HK1257358B - Lipocationic dendrimers and uses thereof - Google Patents
Lipocationic dendrimers and uses thereof Download PDFInfo
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Description
Cross Reference to Related Applications
This application claims priority to U.S. provisional application serial No. 62/218,412, filed on 9/14/2015, which is incorporated by reference herein in its entirety.
Technical Field
The present invention relates generally to the field of dendritic polymers. In particular, it relates to dendrimer nanoparticle compositions comprising nucleic acids. More particularly, it relates to dendrimer nanoparticle compositions for delivery of nucleic acids. More particularly, it relates to dendrimer nanoparticle compositions for the delivery of drugs and other excipients.
Background
Since the discovery of RNAi or other nucleic acid agents and the recognition of their therapeutic potential, effective delivery vehicles have been sought (Whitehead et al, 2009, kanasty et al, 2013, akinc et al, 2008; davis et al, 2010 love et al, 2010 siegwart et al, 2011. Progress has been made with respect to the delivery efficacy of small RNAs to healthy liver, but the existing delivery vehicles currently do not meet the clinically needed combination of high efficacy against tumors and low normal cellular hepatotoxicity. Unfortunately, over the last four years, all five phase III human clinical trials of small molecule drugs for the treatment of hepatocellular carcinoma (HCC) have failed, in part because debilitating advanced liver dysfunction amplifies drug toxicity (Roberts, l.r.,2008, scudelari, m., 2014). Micrornas (mirnas) represent a promising alternative strategy because they can act as tumor suppressants by simultaneously targeting multiple pathways involved in cell differentiation, proliferation and survival, but these therapeutic agents require carriers to be effective (Ventura and Jacks,2009 kasinski and slak, 2011 ling et al, 2013; cheng et al, 2015). The balance of potency and toxicity of a pharmaceutical carrier is a useful criterion, particularly in the case of liver cancer, where toxicity of the carrier itself can diminish the therapeutic efficacy of RNA therapy.
To achieve this balance of low toxicity and high potency, the impact on chemical structure by expanding the structural diversity and molecular size of the delivery vehicle can be used to achieve a therapeutically effective balance. Dendrimers are monodisperse macromolecules consisting of a plurality of fully branched monomers emanating radially from a central core. Dendrimers therefore have the same high degree of molecular homogeneity and chemical tuning as small molecules into a broad theoretical space for polydisperse polymers (Bosman et al, 1999, frchet and Tomalia,2002, gillies and Frechet,2002, grayson and Frechet, 2001. These intrinsic characteristics give dendrimers unique properties for various biomedical applications (stirbiba et al, 2002 lee et al, 2005 wu et al, 2015) (Murat and Grest,1996, percec et al, 2010, duncan and Izzo, 2005. In gene delivery, most studies have used a limited number of commercial dendrimers for further chemical modification. (Kang et al, 2005. Thus, the expansion of dendrimer applications depends on the ability to easily tune the size, chemistry, topology, and physical properties of the final dendrimer by chemical synthesis. Therefore, the development of novel dendrimers that can serve as carriers for nucleic acids and other drugs is clinically useful.
Disclosure of Invention
In some aspects, the present disclosure provides a dendritic polymer of the formula:
nucleus- (repeat unit) n -end capping group (I)
Wherein the core is attached to the repeat unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeat unit, and wherein:
the core has the formula:
wherein:
X 1 is amino or alkylamino (C≤12) Dialkylamino group (b) (C≤12) Heterocycloalkyl group (C≤12) (iii) heteroaryl (C≤12) Or substituted forms thereof;
R 1 is amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (C≤12) Or substituted forms of any of these groups; and is
a is 1,2, 3, 4,5 or 6; or
The core has the formula:
wherein:
X 2 is N (R) 5 ) y ；
R 5 Is hydrogen, alkyl (C≤18) Or substituted alkyl (C≤18) (ii) a And is
y is 0, 1 or 2, provided that the sum of y and z is 3;
R 2 is amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (C≤12) Or substituted versions of any of these groups;
b is 1,2, 3, 4,5 or 6; and is
z is 1,2, 3; provided that the sum of z and y is 3; or alternatively
The core has the formula:
wherein:
X 3 is-NR 6 -, wherein R 6 Is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) -O-, or alkylamino diyl (C≤8) Alkoxy diyl group (C≤8) An aromatic hydrocarbon diyl group (C≤8) Hetero aromatic diyl (C≤8) Heterocyclic alkanediyl group (C≤8) Or substituted versions of any of these groups;
R 3 and R 4 Each independently being amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (b) (C≤12) Or substituted versions of any of these groups; or a group of the formula: - (CH) 2 CH 2 N) e (R c )R d ；
Wherein:
e is 1,2 or 3;
R c and R d Each independently is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
c and d are each independently 1,2, 3, 4,5 or 6; or
The nucleus being an alkylamine (C≤18) Dialkyl amines (C≤36) Heterocyclic alkanes (C≤12) Or substituted versions of any of these groups;
wherein the repeat unit comprises a degradable diacyl and a linker;
the degradable diacyl has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Y 3 Is alkanediyl (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; or a group of the formula:
wherein:
X 3 and X 4 Is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups;
Y 5 is a covalent bond, alkanediyl (C≤12) Olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; and is
R 9 Is an alkyl group (C≤8) Or substituted alkyl (C≤8) ；
The linker group has the formula:
wherein:
Y 1 is an alkanediyl radical (C≤12) Olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; and is
Wherein when the repeating unit comprises a linker group then the linker group is attached to a degradable diacyl group on the nitrogen and sulfur atoms of the linker group, wherein a first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next group comprises two degradable diacyl groups attached to the nitrogen atom of the linker group; and wherein n is the number of linker groups present in the repeat unit; and is
The end capping group has the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or in which alkanediyl (C≤18) One or more hydrogen atoms on the compound have been replaced by-OH-F, -Cl, -Br-I, -SH, -OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) ；
R 10 Is hydrogen, carboxyl, hydroxyl, or
Aryl radicals (C≤12) Alkylamino group (C≤12) Dialkylamino group (C≤12) N-heterocycloalkyl group (C≤12) 、 -C(O)N(R 11 ) -alkanediyl (C≤6) -heterocycloalkyl radical (C≤12) -C (O) -alkylamino (C≤12) -C (O) -dialkylamino (C≤12) -C (O) -N-heterocycloalkyl (C≤12) Wherein:
R 11 is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Wherein the last degradable diacyl group in the chain is attached to a capping group;
n is 0, 1,2, 3, 4,5 or 6;
or a pharmaceutically acceptable salt thereof. In some embodiments, the structure of the dendritic polymer is further defined:
the core has the formula:
wherein:
X 1 is amino or alkylamino (C≤12) Dialkylamino group (C≤12) Heterocycloalkyl group (C≤12) Heteroaryl group (C≤12) Or substituted forms thereof;
R 1 is amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (C≤12) Or substituted versions of any of these groups; and
a is 1,2, 3, 4,5 or 6; and is provided with
Wherein the repeat unit comprises a degradable diacyl and a linker;
the degradable diacyl has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Y 3 Is an alkanediyl radical (C≤12) Olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted forms of any of these groups; or a group of the formula:
wherein:
X 3 and X 4 Is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted versions of any of these groups;
Y 5 is a covalent bond, an alkanediyl (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; and is provided with
R 9 Is an alkyl group (C≤8) Or substituted alkyl (C≤8) ；
The linker group has the formula:
wherein:
Y 1 is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; and is
Wherein when the repeating unit comprises a linker group, then the linker group is attached to a degradable diacyl group on the nitrogen atom and the sulfur atom of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next group comprises two degradable diacyl groups attached to the nitrogen atom of the linker group; and wherein n is the number of linker groups present in the repeat unit; and
an end capping group, wherein the end capping group has the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or in which alkanediyl (C≤18) One or more hydrogen atoms in the group have been replaced by-OH, -F, -Cl,-Br、-I、-SH、-OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) ；
R 10 Is hydrogen, carboxyl, hydroxyl, or
Aryl radical (C≤12) Alkylamino group (C≤12) Dialkylamino group (C≤12) N-heterocycloalkyl, N-heterocycloalkyl (C≤12) 、 -C(O)N(R 11 ) -alkanediyl (C≤6) -heterocycloalkyl radical (C≤12) -C (O) -alkylamino (C≤12) -C (O) -dialkylamino (C≤12) -C (O) -N-heterocycloalkyl (C≤12) Wherein:
R 11 is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Wherein the last degradable diacyl group in the chain is attached to a capping group;
n is 0, 1,2, 3, 4,5 or 6;
or a pharmaceutically acceptable salt thereof. In some embodiments, the dendritic polymer has the formula:
nucleus- (repeat unit) n -end capping group (I)
Wherein the core is attached to the repeat unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeat unit, and wherein:
the core has the formula:
wherein:
X 2 is N (R) 5 ) y ；
R 5 Is hydrogen or alkyl (C≤8) Or substituted alkyl (C≤18) (ii) a And is
y is 0, 1 or 2, provided that the sum of y and z is 3;
R 2 is amino, hydroxy or mercapto, or alkylamino (C≤12) Dialkylamino group (b) (C≤12) Or substituted forms of any of these groups;
b is 1,2, 3, 4,5 or 6; and is provided with
z is 1,2, 3; provided that the sum of z and y is 3;
wherein the repeat unit comprises a degradable diacyl and a linker;
the degradable diacyl has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Y 3 Is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; or a group of the formula:
wherein:
X 3 and X 4 Is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted forms of any of these groups;
Y 5 is a covalent bond, an alkanediyl (C≤12) An olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted versions of any of these groups; and is
R 9 Is an alkyl group (C≤8) Or substituted alkyl (C≤8) ；
The linker group has the formula:
wherein:
Y 1 is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; and is provided with
Wherein when the repeating unit comprises a linker group, then the linker group is attached to a degradable diacyl group on the nitrogen atom and the sulfur atom of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next group comprises two degradable diacyl groups attached to the nitrogen atom of the linker group; and wherein n is the number of linker groups present in the repeat unit; and
an end capping group, wherein the end capping group has the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or in which alkanediyl (C≤18) One or more hydrogen atoms on the surface of the substrate have been replaced by-OH-F, -Cl, -Br, -I, -SH, -OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) ；
R 10 Is hydrogen, carboxyl, hydroxyl, or
Aryl radicals (C≤12) Alkylamino group (C≤12) Dialkylamino group (C≤12) N-heterocycloalkyl group (C≤12) 、 -C(O)N(R 11 ) -alkanediyl (C≤6) -heterocycloalkyl radical (C≤12) -C (O) -alkylamino (C≤12) -C (O) -dialkylamino (C≤12) -C (O) -N-heterocycloalkyl (C≤12) Wherein:
wherein the last degradable diacyl group in the chain is attached to a capping group;
n is 0, 1,2, 3, 4,5 or 6;
or a pharmaceutically acceptable salt thereof. In other embodiments, the dendritic polymer has the formula:
core- (repeating unit) n -end capping group (I)
Wherein the core is attached to the repeat unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeat unit, and wherein:
the core has the formula:
wherein:
X 3 is-NR 6 -, wherein R 6 Is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) -O-, or alkylamino diyl group (C≤8) Alkoxy diyl group (C≤8) An aromatic hydrocarbon diyl group (C≤8) Hetero aromatic diyl (C≤8) Heterocyclic alkanediyl (C≤8) Or substituted versions of any of these groups;
R 3 and R 4 Each independently being amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (b) (C≤12) Or substituted versions of any of these groups; or a group of the formula: - (CH) 2 CH 2 N) e (R c )R d ；
Wherein:
e is 1,2 or 3;
R c and R d Each independently is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
c and d are each independently 1,2, 3, 4,5 or 6; and is
Wherein the repeat unit comprises a degradable diacyl and a linker;
the degradable diacyl has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Y 3 Is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted versions of any of these groups; or a group of the formula:
wherein:
X 3 and X 4 Is an alkanediyl radical (C≤12) Olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups;
Y 5 is a covalent bond, an alkanediyl (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; and is
R 9 Is an alkyl group (C≤8) Or substituted alkyl (C≤8) ；
The linker group has the formula:
wherein:
Y 1 is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted forms of any of these groups; and is
Wherein when the repeating unit comprises a linker group, then the linker group is attached to a degradable diacyl group on the nitrogen atom and the sulfur atom of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next group comprises two degradable diacyl groups attached to the nitrogen atom of the linker group; and wherein n is the number of linker groups present in the repeat unit; and
an end capping group, wherein the end capping group has the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or in which alkanediyl (C≤18) One or more hydrogen atoms on the surface of the substrate have been replaced by-OH-F, -Cl, -Br, -I, -SH, -OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) ；
R 10 Is hydrogen, carboxyl, hydroxyl, or
Aryl radicals (C≤12) Alkylamino group (C≤12) Dialkylamino group (C≤12) N-heterocycloalkyl, N-heterocycloalkyl (C≤12) 、 -C(O)N(R 11 ) -alkanediyl (C≤6) -heterocycloalkyl radical (C≤12) -C (O) -alkylamino (C≤12) -C (O) -dialkylamino (C≤12) -C (O) -N-heterocycloalkyl (C≤12) Wherein:
R 11 is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Wherein the last degradable diacyl group in the chain is attached to a capping group;
n is 0, 1,2, 3, 4,5 or 6;
or a pharmaceutically acceptable salt thereof. In other embodiments, the dendritic polymer has the formula:
nucleus- (repeat unit) n -end capping group (I)
Wherein the core is attached to the repeat unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeat unit, and wherein:
the nucleus being an alkylamine (C≤18) Dialkyl amines (C≤36) Heterocyclic alkanes (C≤12) Or substituted versions of any of these groups; and is
Wherein the repeat unit comprises a degradable diacyl and a linker;
the degradable diacyl has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Y 3 Is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted versions of any of these groups; or a group of the formula:
wherein:
X 3 and X 4 Is alkanediyl (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted forms of any of these groups;
Y 5 is a covalent bond, alkanediyl (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups; and is
R 9 Is an alkyl radical (C≤8) Or substituted alkyl (C≤8) ；
The linker group has the formula:
wherein:
Y 1 is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted forms of any of these groups; and is provided with
Wherein when the repeating unit comprises a linker group, then the linker group is attached to a degradable diacyl group on the nitrogen atom and the sulfur atom of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next group comprises two degradable diacyl groups attached to the nitrogen atom of the linker group; and wherein n is the number of linker groups present in the repeat unit; and
an end capping group, wherein the end capping group has the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or in which alkanediyl (C≤18) One or more hydrogen atoms on the surface of the substrate have been replaced by-OH-F, -Cl, -Br, -I, -SH, -OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) ；
R 10 Is hydrogen, carboxyl, hydroxyl, or
Aryl radicals (C≤12) Alkylamino group (C≤12) Dialkylamino group (C≤12) N-heterocycloalkyl group (C≤12) 、 -C(O)N(R 11 ) -alkanediyl (C≤6) -heterocycloalkyl radical (C≤12) -C (O) -alkylamino (C≤12) -C (O) -dialkylamino (C≤12) -C (O) -N-heterocycloalkyl (C≤12) Wherein:
R 11 is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Wherein the last degradable diacyl group in the chain is attached to a capping group;
n is 0, 1,2, 3, 4,5 or 6;
or a pharmaceutically acceptable salt thereof. In some embodiments, the end capping group is further defined by the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or in which one or more hydrogen atoms have been replaced by-OH-F, -Cl, -Br, -I, -SH, -OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) (ii) a And is
R 10 Is hydrogen.
In other embodiments, the end capping group is further defined by the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) (ii) a And is provided with
R 10 Is hydrogen.
In some embodiments, Y is 4 Is an alkanediyl radical (C4-18) . In other embodiments, the end capping group is further defined by the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or one or more of themThe hydrogen atom has been replaced by-OH, -F, -Cl, -Br, -I, -SH, -OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) ；
R 10 Is alkylamino (C≤12) Dialkylamino group (C≤12) N-heterocycloalkyl group (C≤12) 。
In some embodiments, the end capping group is further defined by the formula:
wherein:
Y 4 is an alkanediyl radical (C≤18) Or in which one or more hydrogen atoms have been replaced by-OH-F, -Cl, -Br, -I, -SH, -OCH 3 、-OCH 2 CH 3 、-SCH 3 or-OC (O) CH 3 Substituted alkanediyl (C≤18) ；
R 10 Is a hydroxyl group.
In some embodiments, the core is further defined by the formula:
wherein:
X 1 is alkylamino (C≤12) Dialkylamino group (C≤12) Heterocycloalkyl group (C≤12) (iii) heteroaryl (C≤12) Or a substituted form thereof;
R 1 is amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (C≤12) Or substituted versions of any of these groups; and is
a is 1,2, 3, 4,5 or 6;
in some embodiments, X 1 Is alkylamino (C≤12) Or substituted alkylamino (C≤12) . In some embodiments, X 1 Is an ethylamino group. In other embodiments, X 1 Is a dialkylamino group (C≤12) Or substituted dialkylamino (C≤12) . In some embodiments, X 1 Is dimethylamino. In other embodiments, X 1 Is heterocycloalkyl (C≤12) Or substituted heterocycloalkyl (C≤12) . In some embodiments, X 1 Is 4-piperidinyl, N-morpholinyl, N-pyrrolidinyl, 2-pyrrolidinyl, N-piperazinyl or N-4-methylpiperazinyl. In other embodiments, X 1 Is heteroaryl (C≤12) Or substituted heteroaryl (C≤12) . In some embodiments, X 1 Is 2-pyridyl or N-imidazolyl. In some embodiments, R 1 Is a hydroxyl group. In other embodiments, R 1 Is an amino group. In other embodiments, R 1 Is alkylamino (C≤12) Or substituted alkylamino (C≤12) . In some embodiments, R 1 Is alkylamino (C≤12) . In some embodiments, R 1 Is methylamino or ethylamino. In some embodiments, a is 1,2, 3, or 4. In some embodiments, a is 2 or 3. In some embodiments, a is 2. In other embodiments, a is 3. In some embodiments, the core is further defined as a compound of the formula:
in some embodiments, the core is further defined as:
in other embodiments, the core is further defined by the formula:
wherein:
X 2 is N (R) 5 ) y ；
R 5 Is hydrogen or alkyl (C≤8) Or substituted alkyl (C≤18) (ii) a And is provided with
y is 0, 1 or 2, provided that the sum of y and z is 3;
R 2 is amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (C≤12) Or substituted versions of any of these groups;
b is 1,2, 3, 4,5 or 6; and is provided with
z is 1,2, 3; provided that the sum of z and y is 3.
In some embodiments, X 2 Is N. In other embodiments, X 2 Is NR 5 Wherein R is 5 Is hydrogen or alkyl (C≤8) . In some embodiments, R 5 Is hydrogen. In other embodiments, R 5 Is methyl. In some embodiments, z is 3. In other embodiments, z is 2. In some embodiments, R 2 Is a hydroxyl group. In other embodiments, R 2 Is an amino group. In other embodiments, R 2 Is alkylamino (C≤12) Or substituted alkylamino (C≤12) . In some embodiments, R 2 Is alkylamino (C≤12) . In some embodiments, R 2 Is a methylamino group. In other embodiments, R 2 Is a dialkylamino group (C≤12) Or substituted dialkylamino (C≤12) . In some embodiments, R 2 Is a dialkylamino group (C≤12) . In some embodiments, R 2 Is dimethylamino. In some embodiments, b is 1,2, 3, or 4. In some embodiments, b is 2 or 3. In some embodiments, b is 2. In other embodiments, b is 3. In some embodiments, the core is further defined as:
in some embodiments, the core is further defined as:
in other embodiments, the core is further defined as:
wherein:
X 3 is-NR 6 -, wherein R 6 Is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) -O-, or alkylamino diyl (C≤8) Alkoxy diyl group (C≤8) An aromatic hydrocarbon diyl group (C≤8) Hetero aromatic diyl (C≤8) Heterocyclic alkanediyl (C≤8) Or substituted forms of any of these groups;
R 3 and R 4 Each independently being amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (C≤12) Or substituted forms of any of these groups; or a group of the formula: - (CH) 2 CH 2 N) e (R c )R d ；
Wherein:
e is 1,2 or 3;
R c and R d Each independently is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
c and d are each independently 1,2, 3, 4,5 or 6.
In some embodiments, X 3 is-O-. In other embodimentsIn, X 3 is-NR 6 -, wherein R 6 Is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) . In some embodiments, X 3 is-NH-or-NCH 3 -. In other embodiments, X 3 Is an alkylamino diyl group (C≤8) Or substituted alkylamino diyl (C≤8) . In some embodiments, X 3 is-NHCH 2 CH 2 NH-or-NHCH 2 CH 2 NHCH 2 CH 2 NH-. In other embodiments, X 3 Is an alkoxy diradical (C≤8) Or substituted alkoxydiyl (C≤8) . In some embodiments, X 3 is-OCH 2 CH 2 O-is added. In other embodiments, X 3 Is an aromatic diyl group (C≤8) Or substituted arenediyl (C≤8) . In some embodiments, X 3 Is a phenyl-diyl group. In other embodiments, X 3 Is a heterocyclic alkanediyl radical (C≤8) Or substituted heterocycloalkane diyl (C≤8) . In some embodiments, X 3 Is N, N' -piperazinediyl.
In some embodiments, R 3 Is an amino group. In other embodiments, R 3 Is a hydroxyl group. In other embodiments, R 3 Is alkylamino (C≤12) Or substituted alkylamino (C≤12) . In some embodiments, R 3 Is alkylamino (C≤12) . In some embodiments, R 3 Is a methylamino group. In other embodiments, R 3 Is a dialkylamino group (C≤12) Or substituted dialkylamino (C≤12) . In some embodiments, R 3 Is a dialkylamino group (C≤12) . In some embodiments, R 3 Is dimethylamino.
In some embodiments, R 4 Is an amino group. In other embodiments, R 4 Is a hydroxyl group. In other embodiments, R 4 Is alkylamino (C≤12) Or substituted alkylamino (C≤12) . In some embodiments, R 4 Is an alkyl groupAmino group (C≤12) . In some embodiments, R 4 Is a methylamino group. In other embodiments, R 4 Is a dialkylamino group (C≤12) Or substituted dialkylamino (C≤12) . In some embodiments, R 4 Is a dialkylamino group (C≤12) . In some embodiments, R 4 Is dimethylamino. In other embodiments, R 4 Is- (CH) 2 CH 2 N) e (R c )R d : wherein: e is 1,2 or 3; and R is c And R d Each independently is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) . In some embodiments, e is 1 or 2. In some embodiments, e is 1. In some embodiments, R c Is hydrogen. In some embodiments, R d Is hydrogen.
In some embodiments, c is 1,2, 3, or 4. In some embodiments, c is 2 or 3. In some embodiments, c is 2. In other embodiments, c is 3. In some embodiments, d is 1,2, 3, or 4. In some embodiments, d is 2 or 3. In some embodiments, d is 2. In other embodiments, d is 3. In some embodiments, the core is further defined as:
in some embodiments, the core is further defined as:
in other embodiments, the core is an alkylamine (C≤18) Dialkyl amines (C≤36) Heterocyclic alkanes (C≤12) Or this isSubstituted forms of any of these groups. In some embodiments, the core is an alkylamine (C≤18) Or substituted alkylamines (C≤18) . In some embodiments, the core is octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine. In other embodiments, the core is a dialkylamine (C≤36) Or substituted dialkylamines (C≤36) . In some embodiments, the core is N-methyl, N-dodecylamine, dioctylamine, or didecylamine. In other embodiments, the nucleus is a heterocycloalkane (C≤12) Or substituted heterocyclic alkanes (C≤12) . In some embodiments, the core is 4-N-methylpiperazinyl. In some embodiments, Y 1 Is an alkanediyl radical (C≤8) Or substituted alkanediyl (C≤8) . In some embodiments, Y is 1 Is alkanediyl (C≤8) . In some embodiments, Y is 1 is-CH 2 CH 2 -. In some embodiments, Y is 3 Is an alkanediyl radical (C≤8) Or substituted alkanediyl (C≤8) . In some embodiments, Y is 3 Is alkanediyl (C≤8) . In some embodiments, Y is 3 is-CH 2 CH 2 -. In other embodiments, Y 3 The method comprises the following steps:
wherein:
X 3 and X 4 Is alkanediyl (C≤12) Olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted versions of any of these groups;
Y 5 is a covalent bond, an alkanediyl (C≤12) Olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted versions of any of these groups.
In some embodiments, X 3 Is an alkanediyl radical (C≤12) Or substituted alkanediyl (C≤12) . In some embodiments, X 3 is-CH 2 CH 2 -. In some embodiments, X 4 Is an alkanediyl radical (C≤12) Or substituted alkanediyl (C≤12) . In some embodiments, X 4 is-CH 2 CH 2 -. In some embodiments, Y 5 Is a covalent bond. In some embodiments, Y is 3 The method comprises the following steps:
wherein:
X 3 and X 4 Is an alkanediyl radical (C≤12) An olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted forms of any of these groups;
Y 5 is a covalent bond, an alkanediyl (C≤12) Olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted versions of any of these groups.
In some embodiments, X 3 Is an alkanediyl radical (C≤12) Or substituted alkanediyl (C≤12) . In some embodiments, X 3 is-CH 2 CH 2 -. In some embodiments, X 4 Is an alkanediyl radical (C≤12) Or substituted alkanediyl (C≤12) . In some embodiments, X 4 is-CH 2 CH 2 -. In some embodiments, Y is 5 Is a covalent bond. In some embodiments, Y is 5 is-CH 2 -or-C (CH) 3 ) 2 -. In some embodiments, a is 1 Is a-O-. In other embodiments, A 1 is-NR a -. In some embodiments, R a Is hydrogen. In some embodiments, a is 2 is-O-. In other embodiments, A 2 is-NR a -. In some embodiments, R a Is hydrogen. In some embodiments, R 9 Is an alkyl group (C≤8) . In some embodiments, R 9 Is methyl. In some embodiments, n is 0, 1,2, 3, or 4. In some embodiments, n is 0, 1,2, or 3. In some embodiments, n is 0. In other embodiments, n is 1. In other embodiments, n is 2. In other embodiments, n is 3.
In another aspect, the present disclosure provides a composition comprising:
(a) A dendritic polymer as described herein; and
(b) A nucleic acid.
In some embodiments, the nucleic acid is a short interfering RNA (e.g., a small interfering RNA) (siRNA), a microrna (miRNA), a pri-miRNA, a messenger RNA (mRNA), a regularly clustered spacer short palindromic repeats (CRISPR) -associated nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), a plasmid DNA (pDNA), a transport RNA (tRNA), an antisense oligonucleotide (ASO), a guide RNA, a double-stranded DNA (dsDNA), a single-stranded DNA (ssDNA), a single-stranded RNA (ssRNA), and a double-stranded RNA (dsRNA). In some embodiments, the nucleic acid is an siRNA, tRNA, or nucleic acid that can be used in a CRISPR process. The nucleic acid may be an siRNA. In other embodiments, nucleic acids useful in CRISPR processes are such as regularly clustered short palindromic repeats (CRISPR) -associated nucleic acids, single guide RNAs (sgrnas), CRISPR-RNAs (crrnas), or trans-activating crrnas (tracrrnas). In some embodiments, the nucleic acid is an siRNA against factor VII comprising the sequence:
5'-GGAucAucucAAGucuuAc [ dT ] [ dT ] -3' (SEQ ID NO: 1); or
3′-GuAAGAcuuGAGAuGAucc[dT][dT]-5′(SEQ ID NO:2)。
In other embodiments, the nucleic acid is a miRNA. In other embodiments, the nucleic acid is mRNA. In other embodiments, the nucleic acid is a tRNA. In other embodiments, the nucleic acid is a guide RNA. In some embodiments, the guide RNA is used in a CRISPR process. In other embodiments, the nucleic acid is pDNA.
In some embodiments, the dendrimer and the nucleic acid are present in a weight ratio of about 100 to about 1:5. In some embodiments, the weight ratio of dendrimer to nucleic acid is about 50 to about 2:1. In some embodiments, the weight ratio of dendrimer to nucleic acid is 25. In other embodiments, the weight ratio of dendrimer to nucleic acid is 7:1. In some embodiments, the composition further comprises one or more helper lipids. In some embodiments, the helper lipid is selected from a steroid, a steroid derivative, a PEG lipid, or a phospholipid. In some embodiments, the helper lipid is a steroid or a steroid derivative. In some embodiments, the steroid is cholesterol. In some embodiments, the steroid or steroid derivative is present in a molar ratio of about 10. In some embodiments, the molar ratio of steroid or steroid derivative to dendrimer is from about 1:1 to about 1. In some embodiments, the molar ratio of steroid or steroid derivative to dendrimer is about 38. In some embodiments, the molar ratio of steroid or steroid derivative to dendrimer is about 1:5.
In other embodiments, the helper lipid is a PEG lipid. In some embodiments, the PEG lipid is a pegylated diacylglycerol, such as a compound of the formula:
wherein:
R 12 and R 13 Each independently is an alkyl group (C≤24) Alkenyl radical (C≤24) Or substituted versions of any of these groups;
R e is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) (ii) a And is provided with
x is 1 to 250.
In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or a compound of the formula:
wherein:
n 1 is 5 to 250; and is
n 2 And n 3 Each independently 2-25.
In some embodiments, the PEG lipid and the dendrimer are present in a molar ratio of about 1:1 to about 1. In some embodiments, the molar ratio of PEG lipid to dendrimer is from about 1. In some embodiments, the molar ratio of PEG lipid to dendrimer is from about 1.
In other embodiments, the helper lipid is a phospholipid. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In other embodiments, the phospholipid is 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE). In some embodiments, the phospholipid and the dendrimer are present in a molar ratio of about 10. In some embodiments, the molar ratio of phospholipid to dendrimer is about 1:1 to about 1. In some embodiments, the molar ratio of phospholipid to dendrimer is about 4:5. In some embodiments, the molar ratio of phospholipid to dendrimer is about 1:5. In some embodiments, the composition consists essentially of the dendrimer, the nucleic acid, and the one or more helper lipids.
In another aspect, the present disclosure provides a pharmaceutical composition comprising:
(a) A composition or dendrimer as described herein; and
(b) A pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutically acceptable carrier is a solvent or solution. In some embodiments, the pharmaceutical composition is formulated for administration by: oral, intralipidic, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical cord, intravaginal, intravenous, intracapsular, intravitreal, liposomal, topical, transmucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, buccal, transdermal, vaginal, in the form of a paste, in the form of a lipid composition, by catheter, by lavage, by continuous infusion, by inhalation, by injection, by local delivery or by local infusion. In some embodiments, the pharmaceutical composition is formulated for intravenous or intra-arterial injection. In some embodiments, the pharmaceutical composition is formulated as a unit dose.
In another aspect, the present disclosure provides a method of modulating gene expression comprising delivering a nucleic acid to a cell, the method comprising contacting the cell with a composition or pharmaceutical composition described herein under conditions sufficient to cause uptake of the nucleic acid into the cell. In some embodiments, the cells are contacted in vitro. In other embodiments, the cells are contacted in vivo. In other embodiments, the cells are contacted ex vivo. In some embodiments, modulation of gene expression is sufficient to treat a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is liver cancer. In some embodiments, the disease or disorder is hepatocellular carcinoma.
In another aspect, the present disclosure provides a method of treating a disease or disorder in a patient comprising administering to a patient in need thereof a pharmaceutically effective amount of a composition or pharmaceutical composition described herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is liver cancer. In some embodiments, the disease or disorder is hepatocellular carcinoma. In some embodiments, the method further comprises administering to the patient one or more additional cancer therapies. In some embodiments, the cancer therapy is a chemotherapeutic compound, surgery, radiation therapy, or immunotherapy. In some embodiments, the composition or pharmaceutical composition is administered to the patient once. In other embodiments, the composition or pharmaceutical composition is administered to the patient two or more times. In some embodiments, the patient is a mammal, e.g., a human.
In another aspect, the present disclosure provides a dendritic polymer of the formula:
nucleus- (repeat unit) n -end capping groups (I)
Wherein the core is attached to the repeat unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeat unit, and wherein:
the core has the formula:
wherein:
X 3 is-NR 6 -, wherein R 6 Is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) -O-or alkylamino diyl group (C≤8) Alkoxy diyl group (C≤8) Aromatic diyl group (C≤8) Hetero arene diyl (C≤8) Heterocyclic alkene diyl (C≤8) Or substituted versions of any of these groups;
R 3 and R 4 Each independently being amino, hydroxy, or mercapto, or alkylamino (C≤12) Dialkylamino group (C≤12) Or substituted forms of any of these groups;
c and d are each independently 1,2, 3, 4,5 or 6; or
Wherein the repeat unit comprises a degradable diacyl and a linker;
the degradable diacyl has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ；
Y 3 Is alkanediyl (C≤12) An olefin diyl group (C≤12) Aromatic diyl group (C≤12) Or substituted versions of any of these groups; and is
R 9 Is an alkyl group (C≤8) Or substituted alkyl (C≤8) ；
The linker group has the formula:
wherein:
Y 1 is an alkanediyl radical (C≤12) Olefin diyl group (C≤12) An aromatic hydrocarbon diyl group (C≤12) Or substituted forms of any of these groups; and is
Wherein when the repeating unit comprises a linker group, then the linker group is attached to a degradable diacyl group on the nitrogen atom and the sulfur atom of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next group comprises two degradable diacyl groups attached to the nitrogen atom of the linker group; and wherein n is the number of linker groups present in the repeat unit; and is provided with
The end capping group has the formula:
wherein:
Y 4 is alkanediyl (C≤18) (ii) a And is
R 10 Is hydrogen;
wherein the last degradable diacyl group in the chain is attached to a capping group;
n is 0, 1,2, 3, 4,5 or 6;
or a pharmaceutically acceptable salt thereof.
The terms "comprising" (and any form of comprising), "having" (and any form of having), "containing" (and any form of containing), and "including" (and any form of including) are open-ended linking verbs. Thus, a method, composition, kit, or system that "comprises," "has," "contains," or "includes" one or more of the listed steps or elements has those listed steps or elements, but is not limited to having only those steps or elements; it may have (i.e., encompass) elements or steps not recited. Likewise, an element of a method, composition, kit, or system that "comprises," "has," "contains," or "includes" one or more of the enumerated features possesses those features, but is not limited to possessing only those features; it may have features not listed.
Any embodiment of any of the present methods, compositions, kits, and systems may consist of or consist essentially of the described steps and/or features, but does not comprise/include/contain/have the described steps and/or features. Thus, in any claim, the term "consisting of … …" or "consisting essentially of … …" may be substituted for any open-link verb listed above, such that the scope of a given claim is altered from its scope in which it would have been used an open-link verb.
The use of the term "or" in the claims is intended to mean "and/or" unless explicitly indicated to refer only to alternatives or alternatives are mutually exclusive, but the present disclosure supports the definition of referring only to alternatives and "and/or".
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It should be noted that merely because a particular compound belongs to one particular formula does not mean that it cannot also belong to another formula.
Drawings
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figures 1A-1D show that small RNA therapy in susceptible liver cancer requires a combination of high efficacy on tumor cells and low toxicity on normal cells. The modular strategy for diversifying the chemical functionality and size of biocompatible ester-based dendrimers allows for the discovery of dendrimers that balance low toxicity and high in vivo small RNA delivery efficacy. Orthogonal reactions accelerate the synthesis of 1,512G 1 modular degradable dendrimers, thereby increasing the number, size and chemical diversity of molecular structures. The inclusion of degradable ester linkages in each step promotes low toxicity. (FIG. 1A) Small RNAs are too large and anionic to enter cells themselves. To effectively utilize the RNAi machinery, the delivery vector must accompany the small RNAs through numerous extracellular and intracellular barriers. A modular design is envisaged that will enable fine tuning of the identity and location of the functional groups in the dendrimer structure. (FIG. 1B) the library was constructed by sequential orthogonal reactions. First, amines with a series of Initial Branching Centers (IBCs) react quantitatively and selectively with the less sterically hindered acrylate group of AEMA containing two degradable ester groups. The product then undergoes DMPP catalyzed reactions with various mercaptans. (FIG. 1C) to identify degradable dendrimers with optimized topology to mediate small RNAs to overcome a variety of extracellular and intracellular delivery barriers, the library was divided into four regions: nuclear binding-peripheral stabilization (zone I), nuclear binding-peripheral binding (zone II), nuclear stabilization-peripheral stabilization (zone III), and nuclear stabilization-peripheral binding (zone IV). (FIG. 1D) dendrimers were independently tuned for core and periphery with chemically diverse amines and thiols. The selected amines are divided into two classes: the ionizable amine for RNA binding that will yield 1 to 6 branched species is labeled 1A-6A, while the hydrophobic amine for NP stabilization is labeled 1H-2H. These amines are expected to increase the effectiveness of higher generation dendrimers. Hydrophobic alkyl amines used for NP stabilization were labeled SC1-SC19 based on hydrocarbon length. Alcohol and carboxylic acid-terminated thiols (SO 1-SO 9) and amine-functionalized thiols (SN 1-SN 11) were included in the library design to increase chemical diversity. Generation amplification reactions were also used to synthesize G2-G4 higher generation dendrimers with multiple branches (see fig. 10B and fig. 11).
FIG. 2 shows the high aza Michael addition selectivity of tris (2-aminoethyl) amine 6A3 to 2- (acryloyloxy) ethyl methacrylate (AEMA) in the presence of 5 mole% Butylated Hydroxytoluene (BHT) at 50 ℃. Without the addition of tris (2-aminoethyl) amine, only AEMA had not reacted after 24 hours and its conversion was 0%. After addition of tris (2-aminoethyl) amine, the conversion of AEMA was about 82% after 2 hours and about 98% after 24 hours, yielding the first generation dendrimers 6A3-G1 with 6 branches. It should be noted that addition of excess EAMA (6X 0.05 equivalents) is intended to pass through EAMA 1 H NMR was followed to easily monitor the reaction. If the EAMA conversion is complete, there should still be 5% H a And H b A signal.
FIG. 3 shows the high aza Michael addition selectivity of long alkyl chain tetradecylamine 2H4 with 2- (acryloyloxy) ethyl methacrylate (AEMA) in the presence of 5 mole% Butylated Hydroxytoluene (BHT) at 50 ℃. After 24 hours only AEMA had not reacted and its conversion was 0%. After addition of tetradecylamine, the conversion of AEMA was about 97% after 24 hours, yielding the first generation dendrimers 2H4-G1 with long alkyl chain cores. It should be noted that addition of excess EAMA (2X 0.05 equivalents) is intended to pass through EAMA 1 H NMR was followed to easily monitor the reaction. If the EAMA conversion is complete, there should still be 5% H a And H b A signal.
FIG. 4 shows the ThiaeMichael addition of 6A3-G1 (125 mM) to 2- (butylamino) ethanethiol (6X 1.2 equivalents) or 1-tetradecanethiol (6X 1.2 equivalents) in 400. Mu.L DMSO-D6 at 60 ℃ for 48 hours. 6A3-G1 was kept unchanged in DMSO-D6 for 48 hours at 60 ℃ without addition of a thiol compound. With the addition (5 mol%) of Dimethylphenylphosphine (DMPP) as catalyst, 6A3-G1 was reacted with 1-tetradecanethiol at 100% conversion in DMSO-D6 at 60 ℃ within 48 hours, whereas the conversion of 6A3-G1 in the absence of DMPP was only 57%. The conversion of 6A3-G1 with 2- (butylamino) ethanethiol with or without DMPP was almost quantitative, probably because the amine groups in 2- (butylamino) ethanethiol could act as catalyst. It should be noted that an excess of thiol (6X 0.2 equivalents) was added because 6A3-G1 contained (6X 0.05 equivalents) EAMA, which consumed the thiol reactant (6X 0.1 equivalents) with its two double bonds.
FIG. 5 shows the ThiaeMichael addition of 2H4-G1 (125 mM) to 2- (butylamino) ethanethiol (2X 1.2 equivalents) or 1-tetradecanethiol (2X 1.2 equivalents) in DMSO-D6 at 60 ℃ for 48 hours. Without addition of a thiol compound, 2H4-G1 remained unchanged in DMSO-D6 for 48 hours at 60 ℃. With the addition (5 mol%) of Dimethylphenylphosphine (DMPP) as catalyst, 2H4-G1 was reacted with 1-tetradecanethiol at 100% conversion in DMSO-D6 at 60 ℃ within 48 hours, whereas the conversion of 2H4-G1 in the absence of DMPP was only 51%. The conversion of 2H4-G1 to 2- (butylamino) ethanethiol with or without DMPP was quantitative, probably because the amine groups in 2- (butylamino) ethanethiol could act as catalyst. It should be noted that an excess of mercaptan (2X 0.2 equivalents) was added because 2H4-G1 contained (2X 0.05 equivalents) EAMA, which consumed the mercaptan reactant (2X 0.1 equivalents) at its two double bonds.
FIGS. 6A and 6B show that a library of 1,512 first generation degradable dendrimers was efficiently established. (FIG. 6A) different amines C with various Initial Branching Centers (IBC) were reacted with 2- (acryloxy) ethyl methacrylate (AEMA) at 50 ℃ in the presence of 5 mole% Butylated Hydroxytoluene (BHT) at exactly 1:1 feed equivalents for 24 hours. Conversion of all 42 reactions was based on 1 H NMR is almost quantitative. (FIG. 6B) each of the 42C-L-G1 species was reacted with each of the 36 thiols (P) in 66. Mu.L DMSO at 5% DMPP on a small scale (average about 20 mg). The thiol concentration is 750mM, and 1An&1Hn、2An&The concentrations of 2Hn, 3An, 4An, 5An and 6An were 750mM, 275mM, 250mM, 187.5mM, 150mM and 125mM, respectively. All 42C-L-G1 were stable at 60 ℃ for 48 hours without any addition of thiol compounds. Each reaction of all 42 species with SC4, SN8 and SO9 had almost quantitative conversion (by 1 H NMR measurement).
FIGS. 7A-7C show that in vitro siRNA delivery screening of 1,512G 1 DDs can find dendrimers that can overcome intracellular barriers and establish structure-activity relationships (FIG. 7A). (FIG. 7B) heatmap of luciferase silencing in HeLa-Luc cells after treatment with dendrimer nanoparticles (33nM siLuc, n = 3) illustrates the regional activity relationship. Luciferase activity and cell viability were measured to identify dendrimers that balanced high delivery potency with low toxicity (see additional data in figure 8). (fig. 7C) analysis of nanoparticle populations capable of greater than 50% silencing identified dendrimers with optimized topologies to overcome intracellular delivery barriers. According to a series of criteria, if the hit rate of a subregion is higher than the hit rate of its parent, the subregion is further analyzed. The hit rate for the mother is marked in orange, while the higher or lower hit rates for its subgroup are marked in green or blue, respectively. About 6% of the entire library was able to achieve >50% gene silencing. Nuclear binding-peripheral stabilization I-region has a hit rate of 10%. Within region I, the subregion with the SC branch accounts for 15%, while the subregion with the SO branch has only a 1% hit rate. In the sub-region with SC branches, dendrimers with three to six branches, either the SC5-8 branch or the SC9-12 branch, have a much higher chance of efficiently mediating siRNA delivery.
Fig. 8 shows cell viability after addition of 1,512 first generation degradable dendrimer (G1 DD) NPs containing siLuc (33 nM siRNA, average value of n = 3). G1DD was formulated as nanoparticles containing firefly luciferase targeting siRNA (siLuc) at a weight ratio of 12.5 (G1 DD: siRNA) and helper lipid cholesterol, 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and lipid PEG2000 at a molar ratio of 50. Cell viability was measured using the ONE-Glo + Tox luciferase reporter gene and cell viability assay (Promega) according to its protocol. Cell viability was obtained by normalization against untreated cells. Untreated control (n = 6). Experimental sample (n = 3).
Fig. 9A and 9B show intracellular siRNA delivery activity of 1,512 first generation degradable dendrimers (G1 DD). G1DD was formulated as nanoparticles containing firefly luciferase-targeting siRNA (siLuc) at a weight ratio of 12.5 (G1 DD: siRNA) and helper lipid cholesterol at a molar ratio of 50. (FIG. 9A) the heat map of the decrease in luciferase activity in HeLa cells stably expressing firefly luciferase after treatment of G1DD nanoparticles with 33nM siRNA was divided into regions and areas to describe the breakdown of the dendron assay process (see FIG. 9B in part). Cell viability and luciferase activity were measured using the ONE-Glo + Tox luciferase reporter gene and cell viability assay (Promega) according to its protocol. Luciferase reduction was obtained by normalizing luciferase activity relative to luciferase activity and viability of untreated cells. Untreated control (n = 6). Experimental sample (n = 3). (fig. 9B) dendrimer inspired tree analysis procedure was used to identify degradable dendrimers with optimized structures to mediate siRNA to overcome intracellular delivery barriers by analyzing their hit rate with more than 50% reduction in luciferase activity. According to a series of criteria, a subregion is further analyzed if its hit rate is higher than its parent. The hit rate for the mother is a black bar graph, while the higher or lower hit rate for its subgroup is a blue or red font. About 6% of the entire library induced >50% gene silencing. The nuclear binding-peripheral stabilization zone (zone I) had a hit rate of 10%. In region I, the subregion with the SC branch has 15%, while the subregion with the SO branch has 1%. In the sub-region with SC branches, dendrimers with 3, 4,5 or 6 branches, i.e., SC5-8 branches or SC9-12 branches, have a much higher chance of efficiently mediating siRNA to overcome intracellular delivery barriers.
Fig. 10A-10C show a systemic in vivo siRNA delivery screen that further identifies dendrimers that can also overcome the extracellular barrier. The analysis provides SAR to design additional dendrimers with predicted activity. (fig. 10A) factor VII knockdown (n = 3) in mice was evaluated for 26 first generation degradable dendrimers with diverse structures at a siRNA dose of 1 mg/kg. PBS control (n = 3). Data are shown as mean ± s.d. (FIG. 10B) rational design of degradable dendrimers with multiple branches was achieved by (I) selection of polyamines with multiple IBCs and (II) addition of branches by generation amplification. Natural polyamines spermidine 5A5 and spermine 6A4 were used. 1A2 (one IBC), 2A2 and 2A11 (two IBCs), 3A3 and 3A5 (three IBCs) and 4A1 and 4A3 (four IBCs) were selected to synthesize degradable dendrimers with multiple branches by surrogate amplification (see also FIG. 11). (fig. 10C) factor VII knockdown (n = 3) in mice was evaluated for 24 degradable dendrimers rationally designed by strategies I and II at a siRNA dose of 1 mg/kg. PBS control (n = 3). Data are shown as mean ± s.d. Rationally designed dendrimers are active at high hit rates.
Fig. 11 shows synthetic routes to make 2A2 and 2a11 (two IBCs), 3A3 and 3A5 (three IBCs) and 4A1 and 4A3 (four IBCs) of degradable dendrimers with multiple branches by a generational amplification strategy.
Fig. 12A-12E show in vivo toxicity assessments of some degradable dendrimers (> 95% in vivo FVII knockdown), which further identify dendrimers that can balance high delivery efficacy with low toxicity. Some degradable dendrimer NPs had similar size (fig. 12A) and net surface charge (fig. 12B) after binding to control siRNA (siCTR) (nanoparticles are depicted from left to right in the figure, based on the legend to fig. 12B). The C12-200 lipid class LNPs provide a challenging comparison, as they represent the best example of a non-hydrolysable system with similar in vivo efficacy. (fig. 12C) wild type mice (p 26) were injected intravenously with some NP (100 mg dendrimer/kg or 28mg control C12-200/kg) at 4mg siCTR/kg (n = 3). Body weight changes varied between different formulations depending on the identity of the dendrimer, but all NPs were essentially non-toxic in normal WT mice. (fig. 12D) body weight change of transgenic mice (p 32) carrying aggressive MYC-driven tumors after injection of 3mg siCTR/kg (75 mg/kg 5A2-SC8 and 6A3-SC12 or 21mg/kg C12-200) (n = 5). (fig. 12E) Kaplan-Meier survival curves (n = 5) of transgenic mice injected with 5A2-SC8 and 6A3-SC12 nanoparticles at 3mg siCTR/kg doses (75 mg dendrimer/kg) on days 32, 36, 40 and 44. In tumor-bearing mice (susceptible hosts), the toxicity of the vector is amplified and only 5A2-SC8 can be well tolerated and does not affect survival. Data are shown as mean ± s.d. Statistical analysis was performed using (e) Mantel-Cox test; n.s.p >0.05; p <0.05.
Fig. 13A and 13B show the selection of an aggressive transgenic MYC-driven liver tumor model to evaluate the toxicity and efficacy of modular degradable dendrimer delivered mirnas for inhibition of tumor growth (Nguyen et al, 2014). (FIG. 13A) schematically illustrates LAP-tTa; TRE-MYC transgenic mouse model. When a LAP-tTA transgene is present, the TRE-MYC is turned on or off by the liver-specific LAP promoter in the absence or presence of doxycycline (Dox). (FIG. 13B) without any treatment, liver tumors were visible at approximately p20-26, then at p32 the liver was full of small tumors, and finally tumors grew and liver size increased dramatically at p42 to p 60.
FIGS. 14A-14C show that fluorescence imaging confirmed siRNA delivery to tumor cells within the liver. (FIG. 14A) gross anatomy and fluorescence imaging of transgenic mice bearing aggressive liver tumors at 41 days of age. Fluorescence imaging showed that 5A2-SC8 nanoparticles formulated with Cy5.5-labeled siRNA mediated large amounts of siRNA accumulation throughout the cancerous liver, with small amounts in the spleen and kidney after 24 hours of intravenous injection at 1mg Cy5.5-siRNA/kg. To further confirm whether 5A2-SC8 NPs can deliver siRNA into tumor cells in vivo, tumor tissues of the liver were collected, embedded in OTC and sections were H & E stained and confocal imaged 24 hours after intravenous injection. (FIG. 14B) H & E staining confirmed that the liver contained tumors. The same tumor tissue sections were scanned using confocal imaging and captured under three channels: DAPI for nuclei (blue), FITC for phalloidin-stained actin (green), and cy5.5 for siRNA (red). (FIG. 14C) confocal imaging of the same area showed that 5A2-SC8 could efficiently deliver siRNA into tumor cells inside the liver.
FIGS. 15A and 15B show the biodistribution of 5A2-SC8NP formulated with Cy5.5-labeled siRNA in normal wild type mice and mice bearing liver tumors (FIG. 15A) and H & E staining images of liver from tumor-bearing mice (FIG. 15B). 5A2-SC8NP mediated accumulation of Cy5.5-labeled siRNA in the whole liver of normal and hepatoma bearing mice 24 hours after intravenous injection of 1mg siRNA/kg. H & E staining images show that the liver of tumor-bearing mice is full of tumors and the slides used for confocal imaging contain tumor cells. It should be noted that the size of the liver increases as the tumor grows (see the isometric box in fig. 15A).
Fig. 16A-16H show that modular degradable dendrimers can deliver therapeutic Let-7g miRNA mimics to clinically relevant and aggressive MYC-driven genetic tumor models, resulting in significant survival benefits. 5A2-SC8NP silenced FVII proteins in transgenic mice bearing MYC-driven liver tumors as measured in blood (FIG. 16A) and in harvested liver tissue (FIG. 16B) (single injection, 1mg/kg, p26 mice, 48 hours post-injection) (siCTR on left and siFVII on right). (FIG. 16C) 5A2-SC8NP enabled delivery of Let-7g into liver tissue of transgenic mice carrying MYC-driven liver tumors (single injection, 1mg/kg, p26 mice, 48 hours post-injection). Let-7g expression was significantly increased, while other Let-7 family members were unaffected (siCTR on the left and siFVII on the right). (FIG. 16D) transgenic mice bearing MYC-driven liver tumors were given 1mg/kg Let-7g intravenous injections once a week, starting on day 26 (after tumor development started) until day 61. Mice receiving Let-7g had significantly smaller abdomens. (FIG. 16E) the abdominal circumference of the treated mice was smaller compared to the control group. (FIG. 16F) representative images of livers from Let-7g mock and control mock-injected mice show reduced tumor burden. (fig. 16G) once weekly delivery of miRNA mimics within 5A2-SC8 NPs did not affect normal weight gain, whereas delivery of miRNA mimics within C12-200 LNPs resulted in weight loss and death. n =5. (FIG. 16H) delivery of Let-7g once a week from day 26 to day 61 prolonged survival. All control mice that received no treatment (n = 9) and mice that received 5A2-SC8 NPs containing control untargeted mimics (n = 5) died around 60 days after birth. Mice injected with C12-200LNP died prematurely (n = 7). The # C12-200+ CTR mimic experiment was stopped because all mice receiving C12-200+ let-7g mimic injection had died (n = 7). Delivery of Let-7g within 5A2-SC8 NPs provided significant survival benefits. Data are shown as mean ± s.d. Performing statistical analysis by using (a, b, c, e) two-tail Student's t test or (h) Mantel-Cox test; n.s.P >0.05; * P <0.05; * P <0.01; * P <0.001; * P <0.0001.
FIGS. 17A-17C show delivery of siLuc using dendrimer nanoparticles formulated with different combinations of cholesterol, phospholipids, and PEG lipids in HeLa-Luc (FIG. 17A), A549-Luc (FIG. 17B), and MDA-MB231-Luc (FIG. 17C).
FIGS. 18A and 18B show (FIG. 18A) a comparison of different composition formulations with DSPC lipids versus DOPE lipids versus PEG-DMG when delivering siLuc to HeLa-Luc. FIG. 18B shows a comparison of different composition formulations with DSPC lipids versus DOPE lipids versus PEG-DHD when delivering siLuc to HeLa-Luc.
Fig. 19 shows delivery of sgrnas using nanoparticle compositions containing dendrimers or Z120 with and without the presence of phospholipid DSPC in the nanoparticle formulation.
Fig. 20A and 20B show the encapsulation percentage of sgrnas (fig. 20A) and delivery in HeLa-Luc-Cas9 cells (fig. 20B).
Fig. 21A and 21B show viability of IGROV cells that have delivered Luc mRNA after 24 hours incubation (fig. 21A) and 48 hours incubation (fig. 21B).
Figure 22 shows a fluorescence micrograph of cells treated with mCherry mRNA showing mRNA delivery to the cells.
Detailed Description
In some aspects, the present disclosure provides lipophilic cationic dendrimers useful as nucleic acid carriers. In some embodiments, the dendritic polymer contains one or more groups that degrade under physiological conditions. In some embodiments, the dendrimer is formulated as a composition comprising the dendrimer and one or more nucleic acids. These compositions may further comprise one or more helper lipids such as cholesterol and/or phospholipids. Finally, in some aspects, the disclosure also provides methods of using the dendrimer compositions to treat one or more diseases treatable with a nucleic acid therapeutic.
A. Chemical definition
When used in the context of chemical groups: "Hydrogen" means-H; "hydroxy" means-OH; "oxo" means = O; "carbonyl" means-C (= O) -; "carboxy" means-C (= O) OH (also written as-COOH or-CO) 2 H) (ii) a "halo" independently means-F, -Cl, -Br, or-I; "amino" means-NH 2 (ii) a "hydroxyamino" refers to-NHOH; "nitro" means-NO 2 (ii) a Imino means = NH; "cyano" means-CN; "isocyanate group" means-N = C = O; "azido" refers to-N 3 (ii) a "phosphate group" in the monovalent context means-OP (O) (OH) 2 Or a deprotonated form thereof; "phosphate group" in the divalent context means-OP (O) (OH) O-or its deprotonated form; "mercapto" means-SH; and "thio" means = S; "sulfonyl" means-S (O) 2 -; "Hydroxysulfonyl" means-S (O) 2 OH; "sulfonamide" means-S (O) 2 NH 2 (ii) a And "sulfinyl" refers to-S (O) -.
In the case of chemical formulae, the symbol "-" means a single bond, "=" means a double bond, and "≡" means a triple bond. SymbolRepresents an optional bond, which when present is a single or double bond. Symbol(s)Represents a single bond or a double bond. Thus, for example, formulaIncludedAnd it is understood that no such ring atom forms part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when linking one or two stereo atoms. Rather, it encompasses all stereoisomers as well as mixtures thereof. SymbolWhen passing through the bond perpendicularly (e.g. methyl)) The point of attachment of the group is indicated when plotted. It should be noted that the point of attachment is typically determined only for the larger group in this manner to assist the reader in unambiguously determining the point of attachment. SymbolRefers to a single bond in which the group attached to the wedge-shaped butt is "out of the page". SymbolRefers to a single bond in which the group attached to the wedge-shaped butt is "in the page". Symbol(s)Refers to single bonds in which the geometry (e.g., E or Z) surrounding the double bond is undefined. Two options and combinations thereof are therefore contemplated. Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to the atom. Black dots on carbon atoms indicate that the hydrogen attached to the carbon is oriented out of the paper.
When the group "R" is described as a "floating group" on a ring system, for example, in the formula:
r may replace any hydrogen atom attached to any ring atom, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When the group "R" is depicted as a "floating group" on a fused ring system, as for example in the formula:
then R can replace any hydrogen attached to any ring atom of any fused ring unless otherwise indicated. Alternative hydrogens include the depicted hydrogen (e.g., a hydrogen attached to a nitrogen in the above formula), implied hydrogens (e.g., a hydrogen not shown in the above formula but understood to be present), explicitly defined hydrogens, and optionally hydrogens, the presence of which depends on the identity of the ring atom (e.g., a hydrogen attached to group X when X is equal to-CH-), so long as a stable structure is formed. In the depicted examples, R may be located on a 5-or 6-membered ring of the fused ring system. In the above formula, the subscript letter "y" immediately following the group "R" enclosed in parentheses represents a numerical variable. Unless otherwise specified, the variable may be 0, 1,2, or any integer greater than 2, limited only by the maximum number of substitutable atoms in the ring or ring system.
For chemical groups and classes of compounds, the number of carbon atoms in the group or class is as follows: "Cn" defines the exact number of carbon atoms (n) in a group/class. "C.ltoreq.n" defines the exact number of possible carbon atoms (n) in a group/class, with the minimum number being as small as the possible number of groups/classes in question, e.g., it is understood that the group "alkenyl (C≤8) "or class" olefins (C≤8) The minimum number of carbon atoms in "is 2. Compared with "alkoxy group (C≤10) ", which indicates an alkoxy group having 1 to 10 carbon atoms. "Cn-n '" defines the minimum (n) and maximum number (n') of carbon atoms in a group. Thus, an "alkyl group (C2-10) "indicates those alkyl groups having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class it modifies, and may or may not beAre included in parentheses and do not imply any change in meaning. Thus, the terms "C5 olefin", "C5-olefin", "olefin (C5) And olefins C5 "is synonymous.
The term "saturated" when used to modify a compound or chemical group means that the compound or chemical group does not have a carbon-carbon double bond and does not have a carbon-carbon triple bond, unless indicated below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted forms of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. And when such a bond is present, carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, this means that no more of the substance can be dissolved in the solution.
The term "aliphatic" when used without the modifier "substituted" means that the compound or chemical group so modified is an acyclic or cyclic but non-aromatic hydrocarbon compound or group. In aliphatic compounds/groups, the carbon atoms may be linked together in straight chain, branched chain or non-aromatic rings (alicyclic rings). Aliphatic compounds/groups may be saturated, i.e. linked by single carbon-carbon bonds (alkane/alkyl), or unsaturated, i.e. having one or more double carbon-carbon bonds (alkene/alkenyl) or having one or more triple carbon-carbon bonds (alkyne/alkynyl).
The term "aromatic" when used to modify a compound or chemical group atom means that the compound or chemical group contains a planar unsaturated ring of atoms stabilized by interaction with bonds forming the ring.
The term "alkyl" when used without the modifier "substituted" refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, having a straight or branched acyclic structure, and no atoms other than carbon and hydrogen. group-CH 3 (Me)、-CH 2 CH 3 (Et)、-CH 2 CH 2 CH 3 (n-Pr or propyl), -CH (CH) 3 ) 2 (i-Pr、 i Pr or isopropyl), -CH 2 CH 2 CH 2 CH 3 (n-Bu)、 -CH(CH 3 )CH 2 CH 3 (sec-butyl), -CH 2 CH(CH 3 ) 2 (isobutyl), -C (CH) 3 ) 3 (tert-butyl, t-Bu or t Bu) and-CH 2 C(CH 3 ) 3 (neopentyl) is a non-limiting example of an alkyl group. The term "alkanediyl" when used without the modifier "substituted" refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as the point of attachment, having a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. group-CH 2 - (methylene), -CH 2 CH 2 -、-CH 2 C(CH 3 ) 2 CH 2 -and-CH 2 CH 2 CH 2 -is a non-limiting example of an alkanediyl group. "alkane" refers to a class of compounds having the formula H-R, where R is alkyl, the term being as defined above. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms having been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、 -CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、 -C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、 -S(O) 2 OH or-S (O) 2 NH 2 Instead of this. The following groups are non-limiting examples of substituted alkyls: -CH 2 OH、-CH 2 Cl、-CF 3 、-CH 2 CN、-CH 2 C(O)OH、-CH 2 C(O)OCH 3 、 -CH 2 C(O)NH 2 、-CH 2 C(O)CH 3 、-CH 2 OCH 3 、-CH 2 OC(O)CH 3 、-CH 2 NH 2 、 -CH 2 N(CH 3 ) 2 and-CH 2 CH 2 And (4) Cl. The term "haloalkyl" is a subset of substituted alkyl wherein hydrogen atom substitution is not limited to halo (i.e., halo)-F, -Cl, -Br or-I) so that no other atoms than carbon, hydrogen and halogen are present. group-CH 2 Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyls, wherein hydrogen atom substitution is not limited to fluoro, and thus no other atoms than carbon, hydrogen, and fluorine are present. group-CH 2 F、-CF 3 and-CH 2 CF 3 Are non-limiting examples of fluoroalkyl groups.
The term "cycloalkyl" when used without the modifier "substituted" refers to a monovalent saturated aliphatic group having as the point of attachment a carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH (CH) 2 ) 2 (cyclopropyl), cyclobutyl, cyclopentyl or cyclohexyl (Cy). The term "cycloalkanediyl," when used without the modifier "substituted," refers to a divalent saturated aliphatic group having two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Radical (I)Are non-limiting examples of cycloalkanediyl groups. "Cycloalkane" refers to a class of compounds having the formula H-R, wherein R is cycloalkyl, the term being as defined above. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms having been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、-CN、-SH、 -OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、 -C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead.
The term "alkenyl" when used without the modifier "substituted" means having a carbon atom as the substituentA linkage point, a monovalent unsaturated aliphatic group having a straight or branched acyclic structure, having at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH = CH 2 (vinyl), -CH = CHCH 3 、-CH＝CHCH 2 CH 3 、-CH 2 CH＝CH 2 (allyl), -CH 2 CH＝CHCH 3 and-CH = CHCH = CH 2 . The term "alkenediyl" when used without the modifier "substituted" refers to a divalent unsaturated aliphatic group having two carbon atoms as points of attachment, having a straight or branched chain, a straight or branched chain acyclic structure, having at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The radicals-CH = CH-, -CH = C (CH) 3 )CH 2 -、-CH＝CHCH 2 -and-CH 2 CH＝CHCH 2 -is a non-limiting example of an alkene diradical. It should be noted that while the alkene diradical is aliphatic, once attached at both ends, it is not excluded that the group forms part of an aromatic structure. The terms "alkene" and "alkene" are synonymous and refer to a class of compounds having the formula H-R, wherein R is alkenyl, the terms being as defined above. Similarly, the terms "terminal olefin" and "alpha-olefin" are synonymous and refer to an olefin having only one carbon-carbon double bond, where the bond is part of a vinyl group at the end of the molecule. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms having been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、-CN、 -SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、 -C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、 -S(O) 2 OH or-S (O) 2 NH 2 Instead. The groups-CH = CHF, -CH = CHCl and-CH = CHBr are non-limiting examples of substituted alkenyl groups.
The term "alkynyl" when used without the modifier "substituted" refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, having a straight or branched chain acyclic structure, having at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The group-C.ident.CH, -C.ident.CCH 3 and-CH 2 C≡CCH 3 Are non-limiting examples of alkynyl groups. "alkyne" refers to a class of compounds having the formula H-R, wherein R is an alkynyl group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms having been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、 -CO 2 H、-CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、 -NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、 -NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead.
The term "aryl," when used without the modifier "substituted," refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment and the carbon atom forms part of one or more six-membered aromatic ring structures, wherein all of the ring atoms are carbon, and wherein the group does not consist of atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (carbon number limitations allow) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl) phenyl, -C 6 H 4 CH 2 CH 3 (ethylphenyl), naphthyl, and monovalent radicals derived from biphenyl. The term "arenediyl" when used without the modifier "substituted" means having two aromatic carbon atoms as the point of attachment and the carbon atoms form a single unitA divalent aromatic group that is part of one or more six-membered aromatic ring structures, wherein all of the ring atoms are carbon, and wherein the monovalent group is not composed of atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl, aryl or aralkyl groups (carbon number limitations allow) attached to the first aromatic ring or any additional aromatic rings present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected by one or more of the following: covalent bonds, alkanediyl or alkenediyl groups (carbon number restrictions allow). Non-limiting examples of arene diyl groups include:
"arene" refers to a class of compounds having the formula H-R, where R is aryl, which term is defined herein. Benzene and toluene are non-limiting examples of aromatic hydrocarbons. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms having been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、 -C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、 -C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead.
The term "aralkyl" when used without the modifier "substituted" refers to a monovalent group-alkanediyl-aryl, wherein the terms alkanediyl and aryl are each used in a manner consistent with the definition provided above. Non-limiting examples are: phenylmethyl (benzyl, bn) and 2-phenyl-ethyl. When the term aralkyl is used in conjunction with the modifier "substituted", one or more hydrogen atoms from the alkanediyl and/or aryl group have been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、 -CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、 -N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、 -NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead. Non-limiting examples of substituted aralkyl groups are: (3-chlorophenyl) -methyl and 2-chloro-2-phenyl-eth-1-yl.
The term "heteroaryl," when used without the modifier "substituted," refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment and the carbon or nitrogen atom forms part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the heteroaryl does not consist of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. Heteroaryl rings may contain 1,2, 3 or 4 ring atoms selected from nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl, aryl and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridyl, oxazolyl, phenylpyridyl, pyridyl (pyridinyl/pyridinyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as the point of attachment. The term "heteroarenediyl," when used without the modifier "substituted," refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the divalent group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected by one or more of the following: covalent bonds, alkanediyl or alkenediyl groups (carbon number restrictions allow). As used herein, the term does not preclude the presence of one or more alkyl, aryl and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroarenediyl groups include:
"heteroarenes" refers to a class of compounds having the formula H-R, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the modifier "substituted", one or more hydrogen atoms have been independently replaced by-OH-F, -Cl, -Br, -I, -NH 2 、-NO 2 、 -CO 2 H、-CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、 -NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、 -NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead.
The term "heterocycloalkyl" when used without the modifier "substituted" refers to a monovalent non-aromatic group having as a point of attachment a carbon or nitrogen atom that forms part of one or more non-aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the heterocycloalkyl is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. The heterocycloalkyl ring may contain 1,2, 3 or 4 ring atoms selected from nitrogen, oxygen or sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitations allow) attached to the ring or ring system. Furthermore, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkane diyl," when used without the modifier "substituted," refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two points of attachment, the atoms forming part of one or more ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the divalent group is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected by one or more of the following: covalent bonds, alkanediyl or alkenediyl groups (carbon number restrictions allow). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitations allow) attached to the ring or ring system. Furthermore, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkane diyl include:
when these terms are used with the modifier "substituted", one or more hydrogen atoms have been independently replaced by-OH-F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、-CN、 -SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、 -C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、 -S(O) 2 OH or-S (O) 2 NH 2 Instead.
The term "acyl" when used without the modifier "substituted" refers to the group-C (O) R, wherein R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, as these terms are defined above. The group-CHO, -C (O) CH 3 (acetyl, ac), -C (O) CH 2 CH 3 、 -C(O)CH 2 CH 2 CH 3 、-C(O)CH(CH 3 ) 2 、-C(O)CH(CH 2 ) 2 、-C(O)C 6 H 5 、 -C(O)C 6 H 4 CH 3 、-C(O)CH 2 C 6 H 5 and-C (O) (imidazolyl) is a non-limiting example of an acyl group. "thioacyl" is defined in a similar manner, except that the oxygen atom of the group-C (O) R has been replaced by a sulfur atom, i.e., -C (S) R. The term "aldehyde" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced by a-CHO group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms (including hydrogen atoms which may be present directly attached to a carbon atom of a carbonyl or thiocarbonyl group) have been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、 -CO 2 H、-CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、 -NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、 -NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead. The radical-C (O) CH 2 CF 3 、-CO 2 H (carboxyl), -CO 2 CH 3 (methyl carboxyl), -CO 2 CH 2 CH 3 、-C(O)NH 2 (carbamoyl) and-CON (CH) 3 ) 2 Are non-limiting examples of substituted acyl groups.
The term "alkoxy" when used without the modifier "substituted" refers to the group-OR, wherein R is alkyl, as that term is defined above. Non-limiting examples include: -OCH 3 (methoxy), -OCH 2 CH 3 (ethoxy), -OCH 2 CH 2 CH 3 、-OCH(CH 3 ) 2 (isopropoxy), -OC (CH) 3 ) 3 (tert-butoxy), -OCH (CH) 2 ) 2 -O-cyclopentyl and-O-cyclohexyl. The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy", and "acyloxy", when used without the modifier "substituted", refer to a group defined as-OR, wherein R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent radicals-O-alkanediyl-, -O-alkanediyl-O-or-alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio" when used without the modifier "substituted" refer to the group-SR, wherein R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced by a hydroxyl group. The term "ether" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced by an alkoxy group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms having been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、 -NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、 -OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead.
The term "alkylamino" when used without the modifier "substituted" refers to the group-NHR, wherein R is alkyl, as that term is defined above. Is notLimiting examples include: -NHCH 3 and-NHCH 2 CH 3 . The term "dialkylamino," when used without the modifier "substituted," refers to the group-NRR ', where R and R ' can be the same or different alkyl groups, or R and R ' can be taken together to represent an alkanediyl group. Non-limiting examples of dialkylamino groups include: -N (CH) 3 ) 2 and-N (CH) 3 )(CH 2 CH 3 ). The terms "cycloalkylamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", "alkoxyamino", and "alkylsulfonylamino", when used without the modifier "substituted", refer to groups defined as — NHR, wherein R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is-NHC 6 H 5 . The term "alkylaminodiyl" refers to the divalent radical-NH-alkanediyl-, -NH-alkanediyl-NH-or-alkanediyl-NH-alkanediyl-. The term "acylamino" (acylamino), when used without the modifier "substituted", refers to the group-NHR, wherein R is acyl, the term being as defined above. A non-limiting example of an amido group is-NHC (O) CH 3 . The term "alkylimino", when used without the modifier "substituted", means a divalent group = NR, wherein R is alkyl, as that term is defined above. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms attached to a carbon atom have been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、 -C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、 -C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Instead. The group-NHC (O) OCH 3 and-NHC (O))NHCH 3 Are non-limiting examples of substituted amido groups.
The use of the words "a" or "an" when used in the claims and/or the specification with the term "comprising" may mean "one", but it is also intended to conform to the meaning of "one or more", "at least one", and "one or more than one".
Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the device, method used to determine the value, or the variation that exists between study objects.
As used herein, the term "average molecular weight" refers to the relationship between the number of moles of each polymeric species and the molar mass of that species. In particular, each polymer molecule may have a different polymerization level and thus a different molar mass. Average molecular weight may be used to represent the molecular weight of a plurality of polymer molecules. Average molecular weight is generally synonymous with average molar mass. Specifically, there are three main types of average molecular weights: number average molar mass, weight (mass) average molar mass and Z-average molar mass. In the context of the present application, the average molecular weight represents the number-average molar mass or the weight-average molar mass of the formula, unless stated otherwise. In some embodiments, the average molecular weight is a number average molar mass. In some embodiments, the average molecular weight can be used to describe the PEG component present in the lipid.
The terms "comprising," "having," and "including" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprising", "having", "including", and "including", is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps, and also encompasses other unlisted steps.
The term "effective" when used in this specification and/or claims means sufficient to achieve a desired, expected, or expected result. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," when used in the context of treating a patient or subject with a compound, refers to the amount of the compound that, when administered to the subject or patient to treat a disease, is sufficient to effect treatment of such disease.
As used herein, the term "IC 50 By "is meant an inhibitory amount that achieves 50% of the maximal response. Such quantitative measurements indicate how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e., enzyme, cell receptor or microorganism) by half.
An "isomer" of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but in which the three-dimensional configuration of these atoms is different.
As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, adolescents, infants and fetuses.
As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
By "pharmaceutically acceptable salt" is meant a salt of a compound of the invention which is pharmaceutically acceptable as defined above and which has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthylsulfonic acid, 3-phenylpropionic acid, 4,4' -methylenebis (3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo [2.2.2] oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono-and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o- (4-hydroxybenzoyl) benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butyl acetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts, which may be formed when an acidic proton present is capable of reacting with an inorganic or organic base. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It should be recognized that the particular anion or cation that forms part of any salt of the invention is not critical, so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and methods of making and using the same are presented in the handbook of pharmaceutically acceptable salts: properties and uses (Handbook of Pharmaceutical Salts: properties, and Use) (eds. P.H.Stahl and C.G.Wermuth, verlag Helvetica Chimica Acta, 2002).
As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting a chemical agent, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
"preventing" includes: (1) Inhibiting the onset of the disease in a subject or patient who may be at risk for and/or susceptible to the disease but does not yet experience or exhibit any or all of the pathologies or symptoms of the disease, and/or (2) slowing the onset of the pathologies or symptoms of the disease in a subject or patient who may be at risk for and/or susceptible to the disease but does not yet experience or exhibit any or all of the pathologies or symptoms of the disease.
The "repeating units" being of a particular materialThe simplest structural entities, such as the backbone and/or the polymer, whether organic, inorganic or organometallic. In the case of polymer chains, the repeating units are linked together in sequence along the chain, just like a bead of a necklace. For example, in polyethylene- [ -CH 2 CH 2 -] n In-is the repeating unit-CH 2 CH 2 -. The subscript "n" indicates the degree of polymerization, i.e., the number of repeat units linked together. When the value of "n" is undefined or where "n" is absent, it simply indicates the repetition of this formula within parentheses as well as the polymeric nature of the material. The concept of repeating units is equally applicable where the connectivity between repeating units extends in three dimensions, for example in metal organic frameworks, modified polymers, thermosetting polymers, etc. In the case of dendrimers, the repeat units may also be described as branching units, inner layers or generations. Similarly, end capping groups may also be described as surface groups.
"stereoisomers" or "optical isomers" are isomers of a given compound in which the same atom is bonded to the same other atom, but in which the three-dimensional configuration of the atoms is different. "enantiomers" are stereoisomers of a given compound that are mirror images of each other (e.g., left and right handed). "diastereoisomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also known as a stereocenter or stereocenter, which is any point in the molecule that bears a group, but not necessarily an atom, such that exchange of any two groups results in a stereoisomer. In organic compounds, the chiral center is usually a carbon, phosphorus or sulfur atom, but other atoms may also be stereocenters in organic and inorganic compounds. A molecule may have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomers are attributed to tetrahedral stereocenters (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2 n Where n is the number of tetrahedral stereocenters. Molecules with symmetry often have less than the maximum number of stereoisomers possible. A 50. Alternatively, antipodeMixtures of isomers may be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Generally, enantiomers and/or diastereomers may be resolved or separated using techniques known in the art. It is contemplated that for any stereocenter or chiral axis for which stereochemistry has not been defined, the stereocenter or chiral axis may exist in its R form, S form, or as a mixture of R and S forms (including racemic and non-racemic mixtures). As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains 15% or less, more preferably 10% or less, even more preferably 5% or most preferably 1% or less of another stereoisomer.
"treating" includes (1) inhibiting the disease (e.g., arresting the further development of the pathology and/or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease, (2) ameliorating the disease (e.g., reversing the pathology and/or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease, and/or (3) effecting any measurable reduction in the disease in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease.
The above definitions override any conflicting definitions in any reference incorporated by reference herein. However, the fact that certain terms are defined should not be taken as indicating that any undefined terms are undefined. Rather, all terms used are to be interpreted as describing the invention in a manner that would enable one of ordinary skill in the art to understand the scope and practice the invention.
B. Dendritic polymer and dendritic structure
In some aspects of the present disclosure, dendrimers containing lipophilic and cationic components are provided. Dendrimers are polymers that exhibit regular dendritic branches, formed by the addition of branching layers into or from the core, either sequentially or in generations, and are characterized by one core, at least one internal branching layer, and one surface branching layer. (see Petar R.Dvornic and Donald A.Tomalia in chem. UK, 641-645, 8.1994.) in other embodiments, the term "dendrimer" as used herein is intended to include, but is not limited to, a molecular structure having an inner core, an inner layer (or "generation") of repeating units regularly linked to the starting core, and an outer surface linked to the terminal groups of the outermost generation. A "dendrimer" is a dendrimer species having branches emanating from a focal point that is attached or can be attached to a core, either directly or through a linking moiety, to form a larger dendrimer. In some embodiments, the dendritic polymer structure has repeating groups radiating from a central core that are doubled with each repeating unit for each branch. In some embodiments, the dendrimers described herein may be described as small molecules, mesosized molecules, lipids, or lipid-like substances. These terms may be used to describe compounds having a dendrimer appearance (e.g., molecules radiating from a single focal point) as described herein.
While dendrimers are polymers, they are superior to traditional polymers in that they have a controlled structure, a single molecular weight, numerous and controllable surface functional groups, and traditionally adopt a globular conformation after reaching a certain number of generations. Dendrimers can be prepared by the sequential reaction of each repeating unit to produce monodisperse, dendritic and/or generational polymer structures. A single dendrimer consists of one central core molecule with dendritic wedges attached to one or more functional sites on the central core. Depending on the assembly monomers used in the preparation process, the dendritic polymer surface layer may have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups.
The physical properties of the core, repeat units, and surface or end capping groups can be adjusted by changing their functional and/or chemical properties. Some properties that may be altered include, but are not limited to, solubility, toxicity, immunogenicity, and bioadhesive capacity. Dendrimers are often described by their algebraic number or number of repeating units in the branch. Dendrimers consisting of only core molecules are referred to as generations 0, while each successive repeat unit along all branches is a generation 1,2, etc. up to an end-cap or surface group. In some embodiments, the half-generations may result solely from a first condensation reaction with an amine, rather than a second condensation reaction with a thiol.
The preparation of dendrimers requires a level of synthetic control achieved through a series of stepwise reactions, including the establishment of the dendrimer through each successive group. Dendrimer syntheses may be convergent or divergent. During the synthesis of a divergent dendrimer, molecules assemble from the core to the periphery in a stepwise process, including linking one generation to the previous, and then changing the functional groups for the next stage of reaction. Functional group conversion is necessary to prevent uncontrolled polymerization. Such polymerization reactions will result in highly branched molecules that are not monodisperse and are otherwise referred to as hyperbranched polymers. Due to steric effects, dendrimer repeat units continue to react to produce spherical or globular molecules until steric crowding prevents complete reaction for a particular generation and disrupts the monodispersity of the molecules. Thus, in some embodiments, dendritic polymers of G1-G10 generations are specifically contemplated. In some embodiments, the dendritic polymer comprises 1,2, 3, 4,5, 6,7, 8,9, or 10 repeating units, or any range derivable therein. In some embodiments, the dendritic polymer used herein is G0, G1, G2 or G3. However, the number of possible generations (e.g., 11, 12, 13, 14, 15, 20, or 25) can be increased by decreasing the spacer units in the branched polymer.
Furthermore, dendrimers have two main chemical environments: the environment created by the capping of specific surface groups, and the interior of the dendritic structure that may shield the bulk medium and surface groups due to higher order structures. Due to these different chemical environments, dendrimers have found many different potential uses, including in therapeutic applications.
In some aspects, the dendritic polymers of the present disclosure are assembled using the differential reactivity of acrylate and methacrylate groups with amines and thiols. Dendrimers that may be used herein include secondary or tertiary amines and thioethers formed from the reaction of acrylate groups with primary or secondary amines and methacrylates with mercapto groups. Furthermore, the repeating units of the dendrimers described herein may contain groups that are degradable under physiological conditions. In some casesIn embodiments, these repeating units may contain one or more germinal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows dendrimer to occur in only one direction. In other embodiments, the core molecule is a polyamine having a plurality of different dendritic branches, each of which may comprise one or more repeat units. The dendrimer may be formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are located on heteroatoms, such as nitrogen atoms. In some embodiments, the end capping group is a lipophilic group such as a long chain alkyl or alkenyl group. In other embodiments, the end capping group is a long chain haloalkyl or haloalkenyl. In other embodiments, the end capping group is one that contains an ionizable group such as an amine (-NH) 2 ) Or carboxylic acids (-CO) 2 H) An aliphatic or aromatic group of (a). In other embodiments, the end capping group is an aliphatic or aromatic group that contains one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.
The dendritic polymers provided by the present disclosure are shown, for example, in the summary above and in the claims below. They can be prepared using the methods outlined in the examples section. These methods can be further modified and optimized using organic chemistry principles and techniques applied by those skilled in the art. For example, higher organic chemistry such as Ma Ji: such principles and techniques are taught in March's Advanced Organic Chemistry: reactions, mechanics, and Structure (2007), which is incorporated herein by reference.
The dendrimers of the present disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic forms, epimeric forms, and all geometric isomeric forms of a formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Dendrimers can exist in the form of racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the dendrimers of the present disclosure may have S or R configurations. Further, it is contemplated that one or more dendrimers may be present as constituent isomers. In some embodiments, the compounds have the same chemical formula but differ in connectivity to the nitrogen atom of the core. Without wishing to be bound by any theory, it is believed that this dendritic polymer is present because the starting monomer reacts first with the primary amine and then statistically with any secondary amines present. Thus, the constituent isomers may present a fully reacted primary amine followed by a mixture of reacted secondary amines.
The chemical formula used to represent the dendrimers of the present disclosure will generally show only one of several different tautomers that are possible. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which tautomer is the most prevalent, all tautomers of a given formula are contemplated.
The dendrimers of the present disclosure may also have the advantage of: they may be more potent, less toxic, longer acting, more potent, produce fewer side effects, be more easily absorbed, and/or have better pharmacokinetic profiles (e.g., higher oral bioavailability and/or lower clearance rates), and/or have other useful pharmacological, physical, or chemical properties than compounds known in the art, whether or not used for the indications or otherwise described herein.
In addition, the atoms comprising the dendrimers of the present disclosure are intended to include all isotopic forms of these atoms. As used herein, isotopes include those atoms having the same atomic number but different mass numbers. As a general example, and not by way of limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C。
it should be recognized that the particular anion or cation forming part of any salt form of the dendrimers provided herein is not critical, so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and methods of making and using the same are presented in the handbook of pharmaceutically acceptable salts: properties and uses (2002), which are incorporated herein by reference.
C. Helper lipids
In some aspects of the disclosure, one or more helper lipids are mixed with the polymers of the disclosure to produce a composition. In some embodiments, the polymer is mixed with 1,2, 3, 4, or 5 different types of helper lipids. It is contemplated that the polymer may be mixed with a single type of a plurality of different lipids. In some embodiments, the lipid may be a steroid or a steroid derivative. In other embodiments, the lipid is a PEG lipid. In other embodiments, the lipid is a phospholipid. In other embodiments, the dendrimer composition comprises a steroid or steroid derivative, a PEG lipid, and a phospholipid.
1. Steroid alcohols and steroid derivatives
In some aspects of the disclosure, the polymer is mixed with one or more steroids or steroid derivatives to produce a dendrimer composition. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17 carbocyclic structure, which may further comprise one or more substitutions including alkyl, alkoxy, hydroxy, oxo, acyl, or a double bond between two or more carbon atoms. In one aspect, the ring structure of the steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the formula:
in some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as:
in some embodiments of the disclosure, the steroid or steroid derivative is a cholestane or a cholestane derivative. In cholestanes, the ring structure is further defined by the formula:
as mentioned above, cholestane derivatives include non-alkyl substitution of one or more of the above ring systems. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol derivative. In other embodiments, the cholestane or cholestane derivatives cholestenes and sterols or derivatives thereof.
In some embodiments, the composition may further comprise a molar ratio of steroid to dendrimer of from about 1. In some embodiments, the molar ratio is from about 1. In some embodiments, the molar ratio is about 38.
PEG or PEGylated lipids
In some aspects of the disclosure, a polymer is mixed with one or more pegylated lipids (or PEG lipids) to produce a dendrimer composition. In some embodiments, the present disclosure includes the use of any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride, which also comprises a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long chain alkyl or alkenyl groups or C6-C24 fatty acid groups attached to a linker group with a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-conjugated ceramides, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropyl-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, the PEG-modified distearoylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The PEG-modified molecular weight is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present invention are taught in us patent 5,820,873, WO 2010/141069, or us patent 8,450,298, which are incorporated herein by reference.
In another aspect, the PEG lipid has the formula:
wherein: r 12 And R 13 Each independently is an alkyl group (C≤24) Alkenyl radical (C≤24) Or substituted versions of any of these groups; r e Is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) (ii) a And x is 1-250. In some embodiments, R e Is an alkyl group (C≤8) Such as methyl. R 12 And R 13 Each independently is an alkyl group (C≤4-20) . In some embodiments, x is 5 to 250. In one embodiment, x is from 5 to 125 or x is from 100 to 250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.
In another aspect, the PEG lipid has the formula:
wherein: n is 1 Is an integer from 1 to 100 and n 2 And n 3 Each independently selected from an integer of 1 to 29. In some embodiments, n is 1 Is 5,10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. In some embodiments, n is 1 Is about 30 to about 50. In some embodiments, n is 2 Is 5 to 23. In some embodiments, n is 2 Is 11 to about 17. In some embodiments, n is 3 Is 5 to 23. In some embodiments, n is 3 Is 11 to about 17.
In some embodiments, the composition may further comprise a molar ratio of PEG lipid to dendrimer of about 1:1 to about 1. In some embodiments, the molar ratio is about 1:1, 1. In some embodiments, the molar ratio is about 1.
3. Phospholipids
In some aspects of the disclosure, a polymer is mixed with one or more phospholipids to produce a dendrimer composition. In some embodiments, any lipid that also comprises a phosphate group. In some embodiments, the phospholipid is a structure containing one or two long chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and optionally a small organic molecule. In some embodiments, the small organic molecule is an amino acid, sugar, or amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.
In some embodiments, the composition may further comprise a phospholipid to dendrimer molar ratio of about 1. In some embodiments, the molar ratio is from about 1. In some embodiments, the molar ratio is about 38.
D. Nucleic acid and nucleic acid-based therapeutics
1. Nucleic acids
In some aspects of the disclosure, the dendrimer composition comprises one or more nucleic acids. In some embodiments, the dendrimer composition comprises one or more nucleic acids present in a weight ratio to dendrimer of about 5:1 to about 1. In some embodiments, the weight ratio of nucleic acid to dendrimer is about 5:1, 2.5, 1:1, 1:5, 1, 10, 1. In some embodiments, the weight ratio is about 1,25 or about 1:7. In addition, it should be clear that the present disclosure is not limited to the specific nucleic acids disclosed herein. The scope of the invention is not limited to any particular source, sequence, or type of nucleic acid, however, as one of ordinary skill in the art can readily identify related homologs in nucleic acids of various other sources, including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat, and other species). It is contemplated that nucleic acids used in the present disclosure may comprise sequences based on naturally occurring sequences. In view of the degeneracy of the genetic code, sequences having at least about 50%, typically at least about 60%, more typically about 70%, most typically about 80%, preferably at least about 90% and most preferably about 95% of the nucleotides are identical to the nucleotide sequence of a naturally occurring sequence. In another embodiment, the nucleic acid is a sequence that is complementary to a naturally occurring sequence, or 75%, 80%, 85%, 90%, 95%, and 100% complementary.
In some aspects, a nucleic acid is a sequence that silences, complements, or replaces another sequence present in vivo. A sequence 17 bases in length should appear only once in the human genome and thus be sufficient to specify a unique target sequence. Although shorter oligomers are easier to prepare and increase in vivo accessibility, many other factors are involved in determining the specificity of hybridization. The binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides having 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are also contemplated. Also contemplated are longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer
The nucleic acid used herein may be derived from genomic DNA, i.e. cloned directly from the genome of a particular organism. However, in a preferred embodiment, the nucleic acid will comprise complementary DNA (cDNA). Also contemplated are cDNA plus a native intron or an intron from another gene; such engineered molecules are sometimes referred to as "mini-genes". At a minimum, these and other nucleic acids of the invention can be used as molecular weight standards, for example, in gel electrophoresis.
The term "cDNA" is intended to mean DNA prepared using messenger RNA (mRNA) as a template. The advantage of using cDNA, in comparison with genomic DNA or DNA polymerized from genomic, non-processed or partially processed RNA templates, is that the cDNA predominantly contains the coding sequence for the corresponding protein. It may sometimes be preferable for all or part of the genomic sequence, for example when a non-coding region is required for optimal expression, or when a non-coding region such as an intron is to be targeted in an antisense strategy.
In some embodiments, the nucleic acid comprises one or more antisense segments that inhibit the expression of a gene or gene product. Antisense approaches exploit the fact that nucleic acids tend to pair with "complementary" sequences. By complementary is meant that the polynucleotide is one that is capable of base pairing according to the standard Watson-Crick complementarity rules. That is, larger purines will base pair with smaller pyrimidines, forming a combination of guanine (G: C) paired with cytosine (G: C) and adenine (A: T) paired with thymine (G: C) in the case of DNA, or adenine (A: U) paired with uracil (G: U) in the case of RNA. The inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. in the hybridizing sequence does not interfere with pairing.
Targeting double-stranded (ds) DNA with a polynucleotide results in triple helix formation; targeting RNA will result in duplex formation. An antisense polynucleotide specifically binds its target polynucleotide when introduced into a target cell and interferes with transcription, RNA processing, transport, translation, and/or stability. Antisense RNA constructs or DNA encoding such antisense RNA can be used to inhibit gene transcription or translation or both in host cells, in vitro or in vivo, e.g., in host animals, including human subjects.
Antisense constructs can be designed to bind to the promoter and other control regions of the gene, exons, introns, or even exon-intron boundaries. It is expected that the most effective antisense construct will include a region complementary to the intron/exon splice junction. Thus, it is proposed that preferred embodiments include antisense constructs having complementarity to a region within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting its target selectivity. The amount of exon material included will vary depending on the particular exon and intron sequences used. One can easily test whether too much exon DNA is included, simply by testing the construct in vitro to determine whether normal cell function is affected or whether expression of the relevant gene with complementary sequence is affected.
As described above, "complementary" or "antisense" refers to a polynucleotide sequence that is substantially complementary over its entire length and has very few base mismatches. For example, sequences 15 bases in length may be said to be complementary when they have complementary nucleotides at 13 or 14 positions. Naturally, a perfectly complementary sequence will be one that is perfectly complementary over its entire length and has no base mismatches. Other sequences with lower homology are also contemplated. For example, antisense constructs can be designed that have a limited region of high homology but also contain non-homologous regions (e.g., ribozymes; see below). These molecules, although having less than 50% homology, bind to the target sequence under appropriate conditions.
It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to form sirnas or to generate specific constructs. For example, when an intron is required for the final construct, it will be necessary to use a genomic clone. The cDNA, siRNA or synthetic polynucleotide may provide a more convenient restriction site for the remainder of the construct and may therefore be used for the remainder of the sequence. Other embodiments include dsRNA or ssRNA that can be used to target genomic sequences or coding/non-coding transcripts.
In other embodiments, the dendrimer composition may comprise a nucleic acid comprising one or more expression vectors, which is used for gene therapy. Expression requires the provision of appropriate signals in the vector and includes various regulatory elements, such as enhancers/promoters from viral and mammalian sources that drive expression of the gene of interest in the host cell. Elements designed to optimize messenger RNA stability and translatability in host cells are also defined. Also provided are conditions for establishing permanently stable cell clones expressing the product using a variety of advantageous drug selection markers, and elements linking expression of the drug selection marker to expression of the polypeptide.
Throughout this application, the term "expression construct" is intended to include any type of genetic construct containing a nucleic acid encoding a gene product, wherein some or all of the nucleic acid coding sequence is capable of being transcribed. Transcripts can be translated into protein, but need not be. In certain embodiments, expression comprises transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression includes transcription of only the nucleic acid encoding the gene of interest.
The term "vector" is used to refer to a vector nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell that can be replicated. The nucleic acid sequence may be "exogenous," meaning that it is foreign to the cell into which the vector is introduced, or the sequence is homologous to a sequence in the cell, but in a position within the host cell nucleic acid in which it is not normally present. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One skilled in the art will be able to construct vectors by standard recombinant techniques, described in Sambrook et al (1989) and Ausubel et al (1994), both of which are incorporated herein by reference.
The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product capable of being transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example in the production of antisense molecules or ribozymes. Expression vectors may contain a variety of "control sequences," which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well, and are described below.
2.siRNA
As noted above, the present invention contemplates the use of one or more inhibitory nucleic acids to reduce the expression and/or activation of a gene or gene product. Examples of inhibitory nucleic acids include, but are not limited to, molecules that target nucleic acid sequences, such as siRNA (small interfering RNA), short hairpin RNA (shRNA), double stranded RNA, antisense oligonucleotides, ribozymes, and molecules that target genes or gene products such as aptamers.
Inhibitory nucleic acids can inhibit transcription of a gene or prevent translation of a gene transcript in a cell. Inhibitory nucleic acids may be 16 to 1000 nucleotides in length, and in certain embodiments may be 18 to 100 nucleotides in length.
Inhibitory nucleic acids are well known in the art. siRNAs, shRNAs, and double stranded RNAs are described, for example, in U.S. Pat. Nos. 6,506,559 and 6,573,099, and U.S. patent publication Nos. 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are incorporated herein by reference in their entirety.
Since Fire and coworkers discovered RNAi in 1998, biochemical mechanisms have been rapidly characterized. Double-stranded RNA (dsRNA) is cleaved by Dicer, which is an RNase III family ribonuclease. This process produces sirnas that are about 21 nucleotides in length. These sirnas are incorporated into a polyprotein RNA-induced silencing complex (RISC), which is directed to a target mRNA. RISC cleaves the target mRNA in the middle of the complementary region. In mammalian cells, the associated micrornas (mirnas) are found as short RNA fragments (about 22 nucleotides). Mirnas are produced following Dicer-mediated cleavage of longer (about 70 nucleotides) precursors with incomplete hairpin RNA structures. The miRNA is incorporated into a miRNA-protein complex (miRNP), which results in translational inhibition of the target mRNA.
In designing a nucleic acid capable of producing an RNAi effect, several factors need to be considered, such as the nature of the siRNA, the persistence of the silencing effect, and the choice of the delivery system. To produce an RNAi effect, siRNA introduced into an organism will typically contain an exon sequence. Furthermore, the RNAi process is homology dependent, and therefore the sequences must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene specific sequences. In particular, the siRNA exhibits greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity between the siRNA sequence and a portion of the EphA nucleotide sequence. Sequences having less than about 80% identity to the target gene are essentially less potent. Thus, the greater the identity between the siRNA and the gene to be inhibited, the less likely the expression of an unrelated gene will be affected.
In addition, the size of the siRNA is an important consideration. In some embodiments, the present disclosure relates to siRNA molecules comprising at least about 19-25 nucleotides and capable of modulating gene expression. In the context of the present disclosure, the siRNA is particularly less than 500, 200, 100, 50, 25 or 20 nucleotides in length. In some embodiments, the siRNA is about 25 nucleotides to about 35 nucleotides or about 19 nucleotides to about 25 nucleotides in length.
To improve the effectiveness of siRNA mediated gene silencing, guidelines for the selection of target sites on mRNA have been developed for the optimal design of sirnas (Soutschek et al, 2004, wadhwa et al, 2004). These strategies may allow rational approaches for selecting siRNA sequences to achieve maximal gene knockdown. To facilitate siRNA entry into cells and tissues, a variety of vectors have been used, including plasmids and viral vectors such as adenovirus, lentivirus and retrovirus (Wadhwa et al, 2004).
Within an inhibitory nucleic acid, the components of the nucleic acid need not be of the same type or be consistently homogeneous (e.g., an inhibitory nucleic acid can comprise nucleotides and nucleic acids or nucleotide analogs). Typically, inhibitory nucleic acids form double-stranded structures; the double stranded structure may be generated from two separate nucleic acids that are partially or fully complementary. In certain embodiments of the invention, an inhibitory nucleic acid may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by being complementary to itself (e.g., forming a hairpin loop). The double-stranded structure of the inhibitory nucleic acid may comprise 16-500 or more consecutive nucleobases, including all ranges derivable therein. The inhibitory nucleic acid may comprise 17 to 35 consecutive nucleobases, more particularly 18 to 30 consecutive nucleobases, more particularly 19 to 25 nucleobases, more particularly 20 to 23 consecutive nucleobases, or 20 to 22 consecutive nucleobases, or 21 consecutive nucleobases, which hybridize to a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double stranded structure.
The siRNA may be obtained from commercial sources, natural sources, or may be synthesized using any of a number of techniques well known to those of ordinary skill in the art. For example, commercial sources of pre-designed siRNA include Stealth from Invitrogen TM Select technology (Carlsbad, CA),(Austin, TX) and(Valencia, CA). Inhibitory nucleic acids useful in the compositions and methods of the invention can be any nucleic acid sequence that has been found by any source to be an effective down-regulator of a gene or gene product.
In some embodiments, the present invention provides an isolated siRNA molecule having at least 19 nucleotides, which has at least one strand that is substantially complementary to at least ten but not more than thirty consecutive nucleotides of a nucleic acid encoding a gene, and which reduces expression of the gene or gene product. In one embodiment of the present disclosure, the siRNA molecule has at least one strand that is substantially complementary to at least ten but not more than thirty consecutive nucleotides of an mRNA encoding a gene or gene product.
In one embodiment, the siRNA molecule has at least 75%, 80%, 85% or 90% homology, particularly at least 95%, 99% or 100% similarity or identity, or any percentage therebetween, to at least 10 contiguous nucleotides of any nucleic acid sequence encoding a target therapeutic protein (e.g., the invention encompasses greater than 75%, greater than 80%, greater than 85%, etc., and the ranges are intended to include all integers therebetween).
The siRNA may also comprise alterations of one or more nucleotides. Such changes may include the addition of non-nucleotide material, for example, to the end or within (at one or more nucleotides of the RNA) 19 to 25 nucleotides of RNA. In certain aspects, the RNA molecule contains a 3' -hydroxyl group. The nucleotides in the RNA molecules of the invention may also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. Double-stranded oligonucleotides may contain modified backbones, such as phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. Other modifications of siRNA (e.g., incorporation of 2' -O-methyl ribonucleotides, 2' -deoxy-2 ' -fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxybase residues) can be found in U.S. publication 2004/0019001 and U.S. patent 6,673,611 (each of which is incorporated herein by reference in its entirety). In summary, all of these altered nucleic acids or RNAs described above are referred to as modified siRNAs.
In one embodiment, the siRNA is capable of reducing the expression of a particular genetic product by at least 10%, at least 20%, at least 30%, or at least 40%, at least 50%, at least 60%, or at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or more, or any range therebetween.
3.CRISPR/CAS
CRISPR (regularly clustered interspaced short palindromic repeats) is a DNA locus containing short repeats of a sequence of bases. Each repetition is followed by a short segment of "spacer DNA" previously exposed to the virus. CRISPR is present in about 40% of sequenced eubacterial genomes and 90% of sequenced archaea. CRISPR is typically associated with cas genes encoding proteins associated with CRISPR. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of adaptive immunity. CRISPR spacers recognize and silence these exogenous genetic elements, such as RNAi, in eukaryotes.
The repetitive sequence of the bacterium Escherichia coli (Escherichia coli) was first described in 1987. In 2000, similar clustered repeats were identified in other bacteria and archaea, and were referred to as Short Regularly Spaced Repeats (SRSR). SRSR was renamed CRISPR in 2002. A panel of genes (some encoding putative nucleases or helicase proteins) were found associated with CRISPR repeats (cas, or CRISPR-associated genes).
In 2005, three independent researchers demonstrated that CRISPR spacer sequences have homology to several phage DNAs and extrachromosomal DNAs such as plasmids. This suggests that the CRISPR/cas system may play a role in adaptive immunity of bacteria. Koonin and colleagues propose that the spacer sequence serves as a template for RNA molecules, similar to eukaryotic cells using a system known as RNA interference.
In 2007, barrangou, horvath (food industry scientist of Danisco), etc. showed that they could alter the resistance of Streptococcus thermophilus (Streptococcus thermophilus) to phage attack by spacer DNA. Doudna and Charpentier have independently studied CRISPR-associated proteins to understand how bacteria deploy spacer sequences in their immune defenses. They have together studied a simpler CRISPR system that relies on a protein called Cas9. They found that bacteria respond to invading phages by transcribing the spacer sequence and palindromic DNA into long RNA molecules, which cells subsequently cleave into fragments called crRNA using tracrRNA and Cas9.
CRISPR first functioned as a genome engineering/editing tool in human cell culture in 2012. It has been widely used in a variety of organisms including baker's yeast (s. Cerevisiae), zebrafish, nematodes (c. In addition, CRISPRs have been modified to be programmable transcription factors enabling scientists to target and activate or silence specific genes. Libraries of thousands of guide RNAs are now available.
Within 3 months 2014 MIT researchers cured a rare liver disorder in mice, demonstrating the first evidence that CRISPR can reverse disease symptoms in live animals. Since 2012, CRISPR/Cas systems have been used for gene editing (silencing, enhancing or altering specific genes), even functioning in eukaryotes such as mice and primates. By inserting a plasmid containing the cas gene and a specially designed CRISPR, the genome of an organism can be cut at any desired location.
CRISPR repeats range in size from 24 to 48 base pairs. They generally exhibit some binary symmetry, meaning that secondary structures such as hairpins are formed, but not true palindromes. The repeated sequences are separated by spacer sequences of similar length. Some CRISPR spacers match perfectly to sequences from plasmids and phages, but some match to the genome of prokaryotes (self-targeting spacers). New spacer sequences can be added quickly in response to phage infection.
CRISPR-associated (cas) genes are commonly associated with CRISPR repeat spacer arrays. By 2013, over forty different Cas protein families have been described. Among these protein families, cas1 appears to be ubiquitous in different CRISPR/Cas systems. Specific combinations of cas genes and repeat structures have been used to define 8 CRISPR isoforms (Ecoli, ypest, nmeni, dvulg, tnepap, hmari, aperrn, and Mtube), some of which are associated with another gene module encoding a mystery protein (RAMP) associated with a repeat. More than one CRISPR subtype may be present in a single genome. Sporadic distribution of CRISPR/Cas subtypes suggests that this system is affected by horizontal gene transfer during microbial evolution.
The foreign DNA is apparently processed into a small element (about 30 base pairs in length) by the protein encoded by the Cas gene and then somehow inserted into the CRISPR locus near the leader sequence. RNA from the CRISPR locus is constitutively expressed and processed by the Cas protein into small RNAs consisting of a single exogenously derived sequence element with flanking repeats. RNA-guided other Cas proteins silence exogenous genetic elements at the RNA or DNA level. There is evidence for functional diversity between CRISPR isoforms. The Cse (Cas subtype E coli) protein (called CasA-E in E coli) forms the functional complex, cascade, which processes the CRISPR RNA transcript into a Cascade-retaining spacer-repeat unit. In other prokaryotes, cas6 processes CRISPR transcripts. Interestingly, CRISPR-based phage inactivation requires Cascade and Cas3, but not Cas1 and Cas2 in e. Cmr (Cas RAMP module) proteins found in pyrenococcus furiosus and other prokaryotes form functional complexes with small CRISPR RNA, which CRISPR RNA recognizes and cleaves complementary target RNA. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.
See also U.S. patent publication 2014/0068797, which is incorporated by reference in its entirety.
i.Cas9
Cas9 is a nuclease, an enzyme dedicated to cutting DNA, with two active cleavage sites, one for each double-helix strand. It has been demonstrated that one or both sites can be disabled while retaining the homing ability of Cas9 to localize its target DNA. Jinek combines the tracrRNA and the spacer RNA into one "single guide RNA" molecule, which when mixed with Cas9 can find and cleave the correct DNA target. Jinek et al suggested that such synthetic guide RNAs might be useful for gene editing.
Cas9 protein is highly enriched for pathogenic and commensal bacteria. CRISPR/Cas mediated gene regulation may be helpful in regulating endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, cas9 protein of new Francisella novivicida uses a unique small CRISPR/Cas-associated RNA (scaRNA) to suppress endogenous transcripts encoding bacterial lipoproteins critical to new Francisella novivicida (f. Novivicida) to suppress host response and promote virulence.
ii gRNA or sgRNA
As an RNA guide protein, cas9 requires a short RNA to direct the recognition of a DNA target (Mali et al, 2013 a). Although Cas9 preferentially interrogates DNA sequences containing the PAM sequence NGG, it can bind without the original spacer sequence target here. However, the Cas9-gRNA complex needs to be closely matched to the gRNA to generate a double strand break (Cho et al, 2013 hsu et al, 2013. CRISPR sequences in bacteria are expressed in a variety of RNAs and then processed to produce the guide strand of the RNA (Bikard et al, 2013). Because eukaryotic systems lack some of the proteins required for processing CRISPR RNA, synthetic constructs grnas were generated to combine the basic fragments of RNA for Cas9 targeting into a single RNA expressed with RNA polymerase type III promoter U6 (Mali et al, 2013a, b). The synthetic gRNA slightly exceeded 100bp in minimum length and contained a portion targeting 20 protospacer nucleotides immediately preceding the PAM sequence NGG; grnas do not contain PAM sequences.
4. Modified nucleobases
In some embodiments, a nucleic acid of the present disclosure comprises one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar modified nucleosides can have a desired property, such as enhanced nuclease stability or increased binding affinity to a target nucleic acid relative to an oligonucleotide comprising only nucleosides containing naturally-occurring sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar substitute. Such sugar substitutes may comprise one or more substitutions corresponding to the substituted sugar moiety.
In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, including but not limited to substituents at the 2 'and/or 5' positions. Examples of suitable sugar substituents at the 2' position includeBut are not limited to: 2'-F, 2' -OCH 3 ("OMe" or "O-methyl") and 2' -O (CH) 2 ) 2 OCH 3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position is selected from the group consisting of allyl, amino, azido, thio, O-allyl, O- -C 1 -C 10 Alkyl, O- -C 1 -C 10 A substituted alkyl group; OCF 3 、O(CH 2 ) 2 SCH 3 、O(CH 2 ) 2 - - -O- -N (Rm) (Rn) and O- -CH 2 -C (= O) - -N (Rm) (Rn), wherein each Rm and Rn is independently H, or substituted or unsubstituted C 1 -C 10 An alkyl group. Examples of sugar substituents at the 5' position include, but are not limited to: 5' -methyl (R or S); 5 '-vinyl and 5' -methoxy. In some embodiments, the substituted saccharide comprises more than one non-bridging saccharide substituent, such as a T-F-5' -methyl saccharide moiety (see, e.g., PCT international application WO 2008/101157 for additional 5',2' -disubstituted saccharide moieties and nucleosides).
Nucleosides that comprise a2 '-substituted sugar moiety are referred to as 2' -substituted nucleosides. In some embodiments, the 2 '-substituted nucleoside comprises a 2' -substituent selected from the group consisting of: halogen, allyl, amino, azido, SH, CN, OCN, CF 3 、OCF 3 O, S or N (R) m ) -an alkyl group; o, S or N (R) m ) -an alkenyl group; o, S or N (R) m ) -an alkynyl group; O-alkylalkenyl-O-alkyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl, O (CH) 2 ) 2 SCH 3 、O(CH 2 ) 2 --O--N(R m )(R n ) Or O- -CH 2 --C(＝O)--N(R m )(R n ) Wherein each R is m And R n Independently is H, an amino protecting group or C, substituted or unsubstituted 1 -C 10 An alkyl group. These 2' -substituents may be further substituted with one or more substituents independently selected from the group consisting of hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO) 2 ) Thiols, thioalkoxy (S-alkyl), halogens, alkyl, aryl, alkenyl, and alkynyl groups.
In some embodiments, the 2' -substituted nucleoside comprises a member selected from the group consisting of2' -substituent: F. NH (NH) 2 、 N 3 、OCF 3 、O--CH 3 、O(CH 2 ) 3 NH 2 、CH 2 —CH＝CH 2 、O--CH 2 —CH＝CH 2 、 OCH 2 CH 2 OCH 3 、O(CH 2 ) 2 SCH 3 、O--(CH 2 ) 2 --O--N(R m )(R n )、 O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2 And N-substituted acetamides (O- -CH) 2 --C(＝O)--N(R m )(R n ) Wherein each R is m And R n Independently is H, an amino protecting group or C, substituted or unsubstituted 1 -C 10 An alkyl group.
In some embodiments, the 2 '-substituted nucleoside comprises a sugar moiety comprising a 2' -substituent selected from the group consisting of F, OCF 3 、O--CH 3 、OCH 2 CH 2 OCH 3 、O(CH 2 ) 2 SCH 3 、 O(CH 2 ) 2 --O--N(CH 3 ) 2 、--O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2 And O- -CH 2 --C(＝O)--N(H)CH 3 。
In some embodiments, the 2 '-substituted nucleoside comprises a sugar moiety comprising a 2' -substituent selected from the group consisting of F, O-CH 3 And OCH 2 CH 2 OCH 3 。
Certain modified sugar moieties comprise a bridging sugar substituent that forms the second ring resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety comprises a bridge between the 4 'and 2' furanose ring atoms. Examples of such 4 'to 2' sugar substituents include, but are not limited to: - - [ C (R) a )(R b )] n --、 --[C(R a )(R b )] n --O--、--C(R a R b ) - - -N (R) - - -O- -or- -C (R) a R b )--O--N(R)--；4'-CH 2 -2'、 4'-(CH 2 ) 2 -2'、4'-(CH 2 )--O-2'(LNA)；4'-(CH 2 )--S-2'；4'-(CH 2 ) 2 --O-2'(ENA)； 4'-CH(CH 3 ) - - -O-2 '(cEt) and 4' -CH (CH) 2 OCH 3 ) -O-2' and its analogs (see, e.g., us patent 7,399,845); 4' -C (CH) 3 )(CH 3 ) -O-2' and analogues thereof (see e.g. WO 2009/006478); 4' -CH 2 --N(OCH 3 ) 2' and analogues thereof (see e.g. WO 2008/150729); 4' -CH 2 --O--N(CH 3 ) -2' (see e.g. US2004/0171570, published in 2004, 9/2); 4' -CH 2 - - -O- -N (R) -2 'and 4' -CH 2 - -N (R) - - -O-2' - -, wherein each R is independently H, a protecting group, or C 1 -C 12 An alkyl group; 4' -CH 2 - - -N (R) - - -O-2', where R is H, C 1 -C 12 Alkyl or protecting groups (see us patent 7,427,672); 4' -CH 2 --C(H)(CH 3 ) -2' (see, e.g., chattopadhyoya et al, journal of organic chemistry (j.org.chem.), 2009,74,118-134); and 4' -CH 2 --C(＝CH 2 ) -2' and analogues thereof (see PCT international application WO 2008/154401).
In some embodiments, such 4 'to 2' bridges independently comprise 1 to 4 linking groups independently selected from: - - [ C (R) a )(R b )] n --、--C(R a )＝C(R b )--、--C(R a )＝N--、--C(＝NR a )--、 --C(＝O)--、--C(＝S)--、--O--、--Si(R a ) 2 --、--S(＝O) x - - -and- -N (R) a ) - -; wherein:
x is 0, 1 or 2;
n is 1,2, 3 or 4;
each R a And R b Independently H, a protecting group, hydroxy, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl radical, C 2 -C 12 Alkenyl, substituted C 2 -C 12 Alkenyl radical, C 2 -C 12 Alkynyl, substituted C 2 -C 12 Alkynyl, C 5 -C 20 Aryl, substituted C 5 -C 20 Aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C 5 -C 7 Alicyclic radical, substituted C 5 -C 7 Alicyclic groupHalogen, OJ 1 、 NJ 1 J 2 、SJ 1 、N 3 、COOJ 1 Acyl (C (= O) - - -H), substituted acyl, CN, sulfonyl (S (= O) 2 -J 1 ) Or a sulfoxide group (S (= O) -J 1 ) (ii) a And is
Each J 1 And J 2 Independently H, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl radical, C 2 -C 12 Alkenyl, substituted C 2 -C 12 Alkenyl radical, C 2 -C 12 Alkynyl, substituted C 2 -C 12 Alkynyl, C 5 -C 20 Aryl, substituted C 5 -C 20 Aryl, acyl (C (= O) - -H), substituted acyl, heterocyclic group, substituted heterocyclic group, C 1 -C 12 Aminoalkyl, substituted C 1 -C 12 Aminoalkyl groups, or protecting groups.
Nucleosides containing a bicyclic sugar moiety are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to: (A) alpha-L-Methyleneoxy (4' -CH) 2 - - -O-2 ') BNA, (B) beta-D-methyleneoxy (4' -CH) 2 - - -O-2 ') BNA (also known as locked nucleic acid or LNA), (C) ethyleneoxy (4' - (CH) 2 ) 2 - -O-2 ') BNA, (D) aminooxy (4' -CH) 2 - - -O- -N (R) -2 ') BNA, (E) oxyamino (4' -CH) 2 - - -N (R) - - -O-2 ') BNA, (F) methyl (methyleneoxy) (4' -CH (CH) 3 ) - - -O-2 ') BNA (also known as constrained ethyl or cEt), (G) methylene-thio (4' -CH) 2 - - -S-2 ') BNA, (H) methylene-amino (4' -CH2- -N (R) -2 ') BNA, (I) methylcarby-yl (4' -CH 2) 2 --CH(CH 3 ) -2 ') BNA, (J) propylidene carbocyclyl (4' - (CH) 2 ) 3 -2 ') BNA, and (K) methoxy (ethyleneoxy) (4' -CH (CH) 2 OMe) -O-2') BNA (also known as restricted MOE or cMOE).
Other bicyclic sugar moieties are known in the art, for example: singh et al, chemical communication (chem. Commun.), 1998,4,455-456; koshkin et al Tetrahedron (Tetrahedron), 1998, 54,3607-3630; wahlestedt et al, proc. Natl. Acad. Sci. U.S.A.) -I. 2000,97,5633-5638; kumar et al, bioorg.Med.chem.Lett., 1998,8,2219-2222; singh et al, J.Org.chem., 1998,63,10035-10039; srivastava et al, "american journal of chemists (j.am. Chem. Soc.), 129 (26) 8362-8379 (4/7 2007); elayadi et al, nov research drugs (curr. Opinion drugs. Drugs), 2001,2,5561; braarch et al, chemi-biological (chem.biol.), 2001,8,1-7; orum et al, current therapy mol, 2001,3,239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461 and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570 and WO 2007/134181; U.S. patent publication Nos. US2004/0171570, US 2007/0287831, and US 2008/0039618; U.S. Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International application Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.
In some embodiments, the bicyclic sugar moiety and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configurations. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge can be in the α -L configuration or in the β -D configuration. Previously, alpha-L-methyleneoxy (4' -CH) 2 - -O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that exhibit antisense activity (Frieden et al, nucleic Acids Research (2003,21,6365-6372).
In some embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5' -substituted and 4' -2' bridging sugars; PCT International application WO 2007/134181, wherein the LNA is substituted with, for example, a 5' -methyl or 5' -vinyl group).
In some embodiments, the modified sugar moiety is a sugar substitute. In some such embodiments, the oxygen atom of the naturally occurring sugar is substituted with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties further comprise bridging and/or non-bridging substituents as described above. For example, certain sugar substitutes comprise a 4' -sulfur atom and a substitution at the 2' -position (see, e.g., published U.S. patent application US 2005/0130923) and/or the 5' -position. As additional examples, carbocyclic bicyclic nucleosides having a 4'-2' bridge have been described (see, e.g., freier et al, "Nucleic Acids Research," 1997,25 (22), 4429-4443; and Albaek et al, "J.Org.Chem.," 2006,71,7731-7740).
In some embodiments, the sugar substitute comprises a ring having not 5 atoms. For example, in some embodiments, the sugar substitute comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol Nucleic Acids (HNA), alunol (anitol) nucleic acids (ANA), mannitol Nucleic Acids (MNA) (see Leumann, C, "bio-organic chemistry and medicinal chemistry communications (j. Bioorg. & med. Chem.) (2002) 10.
In some embodiments, there is provided a modified THP nucleoside of formula VII, wherein q is 1 、q 2 、q 3 、 q 4 、q 5 、q 6 And q is 7 Each is H. In certain embodiments, q is 1 、q 2 、q 3 、q 4 、q 5 、q 6 And q is 7 Is not H. In some embodiments, q is 1 、q 2 、q 3 、q 4 、q 5 、q 6 And q is 7 At least one of which is methyl. In some embodiments, there is provided a THP nucleoside of formula VII, wherein R 1 And R 2 Is F. In certain embodiments, R 1 Is fluorine and R 2 Is H, R 1 Is methoxy and R 2 Is H, and R 1 Is methoxyethoxy and R 2 Is H.
Many other bicyclic and tricyclic sugar substitute ring systems are also known in the art, which can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review articles: leumann, J.C bio-organic & Medicinal Chemistry, 2002,10,841-854).
Combinations of modifications are also provided, not limited to, for example, 2'-F-5' -methyl substituted nucleosides (as to other disclosed 5',2' -bisSubstituted nucleosides, see PCT international application WO 2008/101157) and substitution of the ribosyl epoxy atom with S and further substitution at the 2' position (see US patent publication US 2005/0130923), or alternatively 5' -substitution of bicyclic nucleic acids (see PCT international application WO 2007/134181, wherein 4' -CH is substituted 2 - -O-2 'bicyclic nucleoside further substituted at the 5' position with 5 '-methyl or 5' -vinyl). The synthesis and preparation of carbocyclic bicyclic nucleosides and their oligomerization and biochemical studies have also been described (see, e.g., srivastava et al, 2007).
In some embodiments, the invention provides oligonucleotides comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified nucleobases, and/or modified linkages. The specific modification is selected so that the resulting oligonucleotide has the desired characteristics. In some embodiments, the oligonucleotide comprises one or more RNA-like nucleosides. In some embodiments, the oligonucleotide comprises one or more DNA-like nucleotides.
In some embodiments, the nucleoside of the invention comprises one or more unmodified nucleobases. In certain embodiments, the nucleoside of the invention comprises one or more modified nucleobases.
In some embodiments, the modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-enlarging bases and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines as defined herein, including 2-aminopropyladenine, 5-propynyluracil; 5-propynyl cytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl CH 3 ) Uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo being particularly preferredAre 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-enlarging bases and fluorinated bases. Other modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([ 5,4-b)][1,4]Benzoxazine-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5,4-b)][1,4]Benzothiazin-2 (3H) -one), G clip such as substituted phenoxazine cytidine (e.g. 9- (2-aminoethoxy) -H-pyrimido [5,4-13)][1,4]Benzoxazine-2 (3H) -one), carbazole cytidine: ( 2 H-pyrimido [4,5-b]Indol-2-one), pyridoindocytidine (H-pyrido [3',2':4,5)]Pyrrolo [2,3-d]Pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deaza-guanosine, 2-aminopyridine and 2-pyridone. Other nucleobases include those disclosed in U.S. Pat. No.3,687,808; in brief Encyclopedia Of Polymer Science And Engineering (The circumcise Encyclopedia Of Polymer Science And Engineering), kroschwitz, J.I. eds, john Wiley&Sons,1990, 858-859; those disclosed by Englisch et al, 1991; and those disclosed by Sanghvi, y.s., 1993.
Representative U.S. patents that teach the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Pat. nos. 3,687,808;4,845,205;5,130,302;5,134,066; 5,175,273;5,367,066;5,432,272;5,457,187;5,459,255;5,484,908;5,502,177; 5,525,711;5,552,540;5,587,469;5,594,121;5,596,091;5,614,617;5,645,985; 5,681,941;5,750,692;5,763,588;5,830,653 and 6,005,096, each of which is incorporated herein by reference in its entirety.
In some embodiments, the invention provides oligonucleotides comprising linked nucleosides. In these embodiments, the nucleosides can be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. RepresentExemplary phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters (P = O), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P = S). Representative phosphorus-free internucleoside linking groups include, but are not limited to: methylene methylimino (- -CH) 2 --N(CH 3 )--O--CH 2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -; siloxane (- -O- -Si (H) 2 - -O- -); and N, N' -dimethylhydrazine (- -CH) 2 --N(CH 3 )--N(CH 3 ) - -). Modified linkages can be used to alter (typically increase) nuclease resistance of an oligonucleotide compared to native phosphodiester linkages. In some embodiments, the internucleoside linkage having the chiral atom can be prepared as a racemic mixture or as an individual enantiomer. Representative chiral bonds include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free internucleoside linkages are well known to those skilled in the art.
The oligonucleotides described herein contain one or more asymmetric centers, thus giving rise to enantiomers, diastereomers and other stereoisomeric configurations, which in absolute stereochemistry may be defined as (R) or (S), α or β (e.g., for anomers of sugars), or (D) or (L) (e.g., for amino acids, etc.). Included in the antisense compounds provided herein are all such possible isomers, as well as racemic and optically pure forms thereof.
Neutral internucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, MMI (3' -CH) 2 --N(CH 3 ) - - -O-5 '), amide-3 (3' -CH) 2 - - -C (= O) - - -N (H) -5 '), amide-4 (3' -CH) 2 - - -N (H) - - - -C (= O) -5 '), methylal (3' -O- -CH) 2 - -O-5 ') and thiometals (3' -S- -CH) 2 - -O-5'). Other neutral internucleoside linkages include nonionic linkages including siloxanes (dialkylsiloxanes), carboxylic acid esters, carboxamides, sulfides, sulfonic acid esters and amides (see, e.g., carbohydrate Modifications in Antisense Research; ed. By y.s.sanghvi and p.d.cook, ACS Symposium Series (ACS symposiums Series), 580; chapters 3 and 4, 40-65). Other neutral internucleoside linkages include N, O, S and CH, including mixtures 2 Component partA nonionic bond of (2).
Additional modifications may also be made at other positions on the oligonucleotide, particularly at the 3 'position on the 3' terminal nucleotide of the sugar and at the 5 'position of the 5' terminal nucleotide. For example, one additional modification of the ligand-bound oligonucleotides of the invention involves chemically linking the oligonucleotide to one or more additional non-ligand moieties or conjugates, which enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. These moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, 1989), cholic acid (Manoharan et al, 1994), thiothioethers such as hexyl-5-tritylthiol (Manoharan et al, 1992; manoharan et al, 1993), thiocholesterol (Oberhauser et al, 1992), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, 1991, kabanov et al, 1990; svinarchuk et al, 1993), phospholipids such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonic acid triethylammonium (Manoharan et al, 1995), polyamines or polyethylene glycol chains (Manoharan et al, 1995), or adamantane et al, 1995), palmitoyl moieties (Mishra et al, 1995), or octadecylamine or hexylamino-carbonyloxy-cholesterol moieties (Croo et al, 1996).
Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. patent 4,828,979;4,948,882;5,218,105;5,525,465;5,541,313;5,545,730; 5,552,538;5,578,717;5,580,731;5,580,731;5,591,584;5,109,124;5,118,802; 5,138,045;5,414,077;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044; 4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335; 4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963; 5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873; 5,317,098;5,371,241;5,391,723;5,416,203;5,451,463;5,510,475;5,512,667; 5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587,371;5,595,726; 5,597,696;5,599,923;5,599,928 and 5,688,941, each of which is incorporated herein by reference.
E. Reagent kit
The disclosure also provides kits. Any of the components disclosed herein can be combined in a kit form. In some embodiments, the kit comprises a dendrimer or a composition as described above or in the claims.
The kit will generally comprise at least one vial, test tube, flask, bottle, syringe or other container in which the components may be placed, and preferably suitably aliquoted. Where more than one component is present in a kit, the kit will typically also contain a second, third or other additional container in which the additional components may be placed separately. However, various combinations of components may be contained in the container. In some embodiments, all of the nucleic acid delivery components are combined in a single container. In other embodiments, some or all of the dendrimer delivery component is provided in a separate container from the present polymer.
The kit of the present invention will also typically include a package for holding various containers, which are tightly sealed for commercial sale. Such packaging may include paperboard or injection or blow molded plastic packaging in which the desired container is retained. The kit may also include instructions for using the kit components. The description may include variations that may be implemented.
F. Examples of the invention
The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1: materials and instruments
1. Materials for chemical synthesis
All amines, thiols, and other unspecified chemicals were purchased from Sigma-Aldrich.1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Avanti Lipids. The lipid PEG2000 was chemically synthesized as described below. C12-200 was synthesized following the reported procedure (Love et al, 2010). All organic solvents were purchased from Fisher Scientific and purified using a solvent purification system (Innovative Technology).
2. Nucleic acids and other materials for in vitro and in vivo experiments
All sirnas were purchased from Sigma-Aldrich. Let-7g miRNA mimetics and their control mimetics were purchased from Ambion, life Technologies. Dulbecco's Modified Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from Sigma-Aldrich. OptiMEM, DAPI and Alexa Fluor 488 phalloidin were purchased from Life Technologies. ONE-Glo + Tox was purchased from Promega. Biophen FVII is purchased from Aniara Corporation.
The sequences of the sense and antisense strands of the siRNA are as follows:
siLuc (siRNA against luciferase). dT is a DNA base. All others are RNA bases.
And (3) sense: 5'-GAUUAUGUCCGGUUAUGUA [ dT ] [ dT ] -3' (SEQ ID NO: 3)
Antisense: 3'-UACAUAACCGGACAUAAUC [ dT ] [ dT ] -5' (SEQ ID NO: 4)
siFVII (siRNA against FVII). 2' -fluorine modified nucleotides are lower case letters.
Sense: 5'-GGAucAucucAAGucuuAc [ dT ] [ dT ] -3' (SEQ ID NO: 1)
Antisense: 3'-GuAAGAcuuGAGAuGAucc [ dT ] [ dT ] -5' (SEQ ID NO: 2)
sicTR (siRNA as control)
Sense: 5'-GCGCGAUAGCGCGAAUAUA [ dT ] [ dT ] -3' (SEQ ID NO: 5)
Antisense: 3'-UAUAUUCGCGCUAUCGCGC [ dT ] [ dT ] -5' (SEQ ID NO: 6)
Sigma-Aldrich MISSION siRNA general negative control #1 (catalog number: SIC 001) was used as non-targeted siRNA in control experiments. 2' OMe modified control siRNA (Sigma-Aldrich, proprietary modification) was used in vivo studies to reduce immune stimulation.
Cy5.5 labeled siRNA (siRNA for imaging)
And (3) sense: 5'-Cy5.5-GAUUAUGUCCGGUUAUGUA [ dT ] [ dT ] -3' (SEQ ID NO: 3)
Antisense: 3'-UACAUAACCGGACAUAAUC [ dT ] [ dT ] -5' (SEQ ID NO: 4)
Let-7g miRNA mimetics
Ambion (Life Technologies) mirVana miRNA mimics (catalog number 4464070, product ID: MC11758, name: has-let-7 g). The exact sequence and modifications are not disclosed by Ambion. The mimic matures human Let-7g.
Negative Control (CTR) miRNA mimetics
Ambion (Life Technologies) mirVana miRNA mimic, negative control #1 (Cat # 4464061). The exact sequence and modifications are not disclosed by Ambion.
3. Robot automation
Nanoparticle (NP) formulations and in vitro screening were performed on a Tecan free EVO 200 fluidic handling robot equipped with an 8-channel liquid handling arm (LiHa), a multi-channel arm with 96-channel head (MCA), a robotic manipulator arm (RoMa), and an integrated InfiniTe F/M200Pro microplate reader (Tecan). Two integrated custom heating and stirring chemical reaction stations (V & P Scientific 710E-3HM series drum stirrers) provide reaction and mixing support. All operations are programmed in EVOware Standard software (Tecan).
4. Characterization of the Synthesis
1 H and 13 c NMR was performed on a Varian 500MHz spectrometer. MS was performed on a Voyager DE-Pro MALDI TOF. Flash chromatography was performed on a Teledyne Isco Combiflash Rf-200i chromatography system equipped with a UV-vis and Evaporative Light Scattering Detector (ELSD). Particle size and zeta potential were measured by Dynamic Light Scattering (DLS) using a Malvern Zetasizer Nano ZS (He-Ne laser, λ =632 nm).
5. Nanoparticle formulations for in vivo studies
Formulated dendrimer nanoparticles for in vivo studies were prepared using a microfluidic mixing instrument (Precision Nanosystems nanoassembllr) with chevron rapid mixing characteristics. An ethanol solution of dendrimer, DSPC, cholesterol and lipid PEG2000 was rapidly combined with an acidic solution of siRNA as described below. Typical ratio of aqueous solution to EtOH is 3:1 (volume) and typical flow rate is 12 mL/min.
6. Automated in vitro delivery screening of modular degradable dendrimers
Nanoparticle (NP) formulation and in vitro screening were performed on a Tecan free EVO 200 fluid handling robot equipped with an 8-channel fluid handling arm (LiHa), a multichannel arm with 96-channel head (MCA), a robotic manipulator arm (RoMa), and an integrated InfiniTe F/M200Pro microplate reader (Tecan).
HeLa cells (HeLa-Luc) stably expressing firefly luciferase were obtained from HeLa cells (ATCC) by stable transfection of luciferase gene using lentivirus infection followed by clonal selection. HeLa-Luc cells were seeded (10,000 cells/well) into each well of an opaque white 96-well plate (Corning) and allowed to attach overnight in phenol red-free DMEM supplemented with 5% FBS. The day before the start of transfection, the medium was changed to fresh FBS-containing medium.
G1DD-siLuc nanoparticles were formulated with the aid of an automated fluid handling robot to accelerate the discovery process. All operations are programmed in the EVOware Standard software. First, the dendrimer reaction solution was diluted from the original reaction concentration to 12.5mM in ethanol. Next, the dendrimer solution was diluted a second time in ethanol from 12.5mM to 1mM using a LiHa arm. Then, 89.2 μ L of the lipid mixture in ethanol was added to a 96-well transparent plate. The lipid mixture consisted of DSPC (0.0690 mM), cholesterol (0.2622 mM) and lipid PEG2000 (0.0138 mM) in ethanol. Subsequently, 30.8. Mu.L of each dendrimer (1 mM) was added to the lipid mixture in a 96-well plate by LiHa followed by rapid mixing (15 times; 75. Mu.L mixing volume; speed 250. Mu.L/sec). LiHa was added at once and mixed with 8 tips. To a second clear 96-well plate, 50 μ L of siLuc (20 ng/μ L) in citrate buffer (pH = 4.3) was added by LiHa. Then 30 μ L of ethanol mixture (dendrimer, DSPC, cholesterol, lipid PEG 2000) was added to 50 μ L of the siLuc solution followed by rapid mixing (15 times; 75 μ L mixing volume; speed 250 μ L/sec) to form dendrimer nanoparticles. Is connected withNext, 120 μ L sterile PBS (1X) was added and mixed using LiHa to dilute the NPs and increase the pH. Subsequently, the plates were reformatted to allow easy transfer to the growing cells. Finally, 20 μ L of NP solution was added to the cultured cells through the MCA 96-head using a sterile disposable tip to avoid contamination. Cells eventually received 100ng of siLuc (33 nM). In this screening stage, the molar ratio of dendritic polymer to siLuc is 100. The final composition of the formulation was G1DD cholesterol DSPC lipid PEG2000 = 50. The cells were assayed at 37 ℃ and 5% CO 2 Following incubation for 24 hours, firefly luciferase activity and viability were analyzed using One Glo + Tox assay kit (Promega).
7. Dendrimer-small RNA formulations for in vivo studies
Formulated dendrimer nanoparticles for in vivo studies were prepared using a microfluidic mixing instrument (Precision Nanosystems nanoassembllr) with chevron rapid mixing characteristics. An ethanolic solution of dendrimer, DSPC, cholesterol and lipid PEG2000 (molar ratio 50. Typical ratio of aqueous solution to EtOH is 3:1 (volume) and typical flow rate is 12 mL/min. C12-200LNP was prepared according to the reported procedure (Love et al, 2010). An ethanol solution of C12-200, DSPC, cholesterol and lipid PEG2000 (50 molar ratio 38.5. All formulated NPs were purified by dialysis against sterile PBS with a 3.5kD cut-off and the size was measured by Dynamic Light Scattering (DLS) prior to in vivo studies. Encapsulation of small RNAs was measured with a Ribogreen binding assay (Invitrogen) by taking small amounts of solution and following the protocol, when applicable.
8. Animal research
All experiments were approved by the animal care and use committee of the southwestern medical center of texas university and met applicable local, state and federal regulations. Female C57BL/6 mice were purchased from Harlan Laboratories (Indianapolis, IN). Transgenic mice carrying MYC-driven liver tumors were generated by crossing TRE-MYC strains with LAP-tTA strains. Mice carrying the LAP-tTA and TRE-MYC genotypes were reared with 1mg/mL dox and MYC was induced by withdrawal of dox. Power analysis was performed to predict the number of animals required to achieve statistical significance.
9. In vivo factor VII silencing in mice
For in vivo delivery screening, female C57BL/6 mice received tail intravenous injections of: PBS (negative control, n = 3) or dendrimer NP containing non-targeted siRNA (siCTR, negative control, n = 3) or anti-factor VII siRNA (siFVII, n = 3) diluted in PBS (total volume below 200 μ L). After 48 hours, weight gain/loss was measured and mice were anesthetized by isoflurane inhalation to collect blood samples by retroorbital bleeding. Serum was separated using a serum separator tube (Becton Dickinson) and analyzed for factor VII protein levels by a chromogenic assay (Biophen FVII, ania Corporation). A standard curve was constructed using samples from PBS-injected mice, and relative factor VII expression was determined by comparing treated groups to untreated PBS controls.
For therapeutic studies FVII knockdown in transgenic mice was verified using the blood assay described above and by qPCR using collected liver tissue. To assess statistical significance, a two-tailed Student t-test was performed with 95% confidence.
10. Biodistribution
Female C57BL/6 mice or transgenic mice bearing liver tumors received 200 μ L of dendrimer NP containing Cy5.5-siRNA as 1mg/kg siRNA by tail intravenous injection. At 24 hours post injection, mice were euthanized and organs removed. Biodistribution was assessed by imaging the entire organ with the IVIS luminea system (Caliper Life Sciences) with a cy5.5 filter setting.
For confocal imaging, tissues were frozen sectioned (7 μm) and fixed using 4% paraformaldehyde at room temperature for 10 minutes. Slides were washed three times with PBS and blocked in PBS containing 1% albumin for 30 minutes. Sections were then incubated with Alexa Fluor 488 phalloidin in PBS containing 1% albumin (1 dilution 200, life Technologies) for 30 min. Slides were washed three times with 0.1-vol Tween 20 and fixed using ProLong Gold antibodies (Life Technologies). The sections were imaged using a LSM 700 point scanning confocal microscope (Zeiss) equipped with a 25 x objective.
11. In vivo toxicity assessment and Let-7g treatment study
Wild-type mice or transgenic mice bearing liver tumors were randomly divided into different groups. Mice received tail intravenous injection of dendrimer NPs containing siCTR. Their body weights were monitored daily. For transgenic mice bearing liver tumors, multiple tail vein injections were performed to simulate repeat dosing.
For the Let-7g treatment study, liver tumor-bearing transgenic mice received a tail intravenous injection of dendrimer NP with either a Let-7g mimic or a CTR mimic once a week at a dose of 1mg/kg in 200. Mu.L PBS from 26 to 61 days of age. Process order randomization is used. Blind studies were not performed. Their body weight, abdominal size and survival were carefully monitored. To assess statistical significance, a two-tailed T-test or Mantel-Cox-test with 95% confidence was performed.
Example 2: synthesis and characterization of PEG lipids and dendrimers
1. Synthesis of library containing 1,512 first-generation degradable dendrimers (G1 DD)
G1DD is synthesized by two sequential orthogonal reactions. First, amines having different Initial Branching Centers (IBC) are reacted with the acrylate groups of 2- (acryloxy) ethyl methacrylate (AEMA), respectively, wherein the molar ratio of amine to AEMA is equal to the number of IBCs (e.g., 2A amine added: two equivalents of AEMA; six equivalents of AEMA added). The reaction was carried out at 50 ℃ for 24 hours with the addition of 5 mol% of Butylated Hydroxytoluene (BHT). Next, each first-step adduct is separately reacted with a thiol, the molar ratio of thiol to adduct being equal to the number of amine IBC (e.g. 2A amine first-step adduct: two equivalents of thiol are added; 6A amine first-step adduct: six equivalents of thiol are added). The reaction was carried out at 60 ℃ with the addition of 5 mol% Dimethylphenylphosphine (DMPP) catalyst for 48 hours. Library synthesis of 1,512 members was accelerated by performing the reaction in glass vials and aluminum reaction blocks. A custom heating and stirring chemical reaction station (V & P Scientific 710E-3HM series drum stirrer) was used.
Initial in vitro delivery screening experiments were performed with crude G1DD. Subsequent studies were performed using purified dendrimers to verify activity.
All in vivo animal experiments were performed with purified G1DD. Purified G1DD was obtained by flash column chromatography on neutral alumina column using a Teledyne Isco chromatography system with a gradient of hexane and ethyl acetate eluent.
Higher generation degradable dendrimers were prepared according to the previous method (Ma et al, 2009). 1A2-G1 was prepared directly after 1A2 amine was reacted with 1 equivalent of AEMA in the presence of 5 mol% BHT at 50 ℃ for 24 hours. 1A2-G1 (4.00g, 11.7 mmol) was dissolved in 10mL of DMSO. After 2-aminoethanethiol (1.37g, 17.5 mmol) was added to the above solution, the reaction was stirred at room temperature for 30 minutes. Then 300mL of dichloromethane was immediately added to the reaction solution and washed with cold brine (50 mL. Times.3) to remove excess 2-aminoethanethiol. The organic phase was dried over magnesium sulfate and concentrated by rotary evaporation for direct use in the next step. AEMA (4.75g, 25.8 mmole) and BHT (227mg, 1.08 mmole) were added to the above solution. The reaction is stirred and passed at 50 deg.C 1 H NMR monitoring. After completion of the reaction, the solution was washed repeatedly with 20mL portions of hexane until absence of EAMA was found by TLC plate analysis. The washed solution was dried under vacuum to give viscous liquids 1A3-G2 which were used directly in the next step. The reaction of 1A3-G2 was carried out according to the two-step synthesis procedure described above to give viscous liquids 1A3-G3 which were used directly in the next step. After 1A2-G3 (0.5G, 0.3 mmol) was dissolved in 0.5mL DMSO, 1-octanethiol (216. Mu.L, 1.22 mmol) and Dimethylphenylphosphine (DMPP) (8.6. Mu.L, 0.061 mmol) were added. The reaction was stirred at 60 ℃ for 48 hours and then purified by running a neutral alumina column with a gradient of hexane and ethyl acetate eluent. Obtaining a yellowish viscous massLiquids 1A2-G3-SC8.
3. Synthesis of lipid PEG2000
Mixing PEG 44 -OH (80g, 40 mmol) and pyridine (6.5 mL,80 mmol) were dissolved in 250mL anhydrous DCM and cooled at 0 ℃. Methanesulfonyl chloride (15.5 mL,200 mmol) in 50mL of DCM was added over 30 minutes and the mixture was stirred at room temperature overnight. A further 100mL of DCM were added and the organic phase was saturated with NaHCO 3 The solution (50 mL. Times.3) was washed, then washed with brine (50 mL. Times.3). The resulting solution was concentrated and the residue was recrystallized from isopropanol and dried to yield white powder PEG2000-Ms (74g, 93%).
PEG2000-Ms (35.41g, 17.7 mmol) were dissolved in 250mL DMF. Then, naN is added 3 (12.4 g,19.0 mmol) was added to the solution. The reaction was stirred at 50 ℃ for 2 days under nitrogen. After removal of DMF, the residue was dissolved in 300mL DCM and washed with brine (50 mL. Times.3). After removal of the solvent, the residual oil was dissolved in 50mL of methanol and the product was precipitated three times with 300mL of diethyl ether to give PEG2000-N as a white powder 3 The desired compound (25.55g, 72%).
Propargylamine (0.50g, 9.1 mmol), BHT (191mg, 0.91 mmol), and EAMA (2.73g, 18.2 mmol) were added to a 25mL reaction vial. The mixture was stirred at 50 ℃ for 48 hours. The reaction was cooled to give the product T3-G1 as a colorless oil which was used in the next reaction without purification.
4. Characterization of selected dendrimers
1 H NMR(400MHz,CDCl 3 ,δ):4.38-4.19(br,28H,-OCH 2 CH 2 O-), 2.90-2.80(br,7H,-C(O)CH(CH 3 )CH 2 S-),2.75-2.71(br,14H, -NCH 2 CH 2 C(O)-),2.70-2.49(br,28H,-C(O)CH(CH 3 )CH 2 S-,-SCH 2 -), 2.49-2.39(br,36H,-N(CH 3 ) 2 ,-NCH 2 CH 2 N(CH 2 CH 2 ) 2 NCH 2 -,-CH 2 N(CH 2 -) 2 ), 1.57-1.48(m,8H,-SCH 2 CH 2 CH 2 -),1.37-1.28(br,8H,SCH 2 CH 2 CH 2 -), 1.28-1.16(br,53H,-SCH 2 CH 2 (CH 2 ) 4 CH 3 ,-CHC(CH 3 )CH 2 S-),0.85(t,J＝7.1 Hz,12H,-(CH 2 ) 4 CH 3 )。 13 C NMR(400MHz,CDCl 3 ,δ):174.92,172.03,62.22, 62.17,62.13,62.07,49.06,40.23,40.14,35.36,32.68,32.56,31.76,29.60, 29.14,28.82,22.58,16.85,16.81,14.04。MS(MALDI-TOF,m/z) C 109 H 196 N 6 O 28 S 7 The calculated value of (a): 2261.21, experimental values: 2262.43.
1 H NMR(400MHz,CDCl 3 ,δ):4.34-4.21(br,16H,-OCH 2 CH 2 O-), 2.82-2.76(m,4H,-SCH 2 CH(CH 3 )-),2.73(t,J＝7.1Hz,8H,-C(O)CH 2 CH 2 N-), 2.70-2.64(m,4H,-SCH 2 CH(CH 3 )-),2.58-2.51(m,4H,-SCH 2 CH(CH 3 )-), 2.51-2.46(m,8H,-CH 2 CH 2 S-),2.45-2.40(m,18H, (-C(O)CH 2 CH 2 ) 2 NCH 2 CH 2 CH 2 N(CH 2 -) 2 ),2.35-2.26(br,4H, -CH 2 CH 2 N(CH 2 -) 2 ),1.65-1.58(br,4H,-NCH 2 CH 2 CH 2 N-),1.57-1.49(m,8H, -SCH 2 CH 2 CH 2 -),1.37-1.28(br,8H,-SCH 2 CH 2 CH 2 -),1.28-1.16(br,44H, -SCH 2 CH 2 (CH 2 ) 4 CH 3 ,-CHC(CH 3 )CH 2 S-),0.85(t,J＝7.0Hz,12H, -(CH 2 ) 4 CH 3 )。 13 C NMR(400MHz,CDCl 3 ,δ):174.90,172.18,62.18,62.05, 49.05,40.14,35.37,32.68,32.40,31.76,29.60,29.15,28.83,22.60,16.81, 14.08。MS(MALDI-TOF,m/z)C 78 H 144 N 4 O 16 S 4 the calculated value of (a): 1520.95, experimental values: 1521.32.
1 H NMR(400MHz,CDCl 3 ,δ):4.32-4.21(br,16H,-OCH 2 CH 2 O-), 2.82-2.76(m,4H,-SCH 2 CH(CH 3 )-),2.73(t,J＝7.0Hz,8H,-C(O)CH 2 CH 2 N-), 2.69-2.62(m,4H,-SCH 2 CH(CH 3 )-),2.58-2.50(m,4H,-SCH 2 CH(CH 3 )-), 2.50-2.45(m,8H,-CH 2 CH 2 S-),2.45-2.38(m,12H,(-C(O)CH 2 CH 2 ) 2 NCH 2 -), 2.34-2.24(br,4H,-CH 2 N(CH 3 )CH 2 -),2.24-2.00(br,3H,-CH 2 N(CH 3 )CH 2 -) 1.66-1.57(br,4H,-NCH 2 CH 2 CH 2 N-),1.57-1.48(m,8H,-SCH 2 CH 2 CH 2 -), 1.37-1.28(br,8H,-SCH 2 CH 2 CH 2 -),1.28-1.16(br,45H,-SCH 2 CH 2 (CH 2 ) 4 CH 3 , -CHC(CH 3 )CH 2 S-),0.85(t,J＝7.0Hz,12H,-(CH 2 ) 4 CH 3 )。 13 C NMR(400MHz, CDCl 3 ,δ):174.90,172.18,62.18,62.05,49.00,40.13,35.36,32.68,32.35, 31.76,29.60,29.15,28.83,22.60,16.81,14.04。MS(MALDI-TOF,m/z) C 75 H 139 N 3 O 16 S 4 The calculated value of (a): 1465.90, experimental values: 1465.65.
1 H NMR(400MHz,CDCl 3 ,δ):4.34-4.20(br,20H,-OCH 2 CH 2 O-), 2.82-2.76(m,5H,-SCH 2 CH(CH 3 )-),2.75-2.70(br,10H,-C(O)CH 2 CH 2 N-), 2.69-2.62(m,5H,-SCH 2 CH(CH 3 )-),2.60-2.52(m,5H,-SCH 2 CH(CH 3 )-), 2.52-2.49(m,10H,-CH 2 CH 2 S-),2.49-2.45(br,16H,-NCH 2 CH 2 N-),2.45-2.40 (br,10H,-CH 2 N-),1.57-1.48(br,10H,-SCH 2 CH 2 CH 2 -),1.37-1.28(br,10H, -SCH 2 CH 2 CH 2 -),1.28-1.16(br,55H,-SCH 2 CH 2 (CH 2 ) 4 CH 3 ,-CHC(CH 3 )CH 2 S-), 0.87-0.79(br,15H,-(CH 2 ) 4 CH 3 )。 13 C NMR(400MHz,CDCl 3 ,δ):174.93, 172.13,62.28,62.01,49.04,40.13,35.36,32.68,32.35,31.76,29.60,29.15, 28.83,22.59,16.82,14.05。MS(MALDI-TOF,m/z)C 93 H 173 N 5 O 20 S 5 the calculated value of (c): 1840.13, experimental values: 1841.37.5A2-SC8 has also been prepared with 6 arms (structures shown below).
1 H NMR(400MHz,CDCl 3 ,δ):4.32-4.21(br,20H,-OCH 2 CH 2 O-), 2.82-2.76(m,5H,-SCH 2 CH(CH 3 )-),2.76-2.70(br,10H,-C(O)CH 2 CH 2 N-), 2.69-2.62(m,5H,-SCH 2 CH(CH 3 )-),2.58-2.50(m,5H,-SCH 2 CH(CH 3 )-), 2.50-2.45(m,10H,-CH 2 CH 2 S-),2.45-2.20(br,20H,(-(CH 2 ) 2 NCH 2 -, -CH 2 NHCH 2 -),1.66-1.57(br,6H,-NCH 2 CH 2 CH 2 N-),1.57-1.48(br,10H, -SCH 2 CH 2 CH 2 -),1.37-1.28(br,10H,-SCH 2 CH 2 CH 2 -),1.28-1.16(br,55H, -SCH 2 CH 2 (CH 2 ) 4 CH 3 ,-CHC(CH 3 )CH 2 S-),0.82-0.75(br,15H,-(CH 2 ) 4 CH 3 )。 13 C NMR(400MHz,CDCl 3 ,δ):174.98,172.13,62.28,62.01,49.04,40.13, 35.36,32.68,32.35,31.76,29.60,29.15,28.83,22.61,16.85,14.14。MS (MALDI-TOF,m/z)C 94 H 174 N 4 O 20 S 5 The calculated value of (a): 1839.13, experimental values: 1838.97.
1 H NMR(400MHz,CDCl 3 ,δ):4.33-4.20(br,24H,-OCH 2 CH 2 O-), 2.82-2.77(m,6H,-SCH 2 CH(CH 3 )-),2.77-2.71(br,12H,-C(O)CH 2 CH 2 N-), 2.68-2.62(m,6H,-SCH 2 CH(CH 3 )-),2.60-2.52(m,6H,-SCH 2 CH(CH 3 )-), 2.52-2.48(br,12H,-CH 2 CH 2 S-),2.48-2.46(br,12H,-NCH 2 CH 2 N-),2.45-2.40 (br,12H,(-CH 2 ) 2 N-),1.57-1.47(br,12H,-SCH 2 CH 2 CH 2 -),1.37-1.28(br, 12H,-SCH 2 CH 2 CH 2 -),1.28-1.16(br,108H,-SCH 2 CH 2 (CH 2 ) 8 CH 3 , -CHC(CH 3 )CH 2 S-),0.87-0.80(br,18H,-(CH 2 ) 8 CH 3 )。 13 C NMR(400MHz, CDCl 3 ,δ):174.87,172.07,62.16,62.04,49.48,40.47,40.11,35.34,32.69, 32.42,31.86,29.61,29.58,29.57,29.50,29.29,29.21,28.85,22.62,16.81, 14.06。MS(MALDI-TOF,m/z)C 132 H 246 N 4 O 24 S 6 the calculated value of (a): 2463.65, experimental values: 2464.52.
Example 3: library design and synthesis of first generation degradable dendritic polymer (G1 DD)
Liver cancer is a challenging host for therapeutic intervention because drug-induced hepatotoxicity may exacerbate the underlying liver disease (Boyerinas et al, 2010). To achieve effective RNAi-mediated therapy, a balance of high potency and low toxicity of the vector must therefore be maintained. This requires a general strategy to easily tune the delivery vehicle in terms of size, chemical structure and final physical properties (fig. 1A). In some embodiments, the dendritic polymer is designed to exhibit one or more of the following characteristics: optimal monodisperse materials for chemical and dimensional manipulation (Wu et al, 2004. Sequential reactions with 2- (acryloxy) ethyl methacrylate (AEMA) were carried out using orthogonal reactions to diversify the first generation degradable dendrimers (G1 DD) by the following various parameters: a core (C), a bond or repeat unit (L), and a peripheral or capping group (P) (fig. 1B). In some embodiments, esters are selected as the initial degradable linkage because polyesters are used in FDA approved products with minimal toxicity. In each growth step, the ester number increases, which provides an opportunity to identify degradable dendrimers with balanced potency and toxicity.
Previous results indicate that these orthogonal reactions can build polyester dendrimers with a range of generations (Ma et al, 2009). However, prior to the use of this strategy, it was demonstrated that this method is capable of producing diverse dendrimers using a wide variety of chemically different amine and thiol compounds without purification. To investigate the robustness of this chemistry, the structural limitations of the orthogonal michael addition reaction were tested using the most difficult starting materials, namely tris (2-aminoethyl) amine with six N-H bonds as the Initial Branching Center (IBC) and tetradecylamine with a 14-carbon length alkyl chain. After 24 hours at 50 ℃ in the presence of 5 mol% Butylated Hydroxytoluene (BHT) (to inhibit free radical formation), both tris (2-aminoethyl) amine and tetradecylamine reacted quantitatively and selectively with the acrylate functionality in the AEMA, which itself remained unreacted under these conditions (fig. 2 and 3). In a second orthogonal reaction (Thiamer)Kerr addition), dimethylphenylphosphine is required as a catalyst to achieve a low concentration (minimum 125 mM) or small scale (average about 20 mg) of the final product and to achieve high conversion (in terms of 1 H NMR 100%) so that the material can be used without purification for subsequent testing or passage amplification (fig. 4 and 5). Some dendrimers were re-synthesized on a larger scale and purified by flash chromatography before in vivo studies.
Due to various delivery barriers, the efficacy of small RNA vectors through nanocapsulation is influenced by a variety of factors, including pK a Topology/structure and hydrophobicity (Siegwart et al, 2011 Jayaraman et al, 2012; schaffert et al, 2011 Whitehead et al, 2014). To facilitate the identification of degradable dendrimers with high delivery potency, a G1DD library was designed with four regions by chemically diversifying the core-forming amine C and peripheral-forming thiol P: nuclear binding-peripheral stabilization (zone I), nuclear binding-peripheral binding (zone II), nuclear stabilization-peripheral stabilization (zone III), and nuclear stabilization-peripheral binding (zone IV) (fig. 1C and fig. 1D). In regions I and II, RNA binding is regulated by amines with one (1 An) to six (6 An) Initial Branching Centers (IBC). Thus, the corresponding dendrimers contain one to six branches. In regions III and IV, the stabilization of RNA-dendrimer NPs varies mainly with different lengths of alkyl chains (1 Hn and 2 Hn). In zones II and IV, the binding capacity of the aminothiol (SNn) is largely regulated by the different amines, while in zones I and III, the stabilization varies with the length of the alkylthiol (SCn) and the carboxyl-and hydroxy-alkylthiols (SOn). The efficacy of dendrimers was tested for the entire compound library (fig. 6).
Example 4: in vitro G1DD screening for intracellular delivery of siRNA
The delivery vehicle must overcome a series of extracellular and intracellular barriers to allow the small RNA to be active within the tumor cell. G1DD was identified that can mediate siRNA overcoming intracellular barriers by screening a1,512 member G1DD library for the ability to deliver siRNA in vitro to HeLa cells stably expressing luciferase. G1DD was formulated into Nanoparticles (NP) containing luciferase-targeting siRNA (siLuc) and helper lipid cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and lipid PEG2000 (Akinc et al, 2008, semple et al, 2010. Intracellular delivery potential was assessed by quantifying luciferase reduction and cell viability (figures 7-9).
To extract SAR from in vitro data, we utilized a dendrimer-inspired tree analysis process (fig. 7B and 9B). Of 1,512 dendrimers, 88 mediated luciferase silencing was >50%, and the overall library hit rate was 6%. When the hit rates of all four regions (I-IV) were analyzed, the hit rate of region I was 10%, while the hit rates of regions II, III and IV were 0%, 2% and 3%, respectively. This result indicates that these dendrimers with siRNA-binding cores and stabilized peripheries (region I) have much higher intracellular siRNA delivery potential. In the branching type of region 1, the hit rate of SO peripheral dendrimers is as low as 1%, while the hit rate of SC peripheral dendrimers is as high as 15%. Without wishing to be bound by any theory, it is believed that hydrophobic stabilization from the dendrimer periphery is critical for efficient delivery of siRNA into cells via nano-encapsulation. This may result in an increase in hydrophobic packaging, providing additional NP stability (Leung et al, 2012). After further investigation of the branch number and branch length of these dendrimers with a binding core and SC periphery, dendrimers with a binding core and three, four, five or six SC5-8 branches or SC9-12 branches had >25% chance of delivering siLuc to HeLa cells with luciferase knockdown >50%. By screening the complete G1DD library in vitro and the dendron analysis process, a population of dendrimers showing increased intracellular siRNA delivery was identified: groups with binding nuclei/SC periphery and binding nuclei branching from 3-6 SCs 5-8 or SC 9-12.
Example 5: identification of degradable dendrimers and design of G2-G4 dendrimers for efficient in vivo siRNA delivery
Dendrimers capable of overcoming intracellular barriers have been identified, followed by the identification of dendrimers capable of overcoming extracellular barriers for efficient delivery of siRNA in vivo. By separating these two processes, chemical functions that overcome the barrier can be identified, including blood stability, liver (tumor) localization, cellular uptake, and active siRNA release. The ability of dendrimers to silence factor VII in hepatocytes was evaluated because this coagulation factor can be readily quantified from small serum samples (Akinc et al, 2008, sample et al, 2010). 26 of the hit degradable dendrimers were selected to maximize chemical diversity: 22 based on dendritic analysis process has optimized chemical structure, and the other 4 (2A 2-SC14, 2A6-SC14, 2A9-SC14 and 6A1-SO 9) is based on its high intracellular siRNA delivery capacity selection. Dendrimers were formulated with anti-factor VII siRNA (siFVII) and injected intravenously into mice at a dose of 1mg siFVII/kg. FVII activity was quantified 3 days after injection. Despite the high potency in vitro, 2A2-SC14, 2A6-SC14, 2A9-SC14, 6A1-SO9 and most of the three-branched dendrimers showed only minimal FVII knockdown in vivo (FIG. 10A). Dendrimers comprising a binding core and four, five or six SC8 or SC12 branches show higher knockdown. Based on these studies, SC8 branched dendrimers are generally more effective than SC12 branched compounds.
Based on the in vitro and in vivo high throughput screening results, we asked whether it is now possible to use SAR information to rationally design dendrimers with predicted activity to validate our approach. A series of degradable dendrimers were prepared using the following two strategies: (I) by selecting a polyamine having five or six IBCs; and (II) increasing branching by dendrimer generation amplification (fig. 10). Two natural amines, spermidine (5 IBCs) and spermine (6 IBCs) were selected to implement strategy I. With respect to strategy II, 1A2 (one IBC), 2A2 and 2a11 (two IBCs), 3A3 and 3A5 (three IBCs), and 4A1 and 4A3 (four IBCs) were selected to produce degradable dendrimers with multiple branches by generational amplification (fig. 10C and fig. 11). An additional 24 degradable dendrimers were evaluated (fig. 10C) to further study SAR in vivo. After generation amplification, higher generation dendrimers with four or six SC branches of 1A2 (one IBC), 2A2 (two IBCs) and 3A3 and 3A5 (three IBCs) had good in vivo siRNA delivery to hepatocytes, whereas dendrimers with eight branches were less active. This process converts inactive amine cores in vitro screens, and then rationally designs higher generation dendrimers that exhibit in vivo activity.
Example 6: in vivo toxicity assessment of degradable dendrimers in mice bearing MYC-driven liver tumors
To identify degradable dendrimers with the desired balance of low toxicity and high potency required for liver cancer treatment, degradable dendrimers were selected to assess their in vivo toxicity. In parallel, we analyzed the C12-200 lipid LNP and selected as the best example of a non-hydrolytic system that previously showed strong efficacy in mice and non-human primates (Love et al, 2010). Lipidoids, as a class, are baseline materials at the front of clinical studies (Kanasty et al, 2013 love et al, 2010. Non-immunogenic control siRNA was used to best assess the toxicity of the individual dendrimers themselves. Dendrimer NP was formulated at a weight ratio of 25. C12-200LNP was prepared using the same formulation parameters as previously reported (Love et al, 2010). The size and zeta potential of each NP in PBS buffer were characterized. They all have similar dimensions, are 64-80nm in diameter, and their surface charge is near neutral (fig. 12A and 12B). Each formulated NP was injected intravenously at a dose of 4mg siCTR/kg (100 mg dendrimer/kg or 28mg C12-200/kg) in wild type mice. In many different ways of assessing toxicity in vivo, weight loss can be used as a simple informative parameter. In normal mice, the selected NPs (including C12-200 control LNPs) had minimal weight change. However, among the candidates, mice injected with 5A2-SC8 and 6A3-SC12 experienced faster recovery and normal weight gain after the first day.
Based on these results, 5A2-SC8 and 6A3-SC12 were selected to further evaluate their in vivo toxicity in chronically ill transgenic mice bearing aggressive liver tumors with single and multiple injections. A well-defined Tet-On MYC-inducible transgenic liver cancer model (FIG. 13A) was selected (Nguyen et al, 2014). Since tumors are more aggressive when MYC is overexpressed at early developmental time points, MYC is induced immediately after birth (p 0), which results in rapidly growing liver tumors. At 32 days of age (p 32), these diseased transgenic mice bearing aggressive liver tumors were injected with A3 mg siCTR/kg dose (75 mg dendrimers/kg or 21mg C12-200/kg) of 5A2-SC8 or 6A3-SC12NP. Mice receiving 5A2-SC8 injections lost about 5% of body weight on the first day and returned rapidly to the initial body weight on the second day, while those receiving 6A3-SC12 injections still lost 10% of body weight and failed to recover by the third day (fig. 12D). After multiple injections, these mice died 7 days earlier than untreated mice due to the toxicity of the 6A3-SC12 vector (fig. 12E). Compared to the results of WT mice, while the lipids received were about 3-fold less than the 5A2-SC8 injected mice, C12-200LNP injected mice bearing aggressive tumors lost >20% of body weight after one day (fig. 12D). These data indicate that small changes in chemical structure can produce large toxic changes. It also indicates that tumor-bearing mice are more sensitive to intervention than healthy mice. Based on these results, 5A2-SC8 appeared as a degradable dendrimer with a balance of low toxicity (tolerance up to 75mg/kg in tumor-bearing mice) and efficient in vivo FVII knockdown (> 95% at 1mg siFVII/kg). In addition to being less toxic than the benchmark compounds, 5A2-SC8 NPs are more effective because these dendrimers reduce clinical concern for dose-limiting toxicity and achieve a wider therapeutic window.
Example 7: effective inhibition of liver tumor growth by systemic administration of Let-7g miRNA mimics
To evaluate the ability of degradable dendrimer NPs to deliver therapeutic miRNA mimics without causing additional toxicity, the aggressive MYC transgenic liver cancer model induced at p0 was again used (Nguyen et al, 2014). These mice develop rapidly growing cancers, similar to pediatric Hepatoblastoma (HB), which are of the same type as HCC in many molecular features. After 20 days, abdominal distension was evident from the mass effect and the tumor grew rapidly. Without intervention, mice died within 60 days after birth. Given the speed and fatality of this model, the chances of successful treatment are limited.
Since 5A2-SC8 balances low toxicity in vivo and effectiveness in silencing target FVII of hepatocytes, first, it was investigated whether 5A2-SC8NP could deliver siRNA into tumor cells. At 41 days of age (p 41), the livers of these transgenic mice were confluent with tumors (fig. 14A). Mice were injected intravenously with 5A2-SC8NP with Cy5.5-labeled siRNA at a dose of 1mg siRNA/kg at p 40. Fluorescence imaging showed that 5A2-SC 8-mediated siRNA accumulated in the cancerous liver, but only in small amounts in the spleen and kidney 24 hours after injection (FIGS. 14A-14B). 5A2-SC8NP delivered siRNA to normal and transgenic livers, even though the cancerous liver was larger than the normal liver (FIG. 15A).
To further confirm whether 5A2-SC8NP could deliver siRNA into tumor cells in vivo, tumor tissue of the liver was collected and imaged 24 hours after intravenous injection. H & E staining showed that the tumor tissue was densely packed with nuclei and exhibited a cancerous phenotype (fig. 15B). Confocal imaging confirmed that 5A2-SC8NP was able to efficiently deliver labeled siRNA into tumor cells (fig. 14C).
The therapeutic benefit of 5A2-SC8 mediated small RNA delivery in these chronically diseased transgenic mice was then evaluated. One of the most important mirnas is Let-7, a family of tumor suppressors that is down-regulated in many tumor types (Boyerinas et al, 2010, roush and Slack, 2008). Because endogenous Let-7g is known to be down-regulated in liver HB (Nguyen et al, 2014), tests were performed to determine whether delivery of a Let-7g mimic could inhibit the development of liver cancer in this aggressive, genetically engineered mouse model.
5A2-SC8NP was demonstrated to be able to achieve siRNA delivery in this model. In collected liver tissues, intravenous delivery of a single dose of siFVII using a blood assay (fig. 16A) and by qPCR showed effective silencing of FVII protein (fig. 16B). This silencing is achieved at p26 after initiation of tumor development. Next, 1mg/kg Let-7g was delivered intravenously to tumor-bearing mice as 5A2-SC8NP (p 26). Let-7g expression in liver tissue increased 7-fold 48 hours after injection (FIG. 16C).
Then, the treatment regimen was started from p26 by administering 5A2-SC8NP containing Let-7g of either the mimetic or the control mimetic at 1mg/kg once a week. At p50, mice receiving the Let-7g mimic had significantly smaller abdomens and reduced tumor burden (FIGS. 16D-16F). Let-7g caused a reduction in abdominal circumference and quantified tumor growth (FIG. 16E). The effect on tumor growth was confirmed by ex vivo liver imaging (fig. 16F). Most importantly, delivery of Let-7G once a week from day 26 to day 61 did not affect body weight gain (fig. 16G) and significantly prolonged survival (fig. 16H). All control mice that received no treatment and mice that received 5A2-SC8NP with CTR mimetics died at around 60 days of age. C12-200LNP (Let-7 g or control simulant) caused premature death and required cessation of the experiment. Delivery of Let-7g within 5A2-SC8 NPs provided significant survival benefits, with one mouse alive for 100 days. These results indicate that 5A2-SC8 can balance high delivery efficacy with low toxicity, providing significant therapeutic benefit to chronically ill transgenic mice by effectively inhibiting liver tumor growth.
Example 8: evaluation of different lipid compositions for siRNA delivery
To assess which lipid compositions in dendrimer nanoparticles lead to improved siRNA delivery, changes were made to the identity and concentration of different phospholipids and PEG-lipids. Three different cell lines (HeLa-Luc, A549-Luc and MDA-MB 231-Luc) were used. Cells were present at 10K cells per well and 24 hours incubation. Readings were determined 24 hours after transfection. In the nanoparticles, DSPC and DOPE were used as phospholipids, and PEG-DSPE, PEG-DMG and PEG-DHD were used as PEG-lipids. The composition contains the lipids or dendrimers cholesterol, phospholipid, PEG-lipid in a molar ratio of 50. The molar ratio of lipid/dendrimer to siRNA was 100. RiboGreen, cell-titer Fluor and OneGlo assays were used to determine the effectiveness of these compositions. The results show relative luciferase activities in HeLa-Luc cells (FIG. 17A), A549-Luc (FIG. 17B) and MDA-MB231-Luc (FIG. 17C). Six formulations used in the study included: dendrimer (lipid) + cholesterol + DSPC + PEG-DSPE (formulation 1), dendrimer (lipid) + cholesterol + DOPE + PEG-DSPE (formulation 2), dendrimer (lipid) + cholesterol + DSPC + PEG-DMG (formulation 3), dendrimer (lipid) + cholesterol + DOPE + PEG-DMG (formulation 4), resinoid (lipid) + cholesterol + DSPC + PEG-DSPE (formulation 5), and dendrimer (lipid) + cholesterol + DOPE + PEG-DHD (formulation 6).
Further experiments were performed to determine which phospholipids showed increased delivery of siRNA molecules. HeLa-Luc cell line, 10K cells per well, was used, incubated for 24 hours, and read 24 hours after transfection. The composition contains DOPE or DOPC as a phospholipid and PEG-DHD as a PEG-lipid. Lipid (or dendrimer): cholesterol: phospholipid: PEG-lipid molar ratio 50. These compositions were tested at a dose of 50ng using the Cell-titer Fluor and OneGlo assays. These results are shown in fig. 18A and 18B.
Example 9: evaluation of dendrimer nanoparticles for delivery of sgrnas and other CRISPR nucleic acids
To evaluate composition delivery nucleic acids for CRISPR/Cas gene editing, delivery of sgrnas and mrnas was tested. Cell lines were established that allowed rapid screening of dendrimer NP and Z120 for sgRNA delivery. For example, heLa (cervical cancer) and a549 (lung cancer) cells are established to co-express luciferase and Cas9. Selection and quality control were verified. The guide RNA was designed according to previously reported methods of targeting the first exon of the desired target gene. The target with the highest score indicative of cleavage activity and sequence specificity was used for sgRNA preparation using a defined protocol. DNA oligonucleotides were synthesized commercially, annealed, cloned by BsbI digestion and ligated into Cas 9-containing plasmid backbone. In vitro transcription enables the isolation of sgrnas, which can then be packaged in dendrimer NPs for delivery. A series of 5 different guide sequences were designed for luciferase. These guide sequences were verified by sgLuc-Cas9pDNA transfection using commercial reagents to select the optimal sgRNA sequence. Next, we packaged sgLuc into dendrimer NPs and evaluated delivery of sgrnas in HeLa-Luc-Cas9 cells. After a defined number of hours of exposure, luciferase and viability compared to untreated cells were measured using One Glo + Tox (Promega). In a typical experiment, 10K cells per well were seeded, followed by incubation for 24 hours, addition of dendrimer nanoparticles containing sgLuc, and readout 24-48 hours after transfection. These combinationsThe compositions comprise combinations of dendrimers, DSPC or DOPE, cholesterol and PEG-lipids. In addition, the compositions contain different concentrations of MgCl 2 . Lipid (or dendrimer): cholesterol: PEG-lipid molar ratio 50:38.5, lipid to nucleic acid (sgRNA) molar ratio 200. In addition, results were obtained using Cell-titer Fluor and OneGlo assays. The results in the absence of phospholipids are shown in figure 19. Similar studies were performed with phospholipids present. In these compositions, the phospholipid DSPC is used in the formulation. The phospholipid-containing composition was used in the same ratio as the above composition without phospholipids, in a molar ratio of 50. These compositions were tested using RiboGreen, cell-titer Fluor and OneGlo over two time periods (24 hours and 72 hours). Data obtained at 24 hours is shown in fig. 20A, and data obtained at 72 hours is shown in fig. 20B.
Example 10: evaluation of dendrimer nanoparticles for mRNA delivery
Delivery of mRNA molecules was tested with dendrimers and Z120 described herein, similarly to the studies performed with siRNA. IGROV1 cell lines were used at a concentration of 4K cells per well, incubated for 24 hours, and read at 24 and 48 hours post-transfection. These compositions contain DSPC, DOPE or phospholipid-free and PEG-DHD as PEG-lipids. Lipid (or dendrimer): phospholipid: PEG-lipid molar ratio of 50:50 ng dose and 100ng dose. Results were obtained using the Cell-titer Fluor and OneGlo assays. These results are shown in fig. 21A (24 hours) and fig. 21B (48 hours). In addition, the delivery of mCherry mRNA is shown in figure 22 using a nanoparticle composition with a 20.
*********************
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Reference to the literature
The following references, to the extent that they provide exemplary procedures or other details supplementary to those set forth herein, are expressly incorporated herein by reference.
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Claims (80)
1. A dendrimer of the formula:
core-repeat unit-end capping group (I),
or a pharmaceutically acceptable salt thereof,
wherein the core is attached to one or more of the repeating units by removing one or more hydrogen atoms from the core and replacing the hydrogen with the repeating unit; and wherein:
the core has the formula:
wherein:
X 3 is-NR 6 -, -O-, alkylamino-diyl group C≤8 Alkoxy diyl group C≤8 An aromatic hydrocarbon diyl group C≤8 Hetero arene diyl C≤8 Heterocyclic alkanediyl C≤8 Or substituted forms of any of these groups, wherein R is 6 Is hydrogen, alkyl C≤8 Or substituted alkyl C≤8 ；
R 3 And R 4 Each independently is amino, hydroxy, mercapto, alkylamino C≤12 Dialkylamino group C≤12 Or substituted versions of any of these groups; or a group of the formula:
wherein:
e is 1,2 or 3;
R c and R d Each independently is hydrogen, alkyl C≤6 Or substituted alkyl C≤6 ；
c and d are each independently 1,2, 3, 4,5 or 6;
wherein the repeat unit comprises a degradable diacyl group;
the degradable diacyl group has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl C≤6 Or substituted alkyl C≤6 ；
Y 3 Is an alkanediyl radical C≤12 An olefin diyl group C≤12 An aromatic hydrocarbon diyl group C≤12 Or substituted versions of any of these groups; and is
R 9 Is an alkyl group C≤8 Or substituted alkyl C≤8 ；
The end capping group has the formula:
wherein:
Y 4 is an alkanediyl radical C≤18 (ii) a And is
R 10 Is hydrogen;
wherein the last degradable diacyl group in the chain is attached to the end-capping group.
2. A dendrimer of the formula:
nucleus- (repeat unit) n -a blocking group (I),
or a pharmaceutically acceptable salt thereof,
wherein the core is attached to one or more of the repeating units by removing one or more hydrogen atoms from the core and replacing the hydrogen with the repeating unit; and wherein:
the core has the formula:
wherein:
X 3 is-NR 6 -, -O-, alkylamino-diyl group C≤8 Alkoxy diyl group C≤8 An aromatic hydrocarbon diyl group C≤8 Hetero aromatic diyl C≤8 Heterocyclic alkanediyl C≤8 Or substituted forms of any of these groups, wherein R 6 Is hydrogen, alkyl C≤8 Or substituted alkyl C≤8 ；
R 3 And R 4 Each independently is amino, hydroxy, mercapto, alkylamino C≤12 Dialkylamino group C≤12 Or substituted versions of any of these groups; or a group of the formula:
wherein:
e is 1,2 or 3;
R c and R d Each independently is hydrogen, alkyl C≤6 Or substituted alkyl C≤6 ；
c and d are each independently 1,2, 3, 4,5 or 6;
wherein the repeat unit comprises a degradable diacyl and a linker;
the degradable diacyl has the formula:
wherein:
A 1 and A 2 Each independently is-O-or-NR a -, wherein:
R a is hydrogen, alkyl C≤6 Or substituted alkyl C≤6 ；
Y 3 Is an alkanediyl radical C≤12 An olefin diyl group C≤12 An aromatic hydrocarbon diyl group C≤12 Or substituted versions of any of these groups; and is
R 9 Is an alkyl group C≤8 Or substituted alkyl C≤8 ；
The linker group has the formula:
wherein:
Y 1 is an alkanediyl radical C≤12 Olefin diyl group C≤12 An aromatic hydrocarbon diyl group C≤12 Or substituted versions of any of these groups; and is provided with
Wherein the linker group is linked to a degradable diacyl group through the nitrogen and sulfur atoms of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next group comprises two degradable diacyl groups linked to the nitrogen atom of the linker group; and wherein n is 1,2, 3, 4,5 or 6;
the end capping group has the formula:
wherein:
Y 4 is alkanediyl C≤18 (ii) a And is
R 10 Is hydrogen;
wherein the last degradable diacyl group in the chain is attached to the end-capping group.
3. The dendritic polymer of claim 1 or 2, wherein:
X 3 is-NR 6 -, alkylamino diyl C≤8 Or substituted alkylamino diyl C≤8 Wherein R is 6 Is hydrogen, alkyl C≤8 Or substituted alkyl C≤8 ；
R 3 And R 4 Each independently is amino, hydroxy, or alkylamino C≤12 Or dialkylamino group C≤12 (ii) a Or a group of the formula:
wherein:
e is 1,2 or 3;
R c and R d Each independently is hydrogen, alkyl C≤6 Or substituted alkyl C≤6 ；
c and d are each independently 1,2, 3, 4,5 or 6.
4. The dendritic polymer of claim 1 or 2, wherein:
Y 4 is an alkanediyl radical C4 - 18 (ii) a And R is 10 Is hydrogen.
5. The dendritic polymer of claim 1 or 2, wherein Y is 4 Is an alkanediyl radical C4-18 。
6. The dendritic polymer of claim 1 or 2, wherein X 3 Is an alkylamino diyl group C≤8 Or substituted alkylamino diyl C≤8 。
7. The dendritic polymer of claim 1 or 2, wherein X 3 is-NHCH 2 CH 2 NH-or-NHCH 2 CH 2 NHCH 2 CH 2 NH-。
8. The dendritic polymer of claim 1 or 2, wherein X 3 Is a heterocyclic alkanediyl radical C≤8 Or substituted heterocycloalkane diyl C≤8 。
9. According to claim 1 or 2The dendritic polymer of (1), wherein X 3 Is N, N' -piperazinediyl.
10. The dendritic polymer of claim 1 or 2, wherein R 3 Is an amino group.
11. The dendritic polymer of claim 1 or 2, wherein R 3 Is alkylamino C≤12 Or substituted alkylamino C≤12 。
12. The dendritic polymer of claim 1 or 2, wherein R 3 Is a methylamino group.
13. The dendritic polymer of claim 1 or 2, wherein R 4 Is an amino group.
14. The dendritic polymer of claim 1 or 2, wherein R 4 Is alkylamino C≤12 Or substituted alkylamino C≤12 。
15. The dendritic polymer of claim 1 or 2, wherein R 4 Is a methylamino group.
16. The dendritic polymer of claim 1 or 2, wherein R 4 Is that
17. The dendritic polymer of claim 1 or 2, wherein the core is further defined as:
18. the dendritic polymer of claim 1 or 2, wherein the core is further defined as:
19. the dendritic polymer of claim 1 or 2, wherein the core is further defined as:
20. the dendritic polymer of claim 2, wherein Y 1 Is an alkanediyl radical C≤8 Or substituted alkanediyl C≤8 。
21. The dendritic polymer of claim 2, wherein Y 1 is-CH 2 CH 2 -。
22. The dendritic polymer of claim 1 or 2, wherein Y is 3 Is an alkanediyl radical C≤8 Or substituted alkanediyl C≤8 。
23. The dendritic polymer of claim 1 or 2, wherein Y is 3 is-CH 2 CH 2 -。
24. The dendritic polymer of claim 1 or 2, wherein a 1 is-O-or-NH-.
25. The dendritic polymer of claim 1 or 2, wherein a 2 is-O-or-NH-.
26. The dendritic polymer of claim 1 or 2, wherein R 9 Is an alkyl group C≤8 。
27. The dendritic polymer of claim 1 or 2, wherein R 9 Is methyl.
28. The dendritic polymer of claim 1 or 2, wherein each end capping group is independently selected from:
29. the dendritic polymer of claim 2, wherein n is 1,2 or 3.
30. A composition, comprising:
(A) The dendritic polymer of any one of claims 1 to 29; and
(B) A nucleic acid.
31. The composition of claim 30, wherein the nucleic acid comprises a short interfering ribonucleic acid (siRNA), a microrna (miRNA), a primary miRNA (pri-miRNA), a messenger RNA (mRNA), a single guide RNA (sgRNA), a regularly clustered spacer short palindromic repeats (CRISPR), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), a plasmid DNA (pDNA), a transport RNA (tRNA), an antisense oligonucleotide (ASO), a guide RNA, a double stranded DNA (dsDNA), a single stranded DNA (ssDNA), a single stranded RNA (ssRNA), or a double stranded RNA (dsRNA).
32. The composition of claim 31, wherein the nucleic acid comprises an siRNA, tRNA, sgRNA, crRNA, or tracrRNA.
33. The composition according to claim 31, wherein the dendrimer and the nucleic acid are present in the composition in a weight ratio of 100:1 to 1:5.
34. The composition of claim 30, wherein the composition further comprises one or more helper lipids, wherein the one or more helper lipids comprise a steroid, a polyethylene glycol (PEG) -conjugated lipid, a phospholipid, or a combination thereof.
35. The composition of claim 34, wherein the one or more helper lipids are steroids.
36. The composition of claim 34, wherein the steroid is cholesterol.
37. The composition of claim 35, wherein the steroid and the dendrimer are present in the composition in a molar ratio of from 10:1 to 1:20.
38. The composition of claim 34, wherein the one or more helper lipids are PEG-conjugated lipids.
39. The composition of claim 38, wherein the PEG-conjugated lipid is a pegylated diacylglycerol.
40. The composition of claim 39, wherein the PEG-conjugated lipid is further defined by the formula:
wherein:
R 12 and R 13 Each independently is an alkyl group C≤24 Alkenyl radical C≤24 Or substituted versions of any of these groups;
R e is hydrogen, alkyl C≤8 Or substituted alkyl C≤8 (ii) a And is
x is an integer from 1 to 250.
41. The composition of claim 38, wherein the PEG-conjugated lipid is dimyristoyl-sn-glycerol or a compound of the formula:
wherein:
n 1 is an integer from 5 to 250; and is
n 2 And n 3 Each independently is an integer from 2 to 25.
42. The composition of claim 38, wherein the PEG-conjugated lipid and the dendrimer are present in the composition in a molar ratio of 1:1 to 1:250.
43. The composition of claim 34, wherein the one or more helper lipids are phospholipids.
44. The composition of claim 34, wherein the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
45. The composition according to claim 43, wherein the phospholipid and the dendrimer are present in the composition in a molar ratio of from 10:1 to 1:20.
46. The composition of claim 30, wherein the composition further comprises a steroid, a polyethylene glycol (PEG) conjugated lipid, a phospholipid, or a combination thereof.
47. A pharmaceutical composition comprising:
(A) The dendritic polymer of any one of claims 1 to 29; and
(B) A pharmaceutically acceptable carrier.
48. The pharmaceutical composition according to claim 47, wherein the pharmaceutically acceptable carrier is a solvent or solution.
49. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for administration by: oral, intralipid, intraarterial, intraarticular, intracranial, intradermal, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intraumbilical, intravaginal, intravenous, intravesicular, intravitreal, liposomal, transmucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, buccal, or vaginal.
50. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for intralesional administration.
51. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for intratumoral administration.
52. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for administration by topical administration.
53. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for administration by transdermal administration.
54. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for topical administration.
55. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for administration by: in the form of a paste, by catheter, by lavage, by infusion, by inhalation, by injection or by local infusion.
56. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated for administration by continuous infusion.
57. The pharmaceutical composition of claim 49, wherein the pharmaceutical composition is formulated for intravenous or intra-arterial injection.
58. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is formulated as a unit dose.
59. Use of the composition or pharmaceutical composition of claim 30 or 47 in the preparation of a medicament for modulating gene expression in a cell, wherein the medicament is for use in a method comprising contacting the cell with the medicament under conditions sufficient to cause uptake of the nucleic acid into the cell.
60. The use of claim 59, wherein the contacting is in vitro or ex vivo.
61. The use of claim 59, wherein the contacting is in vivo.
62. The use of claim 59, wherein modulation of gene expression is sufficient to treat a disease or disorder.
63. The use of claim 62, wherein the disease or disorder is cancer.
64. Use of the composition or pharmaceutical composition of claim 30 or 47 in the manufacture of a medicament for treating a disease or disorder in a subject, wherein the medicament is for use in a method comprising administering a pharmaceutically effective amount of the medicament to a subject in need thereof.
65. The use of claim 64, wherein the disease or disorder is cancer.
66. The use of claim 64, wherein the method further comprises administering to the subject one or more additional cancer therapies.
67. The use of claim 66, wherein the cancer therapy is a chemotherapeutic compound, surgery, radiation therapy, or immunotherapy.
68. The use of claim 64, wherein the medicament is administered to the subject once.
69. The use of claim 64, wherein the medicament is administered to the subject two or more times.
70. The use of claim 64, wherein the subject is a mammal.
71. The use of claim 64, wherein the subject is a human.
72. The dendrimer or pharmaceutically acceptable salt thereof according to claim 1, wherein:
the core has the formula:
wherein in formula (IV):
X 3 is-NR 6 -or alkylamino diyl C≤8 Wherein R is 6 Is hydrogen or alkyl C≤8 ；
R 3 And R 4 Each independently is amino, alkylamino C≤12 Dialkylamino group C≤12 Or substituted versions of any of these groups; wherein the substituted form of one or more hydrogen atoms attached to a carbon atom has been independently replaced by-OH, -F, -Cl, -Br, -I, -NH 2 、-NO 2 、-CO 2 H、-CO 2 CH 3 、-CN、-SH、-OCH 3 、-OCH 2 CH 3 、-C(O)CH 3 、-NHCH 3 、-NHCH 2 CH 3 、-N(CH 3 ) 2 、-C(O)NH 2 、-C(O)NHCH 3 、-C(O)N(CH 3 ) 2 、-OC(O)CH 3 、-NHC(O)CH 3 、-S(O) 2 OH or-S (O) 2 NH 2 Replacing;
the degradable diacyl has the formula:
wherein in formula (VII):
A 1 and A 2 is-O-;
Y 3 is an alkanediyl radical C≤12 And is and
R 9 is an alkyl group C≤8 (ii) a And is
Each of the end capping groups is independently selected from:
73. the dendrimer or pharmaceutically acceptable salt thereof according to claim 1, wherein:
the kernel is defined as:
the degradable diacyl has the formula:
wherein in formula (VII):
A 1 and A 2 is-O-;
Y 3 is an alkanediyl radical C≤12 And is and
R 9 is an alkyl group C≤8 (ii) a And is
Each of the end capping groups is independently selected from:
74. the dendrimer or pharmaceutically acceptable salt thereof according to claim 1, wherein:
the kernel is defined as:
and is
Wherein the dendrimer or the pharmaceutically acceptable salt thereof comprises three repeating units.
75. The dendritic polymer of claim 1, or pharmaceutically acceptable salt thereof, wherein:
the kernel is defined as:
and is
Wherein the dendrimer or the pharmaceutically acceptable salt thereof comprises three or four repeating units.
76. The dendrimer or pharmaceutically acceptable salt thereof according to claim 1, wherein:
the kernel is defined as:
and is
Wherein the dendrimer or the pharmaceutically acceptable salt thereof comprises four repeating units.
77. The dendrimer or pharmaceutically acceptable salt thereof according to claim 1, wherein:
the kernel is defined as:
wherein the dendrimer or pharmaceutically acceptable salt thereof comprises four repeating units; and wherein the end capping group is selected from:
78. the dendrimer or pharmaceutically acceptable salt thereof according to claim 1, wherein:
the kernel is defined as:
wherein the dendrimer or pharmaceutically acceptable salt thereof comprises five repeating units; and wherein the end capping group is selected from:
79. the dendrimer or pharmaceutically acceptable salt thereof according to claim 1, wherein:
the kernel is defined as:
wherein the dendrimer or pharmaceutically acceptable salt thereof comprises six repeating units; and wherein the end capping group is selected from:
80. a dendritic polymer selected from the group consisting of:
and pharmaceutically acceptable salts of any of the foregoing.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201562218412P | 2015-09-14 | 2015-09-14 | |
| US62/218,412 | 2015-09-14 | ||
| PCT/US2016/051648 WO2017048789A1 (en) | 2015-09-14 | 2016-09-14 | Lipocationic dendrimers and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1257358A1 HK1257358A1 (en) | 2019-10-18 |
| HK1257358B true HK1257358B (en) | 2023-08-25 |
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