RU2808990C2 - Fusogenic compounds for delivery of biologically active molecules - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to fusogenic compounds, as well as compositions containing such compounds.

EFFECT: fusogenic compounds can be used to prepare nanoparticle compositions to incorporate or encapsulate active agents, to deliver and distribute active agents to cells, tissues, organs and subjects.

20 cl, 53 dwg, 11 tbl, 15 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to the field of biopharmaceuticals and therapeutics. More specifically, this invention relates to compounds, compositions and methods comprising fusogenic molecules that provide nanoparticles for the delivery and distribution of active agents or drug compounds in cells, tissues, organs and subjects.

BACKGROUND OF THE ART

[0002] Therapeutic agents, such as drugs, nucleic acid molecules and other active agents, function after absorption into the cells, tissues and organs of the subject. The transfer of agents and molecules into cells is often the limiting step for their therapeutic activity.

[0003] When the active agent molecules are susceptible to attack or degradation in the serum or other biological environment, it becomes necessary to protect the molecule to achieve its medicinal effect.

[0004] For example, one way to accomplish transfection of nucleic acids is to incorporate or encapsulate the active molecules into nanoparticles. A disadvantage of this methodology may be the low degree of penetration into cells.

[0005] There is a long-standing need for molecules with fusogenic properties that would produce nanoparticles with good transfection properties to increase the penetration and delivery of active agents into cells.

[0006] Compositions and compounds are needed to create nanoparticles for active substances. There is a continuing need for molecules and compositions for efficient transfection and distribution of nucleic acid molecules and other agents into cells and subjects.

SUMMARY OF THE INVENTION

[0007] The present invention relates to molecules and compositions thereof for use in biopharmaceuticals and therapeutics. More specifically, this invention relates to compounds, compositions and methods for producing nanoparticles for the delivery and distribution of active agents or drug compounds in cells, tissues, organs and subjects.

[0008] This invention relates to a number of fusogenic compounds. The fusogenic compounds of this invention can be used to form nanoparticles for the delivery and distribution of active agents.

[0009] Examples of active agents of this invention include biologically active molecules, nucleic acids, DNA, RNA, mRNA, siRNA and microRNA, among other forms.

[0010] Embodiments of the present invention include the following:

[0011] Fusogenic compound having formula I:

Formula I,

wherein each amphiphile independently contains from one to two lipophilic chains, wherein each lipophilic chain independently contains from 8 to 22 carbon atoms;

wherein each AA independently represents an amino acid containing a side chain having an amino group, where the amino acid is attached to the amphiphile at each of its amino groups and is attached to a linker at its C-terminus;

where the linker has the structure

or

,

where Q ¹ is a branched or straight C(2-8)alkanediyl, a branched or straight C(2-8)alkendiyl, a branched or straight C(2-8)alkyndiyl, or

or

,

where Q ² represents

,

where Q ³ represents

,

where X represents -O-, -S- or -NH-;

n, p, q and t independently for each case range from 1 to 3;

m independently ranges from 1 to 10;

r and s independently for each case range from 1 to 5.

[0012] The above fusogenic compound, wherein AA is selected from the following structures and any stereoisomers thereof:

.

[0013] The above fusogenic compound wherein one or two amphiphiles are missing and are replaced by an alkyl group or a pharmaceutically acceptable organic chemical group having 1-400 atoms selected from carbon, oxygen, nitrogen, sulfur, fluorine and hydrogen.

[0014] The above fusogenic compound, wherein the pharmaceutically acceptable organic chemical group is alkyl, alkenyl, alkynyl, acetyl, Boc, Fmoc, TFA or CBZ, preferably alkyl, acetyl, more preferably acetyl.

[0015] The above fusogenic compound, wherein the compound is selected from the following compounds:

[0016] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula II

Formula II,

where R ¹ and R ² represent

R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴

R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ ,

where n and m, each independently, range from 1 to 2; and R ⁴ and R ⁵ independently each represent a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is selected from branched or unbranched C(1-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, substituted or unsubstituted C(3-8)cycloalkanediyl, substituted or unsubstituted arylene, substituted or unsubstituted C(4-8)heteroarylene and substituted or unsubstituted heterocycloalkanediyl and combinations thereof; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0017] R ³ is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3-8)cycloalkanediyl , substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0018] As used herein, the term “and combinations thereof” in relation to the formulas indicates additional changes in structure based on the combination of the listed groups. For example, the combination of C(1-8)alkanediyl and C(4-8)heteroarylene refers to C(1-8)alkanediyl-C(4-8)heteroarylene, as well as C(1-8)alkanediyl-C(4 -8)heteroarylene-C(1-8)alkanediyl.

[0019] The above fusogenic compound, where R ³ is selected from branched or straight-chain C(2-8)alkanediyl,

substituted or unsubstituted C(2-8)alkendiyl,

substituted or unsubstituted C(2-8)alkyndiyl,

substituted or unsubstituted C(3-8)cycloalkanediyl,

substituted or unsubstituted C(4-8)heteroarylene,

;

preferably branched or straight-chain C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0020] The above fusogenic compound, where one or more amphiphiles have the structure shown in formula III:

Formula III,

where R ¹ and R ² represent:

R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴

R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ ,

where n and m, each independently, range from 1 to 2; and R ⁴ and R ⁵ independently each represent a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is selected from:

-alkyl-(C=O)-, which is attached to AA;

-alkyl-O(C=O)-, which is attached to AA;

-alkyl-NH(C=O)-, which is attached to AA;

-alkyl-(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkyl-O(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkyl-NH(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkenyl-(C=O)-, which is attached to AA;

-alkenyl-O(C=O)-, which is attached to AA;

-alkenyl-NH(C=O)-, which is attached to AA;

-alkenyl-(C=O)-alkenyl-(C=O)-, which is attached to AA;

-alkenyl-O(C=O)-alkenyl-(C=O)-, which is attached to AA;

-alkenyl-NH(C=O)-alkenyl-(C=O)-, which is attached to AA;

-alkynyl-(C=O)-, which is attached to AA;

-alkynyl-O(C=O)-, which is attached to AA;

-alkynyl-NH(C=O)-, which is attached to AA;

-alkynyl-(C=O)-alkynyl-(C=O)-, which is attached to AA;

-alkynyl-O(C=O)-alkynyl-(C=O)-, which is attached to AA;

-alkynyl-NH(C=O)-alkynyl-(C=O)-, which is attached to AA,

wherein any alkyl in R ³ is a branched or straight chain C(1-6)alkyl, any alkenyl in R ³ is a branched or straight chain C(2-6)alkenyl, and any alkynyl in R ³ is a branched or straight chain C( 2-6)alkynyl;

and their positional isomers;

,

wherein each ^R6 is independently selected from H, alkyl, alkoxy, and alkoxyalkoxy, with the proviso that one ^R6 is -(C=O)- or -alkyl-(C=O)-, which is attached to AA ;

each R ⁸ is independently selected from H, alkyl, with the proviso that one R ⁸ is -(C=O)- or -alkyl-(C=O)-, which is attached to AA;

q is from zero to four;

Q is O or N.

[0021] The above fusogenic compound, wherein the alkyl in R ⁶ and R ⁸ is each independently branched or straight-chain C(1-6)alkyl, the alkoxy group in R ⁶ is C(1-6)alkoxy, and The alkoxyalkoxy group in R ⁶ is C(1-6)alkoxy-C(1-6)alkoxy.

[0022] The above fusogenic compound, wherein R ⁴ and R ⁵ independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group, preferably a C(14-18)alkenyl group having from 2 to 4 double bonds.

[0023] The above fusogenic compound, wherein the compound is selected from the following compounds:

.

[0024] The above fusogenic compound, where one or more amphiphiles have the structure shown in formula III:

Formula III,

where R ¹ and R ² represent:

R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴

R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ ,

Where

n and m, each independently, range from 1 to 2;

R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group;

R ³ represents a C(1-12)alkyl group or a C(4-12)alkenyl group which is substituted by -(C=O)- or -alkyl-(C=O)- which is attached to AA.

[0025] The above fusogenic compound, wherein R ⁴ and R ⁵ independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group, preferably a C(14-18)alkenyl group having from 2 to 4 double bonds.

[0026] The above fusogenic compound, where one or more amphiphiles have the structure shown in formula IV:

Formula IV,

where R ¹ and R ² represent:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , (C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵

where R ⁴ and R ⁵ represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group;

Z is O or NH; p is from 0 to 5;

where R ³ is selected from branched or unbranched C(1-8)alkendiyl-(C=O)-, which is attached to AA, substituted or unsubstituted C(2-8)alkendiyl-(C=O)-, which is attached to AA , substituted or unsubstituted C(2-8)alkyndiyl-(C=O)-, which is attached to AA, substituted or unsubstituted C(3-8)cycloalkanediyl-(C=O)-, which is attached to AA, substituted or unsubstituted arylene-(C=O)-, which is attached to AA, substituted or unsubstituted C(4-8)heteroarylene-(C=O)-, which is attached to AA, and substituted or unsubstituted heterocycloalkanediyl-(C=O)-, which is affiliated with AA; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0027] R ³ is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3-8)cycloalkanediyl , substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0028] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula IV:

Formula IV,

where R ¹ and R ² represent:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , (C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵ ,

where R ⁴ and R ⁵ represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group;

Z is O or NH; p is from 0 to 5;

where R ³ is selected from a C(1-12)alkyl group or a C(2-12)alkenyl group which is substituted by a -(C=O)-group which is attached to AA,

and their positional isomers;

,

wherein each ^R6 is independently selected from H, alkyl, alkoxy, and alkoxyalkoxy, with the proviso that one ^R6 is -(C=O)- or -alkyl-(C=O)-, which is attached to AA ;

each R ⁸ is independently selected from H, alkyl, with the proviso that one R ⁸ is -(C=O)- or -alkyl-(C=O)-, which is attached to AA;

q is from zero to four;

Q is O or N.

[0029] The above fusogenic compound, wherein R ⁴ and R ⁵ independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group, preferably a C(14-18)alkenyl group having from 2 to 4 double bonds.

[0030] The above fusogenic compound, where the compound is a T10 compound:

.

[0031] The above fusogenic compound, where one or more amphiphiles have the structure shown in formula IV:

Formula IV,

where R ¹ and R ² represent:

R ¹ represents a C(12-20)alkyl group or a C(12-20)alkenyl group;

R ² represents (CH ₂ ) _n XR ⁴ where n is from 0 to 3, X represents O, S, SO, SO ₂ , NH;

where R ⁴ represents a C(12-20)alkyl group or a C(12-20)alkenyl group;

where Z represents O or NH;

where p equals 1;

where R ³ is selected from

a C(1-12)alkyl group or a C(2-12)alkenyl group that is substituted by a -(C=O) group that is attached to AA.

[0032] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in Formula V:

Formula V,

where R ¹ and R ² represent:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , NH(C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵ ,

where R ⁴ and R ⁵ represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is selected from branched or unbranched -O(C=O)-C(1-8)alkanediyl-(C=O)-, which is attached to AA, substituted or unsubstituted -O(C=O)-C(2 -8)alkendiyl-(C=O)-, which is attached to AA, substituted or unsubstituted -O(C=O)-C(2-8)alkyndiyl-(C=O)-, which is attached to AA, substituted or unsubstituted -O(C=O)-C(3-8)cycloalkanediyl-(C=O)-, which is attached to AA, substituted or unsubstituted -O(C=O)-arylene-(C=O)-, which attached to AA, substituted or unsubstituted -O(C=O)-C(4-8)heteroarylene-(C=O)-, which is attached to AA, and substituted or unsubstituted -O(C=O)-heterocycloalkanediyl-( C=O)-, which is attached to AA; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0033] R ³ is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3-8)cycloalkanediyl , substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0034] The above fusogenic compound, wherein R ⁴ and R ⁵ independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group, preferably a C(14-18)alkenyl group having from 2 to 4 double bonds.

[0035] The above fusogenic compound, where the compound is a T12 compound:

[0036] The above fusogenic compound, where one or more amphiphiles have the structure shown in formula V:

Formula V,

where R ¹ and R ² represent:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , NH(C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵ ,

where R ⁴ and R ⁵ represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is selected from:

-(C=O)- or -alkyl-(C=O)-, which is attached to AA;

-O(C=O)- or -alkyl-O(C=O)-, which is attached to AA;

-O(C=O)-alkanediyl-(C=O)-, which is attached to AA;

-O(C=O)-alkendiyl-(C=O)-, which is attached to AA;

-O(C=O)-alkyndiyl-(C=O)-, which is attached to AA;

-NH(C=O)- or -alkyl-NH(C=O)-, which is attached to AA;

-alkyl-(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkyl-O(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkyl-NH(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkenyl-(C=O)-, which is attached to AA;

-alkenyl-O(C=O)-, which is attached to AA;

-alkenyl-NH(C=O)-, which is attached to AA;

-alkenyl-(C=O)-alkendiyl-(C=O)-, which is attached to AA;

-alkenyl-O(C=O)-alkendiyl-(C=O)-, which is attached to AA;

-alkenyl-NH(C=O)-alkendiyl-(C=O)-, which is attached to AA;

-alkynyl-(C=O)-, which is attached to AA;

-alkynyl-O(C=O)-, which is attached to AA;

-alkynyl-NH(C=O)-, which is attached to AA;

-alkynyl-(C=O)-alkyndiyl-(C=O)-, which is attached to AA;

-alkynyl-O(C=O)-alkyndiyl-(C=O)-, which is attached to AA;

-alkynyl-NH(C=O)-alkyndiyl-(C=O)-, which is attached to AA;

and their positional isomers;

wherein any alkyl in R ³ is a branched or straight chain C(1-6)alkyl, any alkenyl in R ³ is a branched or straight chain C(2-6)alkenyl, and any alkynyl in R ³ is a branched or straight chain C( 2-6)alkynyl.

[0037] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula VI:

Formula VI,

where R ¹ and R ² represent:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , NH(C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵ ,

Where

R ⁴ and R ⁵ each represent, independently, a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is selected from branched or unbranched C(1-8)alkendiyl-(C=O)-, which is attached to AA, substituted or unsubstituted C(2-8)alkendiyl-(C=O)-, which is attached to AA , substituted or unsubstituted C(2-8)alkyndiyl-(C=O)-, which is attached to AA, substituted or unsubstituted C(3-8)cycloalkanediyl-(C=O)-, which is attached to AA, substituted or unsubstituted arylene-(C=O)-, which is attached to AA, substituted or unsubstituted C(4-8)heteroarylene-(C=O)-, which is attached to AA, and substituted or unsubstituted heterocycloalkanediyl-(C=O)-, which is affiliated with AA; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0038] R ³ is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3-8)cycloalkanediyl , substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0039] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula VI:

Formula VI,

where R ¹ and R ² represent:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , NH(C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵ ,

Where

R ⁴ and R ⁵ each represent, independently, a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is selected from:

-alkyl-(C=O)-, which is attached to AA;

-alkyl-O(C=O)-, which is attached to AA;

-alkyl-NH(C=O)-, which is attached to AA;

-alkyl-(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkyl-O(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkyl-NH(C=O)-alkyl-(C=O)-, which is attached to AA;

-alkenyl-(C=O)-, which is attached to AA;

-alkenyl-O(C=O)-, which is attached to AA;

-alkenyl-NH(C=O)-, which is attached to AA;

-alkenyl-(C=O)-alkenyl-(C=O)-, which is attached to AA;

-alkenyl-O(C=O)-alkenyl-(C=O)-, which is attached to AA;

-alkenyl-NH(C=O)-alkenyl-(C=O)-, which is attached to AA;

-alkynyl-(C=O)-, which is attached to AA;

-alkynyl-O(C=O)-, which is attached to AA;

-alkynyl-NH(C=O)-, which is attached to AA;

-alkynyl-(C=O)-alkynyl-(C=O)-, which is attached to AA;

-alkynyl-O(C=O)-alkynyl-(C=O)-, which is attached to AA;

-alkynyl-NH(C=O)-alkynyl-(C=O)-, which is attached to AA,

and their positional isomers;

wherein any alkyl in R ³ is a branched or straight chain C(1-6)alkyl, any alkenyl in R ³ is a branched or straight chain C(2-6)alkenyl, and any alkynyl in R ³ is a branched or straight chain C( 2-6)alkynyl.

[0040] The above fusogenic compound, wherein R ⁴ and R ⁵ independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group, preferably a C(14-18)alkenyl group having from 2 to 4 double bonds.

[0041] The above fusogenic compound, where the compound is a T11 compound:

[0042] Fusogenic compound having formula VII:

Formula VII,

wherein each amphiphile independently contains from one to two lipophilic chains, wherein each lipophilic chain independently contains from 8 to 22 carbon atoms;

wherein each AA ^a independently represents an amino acid containing a side chain having an acyl group, wherein the amino acid is attached to the amphiphile at each of its acyl groups and is attached to a linker at its N-terminus;

where the linker has the structure:

or

wherein Q ¹ is a branched or straight-chain C(2-8)alkanediyl, a branched or straight-chain C(2-8)alkendiyl, a branched or straight-chain C(2-8)alkyndiyl, or

or

,

where Q ² represents

,

where Q ³ represents

,

where X represents -O-, -S- or -NH-;

n and p independently for each case range from 1 to 3;

m independently ranges from 1 to 10;

r and s independently for each case range from 1 to 5.

[0043] The above fusogenic compound, where AA ^a is selected from the following structures, and any stereoisomer thereof:

.

[0044] The above fusogenic compound wherein one or two amphiphiles are absent and are replaced by an alkyl group or a pharmaceutically acceptable organic chemical group having 1-400 atoms selected from carbon, oxygen, nitrogen, sulfur, fluorine and hydrogen.

[0045] The above fusogenic compound, wherein the pharmaceutically acceptable organic chemical group is selected from alkyl, alkenyl, alkynyl, alkyl ether, aryl ether, alkoxy and alkoxyakoxy groups.

[0046] The above fusogenic compound, wherein the pharmaceutically acceptable organic chemical group is selected from a methoxy group, an ethoxy group, a tert-butyl ether and a benzyloxy group.

[0047] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula VIII:

Formula VIII,

where R ¹ and R ² represent

R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴

R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ ,

where n and m, each independently, range from 1 to 2;

R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group.

[0048] The above fusogenic compound, where one or more amphiphiles have the structure shown in formula IX:

Formula IX

where R ¹ and R ² represent

R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴

R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ ,

Where

n and m, each independently, range from 1 to 2;

R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is selected from branched or unbranched C(1-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, substituted or unsubstituted C(3-8)cycloalkanediyl, substituted or unsubstituted arylene, substituted or unsubstituted C(4-8)heteroarylene and substituted or unsubstituted heterocycloalkanediyl; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0049] R ³ is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3-8)cycloalkanediyl , substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0050] The above fusogenic compound, wherein R ⁴ and R ⁵ independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group, preferably a C(14-18)alkenyl group having from 2 to 4 double bonds.

[0051] The above fusogenic compound, where the compound is a T9 compound:

.

[0052] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula X:

Formula X,

where R ¹ and R ² represent:

R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴

R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ ,

R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is absent, or it is selected from branched or unbranched *-NH-C(1-8)alkanediyl-(C=O)-, substituted or unsubstituted *-NH-C(2-8)alkendiyl-(C=O )-, substituted or unsubstituted *-NH-C(2-8)alkyndiyl-(C=O)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=O)-, substituted or unsubstituted *-NH-arylene-(C=O)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=O)- and substituted or unsubstituted -NH-heterocycloalkanediyl-(C=O)-, where * denotes the end attached to AA ^a ; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0053] R ³ is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkenediyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3-8)cycloalkanediyl , substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0054] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula XI:

Formula XI,

Where

R ¹ and R ² are:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , NH(C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵

R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is absent, or it is selected from branched or unbranched *-NH-C(1-8)alkanediyl-(C=O)-, substituted or unsubstituted *-NH-C(2-8)alkendiyl-(C=O )-, substituted or unsubstituted *-NH-C(2-8)alkyndiyl-(C=O)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=O)-, substituted or unsubstituted *-NH-arylene-(C=O)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=O)- and substituted or unsubstituted -NH-heterocycloalkanediyl-(C=O)-, where * denotes the end attached to AA ^a ; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0055] R ³ is preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3-8)cycloalkanediyl , substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0056] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in formula III

Formula III,

where R ¹ and R ² represent:

R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴

R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵

where n and m, each independently, range from 1 to 2; and R ⁴ and R ⁵ independently each represent a C(12-20)alkyl group or a C(12-20)alkenyl group;

Where

R ³ is selected from:

-(C=O)-alkyl-NH-, which is attached to AA ^a ;

-(C=O)-alkenyl-NH-, which is attached to AA ^a ;

-(C=O)-alkynyl-NH-, which is attached to AA ^a ;

wherein any alkyl in R ³ is a branched or straight chain C(1-6)alkyl, any alkenyl in R ³ is a branched or straight chain C(2-6)alkenyl, and any alkynyl in R ³ is a branched or straight chain C( 2-6)alkynyl;

and their positional isomers;

,

Where

each R ⁶ is independently selected from H, alkyl, an alkoxy group, and an alkoxyalkoxy group, with the proviso that one R ⁶ is -(C=O)-alkyl-NH-, wherein NH is attached to AA ^a ;

each R ⁸ is independently selected from H, alkyl, with the proviso that one R ⁸ is -(C=O)-alkyl-NH-, wherein NH is attached to AA ^a ;

q is from zero to four;

Q is O or N.

[0057] The above fusogenic compound, wherein one or more amphiphiles have the structure shown in Formula V:

Formula V,

where R ¹ and R ² represent:

R ¹ = (C=O)OR ⁴ , (C=O)NHR ⁴ , O(C=O)R ⁴ , NH(C=O)R ⁴

R ² = (C=O)OR ⁵ , (C=O)NHR ⁵ , O(C=O)R ⁵ , NH(C=O)R ⁵ ,

where R ⁴ and R ⁵ represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group;

where R ³ is absent, or it is selected from branched or unbranched *-NH-C(1-8)alkanediyl-(C=O)-, substituted or unsubstituted *-NH-C(2-8)alkendiyl-(C=O )-, substituted or unsubstituted *-NH-C(2-8)alkyndiyl-(C=O)-, substituted or unsubstituted *-NH-C(3-8)cycloalkanediyl-(C=O)-, substituted or unsubstituted *-NH-arylene-(C=O)-, substituted or unsubstituted *-NH-C(4-8)heteroarylene-(C=O)- and substituted or unsubstituted -NH-heterocycloalkanediyl-(C=O)-, where * denotes the end attached to AA ^a ; where R ³ is optionally interrupted by one or more of the groups -O-, -S-, -SO-, -SO ₂ -, -NH-, -NR ⁶ -, -NH(C=O)-, -O(C= O)-, where R ⁶ represents C(1-6)alkyl-, C(1-6)alkoxy- or C(1-6)alkoxy-C(1-6)alkoxy-.

[0058] R ³ is as described above, preferably branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(2-8)alkendiyl, substituted or unsubstituted C(2-8)alkyndiyl, C(3- 8) cycloalkanediyl, substituted or unsubstituted C(4-8)arylene and, even more preferably, branched or unbranched C(2-8)alkanediyl, substituted or unsubstituted C(3-8)cycloalkanediyl.

[0059] Embodiments of the present invention further provide compositions containing the above fusogenic compound and a pharmaceutically acceptable carrier. The composition may contain nanoparticles or liposomes.

[0060] The pharmaceutical composition of this invention may include a fusogenic compound, an active agent, and a pharmaceutically acceptable carrier. In the composition, the fusogenic compound may comprise from 0.01 to 20 mol% of the lipids in the composition. The composition may contain nanoparticles or liposomes.

[0061] The fusogenic molecules and compositions of the present invention can be used to deliver the active agent.

[0062] In some embodiments, the active agent is one or more nucleic acids.

[0063] In some embodiments, the active agent is one or more DNA, RNA, mRNA, siRNA, or microRNA. The active agent may be one or more RNA molecules.

[0064] The active agent may be one or more RNAi molecules, one or more mRNA molecules, and modified forms thereof.

[0065] Embodiments of the present invention include compositions for use in dispensing an active agent to treat a condition or disease in a subject. The composition may contain an active agent, a fusogenic compound, an ionizable lipid, a structural lipid, a stabilizing lipid, and a lipid to reduce the immunogenicity of the composition.

[0066] The present invention includes methods for preventing, ameliorating, or treating a disease or condition in a subject in need, comprising administering to the subject the above composition. The compositions of this invention can be used in the treatment of a human or animal body.

[0067] Embodiments of this invention also include the following compounds:

compound of formula (A)

formula A,

where the linker is a divalent group containing a PEG region,

X ₁ and X ₂ independently represent a C1-C5 alkanediyl group,

R ₁ , R ₂ , R ₃ and R ₄ are independently:

X ₃ represents a single bond, a C1-C5 alkanediyl group or a C2-C5 alkanediyl group, X ₄ and X ₅ independently represent a C2-5 alkanediyl group,

Z ₁ , Z ₂ and Z ₃ are independently -O-, -S- or -NH-, and

R ₅ and R ₆ independently represent a C11-23 alkyl or a C11-23 alkenyl group.

[0068] Embodiments of the present invention also include the following compounds:

compound according to formula (B):

formula B,

where the linker is a divalent group containing a PEG region,

X ₆ and X ₇ independently represent a C1-C5 alkanediyl group,

X ₈ and X ₉ independently represent a C1-C5 alkanediyl group,

Z ₄ and Z ₅ are independently -O-, -S- or -NH-,

R ₇ , R ₈ , R ₉ and R ₁₀ are independently:

,

X ₄ and X ₅ independently represent a C2-5alkanediyl group,

Z ₂ and Z ₃ are independently -O-, -S- or -NH-, and

R ₅ and R ₆ independently represent a C11-23 alkyl or a C11-23 alkenyl group.

[0069] A compound as defined above, wherein the linker is:

,

where m is an integer from 1 to 12,

Y ₁ represents -O-, -NH- or -NHCH ₂ -,

Y ₂ represents -O-, -NH- or -CH ₂ NH-,

n and q are independently an integer from 1 to 5,

p represents an integer from 1 to 5,

Y ₃ and Y ₅ are independently -O-, -NH- or -NHCH ₂ -, and

Y ₄ and Y ₆ are independently -O-, -NH- or -CH ₂ NH-.

[0070] The compound as above, wherein X ₁ and X ₂ independently represent a C1-C5 straight alkanediyl group, preferably a C2-C4 straight alkanediyl group, more preferably a C4 straight alkanediyl group.

[0071] A compound as defined above, wherein R ₁ , R ₂ , R ₃ and R ₄ are the same groups.

[0072] The compound as above, where X ₃ represents a single bond or a C1-C5 straight alkanediyl group, X ₃ preferably represents a C2-C4 straight alkanediyl group, more preferably ethylene, that is, an ethanediyl group.

[0073] The compound as above, wherein X ₄ and X ₅ are independently a C2-5 straight alkanediyl group, X ₄ and X ₅ are preferably a C2-4 straight alkanediyl group, more preferably ethylene, that is, an ethanediyl group .

[0074] The compound as above, where Z ₁ represents -NH-.

[0075] The compound as above wherein Z ₂ and Z ₃ are -O-.

[0076] A compound as defined above, wherein R ₅ and R ₆ independently represent a C11-23 straight alkenyl group.

[0077] A compound as defined above, wherein R ₅ and R ₆ independently represent a C11-23 straight alkenyl group with 1-6 double bonds, where the number of double bonds is preferably 1-3, more preferably 2-3, more more preferably 2.

[0078] A compound as defined above, wherein R ₅ and R ₆ are independently a C11-23 straight alkenyl group with 2 double bonds.

[0079] A compound as defined above, wherein R ₅ and R ₆ are independently a C13-17 straight alkenyl group, R ₅ and R ₆ are preferably a C15-17 straight alkenyl group, more preferably a C17 straight alkenyl group.

[0080] The compound as defined above, wherein R ₅ and R ₆ independently represent a C17 straight alkenyl group.

[0081] The compound as above, wherein R ₅ and R ₆ represents a heptadeca-8,1-dienyl group.

[0082] The composition mentioned above, which contains a cationic lipid, an ionizable lipid and a lipid of the above compound in a lipid nanoparticle containing a bilayer of lipid molecules.

[0083] The composition specified above, which further contains a nucleic acid.

[0084] The composition as defined above, wherein the nucleic acid is siRNA, mRNA or microRNA.

[0085] The composition as defined above, wherein the composition is a pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 shows a diagram of the preparation of compound R1.

[0087] Figure 2 shows a diagram of the preparation of compound R2.

[0088] FIG. 3A shows a diagram for preparing compound R3.

[0089] Figure 3B shows the structure of connection R3.

[0090] In FIG. 4A shows a diagram of the preparation of compound R4.

[0091] Figure 4B shows the structure of connection R4.

[0092] Figure 5 shows a diagram of the preparation of compound R5.

[0093] Figure 6 shows a diagram of the preparation of compound R6.

[0094] Figure 7 shows an alternative scheme for preparing compound R6.

[0095] FIG. 8 shows a diagram for preparing compound S2.

[0096] FIG. 9 shows a diagram for obtaining connection S3.

[0097] FIG. 10A shows a diagram for preparing compound S4.

[0098] Fig. 10B shows the structure of connection S4.

[0099] FIG. 11 shows a diagram for preparing compound S5.

[00100] FIG. 12A shows a diagram for preparing compound S6.

[00101] FIG. 12B shows the structure of connection S6.

[00102] FIG. 13A shows a diagram for obtaining connection S7.

[00103] Fig. 13B shows the structure of connection S7.

[00104] FIG. 14A shows a diagram of the preparation of compound S8.

[00105] Fig. 14B shows the structure of connection S8.

[00106] FIG. 15A shows a diagram for obtaining a T1 connection.

[00107] FIG. 15B shows the structure of the T1 connection.

[00108] FIG. 16A shows a flow chart for preparing compound T2.

[00109] FIG. 16B shows the structure of connection T2.

[00110] FIG. 17A shows a flow chart for preparing compound T4.

[00111] FIG. 17B shows the structure of the T4 connection.

[00112] FIG. 18A shows a diagram of the preparation of compound T5.

[00113] FIG. 18B shows the structure of the T5 connection.

[00114] FIG. 19A shows a flow chart for preparing compound T6.

[00115] FIG. 19B shows the structure of the T6 connection.

[00116] FIG. 20A shows a flow chart for preparing compound T7.

[00117] FIG. 20B shows the structure of the T7 connection.

[00118] FIG. 21A shows a flow chart for preparing compound T8.

[00119] FIG. 21B shows the structure of a T8 connection.

[00120] FIG. 22A shows a flow chart for preparing compound T9.

[00121] Figure 22B shows the structure of the T9 connection.

[00122] FIG. 23A shows a flow chart for preparing compound T3.

[00123] Figure 23B shows the structure of the T3 connection.

[00124] Figure 24 shows the results of in vitro delivery of biologically active molecules using fusogenic molecules of the present invention. As shown in FIG. 24, liposomal delivery formulation No. 5, which contained 2% (of total lipids) of the R4 fusogenic compound of the present invention, provided an unexpected increase in the activity of the exemplary anti-HSP47 siRNA to knockdown gene expression in stellate cells compared with control composition No. 1, which did not contain the fusogenic compound of the present invention.

[00125] Figure 25 shows the results of in vitro delivery of biologically active molecules using fusogenic molecules of the present invention. The extent of HSP47 gene expression knockdown was measured for several liposomal-delivered siRNA formulations. The formulations provided high activity in suppressing gene expression in stellate cells. The formulations contained 2% (of total lipids) of the fusogenic compound of the present invention and provided high siRNA activity against HSP47.

[00126] Figure 26 shows the results of in vivo delivery of bioactive molecules using fusogenic molecules of the present invention. Liposomally delivered formulations demonstrated gene silencing activity in vivo (in mice) using siRNA directed against HSP47. Active compositions No. 2-No. 8 contained 2% (of total lipids) of the specified fusogenic compound of the present invention.

[00127] FIG. 27 shows the left half of the T10 connection structure.

[00128] FIG. 28 shows the right half of the T10 connection structure.

[00129] FIG. 29 shows a diagram of how connection T10 is made.

[00130] FIG. 30 shows a diagram for obtaining connection T10.

[00131] FIG. 31 shows the left half of the T11 connection structure.

[00132] FIG. 32 shows the right half of the connection structure of T11.

[00133] FIG. 33 shows a diagram for obtaining connection T11.

[00134] FIG. 34 shows a diagram for obtaining connection T11.

[00135] Figure 35 shows the left half of the T12 connection structure.

[00136] FIG. 36 shows the right half of the T12 connection structure.

[00137] FIG. 37 shows a diagram of the preparation of connection T12.

[00138] FIG. 38 shows a diagram of the preparation of connection T12.

[00139] FIG. 39 shows the left half of the T13 connection structure.

[00140] Figure 40 shows the right half of the T13 connection structure.

[00141] FIG. 41 shows a diagram of the preparation of compound T13.

[00142] FIG. 42 shows a diagram of the preparation of compound T13.

[00143] Figure 43 shows the right and left halves of the T14 connection structure.

[00144] FIG. 44 shows a diagram for preparing compound T14.

[00145] FIG. 45 shows a diagram of the preparation of connection T14.

[00146] Figure 46 shows in vitro gene expression knockdown activity using an exemplary siRNA in rat stellate cells for liposomal formulations containing a fusogenic compound of the present invention. (1) Results for a liposomal formulation consisting of HEDC and S104 lipids, which did not contain the fusogenic compound of the present invention. (2) Results for a liposomal formulation similar to (1) except that it contained 2% (of total lipids) fusogenic compound R4. (3) Results for a liposomal formulation similar to (1) except that it contained 10% (of total lipids) fusogenic compound R4. The presence of fusogenic compound R4 in the preparation significantly increased the "delivered" activity of the exemplary siRNA, and the increased activity was directly related to the presence of fusogenic compound R4.

[00147] Figure 47 shows in vitro gene expression knockdown activity using an exemplary siRNA in rat stellate cells using a liposomal formulation containing a fusogenic compound of the present invention. (1) Results for a liposomal formulation consisting of HEDC and S104 lipids that did not contain the fusogenic compound of the present invention. (2) Results for a liposomal formulation similar to (1) except that it contained 2% (of total lipids) fusogenic compound R4. (3) Results for a liposomal formulation similar to (1) except that it contained 2% (of total lipids) fusogenic compound T3. The presence of fusogenic compounds R4 and T3, respectively, in the compositions significantly increased the "delivered" activity of the exemplary siRNA, and this increased activity was directly related to the presence of fusogenic compounds R4 and T3.

[00148] Figure 48 shows the results of delivery and transfection of GFP mRNA at a concentration of 5 nM (top) and 2 nM (bottom) into A549 cells in vitro in LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE -PEG2000: T3 connection. Fluorescent images (left) of GFP expression were obtained using a fluorescence microscope 48 hours after transfection. Overlay (right) of fluorescence images with light field (BF) images demonstrates that almost 100% of the cells were transfected and expressed GFP.

[00149] Figure 49 shows the results of in vivo delivery of GFP mRNA using fusogenic lipid-like molecules of the present invention. Balb/c mice were transfected with GFP mRNA using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:T3 compound. As shown in FIG. 49, mRNA delivery to various tissues and cells was determined using the MAXDISCOVER GFP ELISA. Surprisingly, GFP mRNA was selectively transfected and/or translated in the lung, with lower transfection and/or translation in muscle, liver, heart, and kidney.

[00150] Figure 50 shows the results of in vivo delivery of luciferase mRNA using fusogenic lipid-like molecules of the present invention. Balb/c mice were transfected with luciferase mRNA using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:T3 compound. As shown in Fig. 50, the relative delivery, transfection and/or translation of mRNA in various tissues and cells was determined using the Promega E4510 assay kit. Surprisingly, Fluc (firefly luciferase) mRNA was selectively delivered, transfected and/or translated in the lungs and spleen, with lower delivery, transfection and/or translation in the liver, heart, kidney and muscle.

[00151] Figure 51 shows the results of in vivo delivery of luciferase mRNA using fusogenic lipid-like molecules of the present invention. Balb/c mice were transfected with luciferase mRNA using LNP nanoparticles of the present invention having the composition:

(-01) HE2DC:S104:CH:DOPE:DMPE-PEG2000:T3 connection or

(-02) HEDC:S104:CH:DOPE:DMPE-PEG200:T3 connection,

administered in an amount of 2 mg/kg. As shown in Fig. 51, relative delivery, transfection and/or translation of mRNA in various tissues and cells was determined using the E4510 Promega kit. Fluc mRNA was selectively delivered, transfected, and/or translated into the lungs and spleen, with lower delivery, transfected, and/or translated into the pancreas, kidney, liver, testis, and small intestine.

[00152] Figure 52 shows the results of bioluminescence measurements for in vivo delivery of luciferase mRNA to mice using fusogenic lipid-like molecules of the present invention. As shown in Fig. 52, the relative delivery, transfection and/or translation of mRNA in various tissues was determined by measuring luminescence 7 hours after injection.

[00153] Figure 53 shows the results of delivering luciferase mRNA to mice in vivo using the fusogenic lipid-like molecules of the present invention. As shown in FIG. 53, the relative delivery of mRNA was much higher for formulations containing the fusogenic molecules of the present invention (2035-03-03) than for the same formulations without fusogenic molecules (2035-13-01).

DETAILED DESCRIPTION OF THE INVENTION

[00154] The present invention relates to a number of fusogenic molecules. The fusogenic compounds of the present invention can be used to deliver therapeutic agents to cells, tissues or organs, organisms and subjects.

[00155] In some aspects, the present invention relates to a platform of compounds for the formation of fusogenic molecules. Fusogenic molecules can be formed by attaching one or more neutral molecules, such as hydrocarbon molecules, an aliphatic molecule, a saturated fatty acid molecule, an unsaturated fatty acid molecule, a monounsaturated fatty acid molecule, or a polyunsaturated fatty acid molecule, to a platform structure.

[00156] Examples of the fusogenic compound of the present invention include, but are not limited to, compound R4 shown in Fig. 4B; connection S6 shown in Fig. 12B; connection S7 shown in Fig. 13B; connection S8 shown in Fig. 14B; connection T1 shown in Fig. 15B; connection T2 shown in Fig. 16B; connection T4 shown in Fig. 17B; connection T5 shown in Fig. 18B; connection T6 shown in Fig. 19B; connection T7 shown in Fig. 20B; connection T8 shown in Fig. 21B; connection T9 shown in Fig. 22B; connection T3 shown in Fig. 23B; connection T10 shown in Figs. 27 and 28; connection T11 shown in Figs. 31 and 32; connection T12 shown in Figs. 35 and 36; connection T13 shown in Figs. 39 and 40; connection T14 shown in Fig.43.

[00157] The compounds shown in FIGS. 1, 2, 3A, 3B and 6-11 are provided to illustrate methods for synthesizing the compounds.

[00158] In some aspects, the present invention relates to a series of fusion molecules that can be used in formulations to form and use lipid nanoparticles to deliver active agents to cells and subjects.

[00159] The fusogenic compound of the present invention may have one or two amphiphilic groups (amphiphiles) attached to an amino acid group, which amino acid group is attached by a linker to a separate amino acid group carrying one or two additional amphiphiles.

[00160] The lipophilic chains of the amphiphile may independently contain from 8 to 22 carbon atoms.

[00161] An amphiphilic group can be a lipid-like group having one or two lipophilic chains attached to an organic chemical group. An organic chemical group may have up to 400 atoms, or 20-400 atoms, or 10-400 atoms, or 4-400 atoms, or 3-400 atoms, or 2-400 atoms, or 1-400 atoms selected from carbon, oxygen , nitrogen, sulfur, fluorine and hydrogen, and may have any structure suitable for the attachment of one or two lipophilic chains and attachment to an amino acid group. The organic chemical group may be neutral or zwitterionic, or may provide a hydrophilic nature. In some embodiments, the organic chemical group may be ionized. Examples of the organic chemical group include alkyl, alkenyl, alkynyl and acetyl, as well as protecting groups such as Boc, Fmoc, TFA and CBZ (benzyloxycarbonyl).

[00162] Without wishing to be bound by any particular theory, the authors propose that the amphiphile may have a lipid-like structure such that the amphiphile may enter the lipid bilayer in an orientation similar to the lipid molecules of the bilayer while remaining associated with the larger fusion. The fusogenic compound of the present invention can disrupt the dynamic structure of the bilayer to enhance fusogenicity (ability to fuse) with cells.

[00163] The amino acid group of a fusogenic compound (denoted AA or AA ^a ) may be modified with substituents. The amino acid group of the fusogenic compound can be any D- or L-amino acid group having the formula -NR ^N -CR ¹ R ² -(C=O)-, where R ¹ represents a substituted or unsubstituted side chain of certain natural amino acids. R ² and ^RN may each independently represent hydrogen or an organic group consisting of carbon, oxygen, nitrogen, sulfur and hydrogen atoms and having from 1 to 20 carbon atoms, or may represent C(1-6)alkyl, cycloalkyl, cycloalkylalkyl, C(2-6)alkenyl, C(2-6)alkynyl, C(1-6)alkanoyl, C(1-6)alkanoyloxy, C(1-6)alkoxy, C(1-6) alkoxy-C(1-6)alkyl, C(1-6)alkoxy-C(1-6)alkoxy.

[00164] As used herein, the term “which is attached to an AA” is used to refer to the point at which a group is attached to an AA. For example, the term “-alkyl-(C=O)-that is attached to AA” refers to attachment to AA via a (C=O) group so that -alkyl-(C=O)-AA is formed. Unless otherwise noted, the last group that appears next to the phrase “who is affiliated with A.A.” is assumed to be an A.A. affiliated group.

[00165] This invention may relate to compositions for use in the distribution of an active agent in cells, tissues or organs, organisms and subjects, where the composition includes one or more fusogenic molecules of the present invention.

[00166] The fusogenic compounds of the present invention can provide compositions and formulations useful in delivering therapeutic agents to cells, tissues and subjects, preferably without significant aggregation of the components of the composition.

[00167] The fusogenic compound of the present invention can provide a liposomal composition for use in delivering therapeutic agents to cells, tissues and subjects, preferably without significant aggregation of liposomes in the composition.

[00168] The compositions of the present invention may include one or more fusogenic molecules together with a structural lipid and one or more lipids to reduce the immunogenicity of the composition.

[00169] In some aspects, the present invention provides novel fusogenic lipids that facilitate the delivery of biologically active molecules into cells. Fusogenic lipids can be included in formulations such as nanoparticles or liposomes to deliver therapeutic molecules, including nucleic acids or oligonucleotides, to cells, including tumors. In some embodiments, nanoparticles or liposomes containing fusogenic lipids can fuse with the plasma membrane of a cell or the intracellular membrane of a cell and facilitate the release of therapeutic molecules, as well as increase transfection efficiency.

[00170] In additional embodiments, a variety of novel lipid fusions can be synthesized and incorporated into nanoparticles or liposomes. Nanoparticles or liposomes can include or encapsulate therapeutic molecules, including nucleic acid-based molecules such as siRNA, microRNA or mRNA, as well as small molecule drugs and any active therapeutic agents that can be delivered using nanoparticles or liposomes.

[00171] The particle size for nanoparticles or liposomes can be in the range of 50-200 nm with a polydispersity of less than 0.2. The transfection efficiency of nanoparticles or liposomes in various cell lines can be improved compared to nanoparticles or liposomes that lack one or more of the novel fusogenic lipids.

[00172] In additional embodiments, cellular uptake of nanoparticles or liposomes of the present invention may be enhanced, among other things, through the endocytic pathway or through the mechanism of micropinocytosis.

[00173] Nanoparticles or liposomes of the present invention can also reduce lysosomal degradation of therapeutic molecules upon delivery.

[00174] In some embodiments, the present invention includes compositions containing one or more fusogenic molecules, along with other lipid molecules, to form nanoparticles. In some embodiments, the fusogenic molecules may comprise from 0.1 to 40 mol% of the lipids of the composition. In other embodiments, the fusogenic compound may comprise from 1 to 20 mol% of the lipids of the composition. In additional embodiments, the fusogenic compound may comprise 1 to 10 mol% or 2 to 10 mol% of the lipids of the composition. In other embodiments, the fusogenic molecules may comprise 2 mol% of the lipids of the composition.

[00175] In some embodiments, the fusogenic compound may comprise a fourth or fifth lipid component of the composition, or the fusogenic molecules may replace one of the lipid components of the composition.

[00176] The fusogenic molecules of the present invention may consist of a platform structure and have one to four amphiphilic groups with suitable chemical bonds attached to the platform structure.

[00177] The composition of the present invention may include a fusogenic molecule of the present invention. The fusogenic molecule may comprise 1-10 mol% or more of the composition. The composition may form a nanoparticle or a liposome.

[00178] In some embodiments, the composition of the present invention may include a cationic lipid, an ionizable lipid, and a fusogenic lipid molecule, which may combine to form a lipid nanoparticle. In some embodiments, the lipid nanoparticle may have a bilayer of lipid molecules.

[00179] In some embodiments, one or two amphiphiles may be absent, and if absent, may be replaced by a protecting group R ^p .

[00180] In additional embodiments, one or two amphiphiles may be absent, and if absent, they may be replaced by an alkyl, alkenyl, or alkynyl group or an organic chemical group having up to 400 atoms, or 20-400 atoms, or 10-400 atoms, or 4-400 atoms, or 3-400 atoms, or 2-400 atoms, or 1-400 atoms selected from carbon, oxygen, nitrogen, sulfur, fluorine and hydrogen.

[00181] Methods for preparing various organic groups and protecting groups are known in the art, and their use and modification are generally within the competence of one skilled in the art. See, for example, Stanley R. Sandler and Wolf Karo, Organic Functional Group Preparations (1989); Greg T. Hermanson, Bioconjugate Techniques (1996); Leroy G. Wade, Compendium Of Organic Synthetic Methods (1980); Some examples of protecting groups are given in T. W. Greene and P. G. M. Wuts, Protective Groups In Organic Synthesis (3rd ed. 1991). See, for example, Helmut Vorbrüggen, Handbook of Nucleoside Synthesis (2001).

[00182] Examples of the R ^p protecting group include Fmoc (fluorenylmethyloxycarbonyl).

[00183] Examples of the R ^p protecting group include Boc (tert-butyloxycarbonyl).

[00184] Examples of the R ^p protecting group include OTrt(O-triphenylmethyl).

[00185] Examples of the amino protecting group R ^p include Ac (acetamide) (C=O)CH3).

[00186] Examples of the amino protecting group include Fmoc, Boc, Trt, Dde and Alloc.

[00187] Examples of alkoxy protecting groups include OTrt, OClt, OMmt, OMtt, ODpm and OtBu.

[00188] Examples of the protecting group include tert-butyl ether.

[00189] Examples of the carboxylic acid protecting group include benzyl ester.

[00190] Cationic lipids and ionizable lipids

[00191] Examples of cationic lipids and ionizable lipids of this invention are provided in US20130022665A and US20130330401A.

[00192] The structure of HEDC is given in US2013/0022665A at paragraph [0146].

[00193] The structure of S104 is given in US 2013/0115274A1 at paragraph [0046].

[00194] Compositions with three or more components

[00195] In the context of the present invention, a composition component, such as a “lipid,” may be a single compound or may be a combination of one or more suitable lipid compounds. For example, the term "stabilizing lipid" may refer to a single stabilizing lipid or a combination of one or more suitable stabilizing lipids. One skilled in the art will readily appreciate that certain combinations of compounds described herein can be used without undue experimentation and that the formulation component description covers various combinations of compounds.

[00196] The ionizable compounds of the composition of the present invention can comprise from 20 to 80 mol.% of the lipid components of the composition. In some embodiments, the ionizable molecules of the composition may comprise 55 to 65 mol% of the lipid components of the composition. In other embodiments, the ionizable molecules of the composition may comprise about 60 mol% of the lipid components of the composition.

[00197] The structural lipid of the composition of the present invention may comprise from 20 to 50 mol% of the lipid components of the composition. In some embodiments, the structural lipid of the composition may comprise from 35 to 45 mol% of the lipid components of the composition.

[00198] One or more lipids to reduce the immunogenicity of the composition can comprise from 1 to 8 mol.% of the lipid components of the composition. In some embodiments, one or more lipids to reduce the immunogenicity of the composition may comprise from 1 to 5 mol% of the lipid components of the composition.

[00199] In additional aspects, the composition of the present invention may further include a cationic lipid, which may comprise from 5 to 25 mol% of the lipid components of the composition. In some embodiments, the composition of the present invention may further include a cationic lipid, which may comprise from 5 to 15 mol% of the lipid components of the composition. In these aspects, the molar ratio of the concentrations of cationic lipid and ionizable molecules of the composition of the present invention can be from 5:80 to 25:50.

[00200] In the compositions of the present invention, the set of lipid components may include one or more molecular components of an ionizable compound, a structural lipid, and one or more lipids to reduce the immunogenicity of the composition.

[00201] In addition to the above components, the composition of the present invention may further include a fusogenic molecule of the present invention. The fusogenic molecule may comprise 1-10 mol% of the composition.

[00202] Compositions with four or more components

[00203] The ionizable molecules of the composition of the present invention may comprise from 15 to 40 mol% of the lipid components of the composition. In some embodiments, the ionizable molecules of the composition may comprise from 20 to 35 mol% of the lipid components of the composition. In other embodiments, the ionizable molecules of the composition may comprise 25 to 30 mol% of the lipid components of the composition.

[00204] The structural lipid of the composition of the present invention may comprise from 25 to 40 mol% of the lipid components of the composition. In some embodiments, the structural lipid of the composition may comprise 30 to 35 mol% of the lipid components of the composition.

[00205] The total amount of stabilizing lipids of the composition of the present invention may range from 25 to 40 mol% of the lipid components of the composition. In some embodiments, the total amount of stabilizing lipids in the composition may be 30 to 40 mole percent of the lipid components of the composition.

[00206] In some embodiments, the composition of the present invention may include two or more stabilizing lipids, where each of the stabilizing lipids individually may comprise from 5 to 35 mole percent of the lipid components of the composition. In some embodiments, the composition of the present invention may include two or more stabilizing lipids, where each of the stabilizing lipids individually may comprise from 10 to 30 mole percent of the lipid components of the composition.

[00207] In some embodiments, the total amount of one or more stabilizing lipids may be from 25 to 40 mol% of the lipids of the composition, where each of the stabilizing lipids individually may be from 5 to 35 mol%.

[00208] In some embodiments, the total amount of one or more stabilizing lipids may be from 30 to 40 mol% of the lipids of the composition, where each of the stabilizing lipids individually may be from 10 to 30 mol%.

[00209] One or more lipids to reduce the immunogenicity of the composition can comprise from 1 to 8 mol.% of the lipid components of the composition. In some embodiments, one or more lipids to reduce the immunogenicity of the composition may comprise from 1 to 5 mol% of the lipid components of the composition.

[00210] In additional aspects, the composition of the present invention may further include a cationic lipid, which may comprise from 5 to 25 mol% of the lipid components of the composition. In some embodiments, the composition of the present invention may further include a cationic lipid, which may comprise from 5 to 15 mol% of the lipid components of the composition. In these aspects, the molar ratio of the concentrations of cationic lipid and ionizable molecules of the composition of the present invention can be from 5:35 to 25:15.

[00211] In some embodiments, the set of lipid components of the composition may include one or more of the molecular components of the ionizable compound, a structural lipid, one or more lipids to reduce the immunogenicity of the composition, and one or more stabilizing lipids.

[00212] In addition to the above components, the composition of the present invention may further include a fusogenic molecule of the present invention. The fusogenic molecule may comprise 1-10 mol% of the composition.

[00213] Examples of lipid compositions

[00214] In some embodiments, the composition may contain one or more ionizable molecules, a structural lipid, one or more lipids to reduce the immunogenicity of the composition, and a fusogenic molecule of the present invention, which will represent 100% of the lipid components of the composition. In some embodiments, a cationic lipid may be included.

[00215] Examples of compositions of the present invention are shown in Table 1.

Table 1: Compositions of lipid components (amount of each expressed as mol% of total amount)

Ionizable Cationic Structural To reduce immunogenicity Fusogenic 60 0 thirty 8 2 60 0 33 5 2 55 0 42 1 2 65 0 31 3 1 60 0 33 4 3 65 0 28 3 4 70 0 20 5 5 66 0 20 6 8 73 0 15 2 10 50 10 33 5 2 55 15 23 5 2 55 19 19 5 2

[00216] Examples of compositions of the present invention are shown in Table 2.

Table 2: Compositions of lipid components (amount of each expressed as mol% of total amount)

Ionizable Cationic Structural Stabilizing To reduce immunogenicity Fusogenic 17 0 35 38 8 2 20 0 32 40 5 3 25 0 35 37 1 2 25 0 34 34 5 2 25 0 28 38 5 4 25 0 40 27 5 3 thirty 0 25 38 5 2 35 0 24 34 5 2 40 0 29 24 5 2 25 5 thirty 32 5 3 25 10 28 28 5 4 25 15 25 thirty 3 2

[00217] Structural lipids

[00218] Examples of structural lipids include cholesterols, sterols and steroids.

[00219] Examples of structural lipids include cholanes, cholestanes, ergostanes, campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes, gonanes, estranes, androstanes, pregonanes, and cycloartanes.

[00220] Examples of structural lipids include sterols and zoosterols such as cholesterol, lanosterol, zymosterol, zymostenol, desmosterol, stigmastanol, dihydrolanosterol and 7-dehydrocholesterol.

[00221] Examples of structural lipids include PEGylated cholesterols and 3-oxo-(C1-22) acyl compounds of cholestane, for example, cholesteryl acetate, cholesteryl arachidonate, cholesteryl butyrate, cholesteryl hexanoate, cholesteryl myristate, cholesteryl palmitate, cholesteryl behenate, cholesteryl stearate, cholesteryl caprylate, chole steryl-n-decanoate, cholesteryl dodecanoate , cholesteryl nervonate, cholesteryl pelargonate, cholesteryl n-valerate, cholesteryl oleate, cholesteryl elaidate, cholesteryl erucate, cholesteryl heptanoate, cholesteryl linoleidate and cholesteryl linoleate.

[00222] Examples of structural lipids include sterols such as phytosterols, beta-sitosterol, campesterol, ergosterol, brassicasterol, delta-7-stigmasterol and delta-7-avenasterol.

[00223] Stabilizing lipids

[00224] Examples of stabilizing lipids include zwitterionic lipids.

[00225] Examples of stabilizing lipids include compounds such as phospholipids.

[00226] Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and ordylinoleoyl Phosphatidylcholine.

[00227] Examples of stabilizing lipids include phosphatidylethanolamine compounds and phosphatidylcholine compounds.

[00228] Examples of stabilizing lipids include 1,2-dioleoyl -sn -glycero-3-phosphocholine (DOPC).

[00229] Examples of stabilizing lipids include diphytanoylphosphatidylethanolamine (DPhPE) and 1,2-diphytanoyl- sn -glycero-3-phosphocholine (DPhPC).

[00230] Examples of stabilizing lipids include 1,2-distearoyl- sn -glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl- sn -glycero -3-phosphoethanolamine (DPPE) and 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE).

[00231] Examples of stabilizing lipids include 1,2-dilauroyl- sn -glycerol (DLG); 1,2-dimyristoyl- sn -glycerol (DMG); 1,2-dipalmitoyl- sn -glycerol (DPG); 1,2-distearoyl- sn -glycerol (DSG); 1,2-diarachidoyl- sn -glycero-3-phosphocholine (DAPC); 1,2-dilauroyl- sn -glycero-3-phosphocholine (DLPC); 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC); 1,2-dipalmitoyl- sn -glycero-O-ethyl-3-phosphocholine (DPePC); 1,2-dilauroyl- sn -glycero-3-phosphoethanolamine (DLPE); 1,2-dimyristoyl- sn -glycero-3-phosphoethanolamine (DMPE); 1,2-distearoyl- sn -glycero-3-phosphoethanolamine (DSPE); 1-palmitoyl-2-linoleyl- sn -glycero-3-phosphocholine; 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC); 1-palmitoyl-2-lyso- sn -glycero-3-phosphocholine (P-Lyso-PC); and 1-stearoyl-2-lyso- sn -glycero-3-phosphocholine (S-Lyso-PC).

[00232] Lipids to reduce immunogenicity

[00233] Examples of lipids for reducing immunogenicity include polymer compounds and polymer-lipid conjugates.

[00234] Examples of lipids for reducing immunogenicity include PEGylated lipids containing regions with polyethylene glycol (PEG) molecules. PEG molecules can have any molecular weight. In some embodiments, the PEG molecule may have a molecular weight of 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500, 4000, or 5000 Da.

[00235] Examples of lipids for reducing immunogenicity include compounds having a methoxy polyethylene glycol region.

[00236] Examples of lipids for reducing immunogenicity include compounds having a carbonylmethoxypolyethylene glycol region.

[00237] Examples of lipids for reducing immunogenicity include compounds having a branched PEG region.

[00238] Examples of lipids for reducing immunogenicity include compounds having a polyglycerol region.

[00239] Examples of lipids for reducing immunogenicity include polymeric lipids such as DSPE-mPEG, DMPE-mPEG, DPPE-mPEG and DOPE-mPEG.

[00240] Examples of lipids to reduce immunogenicity include PEG phospholipids and PEG ceramides.

[00241] Cationic lipids

[00242] Examples of cationic lipids include the cationic compounds HEDC (2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N, N-dimethyl-2-oxoethanaminium bromide) described in US2013/0330401A1 . Some examples of cationic lipids are given in US2013/0115274A1.

[00243] Lipid compositions

[00244] In some embodiments, the composition may contain a fusogenic molecule, an ionizable compound, cholesterol, DOPC and DOPE lipids, and DPPE-mPEG. In some embodiments, the fusogenic molecule may comprise 1-20 mol% of the composition, the ionizable molecule may constitute 15 to 25 mol% of the composition; collectively, cholesterol, DOPC and DOPE may constitute 75 to 85 mol% of the composition; and DPPE-mPEG may comprise 2-5 mol% of the composition.

[00245] In one embodiment, the fusogenic molecule may comprise 2 mol% of the composition, the ionizable molecule may constitute 24 mol% of the composition; cholesterol may be 29 mol% of the composition, DOPC may be 20 mol% of the composition, DOPE may be 20 mol% of the composition; and DPPE-mPEG (2000) may be 5 mol% of the composition.

[00246] Nanoparticles

[00247] Embodiments of the present invention may provide liposomal nanoparticle compositions. The fusogenic molecules of the present invention can be used to form liposomal compositions, which can have one or more bilayer structures of lipid-like molecules.

[00248] The nanoparticle composition may contain one or more fusogenic molecules of the present invention in a liposomal structure, bilayer structure, micelle, lamellar structure, or a mixture thereof.

[00249] In some embodiments, the composition may include one or more liquid carrier components. A liquid carrier suitable for delivering the active agents of the present invention may be a pharmaceutically acceptable liquid carrier. The liquid carrier may include an organic solvent or a combination of water and an organic solvent.

[00250] Embodiments of the present invention can provide lipid nanoparticles having a size from 10 to 1000 nm. In some embodiments, liposomal nanoparticles may have a size between 10 and 150 nm.

[00251] Pharmaceutical compositions

[00252] The present invention further provides methods for distributing an active agent to an organ of a subject for treating fibrosis by administering to the subject a composition of the present invention. Organs that are suitable for treatment include the lung, liver, pancreas, kidney, colon, heart, bone marrow, skin, intestines, brain and eye.

[00253] In some embodiments, the present invention provides methods for treating pulmonary fibrosis disease by administering to a subject a composition of the present invention.

[00254] Examples of fibrosis include idiopathic pulmonary fibrosis and liver cirrhosis.

[00255] In additional aspects, the present invention relates to a number of pharmaceutical compositions.

[00256] The pharmaceutical composition herein may include an active agent as well as a drug carrier or lipid of the present invention together with a pharmaceutically acceptable carrier or diluent.

[00257] In general, active agents herein include siRNAs, fibrosis active agents, as well as any small molecule drug. The active agent may be a nucleic acid, siRNA, mRNA or microRNA.

[00258] The pharmaceutical composition of the present invention may contain one or more of the following: a surfactant, a diluent, an excipient, a preservative, a stabilizer, a coloring agent, and a suspending agent.

[00259] Certain pharmaceutical carriers, diluents and components for pharmaceutical composition, as well as methods of formulation and administration of the compounds and compositions of the present invention are described in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Penn. (1990).

[00260] Examples of preservatives include sodium benzoate, ascorbic acid, and p-hydroxybenzoic acid esters.

[00261] Examples of surfactants include alcohols, esters, sulfated aliphatic alcohols.

[00262] Examples of excipients include sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate and calcium carboxymethylcellulose.

[00263] Examples of suspension agents include coconut oil, olive oil, sesame oil, peanut oil, soybean, cellulose acetate phthalate, methyl acetate methacrylate copolymer, and ester phthalates.

[00264] Molecular tail structures

[00265] A compound of the present invention may have one or more lipophilic tails that contain one or more alkyl or alkenyl groups. Examples of lipophilic tails having alkenyl groups include C(14:1(5))alkenyl, C(14:1(9))alkenyl, C(16:1(7))alkenyl, C(16:1(9) )alkenyl, C(18:1(3))alkenyl, C(18:1(5))alkenyl, C(18:1(7))alkenyl, C(18:1(9))alkenyl, C(18 :1(11))alkenyl, C(18:1(12))alkenyl, C(18:2(9.12))alkenyl, C(18:2(9.11))alkenyl, C(18:3) (9,12,15))alkenyl, C(18:3(6,9,12))alkenyl, C(18:3(9,11,13))alkenyl, C(18:4(6,9, 12,15))alkenyl, C(18:4(9,11,13,15))alkenyl, C(20:1(9))alkenyl, C(20:1(11))alkenyl, C(20: 2(8,11))alkenyl, C(20:2(5,8))alkenyl, C(20:2(11,14))alkenyl, C(20:3(5,8,11))alkenyl, C(20:4(5,8,11,14))alkenyl, C(20:4(7,10,13,16))alkenyl, C(20:5(5,8,11,14,17) )alkenyl, C(20:6(4,7,10,13,16,19))alkenyl, C(22:1(9))alkenyl, C(22:1(13))alkenyl, and C(24 :1(9))alkenyl. Some examples of tail structures can be found in Donald Voet and Judith Voet, Biochemistry , 3rd Edition (2005), p. 383.

[00266] Some examples of lipophilic tails include the following structures:

[00267] Any of these exemplary lipophilic tail structures may have one or more additional chemical branches.

[00268] Additional embodiments

[00269] Embodiments of the present invention further include:

[00270] Compound of formula (A)

formula A,

where the linker is a divalent group containing a PEG region,

X ₁ and X ₂ independently represent a C1-C5 alkanediyl group,

R ₁ , R ₂ , R ₃ and R ₄ are independently:

,

X ₃ represents a single bond, a C1-C5alkanediyl group or a C2-C5alkendiyl group,

X ₄ and X ₅ independently represent a C2-5alkanediyl group,

Z ₁ , Z ₂ and Z ₃ are independently -O-, -S- or -NH-, and

R ₅ and R ₆ independently represent a C11-23 alkyl or a C11-23 alkenyl group.

Compound according to formula (B)

formula (B),

where the linker is a divalent group containing a PEG region,

X ₆ and X ₇ independently represent a C1-C5 alkanediyl group,

X ₈ and X ₉ independently represent a C1-C5 alkanediyl group,

Z ₄ and Z ₅ are independently -O-, -S- or -NH-,

R ₇ , R ₈ , R ₉ and R ₁₀ are independently:

,

X ₄ and X ₅ independently represent a C2-5alkanediyl group,

Z ₂ and Z ₃ are independently -O-, -S- or -NH-, and

R ₅ and R ₆ independently represent a C11-23 alkyl or a C11-23 alkenyl group.

The compound specified above, where the linker is:

,

where m is an integer from 1 to 12,

Y ₁ represents -O-, -NH- or -NHCH ₂ -,

Y ₂ represents -O-, -NH- or -CH ₂ NH-,

n and q are independently an integer from 1 to 5,

p represents an integer from 1 to 5,

Y ₃ and Y ₅ are independently -O-, -NH- or -NHCH ₂ -, and

Y ₄ and Y ₆ are independently -O-, -NH- or -CH ₂ NH-.

A compound as defined above, wherein X ₁ and X ₂ independently represent a C1-C5 straight alkanediyl group, preferably a C2-C4 straight alkanediyl group, more preferably a C4 straight alkanediyl group.

A compound as defined above, wherein R ₁ , R ₂ , R ₃ and R ₄ are the same groups.

The compound as above, where X ₃ represents a single bond or a C1-C5 straight alkanediyl group, X ₃ preferably represents a C2-C4 straight alkanediyl group, more preferably ethylene, that is, an ethanediyl group.

The compound as above, wherein X ₄ and X ₅ independently represent a C2-5 straight alkanediyl group, X ₄ and X ₅ preferably represent a C2-4 straight alkanediyl group, more preferably ethylene, that is, an ethanediyl group.

The compound as above wherein Z ₁ is -NH-.

The compound as above where Z ₂ and Z ₃ are -O-.

The compound as defined above, wherein R ₅ and R ₆ independently represent a C11-23 straight alkenyl group.

A compound as defined above, wherein R ₅ and R ₆ independently represent a C11-23 straight alkenyl group with 1-6 double bonds, where the number of double bonds is preferably 1-3, more preferably 2-3, even more preferably, 2.

The compound above wherein R ₅ and R ₆ are independently a C11-23 straight alkenyl group with 2 double bonds.

The compound as above wherein R ₅ and R ₆ independently represent a C13-17 straight alkenyl group, R ₅ and R ₆ preferably represent a C15-17 straight alkenyl group, more preferably a C17 straight alkenyl group.

The compound as defined above wherein R ₅ and R ₆ independently represent a C17 straight alkenyl group.

The compound as above, wherein R ₅ and R ₆ represents a heptadeca-8,1-dienyl group.

The composition mentioned above, which contains a cationic lipid, an ionizable lipid and a lipid of the above compound in a lipid nanoparticle containing a bilayer of lipid molecules.

The composition specified above, which further contains a nucleic acid.

The composition as defined above, wherein the nucleic acid is siRNA, mRNA or microRNA.

The composition as defined above, wherein the composition is a pharmaceutical composition.

[00271] Chemical definitions

[00272] As used herein, the term “alkyl” refers to a hydrocarbyl radical of a saturated aliphatic group, which can be of any length unless otherwise noted. An alkyl group may be a branched or unbranched, substituted or unsubstituted aliphatic group containing from 1 to 22 carbon atoms. This definition also applies to the alkyl moiety of other groups such as, for example, cycloalkyl, alkoxy, alkanoyl and aralkyl.

[00273] For example, as used herein, a term such as "C(1-5)alkyl" includes C(1)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl and C(5)alkyl. Likewise, for example, the term “C(3-22)alkyl” includes C(1)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, C(5)alkyl, C( 6)alkyl, C(7)alkyl, C(8)alkyl, C(9)alkyl, C(10)alkyl, C(11)alkyl, C(12)alkyl, C(13)alkyl, C(14) alkyl, C(15)alkyl, C(16)alkyl, C(17)alkyl, C(18)alkyl, C(19)alkyl, C(20)alkyl, C(21)alkyl and C(22)alkyl.

[00274] As used herein, an alkyl group may be referred to by a term such as Me (methyl, -CH ₃ ), Et (ethyl, -CH ₂ CH ₃ ), Pr (any propyl group), ⁿ Pr (n-Pr, n- propyl), ⁱ Pr (i-Pr, isopropyl), Bu (any butyl group), ⁿ Bu (n-Bu, n-butyl), ⁱ Bu (i-Bu, isobutyl), ^s Bu (s-Bu, sec -butyl) and ^t Bu (t-Bu, tert-butyl).

[00275] As used herein, the term “alkenyl” refers to a hydrocarbyl radical having at least one carbon-carbon double bond. An alkenyl group may be a branched or straight-chain, substituted or unsubstituted hydrocarbyl radical having from 2 to 22 carbon atoms and at least one carbon-carbon double bond. An "alkenyl" group has one or more carbon-carbon double bonds.

[00276] As used herein, the term “substituted” refers to an atom having one or more substituent groups or one or more substituents, which may be the same or different and may include a hydrogen substituent. Thus, the terms alkyl, cycloalkyl, alkenyl, alkoxy, alkanoyl and aryl, for example, refer to groups that may include substituted variants. Substituted variants include linear, branched and cyclic variants and groups having a substituent or substituents substituting one or more hydrogen atoms attached to any carbon atom in the group.

[00277] In general, a compound may contain one or more chiral centers. Compounds containing one or more chiral centers may include compounds described as an "isomer", "stereoisomer", "diastereomer", "enantiomer", "optical isomer" or as a "racemic mixture". Conventions of stereochemical nomenclature are known in the art, such as the Cahn-Ingold-Prelog rules for naming stereoisomers, as well as methods for determining stereochemistry and separating stereoisomers. See, for example, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th edition, 2001. The compounds and structures of the present invention, including the chemical drawings, are intended to cover all possible isomers, chemically acceptable positional isomers, stereoisomers, diastereomers, enantiomers and/or optical isomers that might exist for a particular compound or structure, including any mixture thereof, racemic or otherwise.

[00278] This invention covers any and all tautomeric, solvated or unsolvated, hydrated or unhydrated forms, as well as any isotopic atomic forms of the compounds and compositions disclosed herein.

[00279] The present invention covers any and all crystalline polymorphs or various crystalline forms of the compounds and compositions disclosed herein.

[00280] Abbreviations used:

DMAP - 4-N,N-dimethylaminopyridine

DCM - dichloromethane

TEA - triethylamine

EDC - 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

Na ₂ SO ₄ - sodium sulfate

EtOAc - ethyl acetate

DMF - N,N-dimethylforamide

ELSD - evaporative light scattering detector

NaCl - sodium chloride

K ₂ CO ₃ - potassium carbonate

MeOH - methanol

TFA - trifluoroacetic acid

DIEA - N, N-diisopropylethylamine

PEG/PEG - polyethylene glycol or polyethylene oxide

MgSO ₄ - magnesium sulfate

LCMS - liquid chromatography-mass spectrometry

NaHCO ₃ - sodium bicarbonate

H ₂ O - water

HCl - hydrochloride

KI - potassium iodide

DMSO - dimethyl sulfoxide

TBAF - tetra-N-butylammonium fluoride

NaBH ₄ - sodium borohydride

THF - tetrahydrofuran

TBDMS - tert-butyldimethylsilyl

LiOH - lithium hydroxide

MeI - methyl iodide

BOC - tert-butyloxycarbonyl

Fmoc - fluorenylmethyloxycarbonyl

[00281] EXAMPLES

[00282] Example 1: The fusogenic molecules of the present invention were useful for delivering one or more biologically active agents to cells. In this example, the fusogenic molecules of the present invention have been shown to provide surprisingly effective delivery of an exemplary siRNA targeting HSP47 to knock down gene expression. Delivery of an exemplary siRNA was carried out in a liposomal composition containing fusogenic molecules. The presence of fusogenic molecules of the present invention in the delivery composition unexpectedly provided high activity of the composition for suppressing gene expression using siRNA.

[00283] In vitro activity for knockdown of gene expression using siRNA was measured using rat stellate cells in DMEM according to the following protocol: one day before transfection, cells are seeded in a 96-well plate at 3 x 10 ³ cells per well for stellate cells in 100 μl of medium containing 10% FBS and cultured at 37°C in an incubator in an atmosphere of humidified air containing 5% CO ₂ . Before transfection, the medium is changed to 90 μl of antibiotic-free medium. Prepare appropriate dilutions of the solution collected from the dialysis bags in PBS buffer so that adding 10 µl to each well is sufficient to achieve the desired concentration. 48 hours after transfection, cells are washed once with ice-cold PBS. Cells are lysed in 50 μl of Cell-to-Ct Lysis Buffer for 5-30 minutes at room temperature. Add 5 µl of stop solution and incubate for 2 minutes at ambient room temperature. The mRNA level is measured immediately using qPCR with TAQMAN probes. Alternatively, samples can be frozen at -80°C and analyzed later. For qRT-PCR analysis: thaw all reagents on ice. Mix the combined reagents in 0.2 ml PCR tubes. Distribute the finished mixture into the wells of a 384-well plate, 10 μl per well in triplicate. The plate is sealed with film, and the mixture is dumped by centrifugation to the bottom of the wells. qRT-PCR analysis is performed.

[00284] In this example, HSP47 siRNA was used to knock down the HSP47 gene. For TaqMan analysis of gene expression, a HSP47 gene-specific TaqMan probe was used.

[00285] Experimental results regarding in vitro gene silencing activity in rat stellate cells are shown in Tables 3 and 4.

Table 3: In vitro activity in rat stellate cells

Sample % expression at dosage (nM) % expression at dosage (nM) 50 nM 200 nM 18 nM 75 nM 300 nM Control (PBS) 110 92 80 80 36 Connection R4 44 15 16 10 6 Connection S6 97 80 33 S7 connection 57 48 27 Connection S8 74 64 42 T1 connection 76 68 44 T2 connection 82 70 44 T3 connection 10 8 5 T4 connection 82 70 44

Table 4: In vitro activity in rat stellate cells

Sample % expression at dosage (nM) 18 nM 75 nM 300 nM Control (PBS) 88 88 88 T3 connection 36 15 3 T4 connection 27 10 8 T5 connection 29 13 eleven T6 connection thirty 12 5 T7 connection 51 20 8 T8 connection 29 9 8 T9 connection 100 65 36

[00286] These data indicate that the siRNA preparation containing the fusogenic molecule of the present invention was surprisingly effective in delivering the siRNA active agent into cells. The fusogenic molecules of the present invention have been shown to provide surprisingly effective delivery for knockdown of gene expression using an exemplary siRNA directed against HSP47.

[00287] Example 2: The broad spectrum of fusogenic molecules of the present invention have been shown to be useful for delivering the active agent into cells. In this example, a number of fusogenic molecules of the present invention were shown to provide surprisingly effective delivery of an exemplary siRNA directed against HSP47 to knockdown gene expression. Delivery of an exemplary siRNA was carried out in a liposomal composition containing fusogenic molecules.

[00288] In vitro gene expression knockdown activity measured in stellate cells using an exemplary siRNA in a liposomal composition containing various fusogenic molecules of the present invention is shown in Table 5.

Table 5: % HSP47 expression in stellate cells

Sample siRNA concentration 18 nM 75 nM 300 nM Cells only 108 108 108 Cells+PBS 100 100 100 T10 connection 11.1 4.2 1.9 T11 connection 11.1 4.5 1.8 T12 connection 40.2 8.4 2.7 T13 connection 21.8 6.4 3.1 T14 connection 103.3 59.1 5.2 T3 connection 13.2 4.5 2.3

[00289] As shown in Table 5, the presence of the fusogenic molecules of the present invention in the liposomal delivery formulation unexpectedly provided high potency of the gene expression knockdown formulation using an exemplary siRNA.

[00290] Example 3: The fusogenic molecules of the present invention were unexpectedly active in increasing the efficiency of delivery of the active agent into cells. The activity of the agent delivered in a liposomal composition containing the fusogenic molecule of the present invention was significantly increased compared to the activity of the agent delivered in a liposomal composition that did not contain the fusogenic molecule of the present invention.

[00291] In this example, the gene expression knockdown activity of an exemplary siRNA directed against HSP47 was unexpectedly enhanced using a liposomal delivery composition that included compound R4 of the present invention.

[00292] The liposomal delivery composition was prepared according to the following protocol: HEDC (2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethane-aminium bromide) and S104 (((2-((2-(dimethylamino)ethyl)thio)acetyl)azandiyl)bis(ethane-1,2-diyl)ditetradecanoate) was dissolved in absolute EtOH (100% alcohol content)) in molar ratio 1:1. HSP47 siRNA was solubilized in 50 mM citrate buffer, and the temperature was adjusted to 35–40°C. The ethanol/lipid mixture was then added to the siRNA-containing buffer with stirring to spontaneously form siRNA-loaded liposomes. Lipids were combined with siRNA to achieve a final total lipid to siRNA ratio of 5:1 to 15:1 (w/w). The siRNA-loaded liposomes were diafiltered against 10 volumes of PBS (pH 7.2) to remove ethanol and exchange buffer. The final product was filtered through a 0.22 μm sterilizing PES filter to reduce bioburden.

[00293] In this example, in vitro gene expression knockdown activity using an exemplary siRNA in rat stellate cells was performed in the same manner as Example 1. The results are shown in FIG. 46. The liposomal composition contained HEDC and S104 lipids, as well as a fusogenic compound.

[00294] Figure 46 shows the results for a liposomal composition consisting of HEDC and S104 lipids, which did not contain fusogenic compound (1). Knockdown of HSP47 gene expression for this control composition was only significant at the highest concentration of 300 nM.

[00295] Figure 46 shows the results for a liposomal composition consisting of HEDC and S104 lipids, which contained the fusogenic compound R4 (2). The amount of fusogenic compound R4 in this composition was 2% of the total lipids. Knockdown of HSP47 gene expression for this formulation was significantly and unexpectedly increased at all siRNA concentrations compared to the control formulation that did not contain the fusogenic compound R4. Thus, the presence of the fusogenic compound R4 in the formulation significantly increased the delivered activity of the exemplary siRNA, and the increased activity was directly related to the presence of the fusogenic compound R4.

[00296] Figure 46 shows the results for a liposomal composition consisting of HEDC and S104 lipids, which contained the fusogenic compound R4 (3). The amount of fusogenic compound R4 in this composition was 10% of the total lipids. Knockdown of HSP47 gene expression for this formulation was significantly and unexpectedly increased at all siRNA concentrations compared to the control formulation that did not contain the fusogenic compound R4. Thus, the presence of the fusogenic compound R4 in the formulation significantly increased the delivered activity of the exemplary siRNA, and the increased activity was directly related to the presence of the fusogenic compound R4.

[00297] As shown in FIG. 46, for liposomal delivery compositions containing HEDC and S104 lipids, activity in knocking down gene expression in stellate cells using an exemplary siRNA directed against HSP47 was unexpectedly increased in compositions containing 2 to 10% (of total lipids) fusogenic compound R4.

[00298] Example 4: The fusogenic molecules of the present invention provided unexpectedly increased activity of the active agent, nucleic acid, in cells. The activity of the nucleic acid agent delivered in a liposomal formulation containing the fusogenic molecule of the present invention was significantly increased compared to the activity of a liposomal formulation that did not contain the fusogenic molecule of the present invention.

[00299] In this example, an in vitro activity assay for gene expression knockdown using an exemplary siRNA in rat stellate cells was performed in the same manner as Example 1. The results are shown in FIG. 47. The liposome composition contained HEDC and S104 lipids, as well as a fusogenic compound.

[00300] Figure 47 shows the results for a liposomal composition consisting of HEDC and S104 lipids that did not contain the fusogenic compound of the present invention (1). Knockdown of HSP47 gene expression for this control composition was only significant at the highest siRNA concentration of 300 nM.

[00301] Figure 47 shows the results for a liposomal composition consisting of HEDC and S104 lipids, which contained the fusogenic compound R4 (2). The amount of fusogenic compound R4 in this composition was 2% of the total lipids. Knockdown of HSP47 gene expression for this formulation was significantly and unexpectedly increased at all siRNA concentrations compared to the control formulation that did not contain the fusogenic compound R4. Thus, the presence of the fusogenic compound R4 in the formulation significantly increased the delivered activity of the exemplary siRNA, and the increased activity was directly related to the presence of the fusogenic compound R4.

[00302] Figure 47 shows the results for a liposomal composition consisting of HEDC and S104 lipids, which contained the fusogenic compound T3 (3). The amount of fusogenic compound T3 in this composition was 2% of the total lipids. Knockdown of HSP47 gene expression for this formulation was significantly and unexpectedly increased at all siRNA concentrations compared to the control formulation that did not contain fusogenic T3. Thus, the presence of fusogenic T3 in the formulation significantly increased the delivered activity of the exemplary siRNA, and the increased activity was directly related to the presence of fusogenic T3.

[00303] Example 5: The fusogenic molecules of the present invention were effective in delivering one or more biologically active molecules in vitro . For example, gene silencing activity using siRNA (HSP47 siRNA, see Example 1) was unexpectedly enhanced by the presence of the fusogenic compound of the present invention in the liposomal delivery formulation.

[00304] In this example, liposomal delivery formulations were prepared for the reference compounds as well as the fusogenic compound R4. Compositions for liposomal delivery were prepared in the same manner as in Example 1, with the compositions shown in Table 6 (CH is cholesterol).

Table 6: Fusogenic liposomal formulations

No. Compound Description Type Final conc. siRNA (nM) 1 HEDC:S104:DOPE:CH:DMPE-PEG Large batch Control 2000 2 HEDC:S104:DOPE:CH:DMPE-PEG:R1 Large batch Experimental 2000 3 HEDC:S104:DOPE:CH:DMPE-PEG:R2 Large batch Experimental 2000 4 HEDC:S104:DOPE:CH:DMPE-PEG:R3 Large batch Experimental 2000 5 HEDC:S104:DOPE:CH:DMPE-PEG: R4 Large batch Experimental 2000 6 HEDC:S104:DOPE:CH:DMPE-PEG:R5 Large batch Experimental 2000 7 HEDC:S104:DOPE:CH:DMPE-PEG:S1 Large batch Experimental 2000 8 HEDC:S104:DOPE:CH:DMPE-PEG:S2 Large batch Experimental 2000 9 HEDC:S104:DOPE:CH:DMPE-PEG:S3 Large batch Experimental 2000 10 HEDC:S104:DOPE:CH:DMPE-PEG:S4 Large batch Experimental 2000

[00305] As shown in FIG. 24, for the liposomal delivered formulations of Table 6, the activity for knockdown of gene expression in stellate cells using an exemplary siRNA directed against HSP47 was unexpectedly increased in formulations containing 2% (of total lipids) fusogenic compound R4 of the present invention compared to a control composition that did not contain the fusogenic compound of the present invention. The R4 fusogenic compound structure of the present invention provided surprising delivery efficiency for knockdown of gene expression in stellate cells using exemplary siRNA.

[00306] Example 6: The fusogenic lipid molecules of the present invention were effective in delivering one or more biologically active molecules into cells. In this example, liposomal delivery formulations containing an exemplary siRNA (HSP47 siRNA, see Example 1) as well as a fusogenic compound of the present invention provided effective knockdown of gene expression. In this example, as shown in Table 7, siRNA delivery liposomal formulations containing various compounds T3 to T9 (1 to 7) were prepared, each containing 2% (of total lipids) of the fusogenic compound of the present invention. Compositions for liposomal delivery were prepared in the same manner as in Example 1.

Table 7: Fusogenic liposomal formulations

No. Compound 1 HEDC:S104:DOPE:CH:DMPE-PEG:T4 2 HEDC:S104:DOPE:CH:DMPE-PEG:T5 3 HEDC:S104:DOPE:CH:DMPE-PEG:T6 4 HEDC:S104:DOPE:CH:DMPE-PEG:T7 5 HEDC:S104:DOPE:CH:DMPE-PEG:T8 6 HEDC:S104:DOPE:CH:DMPE-PEG:T9 7 HEDC:S104:DOPE:CH:DMPE-PEG:T3

[00307] As shown in Figure 25, in this measurement of the activity of the siRNA liposomal delivery formulations of Table 7, the formulations provided high gene expression knockdown activity in stellate cells. Thus, formulations containing 2% (of total lipids) of the fusogenic compound of the present invention provided high siRNA activity against HSP47.

[00308] Example 7: In vivo activity of fusogenic formulations. The fusogenic molecules of the present invention were useful for delivering an active agent in vivo . For example, liposomal delivery formulations for knockdown of gene expression using an exemplary siRNA directed against HSP47 were active due to the presence of a fusogenic compound of the present invention.

[00309] As shown in Figure 26, liposomal delivery formulations demonstrated gene expression knockdown activity in vivo (mouse) using siRNA directed against HSP47. The compositions contained 2% (of total lipids) of the specified fusogenic compound of the present invention. The formulations were delivered by infusion bolus. Delivery parameters are given in Table 8.

Table 8: Fusogenic liposomal formulations in vivo

Group number Compound Dosage Conc. (mg/ml) #1 Vehicle, saline 10 mg/kg@day 0-2 #2 HEDC:S104:DOPE:CH:DMPE-PEG:T4 0.5 mg/kg 0.17 #3 HEDC:S104:DOPE:CH:DMPE-PEG:T5 0.5 mg/kg 0.17 #4 HEDC:S104:DOPE:CH:DMPE-PEG:T6 0.5 mg/kg 0.17 #5 HEDC:S104:DOPE:CH:DMPE-PEG:T7 0.5 mg/kg 0.17 #6 HEDC:S104:DOPE:CH:DMPE-PEG:T8 0.5 mg/kg 0.17 #7 HEDC:S104:DOPE:CH:DMPE-PEG:T9 0.5 mg/kg 0.17 #8 HEDC:S104:DOPE:CH:DMPE-PEG:T3 0.5 mg/kg 0.17 #9 Negative control saline solution

[00310] An example protocol for these results is provided below.

[00311] 1.1. Animals

[00312] Eighty male Sprague-Dawley rats, ~49 days old, were purchased from Charles River Laboratories and transported to the testing facility. The average weight of animals upon receipt was about 200-210 grams. Animals were housed in standard cage systems with 2 animals per cage on an alternating light/dark cycle of 12 h. Room temperature was maintained at 64-79°F (18-26°C) and humidity at 30-70% with at least 10 air volume changes per hour using 100% fresh air without recirculation. Animals were provided with certified radiation-sterilized standard fresh rodent chow and ad-libitum tap water.

[00313] Seventy-two animals were administered DMN on days 0-5 and then randomly divided into 9 groups of 8 rats per group based on their body weight, so that there was no significant difference in body weight between groups before siRNA administration. This was confirmed using one-way ANOVA. As expected, animals that received DMN had significantly lower body weight than animals that did not receive DMN (“clean animals”).

[00314] 1.2. Introduction to DMN

[00315] DMN was obtained from Wako (lot number DSP2369) and prepared for intraperitoneal (IP) injection in phosphate-buffered saline (PBS), preparing a solution of the compound at a concentration of 5 mg/ml. Seventy-two rats were administered daily DMN at a dose of 10 mg/kg in a volume of 2 ml/kg from day 0 to day 2 and then 5 mg/kg in a volume of 1 ml/kg from day 3 to day 5 ; eight animals that did not receive DMN were used as negative controls for this procedure. Animals were weighed daily and the DMN dose was adjusted accordingly.

[00316] 1.3. Introduction of siRNA

[00317] On experimental day 5, animals were assigned to different treatment groups and administered drugs according to the appropriate treatment regimen. The tested drugs were used for treatment groups 2-8 (from Nos. 1-8), respectively; whereas animals in group 1 (No. 1) received only vehicle (saline) at a dose of 3 ml/kg by a single intravenous injection into the lateral tail vein. Animals in group 9 (clean group, no. 9) received no treatment.

[00318] 1.4. Euthanasia and autopsy

[00319] On experimental day 6, 24 hours after administration, animals were sacrificed by inhalation overdose of carbon dioxide. The liver was immediately flushed with PBS, pH 7.4 (40 ml at 20 ml/min) through the hepatic portal vein to remove residual blood and blood-associated drug. One 2-mm-thick liver cross-section was taken from the left lateral lobe and immediately immersed in 2 mL of RNAlater buffer in a microcentrifuge tube. Samples were stored at 4°C until further processing for RNA isolation.

[00320] 1.6. RNA analysis

[00321] HSP47 mRNA content in liver samples was assessed. Total RNA was extracted using RNeasy columns (Qiagen) according to the manufacturer's instructions. The RNA concentration for each sample was quantified using a Nanodrop spectrophotometer and then diluted to 10 ng/ml using nuclease-free water. For each PCR reaction, 20 ng of total RNA was used. Briefly, total RNA from the left lobe liver tissue section will be extracted using RNeasy columns (Qiagen) according to the manufacturer's instructions. A Nanodrop spectrophotometer was used for RNA quantification. The RNA concentration was adjusted to 10 ng/μl using nuclease-free water. Real-time PCR was performed on a ViiA7 system in a 96-well format. Each sample was measured in triplicate using TaqMan Gene Expression Master Mix. Cycling parameters were set to 48°C for 15 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The average threshold cycle value for the housekeeping gene, MRP119, was used to normalize the raw threshold cycle data and calculate ΔCt. The ΔΔCt for each gene of interest (GOI) was calculated by subtracting the mean ΔCt for the GOI in the control sample from the ΔCt for each GOI in the test samples. Data for each animal were expressed as both the percentage of the mean for the vehicle group and the fold change compared to the clean group. Differences between the siRNA-treated groups and the vehicle-treated group were analyzed using one-way ANOVA followed by Dunnett's post hoc pairwise multiple comparisons test. For all analyses, a p value of less than 0.05 was considered significant.

[00322] Example 8: Preparation of mRNA nanoparticles. Cationic lipids such as HEDC or HE2DC (2-(bis(2-(palmitoyloxy)ethyl)amino)-N,N-bis(2-hydroxyethyl)-N-methyl-2-oxoethane-1-aminium bromide), ionizable lipids such as S104 or TU104 Dlin ((9Z, 9'Z,12Z,12'Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azandiyl)bis(ethane-1,2, 1-Diyl)bis(octadeca-9,1,2-dienoate)) and auxiliary lipids, cholesterol and DOPE, were dissolved in ethanol. The mRNA was dissolved in 50 mM citrate buffer (pH 3.5). Lipid nanoparticles (LNPs) were prepared by injecting an appropriate amount of ethanol lipid solution into citrate buffer containing mRNA at a flow rate of 25 mL/min at 37 °C. The molar percentages for the LNP composition were 20% HEDC, 20% S104, 30% DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (Avanti Polar Lipid), 25% cholesterol (Puriss grade) (Wilshire Technologies) , 5% DMPE-PEG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) and 2% fusogenic molecule of the present invention, for example, compound T3.

[00323] The high injection rate resulted in a high degree of mRNA encapsidation as well as a controlled particle size distribution. The mixed LNP solution was then diluted to 20 mM HEPES buffer in 9% sucrose (w/v) in a 1:1 ratio, which reduced the ethanol content of the LNP solution from 35% to 17.5%. The diluted LNP solution was subjected to tangential flow filtration (TFF) for ultrafiltration and diafiltration with 20 mM HEPES buffer in 9% sucrose (w/v). A total of 10 volumes of HEPES diafiltration buffer in 9% sucrose was used to remove ethanol from the LNP solution. The concentrated LNP solution after TFF was portioned into filter centrifuge tubes (EMD Millipore) for further concentration, but this step was only required for small batches. After final concentration, LNP was filtered through 0.2 μm pore size filters. Encapsulation efficiency and mRNA yield were calculated using RiboGreen dye.

[00324] Example 9: In vitro transfection delivery of mRNA to cells. Transfection of cells in vitro with mRNA molecules was carried out in three different cell lines: Hek-293, A549 and a lung fibroblast cell line. JET MESSENGER (Polyplus Transfection Company) was used as a positive transfection control. GFP mRNA (TriLink), which was encapsulated in LNP nanoparticles of the present invention or mixed with the control transfection agent JET MESSENGER, was used to transfect cells at various concentrations. At 24, 48 and 72 hours after transfection, the cells were observed under a confocal microscope, and the fluorescence generated by the expressed GFP in the cells was detected and recorded. GFP mRNA was well transfected into all three cell lines using the LNP nanoparticles of the present invention, and mRNA translation occurred.

[00325] Example 10: Transfection-based delivery of mRNA to cells in vivo . The mRNA was transfected into tissues and cells in vivo using LNP nanoparticles of the present invention. Balb/c mice were delivered and transfected with two different mRNAs of different sizes, GFP mRNA and luciferase mRNA (TriLink). In some luciferase mRNA delivery studies, as a positive control, mRNA delivery was also performed using the in vivo mRNA transfection reagent, Viromer. Animals were administered intravenously with a single injection of mRNA encapsulated in LNP nanoparticles of the present invention or positive control particles at a dosage of 1.0, 2.0 or 4.0 mg/kg. Mice were anesthetized 6–8 hours after mRNA injection, and for luciferase mRNA delivery studies, whole body fluorescence was detected and analyzed using an IVIS system. Animals were immediately sacrificed and various organs were removed and stored at -80°C until further analysis. Delivery of mRNA to various tissues and cells was determined using the MAXDISCOVER GFP ELISA kit to analyze GFP protein levels in tissues from GFP mRNA delivery studies. For luciferase mRNA delivery studies, tissues were homogenized in CCLR lysis buffer and centrifuged. The resulting supernatants were used for luciferase activity assays using reagents from the Promega E4510 kit. Surprisingly, transfection and/or translation of both GFP mRNA and luciferase mRNA were predominantly observed in the lungs and spleen, with significantly less transfection and/or translation in other tissues.

[00326] Example 11: In vitro transfection delivery of mRNA to cells.

According to the method described in Examples 9 and 10 above, GFP mRNA (CleanCap EGFP mRNA, 5 moU) was transfected into A549 cells in vitro using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000 :T3 connection. The results after 48 hours of transfection are shown in Fig. 48. The cells were observed under a confocal microscope and the fluorescence generated as a result of GFP expression in the cells was detected. The results showed that GFP mRNA was transfected and translated in A549 cells.

[00327] Example 12: Transfection-based delivery of mRNA to cells in vivo .

In accordance with the method described in Examples 9 and 10 above, GFP mRNA (CleanCap EGFP mRNA, 5 moU) was transfected into Balb/c mice in LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3 as shown in Table 9:

Table 9: In Vivo Transfection in Balb/c Mice

Group Number of animals Introduction Dosage End point 1 4 IV, once a day 1 mg/kg 8 hours post-administration, IVIS imaging, tissue collection (muscle, liver, heart, lung and kidney), GFP protein level measurement using ELISA 2 4 IV, once a day 4 mg/kg 3 3 Not applicable Not applicable

[00328] As shown in Figure 49, delivery of mRNA to various tissues and cells was determined using the MAXDISCOVER GFP ELISA kit. Surprisingly, GFP mRNA was selectively transfected and/or translated in the lung, with lower transfection and/or translation in muscle, liver, heart, and kidney.

[00329] Example 13: Transfection-based delivery of mRNA to cells in vivo .

In accordance with the method described in Examples 9 and 10 above, luciferase mRNA (Fluc mRNA (5meC)) was transfected into Balb/c mice using LNP nanoparticles of the present invention having the composition HEDC:S104:CH:DOPE:DMPE-PEG2000:compound T3 as shown in Table 10:

Table 10: In Vivo Transfection in Balb/c Mice

Group Number of animals Dosage End point 1 3 IVIS imaging, tissue luciferase assay 2 3 1 mg/kg (20 µg/per animal) 3 3 2 mg/kg (40 µg/each animal) 4 1 10 mcg 5 1 30 mcg 6 3 40 mcg

[00330] As shown in Figure 50, the relative delivery, transfection and/or translation of mRNA into various tissues and cells was determined using the Promega E4510 assay kit. Surprisingly, Fluc (firefly luciferase) mRNA was selectively delivered, transfected and/or translated in the lungs and spleen, with lower delivery, transfection and/or translation in the liver, heart, kidney and muscle.

[00331] Example 14: Transfection-based delivery of mRNA to cells in vivo .

In accordance with the method described in Examples 9 and 10 above, luciferase mRNA (Fluc mRNA (5meC)) was transfected into Balb/c mice using LNP nanoparticles of the present invention having the composition:

(-01) HE2DC:S104:CH:DOPE:DMPE-PEG2000:T3 connection or

(-02) HEDC:S104:CH:DOPE:DMPE-PEG2000:T3 connection,

which were administered by injection at 2 mg/kg, with fluorescent imaging 7 hours after injection.

[00332] As shown in Figure 51, the relative delivery, transfection and/or translation of mRNA into various tissues and cells was determined using the Promega E4510 kit. Surprisingly, Fluc mRNA was selectively delivered, transfected, and/or translated in the lungs and spleen, with lower delivery, transfected, and/or translated in the pancreas, kidney, liver, testis, and small intestine.

[00333] As shown in Figure 52, the relative delivery, transfection and/or translation of mRNA in various tissues was determined by fluorescence imaging 7 hours after injection. In FIG. 52, the photographs in the top row show Balb/c mice transfected with LNP(-01) nanoparticles. The photographs in the bottom row show Balb/c mice transfected with LNP(-02) nanoparticles.

[00334] Example 15: Delivery of mRNA to cells in vivo using fusogenic compounds. The fusogenic compounds of the present invention significantly enhance the delivery of active agents to cells, organs and tissues in vivo .

[00335] In this example , in vivo mRNA delivery formulations were formulated with fusogenic compound T3 and compared to the same formulation without fusogenic compound, as shown in Table 11.

Table 11: Formulations for in vivo drug delivery to mice

mRNA Compound Hydrodynamic diameter (PS), nm Polydispersity Index (PDI) Encapsulation efficiency (EE), % 2035-03-03 HEDC:S104:DOPE:CH:DMPE-PEG2K:T3 connection (20:20:30:25:5:5) 81 0.10 93 2035-13-01 HEDC:S104:DOPE:CH:DMPE-PEG2K (20:20:30:25:5) 83 0.148 78

[00336] Figure 53 shows the results of delivering luciferase mRNA to mice in vivo using the fusogenic lipid-like molecules of the present invention. As shown in FIG. 53, the relative delivery of mRNA was much higher in formulations containing the fusogenic molecule of the present invention (2035-03-03) than in the same formulation without the fusogenic molecule (2035-13-01). In all organs observed, including the pancreas, spleen, liver, kidney, lung, testis, and intestine, delivery was preferentially and unexpectedly greater for the fusogenic T3 formulation.

[00337] The embodiments described herein are not limiting, and one skilled in the art can readily appreciate that specific combinations of modifications described herein can be tested without undue experimentation to identify nucleic acid molecules with improved RNA activity. interference.

[00338] All publications, patents and literature specifically referenced herein are incorporated by reference in their entirety for all purposes.

[00339] It is understood that this invention is not limited to the specific methodology, protocols, materials and reagents described, as these may vary. It should also be understood that the terminology used herein is intended only to describe specific embodiments of the invention and is not intended to limit the scope of the present invention. It will be apparent to one skilled in the art that various substitutions and modifications may be made to the specification disclosed herein without departing from the scope and spirit of the specification, and that these embodiments are within the scope of this specification and the appended claims.

[00340] It should be noted that as used herein and in the accompanying claims, the singular number includes the plural unless the context clearly dictates otherwise. Also, the singular terms “one or more” and “at least one” may be used interchangeably herein. It should also be noted that the terms “comprise,” “comprising,” “including,” “including,” and “having” may be used interchangeably and should be construed broadly and without limitation.

[00341] The indication of ranges of values herein is intended merely to serve as a concise means of individually referring to each individual value falling within the range unless otherwise indicated herein, and each individual value is included in the description as if it were individually indicated in this description. For Markush groups, those skilled in the art will understand that this description includes individual members as well as subsets of Markush group members.

[00342] The compound, molecule or composition of the present invention may be in an ionic form for which a corresponding counterion or counterions are not shown. One skilled in the art will readily understand that the counterion or counterions will be present as needed. Examples of counterions include alkali metal ions, Cl ^- and pharmaceutically acceptable counterions.

[00343] For example, when a list of examples or components, such as a list of compounds, molecules or compositions, suitable for this invention is given, it will be apparent to those skilled in the art that mixtures of the listed compounds, molecules or compositions may also be suitable.

[00344] Without further elaboration, it is believed that one skilled in the art can, based on the above description, use the present invention to its fullest extent. Accordingly, the following specific embodiments should be construed merely as illustrative and not in any way limiting the remainder of the disclosure.

[00345] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

[00346] The drawings in the accompanying claims are drawn to fit the page, and the appearance of molecules in the drawings does not necessarily reflect any essential form or properties of the compound depicted.

Claims (125)

1. Fusogenic compound having formula I wherein each AA independently represents an amino acid selected from the following structures, or a stereoisomer thereof: wherein the amino acid is attached to the amphiphile at each of its amino groups and is attached to a linker at its C-terminus; where the linker has the structure or , where Q ² represents , where Q ³ represents , where X represents -O-, -S- or -NH-; where n, p, q and t independently for each case range from 1 to 3; where m independently ranges from 1 to 10; where r and s independently for each case range from 1 to 5; And wherein each amphiphile is independently selected from Formula II, Formula IV, Formula V and Formula VI: where R ¹ in formula II represents CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C= O)OR ⁴ or CH ₂ (CH ₂ ) _n (C=O)NHR ⁴ ; where R ² in formula II represents CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C= O)OR ⁵ or CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ ; where n and m in formula II are each independently from 1 to 2; and R ⁴ and R ⁵ independently each represent a C(12-20)alkyl group or a C(12-20)alkenyl group; And where R ³ in formula II represents a branched or unbranched C(1-8)alkanediyl; where R ¹ in formula IV represents (C=O)R ⁴ ; where R ² in formula IV represents (C=O)OR ⁵ , where R ⁴ and R ⁵ in formula IV represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group; where Z in formula IV represents NH; where p in formula IV is from 1 to 4; where R ³ in formula IV represents a C(1-12)alkyl group, substituted -(C=O)-, which is attached to AA; where R ¹ in formula V represents (C=O)OR ⁴ ; where R ² in formula V represents NH(C=O)R ⁵ ; where R ⁴ and R ⁵ in formula V represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group; where R ³ in formula V represents a branched or unbranched -O(C=O)-C(1-8)alkanediyl-(C=O)-, which is attached to AA; where R ¹ in formula VI represents O(C=O)OR ⁴ ; where R ² in formula VI represents O(C=O)OR ⁵ ; where R ⁴ and R ⁵ in formula VI represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group; where R ³ in formula VI represents ; wherein one or two amphiphiles may be optionally absent and replaced by a pharmaceutically acceptable organic chemical group selected from alkyl, alkenyl, alkynyl, acetyl, Boc, Fmoc, TFA and CBZ having 1-400 atoms selected from carbon, oxygen, nitrogen, sulfur , fluorine and hydrogen. 2. The fusogenic compound according to claim 1, wherein one or two amphiphiles are absent and replaced by a pharmaceutically acceptable organic chemical group. 3. The fusogenic compound according to claim 2, wherein the pharmaceutically acceptable organic chemical group is alkyl, alkenyl, alkynyl, acetyl, Boc, Fmoc, TFA or CBZ. 4. Fusogenic compound according to claim 3, where the compound is selected from the following compounds: . 5. Fusogenic compound according to claim 1, where R ³ in formula II represents a branched or unbranched C(2-8)alkanediyl. 6. Fusogenic compound according to claim 1, where the compound is selected from the following compounds: . 7. Fusogenic compound according to claim 1, where in formula IV R ⁴ and R ⁵ independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group. 8. Fusogenic compound according to claim 1, where the compound is a T10 compound: . 9. The fusogenic compound according to claim 1, wherein R ⁴ and R ⁵ in formula V are independently in each case a C(14-18)alkyl group or a C(14-18)alkenyl group. 10. Fusogenic compound according to claim 1, where the compound is a T12 compound: . 11. The fusogenic compound according to claim 1, wherein R ⁴ and R ⁵ in formula VI independently in each case represent a C(14-18)alkyl group or a C(14-18)alkenyl group. 12. Fusogenic compound according to claim 1, where the compound is compound T11: . 13. Fusogenic compound having formula VII wherein each AA ^a is independently an amino acid selected from the following structures, or a stereoisomer thereof: wherein the amino acid is attached to the amphiphile at each of its carboxyl groups and is attached to a linker at its N-terminus; where the linker has the structure or , where Q ¹ represents a branched or straight-chain C(2-8)alkanediyl, or , where Q ² represents , where Q ³ represents , where X represents -O-, -S- or -NH-; n and p independently for each case range from 1 to 3; m independently ranges from 1 to 10; r and s independently for each case are from 1 to 5, where each amphiphile is independently selected from Formula VIII, Formula IX, Formula XI and Formula V: where R ¹ and R ² represent R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴ , R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ , where n and m are each independently from 1 to 2; R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group; where R ¹ and R ² represent R ¹ =CH ₂ (CH ₂ ) _n O(C=O)R ⁴ , CH ₂ (CH ₂ ) _n NH(C=O)R ⁴ , CH ₂ (CH ₂ ) _n (C=O)OR ⁴ , CH ₂ (CH ₂ ) _n (C=O)NHR ⁴ , R ² =CH ₂ (CH ₂ ) _m O(C=O)R ⁵ , CH ₂ (CH ₂ ) _m NH(C=O)R ⁵ , CH ₂ (CH ₂ ) _m (C=O)OR ⁵ , CH ₂ (CH ₂ ) _m (C=O)NHR ⁵ , where n and m, each independently, range from 1 to 2; R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group; where R ³ represents a branched or unbranched C(1-8)alkanediyl; where R ¹ and R ² represent: R ¹ =(C=O)OR ⁴ , R ² =(C=O)OR ⁵ , R ⁴ and R ⁵ independently in each case represent a C(12-20)alkyl group or a C(12-20)alkenyl group; where R ³ represents branched or unbranched *-NH-C(1-8)alkanediyl-(C=O)-, where * denotes the end attached to AA ^a ; where R ¹ and R ² represent: R ¹ =(C=O)OR ⁴ , R ² =(C=O)OR ⁵ , where R ⁴ and R ⁵ represent, independently in each case, a C(12-20)alkyl group or a C(12-20)alkenyl group; where R ³ represents branched or unbranched *-NH-C(1-8)alkanediyl-(C=O)-, where * denotes the end attached to AA ^a ; wherein one or two amphiphiles may be optionally absent and replaced by a pharmaceutically acceptable organic chemical group selected from alkyl, alkenyl, alkynyl, acetyl, Boc, Fmoc, TFA and CBZ having 1-400 atoms selected from carbon, oxygen, nitrogen, sulfur , fluorine and hydrogen. 14. The fusogenic compound according to claim 13, wherein one or two amphiphiles are absent and replaced by a pharmaceutically acceptable organic chemical group. 15. The fusogenic compound according to claim 14, wherein the pharmaceutically acceptable organic chemical group is selected from alkyl, alkenyl, alkynyl, alkyl ether, aryl ether, alkoxy and alkoxyacoxy groups. 16. The fusogenic compound according to claim 14, wherein the pharmaceutically acceptable organic chemical group is selected from a methoxy group, an ethoxy group, a tert-butyl ether group and a benzyloxy group. 17. The fusogenic compound according to claim 13, wherein R ⁴ and R ⁵ in formula IX are independently in each case a C(14-18)alkyl group or a C(14-18)alkenyl group. 18. Fusogenic compound according to claim 13, where the compound is a T9 compound: . 19. A composition for use in distributing an active agent for treating a condition or disease in a subject, wherein the composition contains the active agent, a fusogenic compound according to any one of claims. 1-18, ionizable lipid, structural lipid, stabilizing lipid and lipid to reduce the immunogenicity of the composition, where the fusogenic compound is 1-10 mol. % of the composition. 20. The composition according to claim 19, where the active agent is one or more nucleic acids.