1, wherein the lipid nanoparticles or lipid nanoparticle formulations further comprise phospholipids, PEG lipids, structured lipids, or any combination thereof.

The lipid nanoparticles and/or lipid nanoparticle formulations are
about 40-60 mol% ionizable lipids;
about 5-15 mol % phospholipids,
about 35-45 mol % structured lipids, or
25. The method of claim 24 , comprising about 0.01-2.0 mol% PEG lipid.

The PEG lipid is
i) selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol , and PEG-modified dialkylglycerol ;
ii) Formula (PL-I):

or a salt thereof, wherein
R ³ is —OR ^O ;
R ^O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;
r is an integer from 1 to 100, inclusive;
L ¹ is optionally substituted C _1-10 alkylene, wherein at least one methylene of said optionally substituted C _1-10 alkylene is optionally substituted carbocyclylene; optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R ^N ), S, C(O), C(O)N(R ^N ), NRNC (O), C(O)O, -OC(O), OC(O)O, OC(O)N(R ^N ⁾ , NRNC ⁽ O)O, or ^NRNC ( independently replaced by O)N(R ^N );
D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;
m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
A is

is the formula for
Each instance of L ² is independently a bond or optionally substituted C _1-6 alkylene, wherein one methylene unit of said optionally substituted C 1-6 alkylene is _O , N(R ^N ), S, C(O), C(O)N(R ^N ⁾ , NRNC (O), C(O)O, OC(O), OC(O)O, OC( optionally replaced by O)N(R ^N ), —NR ^N C(O)O, or NR ^N C(O)N(R ^N );
each instance of R ² is independently optionally substituted C _1-30 alkyl, optionally substituted C _1-30 alkenyl, or optionally substituted C _1-30 alkynyl; optionally, one or more methylene units of R ² are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R ^N ), O, S, C(O), C(O)N(R ^N ), NR ^N C(O), —NR ^N C(O)N(R ^N ), C(O)O, OC(O), OC(O)O, OC(O)N(R ^N ), NR ^N C(O)O, C(O)S, SC(O), -C(= NR ^N ), C(=NR ^N )N(R ^N ), NR ^N C(=NR ^N ), NR ^N C(=NR ^N )N(R ^N ), C(S), C(S)N( R ^N ), NRNC (S), NRNC ^{(S)N(R N} ⁾ , ^S (O), OS(O), S(O)O, OS(O)O, OS(O) ₂ , S(O) ₂ O, OS(O) ₂ O, N(R ^N )S(O), —S(O)N(R ^N ), N(R ^N )S(O)N(R ^N ) , OS(O)N(R ^N ), N(R ^N )S(O)O, S(O) ₂ , N(R ^N )S(O) ₂ , S(O) ₂ N(R ^N ), independently replaced by N(R ^N )S(0) ₂ N(R ^N ), OS(O) ₂ N(R ^N ), or N(R ^N )S(O) ₂ O;
each instance of R ^N is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group;
Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, and
p is 1 or 2, or
iii) Formula (PL-II):

or a salt thereof, wherein
R ³ is —OR ^O ;
R ^O is hydrogen, optionally substituted alkyl or an oxygen protecting group;
r is an integer from 1 to 100,
R ⁵ is optionally substituted C _10-40 alkyl, optionally substituted C _10-40 alkenyl, or optionally substituted C _10-40 alkynyl, and optionally R ⁵ The one or more methylene groups of is optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R ^N ), O, S, C(O), —C(O)N(R ^N ), NR ^N C(O), NR ^N C(O)N(R ^N ), C(O)O, OC(O ), OC(O)O, OC(O)N(R ^N ), —NR ^N C(O)O, C(O)S, SC(O), C(=NR ^N ), C(=NR ^N ) N(R ^N ), NR ^N C(=NR ^N ), NR ^N C(=NR ^N )N(R ^N ), C(S), C(S)N(R ^N ), NR ^N C(S ), NR ^N C(S)N(R ^N ), S(O), OS(O), S(O)O, OS(O)O, OS(O) ₂ , —S(O) ₂ O, OS(O) _2O , N(R ^N )S(O), S(O)N(R ^N ), N(R ^N )S(O)N(R ^N ), OS(O)N(R ^N ), N(R ^N )S(O)O, S(O) ₂ , N(R ^N )S(O) ₂ , S(O) ₂ N(R ^N ), N(R ^N )S(O) ₂ N(R ^N ), OS(O) ₂ N(R ^N ), or N(R ^N )S(O) ₂ O, and
Each instance of R ^N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group, or
iv) Formula (PL-III):

or a salt thereof, wherein s is an integer of 1-100 .

25. The method of claim 24 , wherein said structured lipid is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha- tocopherol . .

The phospholipid is 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-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero -phosphocholine (DUPC), 1-palmitoyl-2-oleyl-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 (C16 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-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 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-didocosahexae selected from noyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin; Item 25. The method of Item 24 .

The ionizable lipid is
i) contains an ionizable amino lipid ;
ii) Formula (IL-1):

or a salt thereof, wherein
R ₁ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R ^* YR", -YR", and -R"M'R';
R ₂ and R ₃ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, —R ^* YR″, —YR″ and —R ^* OR″, or R ₂ and R ₃ together with the atoms to which they are attached form a heterocyclic or carbocyclic ring;
R ₄ is the group consisting of hydrogen, C _3-6 carbocycle, —(CH ₂ ) _n Q, —(CH ₂ ) _n CHQR, —CHQR, —CQ(R) ₂ and unsubstituted C _1-6 alkyl wherein Q is carbocycle, heterocycle, —OR, —O(CH ₂ ) _n N(R) ₂ , —C(O)OR, —OC(O)R, —CX ₃ , —CX ₂ H, —CXH ₂ , —CN, —N(R) ₂ , —C(O)N(R) ₂ , —N(R)C(O)R, —N(R)S(O) ₂ R, —N(R)C(O)N(R) ₂ , —N(R)C(S)N(R) ₂ , —N(R)R ₈ , N(R)S(O) ₂ R ₈ , —O(CH ₂ ) _n OR, —N(R)C(=NR ₉ )N(R) ₂ , —N(R)C(=CHR ₉ )N(R) ₂ , —OC(O )N(R) ₂ , —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O) ₂ R, —N(OR)C(O) OR, —N(OR)C(O)N(R) ₂ , —N(OR)C(S)N(R) ₂ , —N(OR)C(=NR ₉ )N(R) ₂ , — N(OR)C(=CHR9 ₎ N(R) ₂ , -C(=NR9 ₎ N(R) ₂ , -C(=NR9 ₎ R, -C(O)N(R)OR, and -C(R)N(R) _2C (O)OR, and each n is independently selected from 1, 2, 3, 4, and 5;
each R ₅ is independently selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl, and H;
each R ₆ is independently selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl, and H;
M and M' are independently -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R' )-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)- , —P(O)(OR′)O—, —S(O) ₂ —, —S—S—, aryl groups, and heteroaryl groups, wherein M″ is a bond, C _{1- 13} alkyl or C _2-13 alkenyl,
R ₇ is selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl, and H;
R ₈ is selected from the group consisting of C _3-6 carbocycle and heterocycle;
R ₉ is H, CN, NO ₂ , C _1-6 alkyl, —OR, —S(O) ₂ R, —S(O) ₂ N(R) ₂ , C _2-6 alkenyl, C _3-6 selected from the group consisting of carbocycles and heterocycles,
each R is independently selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl, and H;
each R′ is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, —R ^* YR″, —YR″, and H;
each R″ is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;
each R ^* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;
each Y is independently a C _3-6 carbocycle;
each X is independently selected from the group consisting of F, Cl, Br, and I; and
m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, and wherein R ₄ is —(CH ₂ ) _n Q, —(CH ₂ ) _n CHQR, — CHQR, or -CQ(R) ₂ , then (i) if n is 1, 2, 3, 4 or 5, then Q is not -N(R) ₂ , or (ii) n is , 1 or 2, then Q is not a 5-, 6-, or 7-membered heterocycloalkyl;
iii) Formula (IL-II):

or a salt thereof, wherein l is selected from 1, 2, 3, 4 and 5, M ₁ is a bond or M', R ₄ is hydrogen, unsubstituted C _{1- 3} alkyl, or —(CH ₂ ) _n Q, where n is 2, 3, or 4, and Q is —OH, —NHC(S)N(R) ₂ , —NHC( O)N(R) ₂ , —N(R)C(O)R, —N(R)S(O) ₂ R, —N(R)R ₈ , —NHC(=NR ₉ )N(R) ₂ , —NHC(=CHR ₉ )N(R) ₂ , —OC(O)N(R) ₂ , —N(R)C(O)OR, heteroaryl or heterocycloalkyl, and M and M′ are independently —C(O)O—, —OC(O)—, —OC(O)—M″—C(O)O—, —C(O)N(R′)—,— P(O)(OR′)O—, —S—S—, aryl groups and heteroaryl groups, and R ₂ and R ₃ are independently H, C _1-14 alkyl, and C is selected from the group consisting of _2-14 alkenyl, or
iv) Formula (IL-III):

or a salt or isomer thereof, wherein
W is

and
Ring A is

and
t is 1 or 2;
A ₁ and A ₂ are each independently selected from CH or N;
Z is CH2 _or absent, where dashed lines (1) and (2) represent single bonds respectively when Z is CH2 and dashed line (1) when Z is _absent and (2) are both absent,
R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are independently C _5-20 alkyl, C _5-20 alkenyl, —R′MR′, —R ^* YR″, —YR″, and - is selected from the group consisting of R ^* OR";
R _x1 and R _x2 are each independently H or C _1-3 alkyl;
Each M is independently -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C (O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR') selected from the group consisting of O—, —S(O) ₂ —, —C(O)S—, —SC(O)—, aryl groups, and heteroaryl groups;
M ^* is C ₁ -C ₆ alkyl;
W ¹ and W ² are each independently selected from the group consisting of -O- and -N(R ₆ )-;
each R ₆ is independently selected from the group consisting of H and C _1-5 alkyl;
X ¹ , X ² and X ³ are independently a bond, -CH ₂ -, -(CH ₂ ) ₂ -, -CHR-, -CHY-, -C(O)-, -C(O) O—, —OC(O)—, —(CH ₂ ) _n —C(O)—, —C(O)—(CH ₂ ) _n —, —(CH ₂ ) _n —C(O)O—, —OC(O)—(CH ₂ ) _n —, —(CH ₂ ) _n —OC(O)—, —C(O)O—(CH ₂ ) _n —, —CH(OH)—, —C( S)-, and -CH(SH)-,
each Y is independently a C _3-6 carbocycle;
each R ^* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;
each R is independently selected from the group consisting of C _1-3 alkyl and C _3-6 carbocycle;
each R′ is independently selected from the group consisting of C _1-12 alkyl, C _2-12 alkenyl, and H;
each R″ is independently selected from the group consisting of C _3-12 alkyl, C _3-12 alkenyl and —R ^* MR′; and
n is an integer from 1 to 6,
In the formula, ring A is

If it is,
i) at least one of X ¹ , X ² and X ³ is not —CH ₂ — and/or
ii) at least one of R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ is —R″MR′;
25. The method of claim 24 .

The ionizable lipid is 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4- tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriacont-6,9,28,31-tetraene- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1 ,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl- 3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholester-5 -en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R )), and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca -9,12-dien-1-yloxy]propan-1-amine (octyl- CLinDMA (2S ) 2 ).