U.S. patent application number 15/264315 was filed with the patent office on 2017-06-01 for amine-containing lipidoids and uses thereof.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Daniel Griffith Anderson, Joseph A. Dorkin, Robert S. Langer, Arturo Jose Vegas, Kathryn Ann Whitehead, Yunlong Zhang.
Application Number | 20170152213 15/264315 |
Document ID | / |
Family ID | 49004050 |
Filed Date | 2017-06-01 |
United States Patent
Application |
20170152213 |
Kind Code |
A1 |
Anderson; Daniel Griffith ;
et al. |
June 1, 2017 |
AMINE-CONTAINING LIPIDOIDS AND USES THEREOF
Abstract
Provided herein are lipidoids that may be prepared from the
conjugate addition of alkylamines to acrylates. In some
embodiments, provided lipidoids are biodegradable and may be used
in a variety of drug delivery systems. Given the amino moiety of
the lipidoids, they are well-suited for the delivery of
polynucleotides, in addition to other agents. Nanoparticles
containing the inventive lipidoids and polynucleotides have been
prepared and have been shown to be effective in delivering
siRNA.
Inventors: |
Anderson; Daniel Griffith;
(Framingham, MA) ; Whitehead; Kathryn Ann;
(Pittsburgh, PA) ; Dorkin; Joseph A.; (Somerville,
MA) ; Vegas; Arturo Jose; (Belmont, MA) ;
Zhang; Yunlong; (Cambridge, MA) ; Langer; Robert
S.; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
49004050 |
Appl. No.: |
15/264315 |
Filed: |
September 13, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14987717 |
Jan 4, 2016 |
9439968 |
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15264315 |
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14089603 |
Nov 25, 2013 |
9227917 |
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14987717 |
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13966136 |
Aug 13, 2013 |
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14089603 |
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61682468 |
Aug 13, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6925 20170801;
A61K 9/5123 20130101; C07C 229/14 20130101; C07D 295/13 20130101;
A61K 47/542 20170801; C07D 295/15 20130101; C12N 2310/14 20130101;
C07D 211/14 20130101; A61K 31/495 20130101; C07C 229/24 20130101;
C07C 229/26 20130101; A61K 31/4985 20130101; C07D 487/04 20130101;
A61K 31/357 20130101; C07D 319/18 20130101; C07D 211/34 20130101;
C07C 229/28 20130101; C07D 211/26 20130101; A61K 31/4985 20130101;
A61K 31/713 20130101; A61K 47/22 20130101; A61K 31/495 20130101;
C07D 319/16 20130101; A61K 31/445 20130101; A61K 31/40 20130101;
A61K 31/573 20130101; A61K 31/573 20130101; C12N 2320/32 20130101;
C07C 229/34 20130101; C12N 15/111 20130101; A61K 31/704 20130101;
A61K 2300/00 20130101; A61K 9/1272 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; C07D 241/04
20130101; C07C 2601/14 20170501; A61K 31/357 20130101; A61K 31/225
20130101; A61K 31/445 20130101; C07D 295/16 20130101; C12N 15/1137
20130101; A61K 31/40 20130101; A61K 47/18 20130101; A61K 47/543
20170801; C12N 15/88 20130101; A61K 31/704 20130101; C07C 225/06
20130101; A61K 31/225 20130101; A61K 31/713 20130101; A61K 45/06
20130101; C07D 207/09 20130101; C07C 229/12 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
International
Class: |
C07C 225/06 20060101
C07C225/06; C07D 319/16 20060101 C07D319/16; C07D 295/16 20060101
C07D295/16; C07D 487/04 20060101 C07D487/04; C07D 207/09 20060101
C07D207/09; C07D 211/34 20060101 C07D211/34 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Nos. F32-EB009623, EB000244, R01CA115527, and R01CA132091 awarded
by the National Institutes of Health. The government has certain
rights in this invention.
Claims
1-126. (canceled)
127. A compound of formula: ##STR00135## or a salt thereof,
wherein: each R.sup.A is, independently, branched or unbranched,
C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, or branched or unbranched,
C.sub.4-12 cycloalkylalkyl, each of which is, independently,
optionally substituted with one or more fluorine radicals; each R
is, independently, hydrogen or --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B;
and each R.sup.B is, independently, branched or unbranched,
C.sub.10-14 alkyl optionally substituted with one or more fluorine
radicals; provided that at least one R group is
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
128. The compound of claim 127, wherein all R.sup.A groups are the
same.
129. The compound of claim 127, wherein the R.sup.A groups are
different.
130. The compound of claim 127, wherein each R.sup.A is,
independently, branched or unbranched, C.sub.1-6 alkyl.
131-132. (canceled)
133. The compound of claim 127, wherein each R.sup.A is,
independently, branched or unbranched, C.sub.1-3 alkyl.
134. (canceled)
135. The compound of claim 127, wherein each R.sup.A is,
independently, C.sub.3-7 cycloalkyl.
136. The compound of claim 135, wherein R.sup.A is cyclohexyl.
137. The compound of claim 127, wherein each R.sup.A is,
independently, branched or unbranched, C.sub.4-12
cycloalkylalkyl.
138. The compound of claim 127, wherein the compound is of formula:
##STR00136## or a salt thereof, wherein each R.sup.B is,
independently, branched or unbranched, C.sub.10-14 alkyl.
139. The compound of claim 127, wherein at least three R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
140. The compound of claim 127, wherein all R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
141. The compound of claim 127, wherein all R.sup.B groups are the
same.
142. The compound of claim 127, wherein each R.sup.B is,
independently, branched or unbranched, C.sub.10-14 alkyl.
143. The compound of claim 127, wherein each R.sup.B is,
independently, unbranched C.sub.10-14 alkyl.
144-146. (canceled)
147. A compound of formula: ##STR00137## ##STR00138## ##STR00139##
##STR00140## ##STR00141## ##STR00142## or a salt thereof.
148. (canceled)
149. A nanoparticle comprising a compound of claim 127, or a salt
thereof, and one or more agents to be delivered.
150-181. (canceled)
182. A composition comprising one or more compounds of claim 127,
or a salt thereof, and an excipient.
183-194. (canceled)
195. A method of administering an agent, the method comprising:
administering to a subject in need thereof a therapeutically
effective amount of a composition comprising: a compound of claim
127, or a salt thereof; and an agent to be delivered.
196-200. (canceled)
201. The compound of claim 127, wherein the compound is of formula:
##STR00143## or a salt thereof.
202. The compound of claim 127, wherein the compound is of formula:
##STR00144## or a salt thereof.
203. The compound of claim 127, wherein the compound is of formula:
##STR00145## or a salt thereof.
204. The compound of claim 127, wherein the compound is of formula:
##STR00146## or a salt thereof.
205. The compound of claim 127, wherein the compound is of formula:
##STR00147## ##STR00148## or a salt thereof.
Description
RELATED REFERENCES
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. provisional application, U.S. Ser. No.
61/682,468, filed Aug. 13, 2012, which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] The discovery of RNA interference (RNAi) in mammalian cells
(Fire, et al. Nature 391:806-811 (1998)) has allowed for the
development of short interfering RNA (siRNA) therapeutics
(Elbashir, et al. Nature 411:494-8 (2001)), which have the
potential to treat a wide variety of human diseases, including
viral infections and cancer, through genetic modulation.
Theoretically, siRNA can be used to alter the expression of nearly
any gene in the body through the silencing of complementary
messenger RNA. Such precise genetic control offers a broad
therapeutic potential that is typically not attainable using
conventional small molecule drugs. siRNA delivery vehicles must
negotiate a number of obstacles in vivo prior to delivering their
payload to target cells. In addition to escorting therapeutic cargo
through the bloodstream and extracellular matrix, delivery vehicles
must mediate siRNA transport across the cellular membrane of the
target cell as well as to facilitate endosomal escape prior to
lysosomal digestion (Akinc, et al. J. Gene. Med. 7:657-63 (2005)).
It is only once these barriers have been breached that siRNA can
interact with the RNAi machinery within the cytoplasm and trigger
the gene silencing process (Whitehead, et al. Nature Rev. Drug
Discov. 8:129-38 (2009)).
[0004] A select number of delivery systems have previously been
reported to deliver siRNA for the treatment of a variety of disease
targets in vivo, including hypercholesterolemia (Frank-Kamenetsky,
et al. Proc. Natl. Acad. Sci. USA 107:1864-9 (2010); Love, et al.
Proc. Natl. Acad. Sci. USA 26:431-42 (2008)), liver cirrhosis
(Sato, et al. Nature Biotechnol. 26:431-42 (2008)), Ebola virus
(Geisbert, et al. Lancet 375:1896-1905 (2010)), and cancer (Huang,
et al. Proc. Natl. Acad. Sci. USA 106:3426-30 (2009)).
Unfortunately, RNAi success in vivo has not consistently translated
to success in the clinic. Because siRNA must be dosed repeatedly to
achieve therapeutic effect, ideal delivery vehicles will offer a
substantial therapeutic window in order to ensure the broadest
clinical application. Although some materials have been identified
that allow for potent gene silencing at siRNA doses as low as 0.01
mg/kg (Love, et al. Proc. Natl. Acad. Sci. USA 107:1864-9 (2010)),
their clinical potential has been limited due to a lack of delivery
vehicle degradability. There exists a continuing need for
non-toxic, biodegradable, biocompatible lipids that can be used to
transfect nucleic acids and other therapeutic agents. Such lipids
would have several uses, including the delivery of siRNA.
SUMMARY OF THE INVENTION
[0005] The compounds described herein, known as lipidoids for their
lipid-like tails, may be prepared by the addition of a primary or
secondary amine to an acrylate via a Michael addition reaction. The
lipidoids described herein may be used in the delivery of
therapeutic agents to a subject. The inventive lipidoids are
particularly useful in delivering negatively charged agents. For
example, lipidoids described herein may be used to deliver DNA,
RNA, or other polynucleotides to a subject or to a cell. In certain
embodiments, lipidoids of the present invention are used to deliver
siRNA. In certain embodiments, lipidoids described herein are
useful as reagents.
[0006] In one aspect, the present invention provides a compound of
the Formula (I):
##STR00001##
or a salt thereof, wherein L, R, R.sup.A, and q are as defined
herein. In certain embodiments, a provided compound is of the
Formula (I-a), (I-b), (I-c), (I-d), or (I-e):
##STR00002##
or a salt thereof, wherein m, n, R, and R.sup.A are as defined
herein.
[0007] In another aspect, the present invention provides a compound
of the Formula (II):
##STR00003##
or a salt thereof, wherein L, R.sup.C, and R.sup.A are as defined
herein. In certain embodiments, a provided compound is of the
Formula (II-a), (II-b), (II-c), (II-d), (II-e), or (II-f):
##STR00004##
or a salt thereof, wherein v, L, R, R.sup.D, and R.sup.A are as
defined herein.
[0008] In another aspect, the present invention provides a compound
of the Formula (III):
##STR00005##
or a salt thereof, wherein p, R.sup.1, j, and R are as defined
herein. In certain embodiments, a provided compound is of the
Formula (III-a), (III-b), (III-c), (III-d), or (III-e):
##STR00006##
or a salt thereof, wherein w, p, R.sup.1, j, and R are as defined
herein.
[0009] In another aspect, the present invention provides a compound
of Formula (IV)
##STR00007##
or a salt thereof, wherein R, x, and y are as defined herein. In
certain embodiments, a provided compound is of the Formula
(IV-a):
##STR00008##
or a salt thereof, wherein R is as defined herein.
[0010] In another aspect, the present invention provides a compound
of Formula (V):
##STR00009##
or a salt thereof, wherein L, R.sup.2, g, and R are as defined
herein. In certain embodiments, a provided compound is of the
Formula (V-a), (V-b), (V-c), or (V-d):
##STR00010##
or a salt thereof, wherein L, R.sup.2, g, and R are as defined
herein.
[0011] In another aspect, the present invention provides a compound
of formula
##STR00011##
or a salt thereof, wherein R and R.sup.A are as defined herein.
[0012] In another aspect, the present invention provides lipidoids
having certain features. In some embodiments, a lipidoid of the
present invention is prepared from an alkylamine starting material
that has at least one tertiary amine. In some embodiments, a
lipidoid of the present invention has three or more lipid-like
tails. In some embodiments, the lipid-like tails on a lipidoid of
the present invention are between C.sub.12-C.sub.14 in length,
e.g., C.sub.13 (e.g., derived from the O.sub.13 acrylate shown in
FIG. 1). In certain embodiments, a provided lipidoid is prepared
from an alkylamine starting material that has at least one tertiary
amine and has three or more C.sub.13 tails.
[0013] In another aspect, the inventive lipidoids are combined with
an agent to form nanoparticles, microparticles, liposomes, or
micelles. The agent to be delivered by the nanoparticles,
microparticles, liposomes, or micelles may be in the form of a gas,
liquid, or solid, and the agent may be, for example, a
polynucleotide, protein, peptide, or small molecule. In certain
embodiments, inventive lipidoids may be combined with other lipids,
polymers, surfactants, cholesterol, carbohydrates, proteins, etc.
to form the particles. In certain embodiments, the particles may be
combined with an excipient to form pharmaceutical or cosmetic
compositions.
DEFINITIONS
[0014] Definitions of specific functional groups and chemical terms
are described in more detail below. For purposes of this invention,
the chemical elements are identified in accordance with the
Periodic Table of the Elements, CAS version, Handbook of Chemistry
and Physics, 75.sup.th Ed., inside cover, and specific functional
groups are generally defined as described therein. Additionally,
general principles of organic chemistry, as well as specific
functional moieties and reactivity, are described in "Organic
Chemistry", Thomas Sorrell, University Science Books, Sausalito:
1999, the entire contents of which are incorporated herein by
reference.
[0015] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0016] Isomeric mixtures containing any of a variety of isomer
ratios may be utilized in accordance with the present invention.
For example, where only two isomers are combined, mixtures
containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3,
98:2, 99:1, or 100:0 isomer ratios are all contemplated by the
present invention. Those of ordinary skill in the art will readily
appreciate that analogous ratios are contemplated for more complex
isomer mixtures.
[0017] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts are formed with an appropriate
optically-active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0018] Unless otherwise stated, structures depicted herein are also
meant to include compounds that differ only in the presence of one
or more isotopically enriched atoms. For example, compounds having
the present structures except for the replacement of hydrogen by
deuterium or tritium, or the replacement of a carbon by a .sup.13C-
or .sup.14C-enriched carbon are within the scope of this invention.
Such compounds are useful, for example, as analytical tools or
probes in biological assays.
[0019] The term "aliphatic," as used herein, includes both
saturated and unsaturated, nonaromatic, straight chain (i.e.,
unbranched), branched, acyclic, and cyclic (i.e., carbocyclic)
hydrocarbons. In some embodiments, an aliphatic group is optionally
substituted with one or more functional groups. As will be
appreciated by one of ordinary skill in the art, "aliphatic" is
intended herein to include alkyl, alkenyl, alkynyl, cycloalkyl, and
cycloalkenyl moieties.
[0020] The term "alkyl" as used herein refers to saturated,
straight- or branched-chain hydrocarbon radicals derived from a
hydrocarbon moiety containing between one and twenty carbon atoms
by removal of a single hydrogen atom. Examples of alkyl radicals
include, but are not limited to, methyl, ethyl, propyl, isopropyl,
n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl,
n-octyl, n-decyl, n-undecyl, and dodecyl.
[0021] In certain embodiments, the alkyl groups employed in the
inventive lipidoids contain 1-20 aliphatic carbon atoms. In certain
other embodiments, the alkyl groups employed in the inventive
lipidoids contain 1-10 aliphatic carbon atoms. In yet other
embodiments, the alkyl groups contain 1-8 aliphatic carbon atoms.
In still other embodiments, the alkyl groups employed in the
invention contain 1-6 aliphatic carbon atoms. In yet other
embodiments, the alkyl groups contain 1-4 carbon atoms.
Illustrative alkyl groups thus include, but are not limited to, for
example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl,
n-hexyl, and sec-hexyl.
[0022] The terms "alkenyl" and "alkynyl" are given their ordinary
meaning in the art and refer to unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but that contain at least one double or triple
bond respectively.
[0023] The term "cycloalkyl", as used herein, refers saturated,
cyclic hydrocarbon radicals derived from a hydrocarbon moiety
containing between three and seven carbon atoms by removal of a
single hydrogen atom. Suitable cycloalkyls include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
[0024] The term "cycloalkylalkyl," as used herein, refers to a
cycloalkyl group attached via a straight chain or branched alkyl
group. Suitable cycloalkylalkyl groups include, but are not limited
to, --CH.sub.2(cyclopropyl), --CH.sub.2CH.sub.2(cyclopropyl),
--CH.sub.2(cyclobutyl), --CH.sub.2CH.sub.2(cyclobutyl),
--CH.sub.2(cyclopentyl), --CH.sub.2CH.sub.2(cyclopentyl),
--CH.sub.2(cyclohexyl), --CH.sub.2CH.sub.2(cyclohexyl),
--CH.sub.2(cycloheptyl), and --CH.sub.2CH.sub.2(cycloheptyl).
[0025] The term "alkylene" as used herein refers to a bivalent
alkyl group. An "alkylene" group is a polymethylene group, i.e.,
--(CH.sub.2).sub.k--, wherein k is a positive integer, e.g., from 1
to 20, from 1 to 10, from 1 to 6, from 1 to 4, from 1 to 3, from 1
to 2, or from 2 to 3. In some embodiments, one or more hydrogens on
an alkylene group is replaced by a substituent (e.g., fluoro).
[0026] The following are more general terms used throughout the
present application:
[0027] "Animal": The term animal, as used herein, refers to humans
as well as non-human animals, including, for example, mammals,
birds, reptiles, amphibians, and fish. Preferably, the non-human
animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a
monkey, a dog, a cat, a primate, or a pig). An animal may be a
transgenic animal.
[0028] "Associated with": When two entities are "associated with"
one another as described herein, they are linked by a direct or
indirect covalent or non-covalent interaction. Preferably, the
association is covalent. Desirable non-covalent interactions
include hydrogen bonding, van der Waals interactions, hydrophobic
interactions, magnetic interactions, electrostatic interactions,
etc.
[0029] "Biocompatible": The term "biocompatible", as used herein is
intended to describe compounds that are not toxic to cells. In
certain embodiments, compounds are "biocompatible" if their
addition to cells in vitro at a minimum therapeutically effective
dose results in less than or equal to 20% cell death, and their
administration in vivo does not induce inflammation or other such
adverse effects.
[0030] "Biodegradable": As used herein, "biodegradable" compounds
are those that, when introduced into cells, are broken down by the
cellular machinery or by hydrolysis into components that the cells
can either reuse or dispose of without significant long-term toxic
effect on the cells. In certain embodiments, the components do not
induce inflammation or other adverse effects in vivo. In certain
embodiments, the chemical reactions relied upon to break down the
biodegradable compounds are uncatalyzed.
[0031] "Peptide" or "protein": According to the present invention,
a "peptide" or "protein" comprises a string of at least three amino
acids linked together by peptide bonds. The terms "protein" and
"peptide" may be used interchangeably. Peptide may refer to an
individual peptide or a collection of peptides. Inventive peptides
preferably contain only natural amino acids, although non-natural
amino acids (i.e., compounds that do not occur in nature but that
can be incorporated into a polypeptide chain) and/or amino acid
analogs as are known in the art may alternatively be employed.
Also, one or more of the amino acids in an inventive peptide may be
modified, for example, by the addition of a chemical entity such as
a carbohydrate group, a phosphate group, a farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for conjugation,
functionalization, or other modification, etc. In a preferred
embodiment, the modifications of the peptide lead to a more stable
peptide (e.g., greater half-life in vivo). These modifications may
include cyclization of the peptide, the incorporation of D-amino
acids, etc. None of the modifications should substantially
interfere with the desired biological activity of the peptide.
[0032] "Polynucleotide" or "oligonucleotide": Polynucleotide or
oligonucleotide refers to a polymer of nucleotides. Typically, a
polynucleotide comprises at least three nucleotides. The polymer
may include natural nucleosides (i.e., adenosine, thymidine,
guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,
deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g.,
2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,
3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and
2-thiocytidine), chemically modified bases, biologically modified
bases (e.g., methylated bases), intercalated bases, modified sugars
(e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and
hexose), or modified phosphate groups (e.g., phosphorothioates and
5'-N-phosphoramidite linkages).
[0033] "Small molecule": As used herein, the term "small molecule"
refers to organic compounds, whether naturally-occurring or
artificially created (e.g., via chemical synthesis) that have
relatively low molecular weight and that are not proteins,
polypeptides, or nucleic acids. Typically, small molecules have a
molecular weight of less than about 1500 g/mol. Also, small
molecules typically have multiple carbon-carbon bonds. Known
naturally-occurring small molecules include, but are not limited
to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin.
Known synthetic small molecules include, but are not limited to,
ampicillin, methicillin, sulfamethoxazole, sulfonamides,
dexamethasone, and doxorubicin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 displays a subset of the large library of
biodegradable lipidoids that were synthesized combinatorially
through the conjugate addition of alkylamines (in red) to
alkyl-acrylate tails (in blue). The rest of the alkylamines used in
lipidoid library synthesis are shown in FIG. 2.
[0035] FIG. 2 shows additional alkylamines used in the lipidoid
library.
[0036] FIG. 3 shows the evaluation of lipidoids for an ability to
deliver siRNA to HeLa cells. (a) Relative luciferase activity
values (firefly lucifase activity normalized to control Renilla
luciferase activity) are shown for 1400 lipidoids. .about.7% of the
library induced >50% gene silencing (shown in red). The tail
length (b), tail substitution number (c) and alkyl-amine
composition (d) influenced in vitro activity.
[0037] FIG. 4 demonstrates that select lipidoids induced a high
degree silencing of multiple targets in mice. (a) Of the .about.100
lipidoids tested in vivo, 15 induced complete Factor VII knockdown
in mouse hepatocytes at a total siRNA dose of 5 mg/kg (data points
in red). (b) The EC.sub.50 values of these top 15 lipidoids ranged
from 0.05 to 1.5 mg/kg under standard formulation conditions. (c)
The amount of PEG in the lipid nanoparticle formulation had a
dramatic effect on efficacy. Data is shown for the lipidoid
304O.sub.14, which produced the most efficacious formulation of the
study when formulated with 0.75% PEG. (d) Dose response and Factor
VII activity recovery data for the optimized 304O.sub.13 lipid
nanoparticle formulation. 304O.sub.13 also induced CD45 silencing
in monocyte and macrophage (CD11b+) populations in the peritoneal
cavity (e) as well as in dendritic cells (CD11c+) in the spleen 3
days post-injection. In all panels, error bars represent standard
deviation (n=3).
[0038] FIG. 5 shows biodistribution images for Cy5.5-labeled siRNA
delivered with the lipidoid 304O.sub.13. IVIS (a) and Odyssey (b)
imaging show that, while naked siRNA is primarily cleared through
the kidneys, 304O.sub.13 mediates accumulation in the liver and
spleen. (c) Confocal microscopy of 304O.sub.13-treated liver shows
siRNA (red) delivery into nearly all cells, including Kupffer cells
and hepatocytes. In contrast, naked siRNA had a limited penetration
depth from the blood vessels into hepatocellular tissue. (d)
304O.sub.13 lipid nanoparticles were rapidly eliminated from the
bloodstream after tail vein injection. Error bars represent
standard deviation (n=3).
[0039] FIG. 6 shows a comparison of (a) cytokine profiles 4 hours
post-injection and (b) liver histology sections 72 hours
post-injection for degradable (304O.sub.13) and non-degradable
(C12-200) lipid nanoparticles.
[0040] FIG. 7 displays structure-function information of
efficacious lipid nanoparticles. (a) Of the 108 lipid nanoparticles
tested for siRNA delivery to hepatocytes in mice, 66 had 3 or more
tails, 42 had a tertiary amine present in the original alkyl-amine,
and 25 had an O.sub.13 tail length. 88% of the lipid nanoparticles
exhibiting all three "efficacy criteria" achieved complete FVII
knockdown. The percentage of efficacious lipid nanoparticles
decreased precipitously when any criterion were not met. (b) Twelve
second generation lipid nanoparticles were made to meet all
efficacy criteria by first synthesizing custom alkyl-amines and
reacting them with O.sub.13 tails. (c) 83% of second generation
LNPs achieved complete FVII silencing in vivo, and (d) EC.sub.50s
under non-optimized LNP formulating conditions ranged from 0.05 to
1 mg/kg total siRNA. (e) 503O.sub.13 was the most efficacious LNP
upon formulation, with an EC.sub.50 of 0.01 mg/kg. 503O.sub.13
encapsulating control siRNA did not result in FVII knockdown. Error
bars represent standard deviation (n=3).
[0041] FIG. 8 shows that lipid nanoparticles that induced complete
FVII silencing at 5 mg/kg behaved in a dose-dependent manner. Each
lipid nanoparticle was evaluated at three additional doses (2, 0.5,
and 0.1 mg/kg) shown from left to right. Error bars represent
standard deviation (n=3).
[0042] FIG. 9 shows that the lipid nanoparticles 306O.sub.12,
306O.sub.14 and 315O.sub.12 facilitated modest silencing of the
surface receptor CD45 in various white blood cell populations
harvested from the peritoneal cavity (left) and spleen (right) of
B6 mice three days post-injection (dose=2.5 mg/kg total siRNA).
Error bars represent standard deviation (n=3).
[0043] FIG. 10 shows that pKa values significantly influence
delivery efficacy to hepatocytes in vivo. All lipidoid
nanoparticles capable of mediating complete Factor VII gene
silencing had pKa values greater or equal to 5.5.
[0044] FIGS. 11A and 11B show degradation by hydrolysis of the
lipidoid 304O.sub.13. Overlay of .sup.1H NMR spectra of the
starting material 304O.sub.13, the crude reaction mixture, and
authentic 1-tridecanol demonstrated that the 304O.sub.13 had been
consumed and that tridecanol had been formed in significant
quantity under both acidic and basic conditions. FIG. 11A shows
acidic hydrolysis condition, and FIG. 11B shows basic hydrolysis
condition.
[0045] FIG. 12 shows that clinical chemistry parameters were
evaluated for negative control (PBS, black), 304O.sub.13 (blue),
and C12-200 (red) groups of C57BL/6 mice. The mice had been
injected with either a single 3 mg/kg dose of total siRNA or four 3
mg/kg doses (lx per week for four weeks). Blood was drawn for
analysis 72 hours post-final injection. There were no statistically
significant changes in any of the clinical chemistry parameters for
any of the treated groups compared to controls (as evaluated by a
student t-test). Error bars represent standard deviation
(n=3-5).
[0046] FIG. 13 shows that the second generation lipid nanoparticles
(LNPs) facilitated silencing of the surface receptor CD45 in
various white blood cell populations harvested from the peritoneal
cavity (left) and spleen (right) of B6 mice three days
post-injection (dose=2.5 mg/kg total siRNA). Percent silencing was
calculated by comparing to an identically defined cell population
from animals injected with a non-targeting siRNA formulated with
the same LNP. Error bars represent standard deviation (n=3).
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0047] The present invention provides lipidoids and lipidoid-based
delivery systems. The systems described herein may be used in the
pharmaceutical/drug delivery arts to delivery polynucleotides,
proteins, small molecules, peptides, antigen, drugs, etc. to a
patient, tissue, organ, cell, etc.
[0048] The lipidoids of the present invention provide for several
different uses in the drug delivery art. The lipidoids with their
amine-containing hydrophilic portion may be used to complex
polynucleotides and thereby enhance the delivery of polynucleotides
and prevent their degradation. The lipidoids may also be used in
the formation of nanoparticles, microparticles, liposomes, and
micelles containing the agent to be delivered. In certain
embodiments, the lipids are biocompatible and biodegradable, and
particles formed therefrom are also biodegradable and biocompatible
and may be used to provide controlled, sustained release of the
agent. Provided lipidoids and their corresponding particles may
also be responsive to pH changes given that these lipids are
protonated at lower pH.
Lipidoids
[0049] The lipidoids of the present invention contain primary,
secondary, or tertiary amines and salts thereof. In certain
embodiments, the inventive lipidoids are biodegradable. In certain
embodiments, inventive lipidoids are effective at delivering an
agent (e.g., RNA) to a cell.
[0050] In certain embodiments, a lipidoid of the present invention
is prepared from an alkylamine starting material that has at least
one tertiary amine. In some embodiments, a lipidoid of the present
invention has three or more lipid-like tails. In some embodiments,
the lipid-like tails on a lipidoid of the present invention are
between C.sub.10-C.sub.14 in length, e.g., C.sub.12-C.sub.14, e.g.,
C.sub.13. In certain embodiments, a provided lipidoid is prepared
from an alkylamine starting material that has at least one tertiary
amine and the lipidoid formed therefrom has three or more C.sub.13
tails. In certain embodiments, a provided lipidoid is prepared from
an alkylamine starting material that has at least one tertiary
amine, provided that the amine is not amine 110, amine 113, or
amine 115, and the lipidoid formed therefrom has three or more
C.sub.13 tails.
##STR00012##
[0051] In certain embodiments, a lipidoid of the present invention
is of the Formula (I):
##STR00013##
or a salt thereof, wherein
[0052] each L is, independently, branched or unbranched C.sub.1-6
alkylene, wherein L is optionally substituted with one or more
fluorine radicals;
[0053] each R.sup.A is, independently, branched or unbranched
C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, or branched or unbranched
C.sub.4-12 cycloalkylalkyl, wherein R.sup.A is optionally
substituted with one or more fluorine radicals;
[0054] each R is, independently, hydrogen or
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B;
[0055] each R.sup.B is, independently, C.sub.10-14 alkyl, wherein
R.sup.B is optionally substituted with one or more fluorine
radicals; and
[0056] q is 1, 2, or 3.
[0057] In certain embodiments, a lipidoid of formula (I) is not
##STR00014##
[0058] As defined generally above, each L is, independently,
branched or unbranched C.sub.1-6 alkylene, wherein L is optionally
substituted with one or more fluorine radicals. In some
embodiments, L is substituted with one or more fluorine radicals.
In other embodiments, L is unsubstituted. In some embodiments, L is
branched. In other embodiments, L is unbranched. In certain
embodiments, L is C.sub.1-4 alkylene. In certain embodiments, L is
methylene, ethylene, or propylene.
[0059] As defined generally above, each R.sup.A is, independently,
branched or unbranched C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, or
branched or unbranched C.sub.4-12 cycloalkylalkyl, wherein R.sup.A
is optionally substituted with one or more fluorine radicals. In
some embodiments, R.sup.A is substituted with one or more fluorine
radicals. For example, when R.sup.A is methyl, it may be
substituted with one, two, or three fluorine radicals to give
--CH.sub.2F, --CHF.sub.2, or --CF.sub.3. In other embodiments,
R.sup.A is unsubstituted. In some embodiments, all R.sup.A groups
are the same. In other embodiments, the R.sup.A groups are
different. In some embodiments, R.sup.A is branched or unbranched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is branched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is unbranched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is C.sub.1-3
alkyl. In certain embodiments, R.sup.A is methyl, ethyl, or propyl.
In certain embodiments, R.sup.A is C.sub.3-7 cycloalkyl. In certain
embodiments, R.sup.A is cyclohexyl. In certain embodiments, R.sup.A
is cyclopropyl, cyclobutyl, or cyclopentyl. In certain embodiments,
R.sup.A is cycloheptyl. In some embodiments, R.sup.A is branched or
unbranched C.sub.4-12 cycloalkylalkyl.
[0060] As defined generally above, each R is, independently,
hydrogen or --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least three R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
four R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, all R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
[0061] As defined generally above, each R.sup.B is, independently,
C.sub.10-14 alkyl, wherein R.sup.B is optionally substituted with
one or more fluorine radicals. In some embodiments, R.sup.B is
substituted with one or more fluorine radicals. For example, in
some embodiments, R.sup.B may be substituted with one fluoro, or in
other embodiments, may be perfluorinated. In other embodiments,
R.sup.B is unsubstituted. In some embodiments, all R.sup.B groups
are the same. In certain embodiments, R.sup.B is C.sub.10 alkyl. In
some embodiments, R.sup.B is n-decyl. In certain embodiments,
R.sup.B is C.sub.11 alkyl. In some embodiments, R.sup.B is
n-undecyl. In certain embodiments, R.sup.B is C.sub.12 alkyl. In
some embodiments, R.sup.B is n-dodecyl. In certain embodiments,
R.sup.B is C.sub.13 alkyl. In some embodiments, R.sup.B is
n-tridecyl. In certain embodiments, R.sup.B is C.sub.14 alkyl. In
some embodiments, R.sup.B is n-tetradecyl.
[0062] As defined generally above, q is 1, 2, or 3. In some
embodiments, q is 1. In some embodiments, q is 2. In some
embodiments, q is 3.
[0063] In some embodiments, a lipidoid of the present invention is
of the Formula (I-a):
##STR00015##
or a salt thereof, wherein R and R.sup.A are as defined above and
described herein;
[0064] each n is, independently, 0, 1, or 2; and
[0065] m is 0, 1, or 2.
[0066] In some embodiments, m is 0. In some embodiments, m is 1. In
some embodiments, m is 2.
[0067] In some embodiments, n is 0. In some embodiments, n is 1. In
some embodiments, n is 2.
[0068] In some embodiments, m is 0, and n is 0. In some
embodiments, m is 1, and n is 0. In some embodiments, m is 2, and n
is 0. In some embodiments, m is 0, and n is 1. In some embodiments,
m is 0, and n is 2. In some embodiments, m is 1, and n is 1.
[0069] In some embodiments, a lipidoid of the present invention is
of the Formula (I-b):
##STR00016##
or a salt thereof, wherein R and R.sup.A are as defined above and
described herein.
[0070] In some embodiments, a lipidoid of the present invention is
of the Formula (I-c):
##STR00017##
or a salt thereof, wherein R and R.sup.A are as defined above and
described herein.
[0071] In some embodiments, a lipidoid of the present invention is
of the Formula (I-d):
##STR00018##
or a salt thereof, wherein R and R.sup.A are as defined above and
described herein.
[0072] In some embodiments, a lipidoid of the present invention is
of the Formula (I-e):
##STR00019##
or a salt thereof, wherein R and R.sup.A are as defined above and
described herein.
[0073] In some embodiments, a lipidoid of the present invention is
of one of the following formulae:
##STR00020## ##STR00021##
[0074] In certain embodiments, a lipidoid of the present invention
is of the Formula (II):
##STR00022##
or a salt thereof, wherein
[0075] each L is, independently, branched or unbranched C.sub.1-6
alkylene, wherein L is optionally substituted with one or more
fluorine radicals;
[0076] each R.sup.A is, independently, branched or unbranched
C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, or branched or unbranched
C.sub.4-12 cycloalkylalkyl, wherein R.sup.A is optionally
substituted with one or more fluorine radicals;
[0077] each R.sup.C is, independently, -L-N(R.sup.D).sub.2 or
--R;
[0078] each R is, independently, hydrogen or
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B;
[0079] each R.sup.D is, independently, --R.sup.A or --R; and
each R.sup.B is, independently, C.sub.10-14 alkyl, wherein R.sup.B
is optionally substituted with one or more fluorine radicals.
[0080] As defined generally above, each L is, independently,
branched or unbranched C.sub.1-6 alkylene, wherein L is optionally
substituted with one or more fluorine radicals. In some
embodiments, L is substituted with one or more fluorine radicals.
In other embodiments, L is unsubstituted. In some embodiments, L is
branched. In other embodiments, L is unbranched. In certain
embodiments, L is C.sub.1-4 alkylene. In certain embodiments, L is
methylene, ethylene, or propylene.
[0081] As defined generally above, each R.sup.A is, independently,
branched or unbranched C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, or
branched or unbranched C.sub.4-12 cycloalkylalkyl, wherein R.sup.A
is optionally substituted with one or more fluorine radicals. In
some embodiments, R.sup.A is substituted with one or more fluorine
radicals. For example, when R.sup.A is methyl, it may be
substituted with one, two, or three fluorine radicals to give
--CH.sub.2F, --CHF.sub.2, or --CF.sub.3. In other embodiments,
R.sup.A is unsubstituted. In some embodiments, all R.sup.A groups
are the same. In other embodiments, the R.sup.A groups are
different. In some embodiments, R.sup.A is branched or unbranched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is branched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is unbranched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is C.sub.1-3
alkyl. In certain embodiments, R.sup.A is methyl, ethyl, or propyl.
In certain embodiments, R.sup.A is C.sub.3-7 cycloalkyl. In certain
embodiments, R.sup.A is cyclohexyl. In certain embodiments, R.sup.A
is cyclopropyl, cyclobutyl, or cyclopentyl. In certain embodiments,
R.sup.A is cycloheptyl. In some embodiments, R.sup.A is branched or
unbranched C.sub.4-12 cycloalkylalkyl.
[0082] As defined generally above, each R.sup.C is, independently,
-L-N(R.sup.D).sub.2 or --R. In some embodiments, all R.sup.C groups
are --R. In some embodiments, R.sup.C is -L-N(R.sup.D).sub.2.
[0083] As defined generally above, each R.sup.D is, independently,
--R.sup.A or --R. In some embodiments, all R.sup.D groups are --R.
In some embodiments, one R.sup.D on a nitrogen is --R, and the
other is --R.sup.A.
[0084] As defined generally above, each R is, independently,
hydrogen or --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least one R group is
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
two R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least three R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
four R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, all R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
[0085] As defined generally above, each R.sup.B is, independently,
C.sub.10-14 alkyl, wherein R.sup.B is optionally substituted with
one or more fluorine radicals. In some embodiments, R.sup.B is
substituted with one or more fluorine radicals. For example, in
some embodiments, R.sup.B may be substituted with one fluoro, or in
other embodiments, may be perfluorinated. In other embodiments,
R.sup.B is unsubstituted. In some embodiments, all R.sup.B groups
are the same. In certain embodiments, R.sup.B is C.sub.10 alkyl. In
some embodiments, R.sup.B is n-decyl. In certain embodiments,
R.sup.B is C.sub.11 alkyl. In some embodiments, R.sup.B is
n-undecyl. In certain embodiments, R.sup.B is C.sub.12 alkyl. In
some embodiments, R.sup.B is n-dodecyl. In certain embodiments,
R.sup.B is C.sub.13 alkyl. In some embodiments, R.sup.B is
n-tridecyl. In certain embodiments, R.sup.B is C.sub.14 alkyl. In
some embodiments, R.sup.B is n-tetradecyl.
[0086] In certain embodiments, a lipidoid of the present invention
is of the Formula (II-a):
##STR00023##
or a salt thereof, wherein
[0087] each v is, independently, 1, 2, or 3.
[0088] In certain embodiments, v is 1. In certain embodiments, v is
2. In certain embodiments, v is 3.
[0089] In certain embodiments, a lipidoid of the present invention
is of the Formula (II-b):
##STR00024##
or a salt thereof, wherein R.sup.A and R.sup.B are as defined above
and described herein.
[0090] In certain embodiments, a lipidoid of the present invention
is of the formula:
##STR00025##
or a salt thereof, wherein R.sup.B is as defined above and
described herein.
[0091] In certain embodiments, a lipidoid of the present invention
is of the Formula (II-c):
##STR00026##
or a salt thereof, wherein L, R.sup.A, and R.sup.D are as defined
above and described herein.
[0092] In certain embodiments, a lipidoid of the present invention
is of the Formula (II-d):
##STR00027##
or a salt thereof, wherein L, R.sup.A, and R are as defined above
and described herein.
[0093] In certain embodiments, a lipidoid of the present invention
is of the Formula (II-e):
##STR00028##
or a salt thereof, wherein L, R.sup.A, and R are as defined above
and described herein.
[0094] In certain embodiments, a lipidoid of the present invention
is of the Formula (II-f):
##STR00029##
or a salt thereof, wherein R.sup.A and R are as defined above and
described herein.
[0095] In certain embodiments, a lipidoid of the present invention
is of the formula:
##STR00030##
or a salt thereof, wherein R is as defined above and described
herein.
[0096] In certain embodiments, a lipidoid of the present invention
is of the Formula (III):
##STR00031##
or a salt thereof, wherein
[0097] each L is, independently, branched or unbranched C.sub.1-6
alkylene, wherein L is optionally substituted with one or more
fluorine radicals;
[0098] each R is, independently, hydrogen or
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B;
[0099] each R.sup.B is, independently, C.sub.10-14 alkyl, wherein
R.sup.B is optionally substituted with one or more fluorine
radicals;
[0100] each R.sup.1 is, independently, fluoro or C.sub.1-6 alkyl
optionally substituted with one or more fluorine radicals;
[0101] j is 0, 1, 2, 3, or 4; and
[0102] p is 1 or 2.
[0103] In certain embodiments, at least three R groups of formula
(III) are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
[0104] As defined generally above, each L is, independently,
branched or unbranched C.sub.1-6 alkylene, wherein L is optionally
substituted with one or more fluorine radicals. In some
embodiments, L is substituted with one or more fluorine radicals.
In other embodiments, L is unsubstituted. In some embodiments, L is
branched. In other embodiments, L is unbranched. In certain
embodiments, L is C.sub.1-4 alkylene. In certain embodiments, L is
methylene, ethylene, or propylene.
[0105] As defined generally above, each R is, independently,
hydrogen or --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least three R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
four R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, all R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
[0106] As defined generally above, each R.sup.B is, independently,
C.sub.10-14 alkyl, wherein R.sup.B is optionally substituted with
one or more fluorine radicals. In some embodiments, R.sup.B is
substituted with one or more fluorine radicals. For example, in
some embodiments, R.sup.B may be substituted with one fluoro, or in
other embodiments, may be perfluorinated. In other embodiments,
R.sup.B is unsubstituted. In some embodiments, all R.sup.B groups
are the same. In certain embodiments, R.sup.B is C.sub.10 alkyl. In
some embodiments, R.sup.B is n-decyl. In certain embodiments,
R.sup.B is C.sub.11 alkyl. In some embodiments, R.sup.B is
n-undecyl. In certain embodiments, R.sup.B is C.sub.12 alkyl. In
some embodiments, R.sup.B is n-dodecyl. In certain embodiments,
R.sup.B is C.sub.13 alkyl. In some embodiments, R.sup.B is
n-tridecyl. In certain embodiments, R.sup.B is C.sub.14 alkyl. In
some embodiments, R.sup.B is n-tetradecyl.
[0107] In certain embodiments, p is 1. In certain embodiments, p is
2.
[0108] As defined generally above, each R.sup.1 is, independently,
fluoro or C.sub.1-6 alkyl optionally substituted with one or more
fluorine radicals. In some embodiments, R.sup.1 is fluoro. In some
embodiments, R.sup.1 is C.sub.1-6 alkyl optionally substituted with
one or more fluorine radicals. In some embodiments, R.sup.1 is
unsubstituted C.sub.1-6 alkyl. In some embodiments, R.sup.1 is
methyl or ethyl. In some embodiments, R.sup.1 is --CF.sub.3.
[0109] In some embodiments, j is 0. In some embodiments, j is 1. In
some embodiments, j is 2. In some embodiments, j is 3. In some
embodiments, j is 4.
[0110] In certain embodiments, a lipidoid of the present invention
is of the Formula (III-a):
##STR00032##
or a salt thereof, wherein p, R.sup.1, j, and R are as defined
above and described herein, and
[0111] each w is, independently, 1, 2, or 3.
[0112] In certain embodiments, w is 1. In certain embodiments, w is
2. In certain embodiments, w is 3.
[0113] In certain embodiments, a lipidoid of the present invention
is of the Formula (III-b):
##STR00033##
or a salt thereof, wherein R.sup.1, j, w, and R are as defined
above and described herein.
[0114] In certain embodiments, a lipidoid of the present invention
is of the Formula (III-c):
##STR00034##
wherein w and R are as defined above and described herein.
[0115] In certain embodiments, a lipidoid of the present invention
is of the Formula (III-d):
##STR00035##
wherein w, R.sup.1, and R are as defined above and described
herein.
[0116] In certain embodiments, a lipidoid of the present invention
is of the Formula (III-e):
##STR00036##
wherein w, R.sup.1, and R are as defined above and described
herein.
[0117] In certain embodiments, a lipidoid of the present invention
is of the formula:
##STR00037##
wherein R is as defined above and described herein.
[0118] In certain embodiments, a lipidoid of the present invention
is of the Formula (IV):
##STR00038##
or a salt thereof, wherein
[0119] each R is, independently, hydrogen or
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B;
[0120] each R.sup.B is, independently, C.sub.10-14 alkyl, wherein
R.sup.B is optionally substituted with one or more fluorine
radicals;
[0121] x is 1 or 2; and
[0122] y is 1 or 2.
[0123] As defined generally above, each R is, independently,
hydrogen or --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least one R group is
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
two R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least three R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
four R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, all R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
[0124] As defined generally above, each R.sup.B is, independently,
C.sub.10-14 alkyl, wherein R.sup.B is optionally substituted with
one or more fluorine radicals. In some embodiments, R.sup.B is
substituted with one or more fluorine radicals. For example, in
some embodiments, R.sup.B may be substituted with one fluoro, or in
other embodiments, may be perfluorinated. In other embodiments,
R.sup.B is unsubstituted. In some embodiments, all R.sup.B groups
are the same. In certain embodiments, R.sup.B is C.sub.10 alkyl. In
some embodiments, R.sup.B is n-decyl. In certain embodiments,
R.sup.B is C.sub.11 alkyl. In some embodiments, R.sup.B is
n-undecyl. In certain embodiments, R.sup.B is C.sub.12 alkyl. In
some embodiments, R.sup.B is n-dodecyl. In certain embodiments,
R.sup.B is C.sub.13 alkyl. In some embodiments, R.sup.B is
n-tridecyl. In certain embodiments, R.sup.B is C.sub.14 alkyl. In
some embodiments, R.sup.B is n-tetradecyl.
[0125] In some embodiments, x is 1. In some embodiments, x is 2. In
some embodiments, y is 1. In some embodiments, y is 2. In some
embodiments, x is 1 and y is 1. In some embodiments, x is 2 and y
is 2.
[0126] In certain embodiments, a lipidoid of the present invention
is of the Formula (IV-a):
##STR00039##
or a salt thereof, wherein R is as defined above and described
herein.
[0127] In certain embodiments, a lipidoid of the present invention
is of the Formula (V):
##STR00040##
or a salt thereof, wherein
[0128] each L is, independently, branched or unbranched C.sub.1-6
alkylene, wherein L is optionally substituted with one or more
fluorine radicals;
[0129] each R.sup.2 is, independently, halo, C.sub.1-6 aliphatic
optionally substituted with one or more fluorine radicals,
--OR.sup.x, --N(R.sup.y).sub.2, --SR.sup.x, --CN,
--C(.dbd.Z)R.sup.y, --C(.dbd.Z)ZR.sup.y, or
--ZC(.dbd.Z)ZR.sup.y;
[0130] Z is O or N;
[0131] each R.sup.x is, independently, C.sub.1-6 aliphatic;
[0132] each R.sup.y is, independently, hydrogen or C.sub.1-6
aliphatic;
[0133] g is 0, 1, 2, 3, or 4;
[0134] each R is independently hydrogen or
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B; and
[0135] each R.sup.B is independently C.sub.10-14 alkyl, wherein
R.sup.B is optionally substituted with one or more fluorine
radicals.
[0136] As defined generally above, each L is, independently,
branched or unbranched C.sub.1-6 alkylene, wherein L is optionally
substituted with one or more fluorine radicals. In some
embodiments, L is substituted with one or more fluorine radicals.
In other embodiments, L is unsubstituted. In some embodiments, L is
branched. In other embodiments, L is unbranched. In certain
embodiments, L is C.sub.1-4 alkylene. In certain embodiments, L is
methylene, ethylene, or propylene.
[0137] As defined generally above, each R is, independently,
hydrogen or --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least one R group is
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
two R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least three R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
four R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, all R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
[0138] As defined generally above, each R.sup.B is, independently,
C.sub.10-14 alkyl, wherein R.sup.B is optionally substituted with
one or more fluorine radicals. In some embodiments, R.sup.B is
substituted with one or more fluorine radicals. For example, in
some embodiments, R.sup.B may be substituted with one fluoro, or in
other embodiments, may be perfluorinated. In other embodiments,
R.sup.B is unsubstituted. In some embodiments, all R.sup.B groups
are the same. In certain embodiments, R.sup.B is C.sub.10 alkyl. In
some embodiments, R.sup.B is n-decyl. In certain embodiments,
R.sup.B is C.sub.11 alkyl. In some embodiments, R.sup.B is
n-undecyl. In certain embodiments, R.sup.B is C.sub.12 alkyl. In
some embodiments, R.sup.B is n-dodecyl. In certain embodiments,
R.sup.B is C.sub.13 alkyl. In some embodiments, R.sup.B is
n-tridecyl. In certain embodiments, R.sup.B is C.sub.14 alkyl. In
some embodiments, R.sup.B is n-tetradecyl.
[0139] As defined generally above, each R.sup.2 is, independently,
halo, C.sub.1-6 aliphatic optionally substituted with one or more
fluorine radicals, --OR.sup.x, --N(R.sup.y).sub.2, --SR.sup.x,
--CN, --C(.dbd.Z)R.sup.y, --C(.dbd.Z)ZR.sup.y,
--ZC(.dbd.Z)ZR.sup.y; wherein Z is O or N; each R.sup.x is,
independently, C.sub.1-6 aliphatic; and each R.sup.y is,
independently, hydrogen or C.sub.1-6 aliphatic. In some
embodiments, R.sup.2 is halo. In some embodiments, R.sup.2 is
fluoro. In some embodiments, R.sup.2 is C.sub.1-6 aliphatic
optionally substituted with one or more fluorine radicals. In some
embodiments, R.sup.2 is C.sub.1-6 alkyl.
[0140] In some embodiments, g is 0. In some embodiments, g is 1. In
some embodiments, g is 2. In some embodiments, g is 3. In some
embodiments, g is 4.
[0141] In certain embodiments, a lipidoid of the present invention
is of Formula (V-a):
##STR00041##
or a salt thereof, wherein L, R.sup.2, g, and R are as defined
above and described herein.
[0142] In certain embodiments, a lipidoid of the present invention
is of Formula (V-b):
##STR00042##
or a salt thereof, wherein L and R are as defined above and
described herein.
[0143] In certain embodiments, a lipidoid of the present invention
is of Formula (V-c):
##STR00043##
or a salt thereof, wherein L and R are as defined above and
described herein.
[0144] In certain embodiments, a lipidoid of the present invention
is of Formula (V-d):
##STR00044##
or a salt thereof, wherein R is as defined above and described
herein.
[0145] In certain embodiments, a lipidoid of the present invention
is of the formula:
##STR00045##
or a salt thereof, wherein
[0146] each R.sup.A is, independently, branched or unbranched
C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, or branched or unbranched
C.sub.4-12 cycloalkylalkyl, wherein R.sup.A is optionally
substituted with one or more fluorine radicals;
[0147] each R is, independently, hydrogen or
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B; and
[0148] each R.sup.B is, independently, C.sub.10-14 alkyl, wherein
R.sup.B is optionally substituted with one or more fluorine
radicals.
[0149] As defined generally above, each R.sup.A is, independently,
branched or unbranched C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, or
branched or unbranched C.sub.4-12 cycloalkylalkyl, wherein R.sup.A
is optionally substituted with one or more fluorine radicals. In
some embodiments, R.sup.A is substituted with one or more fluorine
radicals. For example, when R.sup.A is methyl, it may be
substituted with one, two, or three fluorine radicals to give
--CH.sub.2F, --CHF.sub.2, or --CF.sub.3. In other embodiments,
R.sup.A is unsubstituted. In some embodiments, all R.sup.A groups
are the same. In other embodiments, the R.sup.A groups are
different. In some embodiments, R.sup.A is branched or unbranched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is branched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is unbranched
C.sub.1-6 alkyl. In certain embodiments, R.sup.A is C.sub.1-3
alkyl. In certain embodiments, R.sup.A is methyl, ethyl, or propyl.
In certain embodiments, R.sup.A is C.sub.3-7 cycloalkyl. In certain
embodiments, R.sup.A is cyclohexyl. In certain embodiments, R.sup.A
is cyclopropyl, cyclobutyl, or cyclopentyl. In certain embodiments,
R.sup.A is cycloheptyl. In some embodiments, R.sup.A is branched or
unbranched C.sub.4-12 cycloalkylalkyl.
[0150] As defined generally above, each R is, independently,
hydrogen or --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least one R group is
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
two R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, at least three R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some embodiments, at least
four R groups are --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B. In some
embodiments, all R groups are
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.B.
[0151] As defined generally above, each R.sup.B is, independently,
C.sub.10-14 alkyl, wherein R.sup.B is optionally substituted with
one or more fluorine radicals. In some embodiments, R.sup.B is
substituted with one or more fluorine radicals. For example, in
some embodiments, R.sup.B may be substituted with one fluoro, or in
other embodiments, may be perfluorinated. In other embodiments,
R.sup.B is unsubstituted. In some embodiments, all R.sup.B groups
are the same. In certain embodiments, R.sup.B is C.sub.10 alkyl. In
some embodiments, R.sup.B is n-decyl. In certain embodiments,
R.sup.B is C.sub.11 alkyl. In some embodiments, R.sup.B is
n-undecyl. In certain embodiments, R.sup.B is C.sub.12 alkyl. In
some embodiments, R.sup.B is n-dodecyl. In certain embodiments,
R.sup.B is C.sub.13 alkyl. In some embodiments, R.sup.B is
n-tridecyl. In certain embodiments, R.sup.B is C.sub.14 alkyl. In
some embodiments, R.sup.B is n-tetradecyl.
[0152] In certain embodiments, a lipidoid of the present invention
is of the formula:
##STR00046##
or a salt thereof, wherein R is as defined above and described
herein.
[0153] In some embodiments, a lipidoid of the present invention is
a compound resulting from a Michael addition between any one of the
amines shown in FIG. 1 or FIG. 2 and an acrylate shown in FIG. 1.
In certain embodiments, the number of equivalents of acrylate can
be controlled to obtain the desired number of lipid tails on the
inventive lipidoid.
[0154] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 113 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 113O.sub.10, 113O.sub.11,
113O.sub.12, 113O.sub.13, or 113O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00047##
wherein z is 11 or 12. In some embodiments, the present invention
provides a composition of one or more of the above lipidoids. In
certain embodiments, an inventive lipidoid is of the formula:
##STR00048##
[0155] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 123 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 123O.sub.10, 123O.sub.11,
123O.sub.12, 123O.sub.13, or 123O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00049##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00050##
[0156] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 154 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 154O.sub.10, 154O.sub.11,
154O.sub.12, 154O.sub.13, or 154O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00051##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00052##
[0157] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 191 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 191O.sub.10, 191O.sub.11,
191O.sub.12, 191O.sub.13, or 191O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00053##
wherein z is 10, 11, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00054##
[0158] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 192 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 192O.sub.10, 192O.sub.11,
192O.sub.12, 192O.sub.13, or 192O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00055##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00056##
[0159] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 193 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 193O.sub.10, 193O.sub.11,
193O.sub.12, 193O.sub.13, or 193O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00057##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00058##
[0160] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 195 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 195O.sub.10, 195O.sub.11,
195O.sub.12, 195O.sub.13, or 195O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00059##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00060##
[0161] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 196 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 196O.sub.10, 196O.sub.11,
196O.sub.12, 196O.sub.13, or 196O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00061##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00062##
[0162] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 200 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 200O.sub.10, 200O.sub.11,
200O.sub.12, 200O.sub.13, or 200O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00063## ##STR00064## ##STR00065##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00066##
[0163] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 205 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 205O.sub.10, 205O.sub.11,
205O.sub.12, 205O.sub.13, or 205O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00067##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00068##
[0164] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 217 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 217O.sub.10, 217O.sub.11,
217O.sub.12, 217O.sub.13, or 217O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00069##
wherein z is 11 or 12. In some embodiments, the present invention
provides a composition of one or more of the above lipidoids. In
certain embodiments, an inventive lipidoid is of the formula:
##STR00070##
[0165] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 218 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 218O.sub.10, 218O.sub.11,
218O.sub.12, 218O.sub.13, or 218O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00071##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00072##
[0166] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 232 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 232O.sub.10, 232O.sub.11,
232O.sub.12, 232O.sub.13, or 232O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00073##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00074##
[0167] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 235 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 235O.sub.10, 235O.sub.11,
235O.sub.12, 235O.sub.13, or 235O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00075##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00076##
[0168] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 302 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 302O.sub.10, 302O.sub.11,
302O.sub.12, 302O.sub.13, or 302O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00077##
wherein z is 12 or 13. In some embodiments, the present invention
provides a composition of one or more of the above lipidoids. In
certain embodiments, an inventive lipidoid is of the formula:
##STR00078##
[0169] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 303 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 303O.sub.10, 303O.sub.11,
303O.sub.12, 303O.sub.13, or 303O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00079##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00080##
[0170] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 304 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 304O.sub.10, 304O.sub.11,
304O.sub.12, 304O.sub.13, or 304O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00081##
wherein z is 11 or 12. In some embodiments, the present invention
provides a composition of one or more of the above lipidoids. In
certain embodiments, an inventive lipidoid is of the formula:
##STR00082##
[0171] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 305 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 305O.sub.10, 305O.sub.11,
305O.sub.12, 305O.sub.13, or 305O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00083##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00084##
[0172] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 306 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 306O.sub.10, 306O.sub.11,
306O.sub.12, 306O.sub.13, or 306O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00085##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00086##
[0173] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 313 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 313O.sub.10, 313O.sub.11,
313O.sub.12, 313O.sub.13, or 313O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00087##
wherein z is 9, 10, 11, or 12. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00088##
[0174] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 315 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 315O.sub.10, 315O.sub.11,
315O.sub.12, 315O.sub.13, or 315O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00089## ##STR00090##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00091##
[0175] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 347 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 347O.sub.10, 347O.sub.11,
347O.sub.12, 347O.sub.13, or 347O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00092##
wherein z is 11 or 13. In some embodiments, the present invention
provides a composition of one or more of the above lipidoids. In
certain embodiments, an inventive lipidoid is of the formula:
##STR00093##
[0176] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 366 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 366O.sub.10, 366O.sub.11,
366O.sub.12, 366O.sub.13, or 366O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00094##
wherein z is 10 or 11. In some embodiments, the present invention
provides a composition of one or more of the above lipidoids. In
certain embodiments, an inventive lipidoid is of the formula:
##STR00095##
[0177] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 371 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 371O.sub.10, 371O.sub.11,
371O.sub.12, 371O.sub.13, or 371O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00096##
In some embodiments, the present invention provides a composition
of one or more of the above lipidoids. In certain embodiments, an
inventive lipidoid is of the formula:
##STR00097##
[0178] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 500 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 500O.sub.10, 500O.sub.11,
500O.sub.12, 500O.sub.13, or 500O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00098##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00099##
[0179] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 501 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 501O.sub.10, 501O.sub.11,
501O.sub.12, 501O.sub.13, or 501O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00100##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00101##
[0180] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 502 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 502O.sub.10, 502O.sub.11,
502O.sub.12, 502O.sub.13, or 502O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00102##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00103##
[0181] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 503 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 503O.sub.10, 503O.sub.11,
503O.sub.12, 503O.sub.13, or 503O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00104##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00105##
[0182] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 504 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 504O.sub.10, 504O.sub.11,
504O.sub.12, 504O.sub.13, or 504O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00106##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00107##
[0183] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 505 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 505O.sub.10, 505O.sub.11,
505O.sub.12, 505O.sub.13, or 505O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00108##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00109##
[0184] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 506 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 506O.sub.10, 506O.sub.11,
506O.sub.12, 506O.sub.13, or 506O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00110##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00111##
[0185] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 507 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 507O.sub.10, 507O.sub.11,
507O.sub.12, 507O.sub.13, or 507O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00112##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00113##
[0186] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 508 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 508O.sub.10, 508O.sub.11,
508O.sub.12, 508O.sub.13, or 508O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00114##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00115##
[0187] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 509 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 509O.sub.10, 509O.sub.11,
509O.sub.12, 509O.sub.13, or 509O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00116##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00117##
[0188] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 510 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 510O.sub.10, 510O.sub.11,
510O.sub.12, 510O.sub.13, or 510O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00118##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00119##
[0189] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 511 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 511O.sub.10, 511O.sub.11,
511O.sub.12, 511O.sub.13, or 511O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00120##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00121##
[0190] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 512 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or 014 to form compound 512O.sub.10, 512O.sub.11,
512O.sub.12, 512O.sub.13, or 512O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00122##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00123##
[0191] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 513 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 513O.sub.10, 513O.sub.11,
513O.sub.12, 513O.sub.13, or 513O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00124## ##STR00125## ##STR00126##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00127## ##STR00128##
[0192] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 514 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 514O.sub.10, 514O.sub.11,
514O.sub.12, 514O.sub.13, or 514O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00129## ##STR00130##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00131##
[0193] In certain embodiments, an inventive lipidoid is prepared by
reacting amine 515 with acrylate O.sub.10, O.sub.11, O.sub.12,
O.sub.13, or O.sub.14 to form compound 515O.sub.10, 515O.sub.11,
515O.sub.12, 515O.sub.13, or 515O.sub.14. In certain embodiments,
an inventive lipidoid is of one of the formulae below:
##STR00132##
wherein z is 9, 10, 11, 12, or 13. In some embodiments, the present
invention provides a composition of one or more of the above
lipidoids. In certain embodiments, an inventive lipidoid is of the
formula:
##STR00133##
Synthesis of Lipids
[0194] Lipidoids described herein may be prepared by any method
known in the art. In certain embodiments, inventive lipidoids are
prepared via the conjugate addition of primary or secondary amines
to acrylates. Such syntheses are described in detail in U.S.
Publication No. 2011/0009641, incorporated herein by reference. In
certain embodiments, inventive lipidoids are prepared from
commercially available starting materials, such acrylates and
amines. In other embodiments, inventive lipidoids are prepared from
easily and/or inexpensively prepared starting materials. As would
be appreciated by one of skill in the art, the lipidoids described
herein can be prepared by total synthesis starting from
commercially available starting materials. A particular lipidoid
may be the desired final product of the synthesis, or a mixture of
lipidoids may be the desired final product.
Polynucleotide Complexes
[0195] The ability of cationic compounds to interact with
negatively charged polynucleotides through electrostatic
interactions is well known. Cationic lipids such as Lipofectamine
have been prepared and studied for their ability to complex and
transfect polynucleotides. The interaction of the lipid with the
polynucleotide is thought to at least partially prevent the
degradation of the polynucleotide. By neutralizing the charge on
the backbone of the polynucleotide, the neutral or
slightly-positively-charged complex is also able to more easily
pass through the hydrophobic membranes (e.g., cytoplasmic,
lysosomal, endosomal, nuclear) of the cell. In certain embodiments,
the complex is slightly positively charged. In certain embodiments,
the complex has a positive .zeta.-potential. In certain
embodiments, the .zeta.-potential is between +1 and +30.
[0196] In certain embodiments, lipidoids of the present invention
possess tertiary amines. Although these amines are hindered, they
are available to interact with a polynucleotide (e.g., DNA, RNA,
synthetic analogs of DNA and/or RNA, DNA/RNA hydrids, etc.). In
certain embodiments, polynucleotides or derivatives thereof are
contacted with the inventive lipidoids under conditions suitable to
form polynucleotide/lipidoid complexes. In certain embodiments, the
lipidoid is at least partially protonated so as to form a complex
with the negatively charged polynucleotide. In certain embodiments,
the polynucleotide/lipidoid complexes form nanoparticles that are
useful in the delivery of polynucleotides to cells. In certain
embodiments, multiple lipidoid molecules may be associated with a
polynucleotide molecule. The complex may include 1-100 lipidoid
molecules, 1-1000 lipidoid molecules, 10-1000 lipidoid molecules,
or 100-10,000 lipidoid molecules. In certain embodiments, the
complex may form a nanoparticle. In certain embodiments, the
diameter of the nanoparticles ranges from 10-500 nm, from 10-1200
nm, or from 50-150 nm. In certain embodiments, nanoparticles may be
associated with a targeting agent as described below.
Polynucleotide
[0197] A polynucleotide to be complexed, encapsulated by the
inventive lipidoids, or included in a composition with the
inventive lipidoids may be any nucleic acid including but not
limited to RNA and DNA. In certain embodiments, the polynucleotide
is DNA. In other embodiments, the polynucleotide is RNA. In certain
embodiments, the polynucleotide is an siRNA. In certain
embodiments, the polynucleotide is an shRNA. In certain
embodiments, the polynucleotide is an mRNA. In certain embodiments,
the polynucleotide is a dsRNA. In certain embodiments, the
polynucleotide is an miRNA. In certain embodiments, the
polynucleotide is an antisense RNA. The polynucleotides may be of
any size or sequence, and they may be single- or double-stranded.
In certain embodiments, the polynucleotide is greater than 100 base
pairs long. In certain other embodiments, the polynucleotide is
greater than 1000 base pairs long and may be greater than 10,000
base pairs long. In certain embodiments, the polynucleotide is
purified and substantially pure. In certain embodiments, the
polynucleotide is greater than 50% pure, greater than 75% pure, or
greater than 95% pure. The polynucleotide may be provided by any
means known in the art. In certain preferred embodiments, the
polynucleotide has been engineered using recombinant techniques
(for a more detailed description of these techniques, please see
Ausubel et al. Current Protocols in Molecular Biology (John Wiley
& Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold
Spring Harbor Laboratory Press: 1989); each of which is
incorporated herein by reference). The polynucleotide may also be
obtained from natural sources and purified from contaminating
components found normally in nature. The polynucleotide may also be
chemically synthesized in a laboratory. In certain embodiments, the
polynucleotide is synthesized using standard solid phase
chemistry.
[0198] The polynucleotide may be modified by chemical or biological
means. In certain embodiments, these modifications lead to
increased stability of the polynucleotide. Modifications include
methylation, phosphorylation, end-capping, etc.
[0199] Derivatives of polynucleotides may also be used in the
present invention. These derivatives include modifications in the
bases, sugars, and/or phosphate linkages of the polynucleotide.
Modified bases include, but are not limited to, those found in the
following nucleoside analogs: 2-aminoadenosine, 2-thiothymidine,
inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, and 2-thiocytidine. Modified sugars include,
but are not limited to, 2'-fluororibose, ribose, 2'-deoxyribose,
3'-azido-2',3'-dideoxyribose, 2',3'-dideoxyribose, arabinose (the
2'-epimer of ribose), acyclic sugars, and hexoses. The nucleosides
may be strung together by linkages other than the phosphodiester
linkage found in naturally occurring DNA and RNA. Modified linkages
include, but are not limited to, phosphorothioate and
5'-N-phosphoramidite linkages. Combinations of the various
modifications may be used in a single polynucleotide. These
modified polynucleotides may be provided by any means known in the
art; however, as will be appreciated by those of skill in this art,
the modified polynucleotides are preferably prepared using
synthetic chemistry in vitro. The polynucleotides to be delivered
may be in any form. For example, the polynucleotide may be a
circular plasmid, a linearized plasmid, a cosmid, a viral genome, a
modified viral genome, an artificial chromosome, etc.
[0200] The polynucleotide may be of any sequence. In certain
preferred embodiments, the polynucleotide encodes a protein or
peptide. The encoded proteins may be enzymes, structural proteins,
receptors, soluble receptors, ion channels, pharmaceutically active
proteins, cytokines, interleukins, antibodies, antibody fragments,
antigens, coagulation factors, albumin, growth factors, hormones,
insulin, etc. The polynucleotide may also comprise regulatory
regions to control the expression of a gene. These regulatory
regions may include, but are not limited to, promoters, enhancer
elements, repressor elements, TATA box, ribosomal binding sites,
stop site for transcription, etc. In other particularly preferred
embodiments, the polynucleotide is not intended to encode a
protein. For example, the polynucleotide may be used to fix an
error in the genome of the cell being transfected.
[0201] The polynucleotide may also be provided as an antisense
agent or RNA interference (RNAi) (Fire et al. Nature 391:806-811,
1998; incorporated herein by reference). Antisense therapy is meant
to include, e.g., administration or in situ provision of single- or
double-stranded oligonucleotides or their derivatives which
specifically hybridize, e.g., bind, under cellular conditions, with
cellular mRNA and/or genomic DNA, or mutants thereof, so as to
inhibit expression of the encoded protein, e.g., by inhibiting
transcription and/or translation (Crooke "Molecular mechanisms of
action of antisense drugs" Biochim. Biophys. Acta 1489(1):31-44,
1999; Crooke "Evaluating the mechanism of action of
antiproliferative antisense drugs" Antisense Nucleic Acid Drug Dev.
10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes
313-314, 1999; each of which is incorporated herein by reference).
The binding may be by conventional base pair complementarity, or,
for example, in the case of binding to DNA duplexes, through
specific interactions in the major groove of the double helix
(i.e., triple helix formation) (Chan et al. J. Mol. Med.
75(4):267-282, 1997; incorporated herein by reference).
[0202] In certain embodiments, the polynucleotide to be delivered
comprises a sequence encoding an antigenic peptide or protein.
Nanoparticles containing these polynucleotides can be delivered to
an individual to induce an immunologic response sufficient to
decrease the chance of a subsequent infection and/or lessen the
symptoms associated with such an infection. The polynucleotide of
these vaccines may be combined with interleukins, interferon,
cytokines, and adjuvants such as cholera toxin, alum, Freund's
adjuvant, etc. A large number of adjuvant compounds are known; a
useful compendium of many such compounds is prepared by the
National Institutes of Health and can be found on the internet
(http:/www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf,
incorporated herein by reference; see also Allison Dev. Biol.
Stand. 92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol.
6:251-281, 1998; and Phillips et al. Vaccine 10:151-158, 1992, each
of which is incorporated herein by reference).
[0203] An antigenic protein or peptides encoded by a polynucleotide
may be derived from such bacterial organisms as Streptococccus
pneumoniae, Haemophilus influenzae, Staphylococcus aureus,
Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria
monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium
botulinum, Clostridium perfringens, Neisseria meningitidis,
Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas
aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,
Bordetella pertussis, Francisella tularensis, Yersinia pestis,
Vibrio cholerae, Legionella pneumophila, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum,
Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter
jejuni, and the like; from such viruses as smallpox, influenza A
and B, respiratory syncytial virus, parainfluenza, measles, HIV,
varicella-zoster, herpes simplex 1 and 2, cytomegalovirus,
Epstein-Barr virus, rotavirus, rhinovirus, adenovirus,
papillomavirus, poliovirus, mumps, rabies, rubella,
coxsackieviruses, equine encephalitis, Japanese encephalitis,
yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus,
and the like; and from such fungal, protozoan, and parasitic
organisms such as Cryptococcus neoformans, Histoplasma capsulatum,
Candida albicans, Candida tropicalis, Nocardia asteroides,
Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae,
Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,
Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,
Trichomonas vaginalis, Schistosoma mansoni, and the like.
Microparticles and Nanoparticles
[0204] The lipidoids of the present invention may also be used to
form drug delivery devices. Inventive lipidoids may be used to
encapsulate agents including polynucleotides, small molecules,
proteins, peptides, metals, organometallic compounds, etc.
Lipidoids described herein have several properties that make them
particularly suitable in the preparation of drug delivery devices.
These include 1) the ability of the lipid to complex and "protect"
labile agents; 2) the ability to buffer the pH in the endosome; 3)
the ability to act as a "proton sponge" and cause endosomolysis;
and 4) the ability to neutralize the charge on negatively charged
agents. In certain embodiments, the diameter of the particles range
from between 1 micrometer to 1,000 micrometers. In certain
embodiments, the diameter of the particles range from between from
1 micrometer to 100 micrometers. In certain embodiments, the
diameter of the particles range from between from 1 micrometer to
10 micrometers. In certain embodiments, the diameter of the
particles range from between from 10 micrometer to 100 micrometers.
In certain embodiments, the diameter of the particles range from
between from 100 micrometer to 1,000 micrometers. In certain
embodiments, the particles range from 1-5 micrometers. In certain
embodiments, the diameter of the particles range from between 1 nm
to 1,000 nm. In certain embodiments, the diameter of the particles
range from between from 1 nm to 100 nm. In certain embodiments, the
diameter of the particles range from between from 1 nm to 10 nm. In
certain embodiments, the diameter of the particles range from
between from 10 nm to 100 nm. In certain embodiments, the diameter
of the particles range from between from 100 nm to 1,000 nm. In
certain embodiments, the diameter of the particles range from
between from 20 nm to 2,000 nm. In certain embodiments, the
particles range from 1-5 nm. In certain embodiments, the diameter
of the particles range from between 1 pm to 1,000 pm. In certain
embodiments, the diameter of the particles range from between from
1 pm to 100 pm. In certain embodiments, the diameter of the
particles range from between from 1 pm to 10 pm. In certain
embodiments, the diameter of the particles range from between from
10 pm to 100 pm. In certain embodiments, the diameter of the
particles range from between from 100 pm to 1,000 pm. In certain
embodiments, the particles range from 1-5 pm.
[0205] The inventive particles may be prepared using any method
known in this art. These include, but are not limited to, spray
drying, single and double emulsion solvent evaporation, solvent
extraction, phase separation, simple and complex coacervation, and
other methods well known to those of ordinary skill in the art. In
certain embodiments, methods of preparing the particles are the
double emulsion process and spray drying. The conditions used in
preparing the particles may be altered to yield particles of a
desired size or property (e.g., hydrophobicity, hydrophilicity,
external morphology, "stickiness", shape, etc.). The method of
preparing the particle and the conditions (e.g., solvent,
temperature, concentration, air flow rate, etc.) used may also
depend on the agent being encapsulated and/or the composition of
the matrix. Methods developed for making particles for delivery of
encapsulated agents are described in the literature (for example,
please see Doubrow, M., Ed., "Microcapsules and Nanoparticles in
Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz and
Langer, J. Controlled Release 5:13-22, 1987; Mathiowitz et al.,
Reactive Polymers 6:275-283, 1987; Mathiowitz et al., J. Appl.
Polymer Sci. 35:755-774, 1988; each of which is incorporated herein
by reference).
[0206] If the particles prepared by any of the above methods have a
size range outside of the desired range, the particles can be
sized, for example, using a sieve. The particle may also be coated.
In certain embodiments, the particles are coated with a targeting
agent. In other embodiments, the particles are coated to achieve
desirable surface properties (e.g., a particular charge).
[0207] In certain embodiments, the present invention provides a
nanoparticle comprising an inventive lipidoid and one or more
agents to be delivered. In certain embodiments, the agent is a
polynucleotide, drug, protein or peptide, small molecule, or gas.
In certain embodiments, the agent is RNA (e.g. mRNA, RNAi, dsRNA,
siRNA, shRNA, miRNA, or antisense RNA). In certain embodiments, the
nanoparticle further comprises cholesterol or a derivative thereof,
such as
3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
(DC-cholesterol). In certain embodiments, the nanoparticle further
comprises a PEG-based material. In certain embodiments, the
PEG-based material is PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, or
DSPE carboxy PEG. For instance, in certain embodiments, the
PEG-based material is C14 PEG2000 DMG, C15 PEG2000 DMG, C16 PEG2000
DMG, C18 PEG2000 DMG, C14 PEG 2000 ceramide, C15 PEG2000 ceramide,
C16 PEG2000 ceramide, C18 PEG2000 ceramide, C14 PEG2000 PE, C15
PEG2000 PE, C16 PEG2000 PE, C18 PEG2000 PE, C14 PEG350 PE, C14
PEG5000 PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, or
DSPE carboxy PEG. In certain embodiments, the nanoparticle further
comprises a lipid. For example, in certain embodiments, the
nanoparticle further comprises DSPC, DOPC, or DOPE. In certain
embodiments, the nanoparticle comprises a lipidoid, an agent (e.g.,
RNA), a lipid, cholesterol or a derivative thereof, and a PEG-based
material.
Micelles, Liposomes, and Lipoplexes
[0208] Lipidoids described herein may also be used to prepare
micelles or liposomes. In addition, any agent may be included in a
micelle or liposome. Micelles and liposomes are particularly useful
in delivering hydrophobic agents such as hydrophobic small
molecules. When the micelle or liposome is complexed with (e.g.,
encapsulates or covers) a polynucleotide it is referred to as a
"lipoplex." Many techniques for preparing micelles, liposomes, and
lipoplexes are known in the art, and any method may be used with
the inventive lipidoids to make micelles and liposomes.
[0209] In certain embodiments, liposomes (lipid vesicles) are
formed through spontaneous assembly. In other embodiments,
liposomes are formed when thin lipid films or lipid cakes are
hydrated and stacks of lipid crystalline bilayers become fluid and
swell. The hydrated lipid sheets detach during agitation and
self-close to form large, multilamellar vesicles (LMV). This
prevents interaction of water with the hydrocarbon core of the
bilayers at the edges. Once these particles have formed, reducing
the size of the particle can be modified through input of sonic
energy (sonication) or mechanical energy (extrusion). See Walde, P.
"Preparation of Vesicles (Liposomes)" In Encylopedia of Nanoscience
and Nanotechnology; Nalwa, H. S. Ed. American Scientific
Publishers: Los Angeles, 2004; Vol. 9, pp. 43-79; Szoka et al.
"Comparative Properties and Methods of Preparation of Lipid
Vesicles (Liposomes)" Ann. Rev. Biophys. Bioeng. 9:467-508, 1980;
each of which is incorporated herein. The preparation of liposomes
involves preparing the lipid for hydration, hydrating the lipid
with agitation, and sizing the vesicles to achieve a homogenous
distribution of liposomes. Lipids are first dissolved in an organic
solvent to assure a homogeneous mixture of lipids. The solvent is
then removed to form a lipid film. This film is thoroughly dried to
remove residual organic solvent by placing the vial or flask on a
vacuum pump overnight. Hydration of the lipid film/cake is
accomplished by adding an aqueous medium to the container of dry
lipid and agitating the mixture. Disruption of LMV suspensions
using sonic energy typically produces small unilamellar vesicles
(SUV) with diameters in the range of 15-50 nm. Lipid extrusion is a
technique in which a lipid suspension is forced through a
polycarbonate filter with a defined pore size to yield particles
having a diameter near the pore size of the filter used. Extrusion
through filters with 100 nm pores typically yields large,
unilamellar vesicles (LUV) with a mean diameter of 120-140 nm.
[0210] In certain embodiments, liposomes are formed comprising an
inventive lipid, a PEG-based material, cholesterol or a derivative
thereof, and a polynucleotide. In certain embodiments, the
polynucleotide is an RNA molecule (e.g., an siRNA). In other
embodiments, the polynucleotide is a DNA molecule. In certain
embodiments, the amount of lipidoid in the liposome ranges from
30-80 mol %, 40-70 mol %, or 60-70 mol %. In certain embodiments,
the liposome comprises a PEG-based material. In certain
embodiments, the amount of PEG-based material in the liposomes
ranges from 5-20 mol %, 10-15 mol %, or 10 mol %. In certain
embodiments, the liposome comprises cholesterol or a derivative
thereof. In certain embodiments, the amount of cholesterol in the
liposome ranges from 5-25 mol %, 10-20 mol %, or 15 mol %. In
certain embodiments, the amount of cholesterol in the liposome is
approximately 20 mol %. These liposomes may be prepared using any
method known in the art. In certain embodiments (e.g., liposomes
containing RNAi molecules), the liposomes are prepared by lipid
extrusion.
[0211] Certain lipidoids can spontaneously self assemble around
certain molecules, such as DNA and RNA, to form liposomes. In some
embodiments, the application is the delivery of polynucleotides.
Use of these lipidoids allows for simple assembly of liposomes
without the need for additional steps or devices such as an
extruder.
The following scientific papers described other methods for
preparing liposomes and micelles: Narang et al. "Cationic Lipids
with Increased DNA Binding Affinity for Nonviral Gene Transfer in
Dividing and Nondividing Cells" Bioconjugate Chem. 16:156-68, 2005;
Hofland et al. "Formation of stable cationic lipid/DNA complexes
for gene transfer" Proc. Natl. Acad. Sci. USA 93:7305-7309, July
1996; Byk et al. "Synthesis, Activity, and Structure--Activity
Relationship Studies of Novel Cationic Lipids for DNA Transfer" J.
Med. Chem. 41(2):224-235, 1998; Wu et al. "Cationic Lipid
Polymerization as a Novel Approach for Constructing New DNA
Delivery Agents" Bioconjugate Chem. 12:251-57, 2001; Lukyanov et
al. "Micelles from lipid derivatives of water-soluble polymers as
delivery systems for poorly soluble drugs" Advanced Drug Delivery
Reviews 56:1273-1289, 2004; Tranchant et al. "Physicochemical
optimisation of plasmid delivery by cationic lipids" J. Gene Med.
6:S24-S35, 2004; van Balen et al. "Liposome/Water Lipophilicity:
Methods, Information Content, and Pharmaceutical Applications"
Medicinal Research Rev. 24(3):299-324, 2004; each of which is
incorporated herein by reference.
Agent
[0212] The agents to be delivered by the system of the present
invention may be therapeutic, diagnostic, or prophylactic agents.
Any chemical compound to be administered to an individual may be
delivered using the inventive complexes, picoparticles,
nanoparticles, microparticles, micelles, or liposomes. The agent
may be a small molecule, organometallic compound, nucleic acid,
protein, peptide, polynucleotide, targeting agent, an isotopically
labeled chemical compound, drug, vaccine, immunological agent,
etc.
In certain embodiments, the agents are organic compounds with
pharmaceutical activity. In another embodiment of the invention,
the agent is a clinically used drug. In a particularly preferred
embodiment, the drug is an antibiotic, chemotherapeutic, anti-viral
agent, anesthetic, steroidal agent, anti-inflammatory agent,
anti-neoplastic agent, antigen, vaccine, antibody, decongestant,
antihypertensive, sedative, birth control agent, progestational
agent, anti-cholinergic, analgesic, anti-depressant,
anti-psychotic, .beta.-adrenergic blocking agent, diuretic,
cardiovascular active agent, vasoactive agent, non-steroidal
anti-inflammatory agent, nutritional agent, etc.
[0213] In certain embodiments, the agent to be delivered may be a
mixture of agents.
[0214] Diagnostic agents include gases; metals; commercially
available imaging agents used in positron emissions tomography
(PET), computer assisted tomography (CAT), single photon emission
computerized tomography, x-ray, fluoroscopy, and magnetic resonance
imaging (MRI); and contrast agents. Examples of suitable materials
for use as contrast agents in MRI include gadolinium chelates, as
well as iron, magnesium, manganese, copper, and chromium. Examples
of materials useful for CAT and x-ray imaging include iodine-based
materials. Prophylactic agents include, but are not limited to,
antibiotics, nutritional supplements, and vaccines. Vaccines may
comprise isolated proteins or peptides, inactivated organisms and
viruses, dead organisms and viruses, genetically altered organisms
or viruses, and cell extracts. Prophylactic agents may be combined
with interleukins, interferon, cytokines, and adjuvants such as
cholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents
include antigens of such bacterial organisms as Streptococccus
pneumoniae, Haemophilus influenzae, Staphylococcus aureus,
Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria
monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium
botulinum, Clostridium perfringens, Neisseria meningitidis,
Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas
aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,
Bordetella pertussis, Francisella tularensis, Yersinia pestis,
Vibrio cholerae, Legionella pneumophila, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum,
Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter
jejuni, and the like; antigens of such viruses as smallpox,
influenza A and B, respiratory syncytial virus, parainfluenza,
measles, HIV, varicella-zoster, herpes simplex 1 and 2,
cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,
adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,
coxsackieviruses, equine encephalitis, Japanese encephalitis,
yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus,
and the like; antigens of fungal, protozoan, and parasitic
organisms such as Cryptococcus neoformans, Histoplasma capsulatum,
Candida albicans, Candida tropicalis, Nocardia asteroides,
Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae,
Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,
Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,
Trichomonas vaginalis, Schistosoma mansoni, and the like. These
antigens may be in the form of whole killed organisms, peptides,
proteins, glycoproteins, carbohydrates, or combinations
thereof.
Targeting Agents
[0215] The inventive lipidoids, and the complexes, liposomes,
micelles, microparticles, picoparticles and nanoparticles prepared
therefrom, may be modified to include targeting agents since it is
often desirable to target a particular cell, collection of cells,
or tissue. A variety of targeting agents that direct pharmaceutical
compositions to particular cells are known in the art (see, for
example, Cotten et al. Methods Enzym. 217:618, 1993; incorporated
herein by reference). The targeting agents may be included
throughout the particle or may be only on the surface. The
targeting agent may be a protein, peptide, carbohydrate,
glycoprotein, lipid, small molecule, etc. The targeting agent may
be used to target specific cells or tissues or may be used to
promote endocytosis or phagocytosis of the particle. Examples of
targeting agents include, but are not limited to, antibodies,
fragments of antibodies, low-density lipoproteins (LDLs),
transferrin, asialycoproteins, gp120 envelope protein of the human
immunodeficiency virus (HIV), carbohydrates, receptor ligands,
sialic acid, etc. If the targeting agent is included throughout the
particle, the targeting agent may be included in the mixture that
is used to form the particles. If the targeting agent is only on
the surface, the targeting agent may be associated with (i.e., by
covalent, hydrophobic, hydrogen bonding, van der Waals, or other
interactions) the formed particles using standard chemical
techniques.
Compositions
[0216] In certain embodiments, an inventive lipidoid is a component
of a composition which may be useful in a variety of medical and
non-medical applications. For example, pharmaceutical compositions
comprising an inventive lipidoid may be useful in the delivery of
an effective amount of an agent to a subject in need thereof.
Nutraceutical compositions comprising an inventive lipidoid may be
useful in the delivery of an effective amount of a nutraceutical,
e.g., a dietary supplement, to a subject in need thereof. Cosmetic
compositions comprising an inventive lipidoid may be formulated as
a cream, ointment, balm, paste, film, or liquid, etc., and may be
useful in the application of make-up, hair products, and materials
useful for personal hygiene, etc.
[0217] In certain embodiments, the composition comprises one or
more lipidoids of the present invention. "One or more lipidoids"
refers to one or more different types of lipidoids included in the
composition, and encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
different types of lipidoids.
[0218] In certain embodiments, the inventive lipidoids are useful
in compositions, either for delivery of an effective amount of an
agent to a subject in need thereof (e.g., a pharmaceutical
composition, a cosmetic composition) or for use as an excipient.
For example, cosmetic compositions may further use the inventive
lipidoids as excipients rather than as a delivery system
encapsulating an agent to be delivered. In certain embodiments, the
composition is a pharmaceutical composition. In certain
embodiments, the composition is a cosmetic composition.
[0219] In certain embodiments, the composition further comprises an
agent, as described herein. For example, in certain embodiments,
the agent is a small molecule, organometallic compound, nucleic
acid, protein, peptide, polynucleotide, metal, targeting agent, an
isotopically labeled chemical compound, drug, vaccine, or
immunological agent. In certain embodiments, the agent is a
polynucleotide. In certain embodiments, the polynucleotide is DNA
or RNA. In certain embodiments, the RNA is mRNA, RNAi, dsRNA,
siRNA, shRNA, miRNA, or antisense RNA.
[0220] In certain embodiments, the polynucleotide and the one or
more lipidoids are not covalently attached.
[0221] In certain embodiments, the one or more lipidoids are in the
form of a particle. In certain embodiments, the particle is a
nanoparticle or microparticle. In certain embodiments, the one or
more conjugated lipidoids are in the form of liposomes or micelles.
It is understood that, in certain embodiments, these lipidoids
self-assemble to provide a particle, micelle or liposome. In
certain embodiments, the particle, liposome, or micelle
encapsulates an agent. The agent to be delivered by the particles,
liposomes, or micelles may be in the form of a gas, liquid, or
solid. The inventive lipidoids may be combined with polymers
(synthetic or natural), surfactants, cholesterol, carbohydrates,
proteins, lipids etc. to form the particles. These particles may be
combined with an excipient to form pharmaceutical and cosmetic
compositions.
Once the complexes, micelles, liposomes, or particles have been
prepared, they may be combined with one or more excipients to form
a composition that is suitable to administer to animals including
humans.
[0222] As would be appreciated by one of skill in this art, the
excipients may be chosen based on the route of administration as
described below, the agent being delivered, time course of delivery
of the agent, etc.
[0223] In certain embodiments, provided is a composition comprising
an inventive lipidoids and an excipient. As used herein, the term
"excipient" means a non-toxic, inert solid, semi-solid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type. Some examples of materials which can serve as excipients
include, but are not limited to, sugars such as lactose, glucose,
and sucrose; starches such as corn starch and potato starch;
cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose, and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil;
safflower oil; sesame oil; olive oil; corn oil and soybean oil;
glycols such as propylene glycol; esters such as ethyl oleate and
ethyl laurate; agar; detergents such as Tween 80; buffering agents
such as magnesium hydroxide and aluminum hydroxide; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator. The compositions of this
invention can be administered to humans and/or to animals, orally,
rectally, parenterally, intracisternally, intravaginally,
intranasally, intraperitoneally, topically (as by powders, creams,
ointments, or drops), bucally, or as an oral or nasal spray.
[0224] Liquid dosage forms for oral administration include
emulsions, microemulsions, solutions, suspensions, syrups, and
elixirs. In addition to the active ingredients (i.e.,
microparticles, nanoparticles, liposomes, micelles,
polynucleotide/lipid complexes), the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0225] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In certain
embodiments, the particles are suspended in a carrier fluid
comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v)
Tween 80.
[0226] The injectable formulations can be sterilized, for example,
by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0227] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
particles with suitable non-irritating excipients or carriers such
as cocoa butter, polyethylene glycol, or a suppository wax which
are solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
particles.
[0228] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the particles are mixed with at least one inert, pharmaceutically
acceptable excipient or carrier such as sodium citrate or dicalcium
phosphate and/or a) fillers or extenders such as starches, lactose,
sucrose, glucose, mannitol, and silicic acid, b) binders such as,
for example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as
glycerol, d) disintegrating agents such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets,
and pills, the dosage form may also comprise buffering agents.
[0229] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0230] The solid dosage forms of tablets, dragees, capsules, pills,
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes.
[0231] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0232] Dosage forms for topical or transdermal administration of an
inventive pharmaceutical composition include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants, or
patches. The particles are admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, ear drops, and
eye drops are also contemplated as being within the scope of this
invention.
[0233] The ointments, pastes, creams, and gels may contain, in
addition to the particles of this invention, excipients such as
animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc, and zinc oxide, or mixtures
thereof.
[0234] Powders and sprays can contain, in addition to the particles
of this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates, and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons.
[0235] Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the microparticles or
nanoparticles in a proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
can be controlled by either providing a rate controlling membrane
or by dispersing the particles in a polymer matrix or gel.
Methods of Use
[0236] In another aspect, provided are methods of using the
inventive lipidoids, e.g., for the treatment of a disease, disorder
or condition from which a subject suffers. It is contemplated that
the inventive lipidoids will be useful in the treatment of a
variety of diseases, disorders or conditions, especially as a
system for delivering agents useful in the treatment of that
particular disease, disorder or condition.
[0237] For example, in one aspect, provided is a method of treating
cancer comprising administering to a subject in need thereof an
effective amount of a lipidoid of the present invention, or salt
thereof, or a composition thereof. In certain embodiments, the
method further comprises administering an anti-cancer agent. In
certain embodiments, the lipidoid encapsulates the anti-cancer
agent. In certain embodiments, the lipidoid and the anti-cancer
agent form a particle (e.g., a nanoparticle, a microparticle, a
micelle, a liposome, a lipoplex).
[0238] A "subject" to which administration is contemplated
includes, but is not limited to, humans (i.e., a male or female of
any age group, e.g., a pediatric subject (e.g, infant, child,
adolescent) or adult subject (e.g., young adult, middle-aged adult
or senior adult)) and/or other non-human animals, for example
mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys);
commercially relevant mammals such as cattle, pigs, horses, sheep,
goats, cats, and/or dogs), birds (e.g., commercially relevant birds
such as chickens, ducks, geese, and/or turkeys), reptiles,
amphibians, and fish. In certain embodiments, the non-human animal
is a mammal. The non-human animal may be a male or female and at
any stage of development. A non-human animal may be a transgenic
animal.
[0239] As used herein, and unless otherwise specified, the terms
"treat," "treating" and "treatment" contemplate an action that
occurs while a subject is suffering from the specified disease,
disorder or condition, which reduces the severity of the disease,
disorder or condition, or retards or slows the progression of the
disease, disorder or condition ("therapeutic treatment"), and also
contemplates an action that occurs before a subject begins to
suffer from the specified disease, disorder or condition
("prophylactic treatment").
[0240] In general, the "effective amount" of a compound refers to
an amount sufficient to elicit the desired biological response. As
will be appreciated by those of ordinary skill in this art, the
effective amount of a compound of the invention may vary depending
on such factors as the desired biological endpoint, the
pharmacokinetics of the compound, the disease being treated, the
mode of administration, and the age, health, and condition of the
subject. An effective amount encompasses therapeutic and
prophylactic treatment.
[0241] As used herein, and unless otherwise specified, a
"therapeutically effective amount" of a compound is an amount
sufficient to provide a therapeutic benefit in the treatment of a
disease, disorder or condition, or to delay or minimize one or more
symptoms associated with the disease, disorder or condition. A
therapeutically effective amount of a compound means an amount of
therapeutic agent, alone or in combination with other therapies,
which provides a therapeutic benefit in the treatment of the
disease, disorder or condition. The term "therapeutically effective
amount" can encompass an amount that improves overall therapy,
reduces or avoids symptoms or causes of disease or condition, or
enhances the therapeutic efficacy of another therapeutic agent.
[0242] As used herein, and unless otherwise specified, a
"prophylactically effective amount" of a compound is an amount
sufficient to prevent a disease, disorder or condition, or one or
more symptoms associated with the disease, disorder or condition,
or prevent its recurrence. A prophylactically effective amount of a
compound means an amount of a therapeutic agent, alone or in
combination with other agents, which provides a prophylactic
benefit in the prevention of the disease, disorder or condition.
The term "prophylactically effective amount" can encompass an
amount that improves overall prophylaxis or enhances the
prophylactic efficacy of another prophylactic agent.
[0243] Exemplary cancers include, but are not limited to, acoustic
neuroma, adenocarcinoma, adrenal gland cancer, anal cancer,
angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma,
hemangiosarcoma), appendix cancer, benign monoclonal gammopathy,
biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast
cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of
the breast, mammary cancer, medullary carcinoma of the breast),
brain cancer (e.g., meningioma; glioma, e.g., astrocytoma,
oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid
tumor, cervical cancer (e.g., cervical adenocarcinoma),
choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer
(e.g., colon cancer, rectal cancer, colorectal adenocarcinoma),
epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's
sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial
cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer
(e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),
Ewing sarcoma, eye cancer (e.g., intraocular melanoma,
retinoblastoma), familiar hypereosinophilia, gall bladder cancer,
gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal
stromal tumor (GIST), head and neck cancer (e.g., head and neck
squamous cell carcinoma, oral cancer (e.g., oral squamous cell
carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal
cancer, nasopharyngeal cancer, oropharyngeal cancer)),
hematopoietic cancers (e.g., leukemia such as acute lymphocytic
leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic
leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic
leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic
lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma
such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and
non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large
cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)),
follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic
lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone
B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT)
lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal
zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt
lymphoma, lymphoplasmacytic lymphoma (i.e., "Waldenstrom's
macroglobulinemia"), hairy cell leukemia (HCL), immunoblastic large
cell lymphoma, precursor B-lymphoblastic lymphoma and primary
central nervous system (CNS) lymphoma; and T-cell NHL such as
precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell
lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,
mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell
lymphoma, extranodal natural killer T-cell lymphoma, enteropathy
type T-cell lymphoma, subcutaneous panniculitis-like T-cell
lymphoma, anaplastic large cell lymphoma); a mixture of one or more
leukemia/lymphoma as described above; and multiple myeloma (MM)),
heavy chain disease (e.g., alpha chain disease, gamma chain
disease, mu chain disease), hemangioblastoma, inflammatory
myofibroblastic tumors, immunocytic amyloidosis, kidney cancer
(e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma),
liver cancer (e.g., hepatocellular cancer (HCC), malignant
hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell
lung cancer (SCLC), non-small cell lung cancer (NSCLC),
adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis
(e.g., systemic mastocytosis), myelodysplastic syndrome (MDS),
mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia
Vera (PV), essential thrombocytosis (ET), agnogenic myeloid
metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic
myelofibrosis, chronic myelocytic leukemia (CML), chronic
neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)),
neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or
type 2, schwannomatosis), neuroendocrine cancer (e.g.,
gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid
tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma,
ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary
adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma,
intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors),
penile cancer (e.g., Paget's disease of the penis and scrotum),
pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer
(e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma,
salivary gland cancer, skin cancer (e.g., squamous cell carcinoma
(SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)),
small bowel cancer (e.g., appendix cancer), soft tissue sarcoma
(e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant
peripheral nerve sheath tumor (MPNST), chondrosarcoma,
fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland
carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular
embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of
the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid
cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g.,
Paget's disease of the vulva).
[0244] Anti-cancer agents encompass biotherapeutic anti-cancer
agents as well as chemotherapeutic agents.
[0245] Exemplary biotherapeutic anti-cancer agents include, but are
not limited to, interferons, cytokines (e.g., tumor necrosis
factor, interferon .alpha., interferon .gamma.), vaccines,
hematopoietic growth factors, monoclonal serotherapy,
immunostimulants and/or immunodulatory agents (e.g., IL-1, 2, 4, 6,
or 12), immune cell growth factors (e.g., GM-CSF) and antibodies
(e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab),
ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab),
BEXXAR (tositumomab)). Exemplary chemotherapeutic agents include,
but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene,
and megestrol), LHRH agonists (e.g. goscrclin and leuprolide),
anti-androgens (e.g. flutamide and bicalutamide), photodynamic
therapies (e.g. vertoporfin (BPD-MA), phthalocyanine,
photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)),
nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide,
chlorambucil, estramustine, and melphalan), nitrosoureas (e.g.
carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.
busulfan and treosulfan), triazenes (e.g. dacarbazine,
temozolomide), platinum containing compounds (e.g. cisplatin,
carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine,
vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel
or a paclitaxel equivalent such as nanoparticle albumin-bound
paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel
(DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel
(PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the
tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three
molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the
erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel,
e.g., 2'-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel,
taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,
teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,
irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR
inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate,
edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid,
tiazofurin, ribavirin, and EICAR), ribonucleotide reductase
inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs
(e.g. 5-fluorouracil (5-FU), floxuridine, doxifluridine,
ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g.
cytarabine (ara C), cytosine arabinoside, and fludarabine), purine
analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs
(e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors
(e.g. lovastatin), dopaminergic neurotoxins (e.g.
1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.
staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),
bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin),
anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal
doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin,
mitoxantrone), MDR inhibitors (e.g. verapamil), Ca.sup.2+ ATPase
inhibitors (e.g. thapsigargin), imatinib, thalidomide,
lenalidomide, tyrosine kinase inhibitors (e.g., axitinib
(AG013736), bosutinib (SKI-606), cediranib (RECENTIN.TM., AZD2171),
dasatinib (SPRYCEL.RTM., BMS-354825), erlotinib (TARCEVA.RTM.),
gefitinib (IRESSA.RTM.), imatinib (Gleevec.RTM., CGP57148B,
STI-571), lapatinib (TYKERB.RTM., TYVERB.RTM.), lestaurtinib
(CEP-701), neratinib (HKI-272), nilotinib (TASIGNA.RTM.), semaxanib
(semaxinib, SU5416), sunitinib (SUTENT.RTM., SU11248), toceranib
(PALLADIA.RTM.), vandetanib (ZACTIMA.RTM., ZD6474), vatalanib
(PTK787, PTK/ZK), trastuzumab (HERCEPTIN.RTM.), bevacizumab
(AVASTIN.RTM.), rituximab (RITUXAN.RTM.), cetuximab (ERBITUX.RTM.),
panitumumab (VECTIBIX.RTM.), ranibizumab (Lucentis.RTM.), nilotinib
(TASIGNA.RTM.), sorafenib (NEXAVAR.RTM.), everolimus
(AFINITOR.RTM.), alemtuzumab (CAMPATH.RTM.), gemtuzumab ozogamicin
(MYLOTARG.RTM.), temsirolimus (TORISEL.RTM.), ENMD-2076, PCI-32765,
AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK.TM.),
SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869,
MP470, BIBF 1120 (VARGATEF.RTM.), AP24534, JNJ-26483327, MGCD265,
DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930,
MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g.,
bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin,
temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus,
AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226
(Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980
(Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen,
gemcitabine, carminomycin, leucovorin, pemetrexed,
cyclophosphamide, dacarbazine, procarbizine, prednisolone,
dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,
methopterin, porfiromycin, melphalan, leurosidine, leurosine,
chlorambucil, trabectedin, procarbazine, discodermolide,
carminomycin, aminopterin, and hexamethyl melamine.
Examples
[0246] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
Lipidoid Synthesis
[0247] Lipidoids were synthesized through the conjugate addition of
alkyl-acrylates to amines. Amines were purchased from Sigma Aldrich
(St. Louis, Mo.), Alfa Aesar, Acros Organics, and CHESS Organics.
Acrylates were purchased from Scientific Polymer Products (Ontario,
N.Y.) and Hampford Research, Inc. (Stratford, Conn.). Amines were
combined with acrylates stoichiometrically in a glass scintillation
vial and were stirred at 90.degree. C. for either for 3 days. In
vitro experiments were conducted with crude materials, and in vivo
experiments were performed with lipidoids purified via a Teledyne
Isco Chromatography system (Lincoln, Nebr.).
Lipidoid Hydrolysis
[0248] To a 25 ml round bottom flask was added 304O.sub.13 (0.250
g, 0.263 mmol, 1 equiv). For acidic hydrolysis, 10 ml of a solution
of 6 N HCl was added to the flask to afford a cloudy heterogeneous
solution. The reaction was heated to reflux to afford a clear,
homogeneous solution and was stirred at reflux for 24 hours. For
basic hydrolysis, 10 ml of a solution of KOH in EtOH/H.sub.2O
(solution=5.61 g KOH in 47.5 ml EtOH w/2.5 ml distilled H.sub.2O)
was added to the flask to afford a clear colorless solution. The
reaction was heated to reflux and stirred for 41 h. Both acidic and
basic reactions were cooled to room temperature and TLC analysis
showed the presence of tridecanol (17.5% EtOAC/Hexanes) and the
consumption of 304O.sub.13. Reactions were concentrated to dryness
under reduced pressure and diluted with CDCl.sub.3. The basic
reaction was filtered to remove excess KOH. Proton NMR analysis was
performed in CDCl.sub.3. Proton nuclear magnetic resonance spectra
were recorded with a Bruker Avance 400 spectrometer, are depicted
in parts per million on the .delta. scale, and are referenced from
the residual protium in the NMR solvent (CDCl.sub.3: .delta. 7.26
(CHCl.sub.3).
Formulation of Lipid Nanoparticles
[0249] Lipidoids were formulated into nanoparticles for all studies
described in the Examples. Nanoparticles were formed by mixing
lipidoids, cholesterol (Sigma Aldrich), DSPC (Avanti Polar Lipids,
Alabaster, Ala.) and mPEG2000-DMG (MW 2660, gift from Alnylam
Pharamceuticals, Cambridge, Mass.) at a molar ratio of
38.5:50:(11.5-X):X in a solution of 90% ethanol and 10% 10 mM
sodium citrate (by volume). An siRNA solution was prepared by
diluting siRNA in 10 mM sodium citrate such that the final weight
ratio of total lipid (lipidoid+cholesterol+DSPC+PEG):siRNA was
10:1. Equal volumes of lipid solution and siRNA solution were
rapidly mixed together using either a microfluidic device (Chen, D.
et al. J. Am. Chem. Soc. 134, 120410134818007 (2012)) or by pipet
to form nanoparticles. Particles were diluted in phosphate buffered
saline (PBS, Invitrogen) and then dialyzed against PBS for 90
minutes in 3500 MWCO cassettes (Pierce/Thermo Scientific, Rockford,
Ill.).
In Vitro Transfection of Cell Lines with Lipid Nanoparticles
[0250] HeLa cells stably modified to express both firefly and
Renilla luciferase were maintained at 37.degree. C. in high glucose
Dulbecco's Modified Eagles Medium without phenol red (Invitrogen,
Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS,
Invitrogen). 12-16 hours prior to transfection, cells were seeded
in white 96-well plates at a density of 15,000 cells per well.
Cells were transfected with a 40 nM concentration of anti-firefly
luciferase siRNA (Dharmacon, Lafayette, Colo.) that had been
formulated with lipidoids into nanoparticles. Firefly luciferase
silencing was assessed with a Dual-Glo.RTM. Luciferase Assay kit
(Promega, Madison, Wis.). Renilla luciferase activity served as a
control. Data for certain lipidoids are shown in Table 1 below.
In Vivo Gene Silencing
[0251] All animal experiments were conducted using
institutionally-approved protocols. Female C57BL/6 mice (Charles
River Laboratories, Wilmington, Mass.) received injections through
the lateral tail vein injections of PBS (negative control), or
lipidoid nanoparticles containing either non-targeting siRNA
(negative control) or anti-Factor VII siRNA diluted in PBS at a
volume of 0.01 ml/g. The sequence of the siFVII, provided by
Alnylam Pharmaceuticals, was:
TABLE-US-00001 sense: (SEQ ID NO.: 1) 5'-GGAucAucucAAGucuuAcT*T-3'
antisense: (SEQ ID NO.: 2) 5'-GuAAGAcuuGAGAuGAuccT*T-3'
where 2'-fluoro-modified nucleotides are in lower case and
phosphorothioate linkages are represented by asterisks. Two days
post-injection, a 100 ul blood sample was obtained from mice and
centrifuged at 13,000 rpm in serum separator tubes (Becton
Dickinson). Serum levels of Factor VII were analyzed using a
Biophen FVII assay kit as described previously (Aniara Corporation,
Mason, Ohio) Semple, S. C. et al. Nature Biotechnology 1-7 (2010).
Results shown in Table 2.
TABLE-US-00002 TABLE 2 Original Library FVII Activity Data 5 mg/kg
2 mg/kg 0.5 mg/kg 0.1 mg/kg Relative Relative Relative Relative
FVII Standard FVII Standard FVII Standard FVII Standard Lipidoid
Activity Deviation Activity Deviation Activity Deviation Activity
Deviation 64O14 0.92 0.05 68O10 0.91 0.20 68O11 1.03 0.11 77O13
0.85 0.10 77O14 0.74 0.02 80O13 0.66 0.23 81O13 0.43 0.18 86O12
0.87 0.07 87O13 0.77 0.03 94O14 0.13 0.06 99O11 0.53 0.07 109O11
0.85 0.05 109O12 0.82 0.06 109O13 0.38 0.09 110O10 0.35 0.11 110O13
0.75 0.07 113O10 0.15 0.06 113O11 0.57 0.20 113O12 0.01 0.00 0.09
0.10 0.49 0.08 0.59 0.08 113O13 0.00 0.00 0.00 0.00 0.04 0.11 0.48
0.12 113O14 0.75 0.14 120O11 0.93 0.04 120O12 0.88 0.15 120O13 0.92
0.08 120O14 0.79 0.05 122O10 0.13 0.06 122O12 0.82 0.06 123O12 0.93
0.07 123O13 0.00 0.00 0.00 0.00 0.50 0.22 0.86 0.13 134O13 0.89
0.16 144O13 0.93 0.17 154O12 0.99 0.11 156O11 0.77 0.09 156O12 1.06
0.06 158O14 0.91 0.04 159O14 0.80 0.01 161O14 0.85 0.05 164O14 0.90
0.08 166O10 0.99 0.03 166O14 0.83 0.08 191O11 0.61 0.19 191O12 0.80
0.19 191O14 0.61 0.10 193O10 0.70 0.12 193O11 0.61 0.13 193O12 0.70
0.09 195O12 0.43 0.07 195O13 0.56 0.04 196O13 0.80 0.01 196O14 0.82
0.06 200O10 0.56 0.17 200O11 0.67 0.07 200O12 0.62 0.00 200O13 0.40
0.10 200O14 0.91 0.20 205O12 0.78 0.10 205O14 0.83 0.09 217O12 0.58
0.13 217O13 0.02 0.01 0.33 0.28 0.85 0.13 0.92 0.12 218O13 0.73
0.16 219O13 0.00 0.00 235O13 0.39 0.07 25O13 0.25 0.23 302O13 1.03
0.22 302O14 0.84 0.29 303O10 0.95 0.26 303O12 0.01 0.01 0.20 0.09
0.91 0.04 0.95 0.05 304O11 0.57 0.02 304O12 0.81 0.24 304O13 0.01
0.00 0.02 0.01 0.23 0.06 0.54 0.26 304O14 0.58 0.13 305O12 0.75
0.11 305O13 0.00 0.00 0.06 0.04 0.25 0.06 0.96 0.17 306O10 0.00
0.00 0.00 0.01 0.23 0.07 0.69 0.13 306O11 0.02 0.01 0.00 0.00 0.40
0.15 0.59 0.17 306O12 0.00 0.00 0.00 0.00 0.20 0.15 0.37 0.02
306O13 0.00 0.00 0.00 0.01 0.04 0.07 0.71 0.14 306O14 0.85 0.05
313O10 0.00 0.00 0.21 0.11 0.74 0.10 1.04 0.13 313O11 0.01 0.02
0.38 0.16 0.75 0.05 0.73 0.07 313O12 0.01 0.04 0.52 0.25 0.89 0.33
0.95 0.26 313O13 0.01 0.01 0.09 0.04 0.09 0.08 0.86 0.24 313O14
0.67 0.04 315O12 0.99 0.39 31O14 0.88 0.14 32O14 0.80 0.06 347O10
0.92 0.08 347O11 0.85 0.16 347O12 0.15 0.08 347O13 0.67 0.22 36O14
0.69 0.25 371O11 0.78 0.09 371O12 0.78 0.06 371O14 0.85 0.03
Biodistribution and Immunostaining
[0252] Female C57BL/6 mice received tail vein injections of lipid
nanoparticles containing siRNA that had been labeled with Cy5.5 on
the 5' end of the sense strand (provided by Alnylam
Pharmaceuticals). Animals were dosed at 1 mg/kg of siRNA and volume
of 0.01 ml/g. At one hour post-injection, mice were euthanized and
organs were removed. Body-wide biodistribution was assessed by
imaging whole organs with an IVIS.RTM. Spectrum system (Caliper
Life Sciences, Hopkinton, Mass.) at excitation and emission
wavelengths of 675 nm and 720 nm, respectively. Cell-specific
distribution within hepatocytes was assessed by embedding,
sectioning, and staining the whole liver with antibodies. Imaging
was conducted on a LSM 700 confocal microscope (Carl Zeiss, Inc.,
Peabody, Mass.). For Odyssey and confocal imaging, organs were snap
frozen on dry ice and embedded in optimal cutting temperature
compound (OCT, Life Technologies, Grand Island, N.Y.). Cryostat
sections were cut and collected on superfrost plus treated slides.
Prepared frozen sections where kept at -20.degree. C. until needed.
Odyssey imaging was conducted on 20 .mu.m thick cryosections of
tissue at a resolution of 21 .mu.m (Lee, M. J.-E. et al., Rapid
Pharmacokinetic and Biodistribution Studies Using
Cholorotoxin-Conjugated Iron Oxide Nanoparticles: A Novel
Non-Radioactive Method. PLoS ONE 5, e9536-e9536 (2010)).
[0253] For confocal imaging, liver tissue was cryosectioned (12
.mu.m) and fixed using 4% paraformaldehyde at room temperature for
30 min. All solutions were prepared in PBS. Sections were washed
2.times. with PBS, permeabilized for 30 min with 0.1% Triton X100,
and blocked for 1 hour with 5% normal goat serum. Sections then
incubated for 1 hour in an immunostaining cocktail solution
consisting of DAPI (3 .mu.M), Alexa Fluor 488 conjugated anti-mouse
F4/80 (1:200 dilution, BioLegend, San Diego, Calif.), Alexa
Fluor.RTM. 555 Phalloidin (1:200 dilution, Life Technologies), and
5% normal goat serum. Slides were washed 3.times. with 0.1% Tween
20 and mounted using ProLong.RTM. Gold Antifade (Life
Technologies). Sections were imaged using an LSM 700 point scanning
confocal microscope (Carl Zeiss, Inc, Jena Germany) equipped with a
40.times. oil immersion objective.
Blood Clearance
[0254] Blood clearance experiments were conducted by injecting LNPs
containing Cy5.5-labeled siRNA at an siRNA dose of 0.5 mg/kg. Blood
samples were collected as a function of time via the retroorbital
vein, with the exception of final time points, which were collected
via cardiac puncture. Serum, obtained by centrifugation, was
diluted 1:30 in PBS and imaged and quantified using an Odyssey CLx
imaging system (LI-COR Biosciences, Lincoln, Nebr.).
Histology
[0255] Organs were harvested from animals that had received various
doses of either 304O.sub.13 or C12-200 lipid nanoparticles (C12-200
is a control non-degradable lipidoid shown below). Organs were
fixed overnight in 4% paraformaldehyde and transferred to 70%
ethanol prior to paraffin embedding, sectioning, and H & E
staining.
##STR00134##
Serum Chemistry and Hematology Analysis
[0256] Post-sacrifice, cardiac sticks were immediately performed on
animals that had been dosed with either 304O.sub.13 or C12-200
lipid nanoparticles. Blood was centrifuged in serum separator tubes
at 5,000 rpm for 10 minutes, and serum was analyzed for various
hematological parameters. Serum chemistry was evaluated on a
Beckman Olympus AU400 Serum Chemistry Analyzer. Cytokines were
analyzed using Bio-Plex Pro Mouse Cytokine 23-Plex Assay kits
(Luminex Corporation, Austin, Tex.) on the Bio-Plex 200 system,
according to manufacturer instructions.
Cytokine Profiling
[0257] Cytokine analysis was done by injecting either 304O.sub.13
or C12-200 nanoparticles at an siRNA dose of 3 mg/kg. Four hours
post-injection, blood was harvested via cardiac stick and serum was
isolated. Cytokine levels were quantified using an ELISA assay.
Nanoparticle Characterization
[0258] Lipid nanoparticles were diluted to an siRNA concentration
of .about.5 ug/ml in 0.1.times.PBS, pH 7.3. siRNA entrapment
efficiency was determined using the Quant-iT.TM. RiboGreen.RTM. RNA
assay (Invitrogen). Particle sizes were measured with a ZETAPals
analyzer (Brookhaven Instruments, Holtsville, N.Y.). Sizes reported
are the average effective diameter of each LNP. Zeta potential
measurements were acquired on a Zetasizer Nano ZS (Malvern,
Westborough, Mass.), and reported values were the average of 10-25
runs.
TABLE-US-00003 TABLE 3 Characterization Parameters for 304O.sub.13
siRNA Entrapment Diameter Zeta Potential (%) (nm) (mV) pKa
304O.sub.13 84.2 86.0 13.7 6.8 306O12 79.0 98.2 12.5 6.8 113O13
75.8 91.1 16.5 6.0
Results and Discussion
[0259] Michael addition chemistry was employed to rapidly
synthesize a library of 1400 lipid-like materials with the
potential to serve as effective, biodegradable delivery vehicles
(FIG. 1). 280 alkyl-amines (FIG. 2) were reacted combinatorially
with 5 alkyl-acrylates to form lipidoids consisting of a polar,
ionizable core surrounded by hydrophobic carbon tails.
Alkyl-amines, which were taken from commercially available supply,
were chosen to maximize structural diversity and reactivity within
a Michael addition scheme. We chose to work with alkyl-acrylate
tails of intermediate length (10-14 carbon chain length), as
previous studies indicated that shorter tails often lack efficacy
while longer tails may cause insolubility during the nanoparticle
formulation process (Akinc, A. et al. Nature Biotechnology 26,
561-569 (2008); Love, K. T. et al. Proc. Natl. Acad. Sci. USA 107,
1864-1869 (2010)).
[0260] The acrylate-based lipidoids provided herein also contain
hydrolysable ester moieties, functional groups which are commonly
incorporated into delivery vehicles to promote physiological
degradation (Staubli, A., Ron, E. & Langer, R. J. Am. Chem.
Soc. 112, 4419-4424 (1990); van Dijkhuizen-Radersma, et al.
Biomaterials 23, 4719-4729 (2002); Geng, Y. & Discher, D. E. J.
Am. Chem. Soc. 127, 12780-12781 (2005)). Proton NMR analysis
indicated that a representative lipidoid, 304O.sub.13, degraded to
the anticipated alkyl-alcohol product under hydrolytic conditions
(FIGS. 11A and 11B).
[0261] To determine the transfection ability of lipidoids, they
were first formulated into lipid nanoparticles (LNPs) containing
siRNA, cholesterol and the helper lipids, DSPC and PEG(MW2000)-DMG.
The delivery potential of lipidoids was assessed by applying LNPs
to HeLa cells that had been genetically modified to stably express
two reporter luciferase proteins: firefly and Renilla. Firefly
luciferase served as the target gene while Renilla luciferase
served as a built-in control for toxicity and off-targeting
effects. Relative luciferase activity, which is the ratio of
firefly to Renilla activity, is shown in FIG. 3a after treatment
with each LNP at an siRNA concentration of 40 nM. Of the 1400
members of the lipidoid library, .about.7% mediated target gene
silencing of >50% (shown in red circles).
[0262] In order to extract structure-function information from the
in vitro data, we asked whether various structural properties were
more or less common within the group of efficacious lipidoids (red
data points) compared to the bulk library. FIG. 3b examines the
importance of tail length on transfection. Because there were five
tails used in this library, each tail length made up 20% of the
library. Of the LNPs that were effective in vitro, however, only
12% contained an O.sub.10 tail. Occurrence rate (the y-axis value)
was calculated as (the occurrence rate in the library)-(the
occurrence rate in the group with >50% silencing). Therefore,
the occurrence rate for O.sub.10 is 12%-20%=-8%, indicating that it
was significantly underrepresented among materials with
transfection potential. On the other hand, O.sub.12 and O.sub.13
tails were overrepresented in the efficacious group compared to the
library at large, suggesting such tail lengths are associated with
efficacious lipidoids. FIG. 3c suggests that lipidoids with the
greatest transfection potential were synthesized from alkyl-amines
with three or more substitution sites. The effect of various
functional groups within the alkyl-amine is analyzed in FIG. 3d.
The presence of tertiary and secondary amines, alcohols, and
branched or linear chains conferred efficacy, while ethers and
rings generally did not. Piperazine rings, however, were an
exception, and generally produced efficacious materials.
[0263] Previous studies have indicated that materials capable of
conferring >50% luciferase silencing activity in cell culture
have the potential to mediate siRNA delivery in vivo (Whitehead, K.
A. et al. In Vitro--In Vivo Translation of Lipid Nanoparticles for
Hepatocellular siRNA Delivery. ACS Nano 120706143602000
(2012).doi:10.1021/nn301922x). Selected lipidoids (those data
points shown in red in FIG. 3a) were analyzed for siRNA delivery to
hepatocytes in a murine model of the blood coagulation Factor VII.
The Factor VII model, which has been well-validated in the
literature (Akinc, A. et al. Nature Biotechnology 26, 561-569
(2008); John, M. et al. Nature 449, 745-747 (2007); Semple, S. C.
et al. Nature Biotechnology 1-7 (2010)), allows silencing to be
assessed from a few drops of blood using a commercially-available
assay. In these experiments, LNPs containing anti-Factor VII siRNA
were injected intravenously into mice, and Factor VII activity
levels were quantified two days post-injection. Fifteen of the 108
lipidoids analyzed in vivo mediated complete knockdown of Factor
VII protein levels at an siRNA dose of 5 mg/kg (FIG. 4a). For these
top LNP candidates, control experiments conducted using
non-targeting siRNA at 5 mg/kg resulted in no FVII knockdown and
suggested that reductions in protein activity were not due to
off-targeting or toxicity-mediated gene downregulation. Silencing
for these top candidates was dose dependent (FIG. 8), with
EC.sub.50 values ranging from 0.05 to 2 mg/kg when LNPs were
formulated at a lipidoid:cholesterol:DSPC:PEG standard testing
molar ratio of 50:38.5:10:1.5.
[0264] While seeking an optimal molar ratio for the top LNPs (e.g.
306O.sub.12, 113O.sub.13, and 304O.sub.13), the PEG molar
percentage was found to have an effect on LNP efficacy. FIG. 4c
reveals that, for the lipidoid 304O.sub.13, there is a range of PEG
% between 0.5 and 1.0 where optimal hepatocellular delivery is
achieved. The optimized 304O.sub.13 formulation (PEG %=0.75) has an
EC50 value, 0.01 mg/kg, that is a full order of magnitude lower
than when using 1.5% PEG. Optimized 304O.sub.13 behaved in a dose
dependent fashion (FIG. 4d), and after a single injection at 0.1
mg/kg, Factor VII levels returned to baseline within 18 days.
[0265] In addition to examining hepatocellular delivery, we also
explored the ability of biodegradable lipidoid materials to deliver
siRNA to leukocyte populations in vivo. Immune cells are attractive
targets for RNA interference therapy, as they have been implicated
in various aspects of disease initiation and progression, including
inflammation and autoimmune responses (Geissmann, F. et al. Science
327, 656-661 (2010); Grivennikov, et al. Cell 140, 883-899 (2010)).
Although moderate levels of gene silencing have been achieved
recently in leukocytes (Leuschner, F. et al. Nature Biotechnology
29, 1005-1010 (2011); Novobrantseva, T. I. et al. Molecular
Therapy--Nucleic Acids 1, e4 (2012)), it will be important
clinically that compounds can be degraded and eliminated from the
body. In these experiments, LNPs were formulated with siRNA
specific against CD45, which is a tyrosine phosphatase protein
found on the surface of all white blood cells. Three days following
the intravenous delivery of LNPs in mice, immune cells were
harvested from the peritoneal cavity and spleen. Cells were stained
with fluorescent antibodies, and CD45 protein silencing was
quantified in specific immune cell subsets via flow cytometry
analysis. Results were normalized to CD45 levels after delivery of
the same LNP containing a non-targeting siRNA. Of the five lipidoid
materials evaluated in this model, 304O.sub.13 and 306O.sub.13
mediated the most robust CD45 silencing in immune cells isolated
from both the peritoneal cavity and the spleen (FIGS. 4e and f).
CD11b+ and CD11c+ populations (monocyte/macrophages and dendritic
cells, respectively) were subject to high levels of knockdown
within the peritoneal cavity (up to 90%) and to a lesser degree
within the spleen (up to 40%). The lipidoids 306O.sub.12,
306O.sub.14, and 315O.sub.12 also offered modest CD45 silencing in
several immune cell subpopulations (FIG. 9).
[0266] Nanoparticle characterization parameters for three of the
top LNP candidates were similar (Table 1). Entrapment of siRNA
refers to the percentage of siRNA in solution that is incorporated
into the nanoparticle during formulation, as measured by an RNA
dye-binding assay (Nolan, T., Hands, R. E. & Bustin, S. A.
Quantification of mRNA using real-time RT-PCR. Nat. Protoc 1,
1559-1582 (2006)). These results are in keeping with a previous
finding that efficacious lipidoid nanoparticles often have
entrapment values of approximately 75%17. Zeta potential
measurements were conducted under neutral pH conditions. pKa
values, which were obtained using a toluene nitrosulphonic acid
(TNS) assay, evaluated the pKa of the nanoparticle surface (Heyes,
J., Palmer, L., Bremner, K. & MacLachlan, I. Cationic lipid
saturation influences intracellular delivery of encapsulated
nucleic acids. J Control Release 107, 276-287 (2005)). The pKa
values of top LNP candidates corroborate the results of another
study in which surface pKa values in the 6-7 range conveyed
efficacy in vivo (Jayaraman, M. M. et al., Maximizing the Potency
of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo.
Angew. Chem. Int. Ed. 51, 8529-8533 (2012)).
[0267] Several analyses were performed to assess the
biodistribution of the lead compound, 304O.sub.13, in mice. For
these experiments, nanoparticles were formulated with Cy5.5-labeled
siRNA. Whole organ IVIS images (FIG. 5a) and Odyssey scans (FIG.
5b) showed that naked siRNA accumulated in the kidneys at 1 hour
post-injection, suggesting rapid renal clearance. Quantification of
IVIS signal indicated that 14%, 1%, and 71% of naked siRNA signal
appeared in the liver, spleen, and kidneys, respectively. In
contrast, at 1 hour post injection, 304O.sub.13 localized primarily
within the liver (42%) and spleen (24%), with only 18% distributing
to the kidneys.
[0268] Given their effectiveness for silencing the hepatocellular
target, FVII, we examined how 304O.sub.13 nanoparticles were
distributing within the liver. Confocal imaging was performed on
liver tissues harvested one hour post-injection and stained with
nuclear, actin, and macrophage markers (FIG. 7c). Images were taken
near the central vein in liver lobules (black void near the center
of images). Hepatocytes are outlined in green and macrophages,
which appear sporadically, are colored magenta. Only 304O.sub.13
was able to mediate siRNA accumulation throughout nearly all
hepatocellular tissue (in red).
[0269] Serum clearance kinetics were assessed by measuring Cy5.5
signal in the mouse bloodstream as a function of time (FIG. 5d). It
should be noted that, while the first blood sample was drawn as
quickly as possible (20 seconds), maximum signal may have occurred
even earlier. Half of the material initially detected at 20 seconds
had distributed to tissues by 6 minutes. At 90 minutes
post-injection, only 4% of signal remained.
[0270] A preliminary safety assessment was conducted on the lead
LNP, 304O.sub.13, and it was compared to another
previously-discovered LNP formulation, C12-200 (Love, K. T. et al.
Lipid-like materials for low-dose, in vivo gene silencing. PNAS
107, 1864-1869 (2010)). C12-200 is a 5-tailed, lipidoid that has
the same EC.sub.50 as 304O.sub.13 (0.01 mg/kg). It was chosen for
comparison purposes because it does not contain any functional
groups that are overtly sensitive to hydrolysis. We chose to
examine the effect of doses that were at least 100-fold higher than
the EC.sub.50. Serum cytokine levels for both materials were
assessed in mice four hours after a 3 mg/kg IV bolus injection
(total siRNA). IL-6, IP-10, KC, and MCP-1 were elevated in the
C12-200 group compared to both PBS negative control and 304O.sub.13
groups under these conditions (FIG. 6). Clinical chemistry
parameters were evaluated for both materials 72 hours after a
single dose of 3 mg/kg and after four once weekly doses of 3 mg/kg
each. There were no toxicologically significant increases in
albumin, ALT, AST, ALP, total bilirubin, or GGT for either
304O.sub.13 or C12-200 after single or multiple doses (FIG.
12).
[0271] Histological analysis was performed through H&E staining
on sections from the liver, spleen, kidneys and pancreas. In
single-dose studies (0, 1, 2, 3, 5, 7.5, 10 mg/kg), liver necrosis
was observed in mice administered .gtoreq.7.5 mg/kg of C12-200 and
at 10 mg/kg of 304O.sub.13. Pancreatic inflammation and islet cell
enlargement were detected at C12-200 doses .gtoreq.2 mg/kg. A small
amount of apoptosis in splenic red pulp was observed at 10 mg/kg
for 304O.sub.13. Multi-dose studies were also conducted in which
mice received four injections of 0.3, 1, 2, 3, or 5 mg/kg, once per
week for four weeks. Liver necrosis and inflammation were observed
in mice administered .gtoreq.1 mg/kg of C12-200. There was no sign
of liver toxicity in any of the 304O.sub.13 groups up to 5 mg/kg.
Based on this limited evaluation, the collective data suggest an
improved toxicity profile for 304O.sub.13 compared to C12-200 in
mice.
[0272] The data from the 108 materials tested in vivo at a total
siRNA dose of 5 mg/kg are shown in FIG. 7a. Of the 108 materials
tested in mice, 25 of them contained an O.sub.13 tail, 66 of them
had three or more tails, and 42 of them had been synthesized from
an alkyl-amine that contained at least one tertiary amine.
[0273] FIG. 7b shows a second generation library of lipidoids from
certain amines conjugated to an O.sub.13 tail. When tested in vivo,
10 out of 12 of these materials mediated 100% Factor VII silencing
at a dose of 5 mg/kg (FIG. 7c). Knockdown was dose-dependent, with
EC50 values varying from 0.05-1 mg/kg (FIG. 7d). Formulation
optimization of the best second generation material, 503O.sub.13,
markedly decreased the EC.sub.50 value to 0.01 mg/kg (FIG. 7e).
Several second generation materials also facilitated significant
CD45 knockdown in monocyte, macrophage, dendritic cell, and B cell
populations (FIG. 13).
[0274] Since the ability of materials to take on a positive charge
with decreasing pH has been shown to confer transfection efficacy
(Zhang, J. J., Fan, H. H., Levorse, D. A. D. & Crocker, L. S.
L. Ionization behavior of amino lipids for siRNA delivery:
determination of ionization constants, SAR, and the impact of lipid
pKa on cationic lipid-biomembrane interactions. Langmuir 27,
1907-1914 (2011)), the surface pKa values of 59 distinct lipidoid
nanoparticles were measured. The data in FIG. 10 indicate that pKa
values play a decisive role in this LNP delivery system, with a
critical pKa value of approximately 5.5. Materials demonstrating
considerable in vivo efficacy (red data points) had surface pKa
values of approximately 5.5 or higher. For values less than
approximately 5.5, average efficacy decreased monotonically with
pKa. Therefore, surface pKa can be used as an indicator of in vivo
potency, improving our predictive capability for this data set.
OTHER EMBODIMENTS
[0275] All patents, patent applications, and literature references
cited herein are incorporated herein by reference.
[0276] Having now described some illustrative embodiments of the
invention, it should be apparent to those skilled in the art that
the foregoing is merely illustrative and not limiting, having been
presented by way of example only. Numerous modifications and other
illustrative embodiments are within the scope of one of ordinary
skill in the art and are contemplated as falling within the scope
of the invention. In particular, although many of the examples
presented herein involve specific combinations of method acts or
system elements, it should be understood that those acts and those
elements may be combined in other ways to accomplish the same
objectives. Acts, elements, and features discussed only in
connection with one embodiment are not intended to be excluded from
a similar role in other embodiments. Further, for the one or more
means-plus-function limitations recited in the following claims,
the means are not intended to be limited to the means disclosed
herein for performing the recited function, but are intended to
cover in scope any means, known now or later developed, for
performing the recited function. Use of terms such as "first",
"second", "third", etc., in the claims to modify a claim element
does not by itself connote any priority, precedence, or order of
one claim element over another or the temporal order in which acts
of a method are performed, but are used merely as labels to
distinguish one claim element having a certain name from another
element having a same name (but for use of the ordinal term) to
distinguish the claim elements. Similarly, use of a), b), etc., or
i), ii), etc. does not by itself connote any priority, precedence,
or order of steps in the claims. Similarly, the use of these terms
in the specification does not by itself connote any required
priority, precedence, or order.
[0277] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the invention.
Sequence CWU 1
1
2121DNAArtificial SequenceSynthetic Polynucleotide 1ggaucaucuc
aagucuuact t 21221DNAArtificial SequenceSynthetic Polynucleotide
2guaagacuug agaugaucct t 21
* * * * *
References