U.S. patent application number 17/496530 was filed with the patent office on 2022-06-30 for lipids and lipid nanoparticle formulations for delivery of nucleic acids.
The applicant listed for this patent is Acuitas Therapeutics, Inc.. Invention is credited to Steven M. Ansell, Xinyao Du.
Application Number | 20220204439 17/496530 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220204439 |
Kind Code |
A1 |
Du; Xinyao ; et al. |
June 30, 2022 |
LIPIDS AND LIPID NANOPARTICLE FORMULATIONS FOR DELIVERY OF NUCLEIC
ACIDS
Abstract
Compounds are provided having the following structure:
##STR00001## or a pharmaceutically acceptable salt, tautomer or
stereoisomer thereof, wherein R.sup.1a, R.sup.1b, R.sup.2a,
R.sup.2b, R.sup.3a, R.sup.3b, R.sup.4a, R.sup.4b, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, L.sup.1, L.sup.2, G.sup.1, G.sup.2,
G.sup.3, a, b, c and d are as defined herein. Use of the compounds
as a component of lipid nanoparticle formulations for delivery of a
therapeutic agent, compositions comprising the compounds and
methods for their use and preparation are also provided.
Inventors: |
Du; Xinyao; (Richmond,
CA) ; Ansell; Steven M.; (Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Acuitas Therapeutics, Inc. |
Vancouver |
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CA |
|
|
Appl. No.: |
17/496530 |
Filed: |
October 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16265234 |
Feb 1, 2019 |
11168051 |
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17496530 |
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15196582 |
Jun 29, 2016 |
10221127 |
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16265234 |
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62186210 |
Jun 29, 2015 |
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International
Class: |
C07C 233/36 20060101
C07C233/36; C07D 295/13 20060101 C07D295/13; C07C 211/09 20060101
C07C211/09; C07C 235/10 20060101 C07C235/10; A61K 9/51 20060101
A61K009/51; A61K 31/713 20060101 A61K031/713; A61K 9/14 20060101
A61K009/14; A61K 31/7105 20060101 A61K031/7105; A61K 47/18 20060101
A61K047/18; A61K 47/24 20060101 A61K047/24; C07C 233/38 20060101
C07C233/38; C07D 207/09 20060101 C07D207/09; C12N 15/113 20060101
C12N015/113 |
Claims
1-55. (canceled)
56. A compound having a structure of Formula I: ##STR00098## or a
pharmaceutically acceptable salt, tautomer or stereoisomer thereof,
wherein: L.sup.1 and L.sup.2 are each a direct bond; G.sup.1 is
--(C.dbd.O)-- or a direct bond; G.sup.2 is --C(.dbd.O)-- or a
direct bond; G.sup.3 is saturated C.sub.1-C.sub.6 alkylene;
R.sup.1a and R.sup.1b are, at each occurrence, independently
either: (a) H or saturated C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a
is H or saturated C.sub.1-C.sub.12 alkyl, and R.sup.1b together
with the carbon atom to which it is bound is taken together with an
adjacent R.sup.1b and the carbon atom to which it is bound to form
a carbon-carbon double bond, wherein for at least one occurrence of
R.sup.1a and R.sup.1b, R.sup.1a is H or saturated C.sub.1-C.sub.12
alkyl, and R.sup.1b together with the carbon atom to which it is
bound is taken together with an adjacent R.sup.1b and the carbon
atom to which it is bound to form a carbon-carbon double bond;
R.sup.2a and R.sup.2b are, at each occurrence, independently
either: (a) H or saturated C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a
is H or saturated C.sub.1-C.sub.12 alkyl, and R.sup.2b together
with the carbon atom to which it is bound is taken together with an
adjacent R.sup.2b and the carbon atom to which it is bound to form
a carbon-carbon double bond; R.sup.3a and R.sup.3b are, at each
occurrence, independently either (a): H or saturated
C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or saturated
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond; R.sup.4a and R.sup.4b are, at each occurrence,
independently either: (a) H or saturated C.sub.1-C.sub.12 alkyl; or
(b) R.sup.4a is H or saturated C.sub.1-C.sub.12 alkyl, and R.sup.4b
together with the carbon atom to which it is bound is taken
together with an adjacent R.sup.4b and the carbon atom to which it
is bound to form a carbon-carbon double bond, wherein for at least
one occurrence of R.sup.4a and R.sup.4b, R.sup.4a is H or saturated
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond; R.sup.5 and R.sup.6 are each independently H or
methyl; R.sup.7 is saturated or unsaturated C.sub.4-C.sub.20 alkyl;
R.sup.8 and R.sup.9 are each independently saturated
C.sub.1-C.sub.12 alkyl; or R.sup.8 and R.sup.9, together with the
nitrogen atom to which they are attached, form a 5, 6 or 7-membered
heterocyclic ring; a, b, c and d are each independently an integer
from 1 to 24.
57. The compound of claim 56, having one of the following
structures (IA) or (IB): ##STR00099##
58. The compound of claim 56, wherein for at least one occurrence
of R.sup.2a and R.sup.2b, R.sup.2a is H or saturated
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
59. The compound of claim 56, wherein for at least one occurrence
of R.sup.3a and R.sup.3b, R.sup.3a is H or saturated
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
60. The compound of claim 56, having one of the following
structures (IC) or (ID): ##STR00100## wherein e, f, g and h are
each independently an integer from 1 to 12.
61. The compound of claim 60, wherein e, f, g and h are each
independently an integer from 4 to 10.
62. The compound of claim 56, wherein a, b, c and d are each
independently an integer from 2 to 12.
63. The compound of claim 62, wherein a, b, c and d are each
independently an integer from 5 to 9.
64. The compound of claim 56, wherein R.sup.1a, R.sup.2a, R.sup.3a
and R.sup.4a are H at each occurrence.
65. The compound of claim 56, wherein at least one of R.sup.1a,
R.sup.2a, R.sup.3a and R.sup.4a is saturated C.sub.1-C.sub.8
alkyl.
66. The compound of claim 65, wherein saturated C.sub.1-C.sub.8
alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,
tert-butyl, n-hexyl or n-octyl.
67. The compound of claim 56, wherein at least one of R.sup.1b,
R.sup.2b, R.sup.3b and R.sup.4b is H.
68. The compound of claim 56, wherein one of R.sup.5 or R.sup.6 is
methyl.
69. The compound of claim 56, wherein each of R.sup.5 and R.sup.6
is methyl.
70. The compound of claim 56, wherein R.sup.7 is saturated or
unsaturated C.sub.6-C.sub.16 alkyl.
71. The compound of claim 56, wherein R.sup.7 is saturated or
unsaturated C.sub.6-C.sub.9 alkyl.
72. The compound of claim 56, wherein R.sup.7 is substituted with
--(C.dbd.O)OR.sup.b, --O(C.dbd.O)R.sup.b, --C(.dbd.O)R.sup.b,
--OR.sup.b, --S(O).sub.xR.sup.b, --S--SR.sup.b,
--C(.dbd.O)SR.sup.b, --SC(.dbd.O)R.sup.b, --NR.sup.aR.sup.b,
--NR.sup.aC(.dbd.O)R.sup.b, --C(.dbd.O)NR.sup.aR.sup.b,
--NR.sup.aC(.dbd.O)NR.sup.aR.sup.b, --OC(.dbd.O)NR.sup.aR.sup.b,
--NR.sup.aC(.dbd.O)OR.sup.b, --NR.sup.aS(O).sub.xNR.sup.aR.sup.b,
--NR.sup.aS(O).sub.xR.sup.b or --S(O).sub.xNR.sup.aR.sup.b,
wherein: R.sup.a is H or saturated C.sub.1-C.sub.12 alkyl; R.sup.b
is C.sub.1-C.sub.15 alkyl; and x is 0, 1 or 2.
73. The compound of claim 72, wherein R.sup.7 is substituted with
--(C.dbd.O)OR.sup.b or --O(C.dbd.O)R.sup.b.
74. The compound of claim 72, wherein R.sup.b is branched saturated
C.sub.1-C.sub.15 alkyl.
75. The compound of claim 74, wherein R.sup.b has one of the
following structures: ##STR00101##
76. The compound of claim 56, wherein at least one of R.sup.8 or
R.sup.9 is methyl.
77. The compound of claim 76, wherein each of R.sup.8 and R.sup.9
is methyl.
78. The compound of claim 56, wherein R.sup.8 and R.sup.9, together
with the nitrogen atom to which they are attached, form a 5, 6 or
7-membered heterocyclic ring.
79. The compound of claim 78, wherein the heterocyclic ring is
pyrrolidinyl or piperazinyl.
80. The compound of claim 56, wherein G.sup.3 is saturated
C.sub.2-C.sub.4 alkylene.
81. The compound of claim 56, wherein G.sup.3 is saturated C.sub.3
alkylene.
82. The compound of claim 56, having one of the following
structures: ##STR00102## ##STR00103##
83. A composition comprising the compound of claim 56 and a
therapeutic agent.
84. The composition of claim 83, wherein the therapeutic agent
comprises a nucleic acid.
85. The composition of claim 84, wherein the nucleic acid is
selected from antisense and messenger RNA.
86. The composition of claim 83, wherein the composition comprises
lipid nanoparticles.
87. A lipid nanoparticle comprising the compound of claim 56.
Description
BACKGROUND
Technical Field
[0001] The present invention generally relates to novel cationic
lipids that can be used in combination with other lipid components,
such as neutral lipids, cholesterol and polymer conjugated lipids,
to form lipid nanoparticles with oligonucleotides, to facilitate
the intracellular delivery of therapeutic nucleic acids (e.g.
oligonucleotides, messenger RNA) both in vitro and in vivo.
Description of the Related Art
[0002] There are many challenges associated with the delivery of
nucleic acids to effect a desired response in a biological system.
Nucleic acid based therapeutics have enormous potential but there
remains a need for more effective delivery of nucleic acids to
appropriate sites within a cell or organism in order to realize
this potential. Therapeutic nucleic acids include, e.g., messenger
RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes,
plasmids, immune stimulating nucleic acids, antagomir, antimir,
mimic, supermir, and aptamers. Some nucleic acids, such as mRNA or
plasmids, can be used to effect expression of specific cellular
products as would be useful in the treatment of, for example,
diseases related to a deficiency of a protein or enzyme. The
therapeutic applications of translatable nucleotide delivery are
extremely broad as constructs can be synthesized to produce any
chosen protein sequence, whether or not indigenous to the system.
The expression products of the nucleic acid can augment existing
levels of protein, replace missing or non-functional versions of a
protein, or introduce new protein and associated functionality in a
cell or organism.
[0003] Some nucleic acids, such as miRNA inhibitors, can be used to
effect expression of specific cellular products that are regulated
by miRNA as would be useful in the treatment of, for example,
diseases related to deficiency of protein or enzyme. The
therapeutic applications of miRNA inhibition are extremely broad as
constructs can be synthesized to inhibit one or more miRNA that
would in turn regulate the expression of mRNA products. The
inhibition of endogenous miRNA can augment its downstream target
endogenous protein expression and restore proper function in a cell
or organism as a means to treat disease associated to a specific
miRNA or a group of miRNA.
[0004] Other nucleic acids can down-regulate intracellular levels
of specific mRNA and, as a result, down-regulate the synthesis of
the corresponding proteins through processes such as RNA
interference (RNAi) or complementary binding of antisense RNA. The
therapeutic applications of antisense oligonucleotide and RNAi are
also extremely broad, since oligonucleotide constructs can be
synthesized with any nucleotide sequence directed against a target
mRNA. Targets may include mRNAs from normal cells, mRNAs associated
with disease-states, such as cancer, and mRNAs of infectious
agents, such as viruses. To date, antisense oligonucleotide
constructs have shown the ability to specifically down-regulate
target proteins through degradation of the cognate mRNA in both in
vitro and in vivo models. In addition, antisense oligonucleotide
constructs are currently being evaluated in clinical studies.
[0005] However, two problems currently face using oligonucleotides
in therapeutic contexts. First, free RNAs are susceptible to
nuclease digestion in plasma. Second, free RNAs have limited
ability to gain access to the intracellular compartment where the
relevant translation machinery resides. Lipid nanoparticles formed
from cationic lipids with other lipid components, such as neutral
lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides
have been used to block degradation of the RNAs in plasma and
facilitate the cellular uptake of the oligonucleotides.
[0006] There remains a need for improved cationic lipids and lipid
nanoparticles for the delivery of oligonucleotides. Preferably,
these lipid nanoparticles would provide optimal drug:lipid ratios,
protect the nucleic acid from degradation and clearance in serum,
be suitable for systemic delivery, and provide intracellular
delivery of the nucleic acid. In addition, these lipid-nucleic acid
particles should be well-tolerated and provide an adequate
therapeutic index, such that patient treatment at an effective dose
of the nucleic acid is not associated with unacceptable toxicity
and/or risk to the patient. The present invention provides these
and related advantages.
BRIEF SUMMARY
[0007] In brief, embodiments of the present invention provide lipid
compounds, including stereoisomers, pharmaceutically acceptable
salts or tautomers thereof, which can be used alone or in
combination with other lipid components such as neutral lipids,
charged lipids, steroids (including for example, all sterols)
and/or their analogs, and/or polymer conjugated lipids to form
lipid nanoparticles for the delivery of therapeutic agents. In some
instances, the lipid nanoparticles are used to deliver nucleic
acids such as antisense and/or messenger RNA. Methods for use of
such lipid nanoparticles for treatment of various diseases or
conditions, such as those caused by infectious entities and/or
insufficiency of a protein, are also provided.
[0008] In one embodiment, compounds having the following Formula
(I) are provided:
##STR00002##
or a pharmaceutically acceptable salt, tautomer or stereoisomer
thereof, wherein R.sup.1a, R.sup.1b, R.sup.2a, R.sup.2b, R.sup.3a,
R.sup.3b, R.sup.4a, R.sup.4b, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, L.sup.1, L.sup.2, G.sup.1, G.sup.2, G.sup.3, a, b, c and d
are as defined herein.
[0009] Pharmaceutical compositions comprising one or more of the
foregoing compounds of Formula (I) and a therapeutic agent are also
provided. In some embodiments, the pharmaceutical compositions
further comprise one or more components selected from neutral
lipids, charged lipids, steroids and polymer conjugated lipids.
Such compositions are useful for formation of lipid nanoparticles
for the delivery of the therapeutic agent.
[0010] In other embodiments, the present invention provides a
method for administering a therapeutic agent to a patient in need
thereof, the method comprising preparing a composition of lipid
nanoparticles comprising the compound of Formula (I) and a
therapeutic agent and delivering the composition to the
patient.
[0011] These and other aspects of the invention will be apparent
upon reference to the following detailed description.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0012] In the figures, identical reference numbers identify similar
elements. The sizes and relative positions of elements in the
figures are not necessarily drawn to scale and some of these
elements are arbitrarily enlarged and positioned to improve figure
legibility. Further, the particular shapes of the elements as drawn
are not intended to convey any information regarding the actual
shape of the particular elements, and have been solely selected for
ease of recognition in the figures.
[0013] FIG. 1 shows time course of luciferase expression in mouse
liver.
[0014] FIG. 2 illustrates the calculation of pKa for MC3 as a
representative example relevant to the disclosed lipids.
[0015] FIG. 3 provides comparative luciferase activity data for
different lipids.
DETAILED DESCRIPTION
[0016] In the following description, certain specific details are
set forth in order to provide a thorough understanding of various
embodiments of the invention. However, one skilled in the art will
understand that the invention may be practiced without these
details.
[0017] The present invention is based, in part, upon the discovery
of novel cationic (amino) lipids that provide advantages when used
in lipid nanoparticles for the in vivo delivery of an active or
therapeutic agent such as a nucleic acid into a cell of a mammal.
In particular, embodiments of the present invention provide nucleic
acid-lipid nanoparticle compositions comprising one or more of the
novel cationic lipids described herein that provide increased
activity of the nucleic acid and improved tolerability of the
compositions in vivo, resulting in a significant increase in the
therapeutic index as compared to nucleic acid-lipid nanoparticle
compositions previously described.
[0018] In particular embodiments, the present invention provides
novel cationic lipids that enable the formulation of improved
compositions for the in vitro and in vivo delivery of mRNA and/or
other oligonucleotides. In some embodiments, these improved lipid
nanoparticle compositions are useful for expression of protein
encoded by mRNA. In other embodiments, these improved lipid
nanoparticle compositions are useful for upregulation of endogenous
protein expression by delivering miRNA inhibitors targeting one
specific miRNA or a group of miRNA regulating one target mRNA or
several mRNA. In other embodiments, these improved lipid
nanoparticle compositions are useful for down-regulating (e.g.,
silencing) the protein levels and/or mRNA levels of target genes.
In some other embodiments, the lipid nanoparticles are also useful
for delivery of mRNA and plasmids for expression of transgenes. In
yet other embodiments, the lipid nanoparticle compositions are
useful for inducing a pharmacological effect resulting from
expression of a protein, e.g., increased production of red blood
cells through the delivery of a suitable erythropoietin mRNA, or
protection against infection through delivery of mRNA encoding for
a suitable antibody.
[0019] The lipid nanoparticles and compositions of embodiments of
the present invention may be used for a variety of purposes,
including the delivery of encapsulated or associated (e.g.,
complexed) therapeutic agents such as nucleic acids to cells, both
in vitro and in vivo. Accordingly, embodiments of the present
invention provide methods of treating or preventing diseases or
disorders in a subject in need thereof by contacting the subject
with a lipid nanoparticle that encapsulates or is associated with a
suitable therapeutic agent, wherein the lipid nanoparticle
comprises one or more of the novel cationic lipids described
herein.
[0020] As described herein, embodiments of the lipid nanoparticles
of the present invention are particularly useful for the delivery
of nucleic acids, including, e.g., mRNA, antisense oligonucleotide,
plasmid DNA, microRNA (miRNA), miRNA inhibitors
(antagomirs/antimirs), messenger-RNA-interfering complementary RNA
(micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary
DNA (cDNA), etc. Therefore, the lipid nanoparticles and
compositions of certain embodiments of the present invention may be
used to induce expression of a desired protein both in vitro and in
vivo by contacting cells with a lipid nanoparticle comprising one
or more novel cationic lipids described herein, wherein the lipid
nanoparticle encapsulates or is associated with a nucleic acid that
is expressed to produce the desired protein (e.g., a messenger RNA
or plasmid encoding the desired protein). Alternatively, the lipid
nanoparticles and compositions of some embodiments of the present
invention may be used to decrease the expression of target genes
and proteins both in vitro and in vivo by contacting cells with a
lipid nanoparticle comprising one or more novel cationic lipids
described herein, wherein the lipid nanoparticle encapsulates or is
associated with a nucleic acid that reduces target gene expression
(e.g., an antisense oligonucleotide or small interfering RNA
(siRNA)). The lipid nanoparticles and compositions of embodiments
of the present invention may also be used for co-delivery of
different nucleic acids (e.g. mRNA and plasmid DNA) separately or
in combination, such as may be useful to provide an effect
requiring colocalization of different nucleic acids (e.g. mRNA
encoding for a suitable gene modifying enzyme and DNA segment(s)
for incorporation into the host genome).
[0021] Nucleic acids for use in embodiments of the invention may be
prepared according to any available technique. For mRNA, the
primary methodology of preparation is, but is not limited to,
enzymatic synthesis (also termed in vitro transcription) which
currently represents the most efficient method to produce long
sequence-specific mRNA. In vitro transcription describes a process
of template-directed synthesis of RNA molecules from an engineered
DNA template comprised of an upstream bacteriophage promoter
sequence (e.g. including but not limited to that from the T7, T3
and SP6 coliphage) linked to a downstream sequence encoding the
gene of interest. Template DNA can be prepared for in vitro
transcription from a number of sources with appropriate techniques
which are well known in the art including, but not limited to,
plasmid DNA and polymerase chain reaction amplification (see
Linpinsel, J. L and Conn, G. L., General protocols for preparation
of plasmid DNA template and Bowman, J. C., Azizi, B., Lenz, T. K.,
Ray, P., and Williams, L. D. in RNA in vitro transcription and RNA
purification by denaturing PAGE in Recombinant and in vitro RNA
syntheses Methods v. 941 Conn G. L. (ed), New York, N.Y. Humana
Press, 2012)
[0022] Transcription of the RNA occurs in vitro using the
linearized DNA template in the presence of the corresponding RNA
polymerase and adenosine, guanosine, uridine and cytidine
ribonucleoside triphosphates (rNTPs) under conditions that support
polymerase activity while minimizing potential degradation of the
resultant mRNA transcripts. In vitro transcription can be performed
using a variety of commercially available kits including, but not
limited to RiboMax Large Scale RNA Production System (Promega),
MegaScript Transcription kits (Life Technologies) as well as with
commercially available reagents including RNA polymerases and
rNTPs. The methodology for in vitro transcription of mRNA is well
known in the art. (see, e.g. Losick, R., 1972, In vitro
transcription, Ann Rev Biochem v.41 409-46; Kamakaka, R. T. and
Kraus, W. L. 2001. In Vitro Transcription. Current Protocols in
Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B.,
(2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods
in Molecular Biology v. 703 (Neilson, H. Ed), New York, N.Y. Humana
Press, 2010; Brunelle, J. L. and Green, R., 2013, Chapter Five--In
vitro transcription from plasmid or PCR-amplified DNA, Methods in
Enzymology v. 530, 101-114; all of which are incorporated herein by
reference).
[0023] The desired in vitro transcribed mRNA is then purified from
the undesired components of the transcription or associated
reactions (including unincorporated rNTPs, protein enzyme, salts,
short RNA oligos etc). Techniques for the isolation of the mRNA
transcripts are well known in the art. Well known procedures
include phenol/chloroform extraction or precipitation with either
alcohol (ethanol, isopropanol) in the presence of monovalent
cations or lithium chloride. Additional, non-limiting examples of
purification procedures which can be used include size exclusion
chromatography (Lukavsky, P. J. and Puglisi, J. D., 2004,
Large-scale preparation and purification of polyacrylamide-free RNA
oligonucleotides, RNA v.10, 889-893), silica-based affinity
chromatography and polyacrylamide gel electrophoresis (Bowman, J.
C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. in RNA in
vitro transcription and RNA purification by denaturing PAGE in
Recombinant and in vitro RNA syntheses Methods v. 941 Conn G. L.
(ed), New York, N.Y. Humana Press, 2012). Purification can be
performed using a variety of commercially available kits including,
but not limited to SV Total Isolation System (Promega) and In Vitro
Transcription Cleanup and Concentration Kit (Norgen Biotek).
[0024] Furthermore, while reverse transcription can yield large
quantities of mRNA, the products can contain a number of aberrant
RNA impurities associated with undesired polymerase activity which
may need to be removed from the full-length mRNA preparation. These
include short RNAs that result from abortive transcription
initiation as well as double-stranded RNA (dsRNA) generated by
RNA-dependent RNA polymerase activity, RNA-primed transcription
from RNA templates and self-complementary 3' extension. It has been
demonstrated that these contaminants with dsRNA structures can lead
to undesired immunostimulatory activity through interaction with
various innate immune sensors in eukaryotic cells that function to
recognize specific nucleic acid structures and induce potent immune
responses. This in turn, can dramatically reduce mRNA translation
since protein synthesis is reduced during the innate cellular
immune response. Therefore, additional techniques to remove these
dsRNA contaminants have been developed and are known in the art
including but not limited to scaleable HPLC purification (see e.g.
Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011,
Generating the optimal mRNA for therapy: HPLC purification
eliminates immune activation and improves translation of
nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39
e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC
Purification of in vitro transcribed long RNA in Synthetic
Messenger RNA and Cell Metabolism Modulation in Methods in
Molecular Biology v.969 (Rabinovich, P. H. Ed), 2013). HPLC
purified mRNA has been reported to be translated at much greater
levels, particularly in primary cells and in vivo.
[0025] A significant variety of modifications have been described
in the art which are used to alter specific properties of in vitro
transcribed mRNA, and improve its utility. These include, but are
not limited to modifications to the 5' and 3' termini of the mRNA.
Endogenous eukaryotic mRNA typically contain a cap structure on the
5'-end of a mature molecule which plays an important role in
mediating binding of the mRNA Cap Binding Protein (CBP), which is
in turn responsible for enhancing mRNA stability in the cell and
efficiency of mRNA translation. Therefore, highest levels of
protein expression are achieved with capped mRNA transcripts. The
5'-cap contains a 5'-5'-triphosphate linkage between the 5'-most
nucleotide and guanine nucleotide. The conjugated guanine
nucleotide is methylated at the N7 position. Additional
modifications include methylation of the ultimate and penultimate
most 5'-nucleotides on the 2'-hydroxyl group.
[0026] Multiple distinct cap structures can be used to generate the
5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of
synthetic mRNA can be performed co-transcriptionally with chemical
cap analogs (i.e. capping during in vitro transcription). For
example, the Anti-Reverse Cap Analog (ARCA) cap contains a
5'-5'-triphosphate guanine-guanine linkage where one guanine
contains an N7 methyl group as well as a 3'-O-methyl group.
However, up to 20% of transcripts remain uncapped during this
co-transcriptional process and the synthetic cap analog is not
identical to the 5'-cap structure of an authentic cellular mRNA,
potentially reducing translatability and cellular stability.
Alternatively, synthetic mRNA molecules may also be enzymatically
capped post-transcriptionally. These may generate a more authentic
5'-cap structure that more closely mimics, either structurally or
functionally, the endogenous 5'-cap which have enhanced binding of
cap binding proteins, increased half life, reduced susceptibility
to 5' endonucleases and/or reduced 5' decapping. Numerous synthetic
5'-cap analogs have been developed and are known in the art to
enhance mRNA stability and translatability (see eg.
Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A. N.,
Slepenkov, S. V., Darynkiewicz, E., Sahin, U., Jemielity, J., and
Rhoads, R. E., Synthetic mRNAs with superior translation and
stability properties in Synthetic Messenger RNA and Cell Metabolism
Modulation in Methods in Molecular Biology v.969 (Rabinovich, P. H.
Ed), 2013).
[0027] On the 3'-terminus, a long chain of adenine nucleotides
(poly-A tail) is normally added to mRNA molecules during RNA
processing. Immediately after transcription, the 3' end of the
transcript is cleaved to free a 3' hydroxyl to which poly-A
polymerase adds a chain of adenine nucleotides to the RNA in a
process called polyadenylation. The poly-A tail has been
extensively shown to enhance both translational efficiency and
stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),
poly (A) binding protein and the regulation of mRNA stability,
Trends Bio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001,
Regulation of mRNA stability in mammalian cells, Gene, v. 265,
11-23; Dreyfus, M. And Regnier, P., 2002, The poly (A) tail of
mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111,
611-613).
[0028] Poly (A) tailing of in vitro transcribed mRNA can be
achieved using various approaches including, but not limited to,
cloning of a poly (T) tract into the DNA template or by
post-transcriptional addition using Poly (A) polymerase. The first
case allows in vitro transcription of mRNA with poly (A) tails of
defined length, depending on the size of the poly (T) tract, but
requires additional manipulation of the template. The latter case
involves the enzymatic addition of a poly (A) tail to in vitro
transcribed mRNA using poly (A) polymerase which catalyzes the
incorporation of adenine residues onto the 3'termini of RNA,
requiring no additional manipulation of the DNA template, but
results in mRNA with poly(A) tails of heterogenous length.
5'-capping and 3'-poly (A) tailing can be performed using a variety
of commercially available kits including, but not limited to Poly
(A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra
kit and Poly (A) Tailing kit (Life Technologies) as well as with
commercially available reagents, various ARCA caps, Poly (A)
polymerase, etc.
[0029] In addition to 5' cap and 3' poly adenylation, other
modifications of the in vitro transcripts have been reported to
provide benefits as related to efficiency of translation and
stability. It is well known in the art that pathogenic DNA and RNA
can be recognized by a variety of sensors within eukaryotes and
trigger potent innate immune responses. The ability to discriminate
between pathogenic and self DNA and RNA has been shown to be based,
at least in part, on structure and nucleoside modifications since
most nucleic acids from natural sources contain modified
nucleosides In contrast, in vitro synthesized RNA lacks these
modifications, thus rendering it immunostimulatory which in turn
can inhibit effective mRNA translation as outlined above. The
introduction of modified nucleosides into in vitro transcribed mRNA
can be used to prevent recognition and activation of RNA sensors,
thus mitigating this undesired immunostimulatory activity and
enhancing translation capacity (see e.g., Kariko, K. And Weissman,
D. 2007, Naturally occurring nucleoside modifications suppress the
immunostimulatory activity of RNA: implication for therapeutic RNA
development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N.,
Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of
long RNA containing modified nucleosides in Synthetic Messenger RNA
and Cell Metabolism Modulation in Methods in Molecular Biology
v.969 (Rabinovich, P. H. Ed), 2013); Kariko, K., Muramatsu, H.,
Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008,
Incorporation of Pseudouridine Into mRNA Yields Superior
Nonimmunogenic Vector With Increased Translational Capacity and
Biological Stability, Mol Ther v.16, 1833-1840. The modified
nucleosides and nucleotides used in the synthesis of modified RNAs
can be prepared monitored and utilized using general methods and
procedures known in the art. A large variety of nucleoside
modifications are available that may be incorporated alone or in
combination with other modified nucleosides to some extent into the
in vitro transcribed mRNA (see e.g., US2012/0251618). In vitro
synthesis of nucleoside-modified mRNA have been reported to have
reduced ability to activate immune sensors with a concomitant
enhanced translational capacity.
[0030] Other components of mRNA which can be modified to provide
benefit in terms of translatability and stability include the 5'
and 3' untranslated regions (UTR). Optimization of the UTRs
(favorable 5' and 3' UTRs can be obtained from cellular or viral
RNAs), either both or independently, have been shown to increase
mRNA stability and translational efficiency of in vitro transcribed
mRNA (see e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K.,
In vitro transcription of long RNA containing modified nucleosides
in Synthetic Messenger RNA and Cell Metabolism Modulation in
Methods in Molecular Biology v.969 (Rabinovich, P. H. Ed),
2013).
[0031] In addition to mRNA, other nucleic acid payloads may be used
for embodiments of this invention. For oligonucleotides, methods of
preparation include but are not limited to chemical synthesis and
enzymatic, chemical cleavage of a longer precursor, in vitro
transcription as described above, etc. Methods of synthesizing DNA
and RNA nucleotides are widely used and well known in the art (see,
e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical
approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984;
and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and
applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.)
Totowa, N.J.: Humana Press, 2005; both of which are incorporated
herein by reference).
[0032] For plasmid DNA, preparation for use with embodiments of
this invention commonly utilizes, but is not limited to, expansion
and isolation of the plasmid DNA in vitro in a liquid culture of
bacteria containing the plasmid of interest. The presence of a gene
in the plasmid of interest that encodes resistance to a particular
antibiotic (penicillin, kanamycin, etc.) allows those bacteria
containing the plasmid of interest to selective grow in
antibiotic-containing cultures. Methods of isolating plasmid DNA
are widely used and well known in the art (see, e.g. Heilig, J.,
Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of
Plasmid DNA. Current Protocols in Molecular Biology.
41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S.,
Bjornestedt, R. and Schmidt, S. R. (2008), Large-scale production
of endotoxin-free plasmids for transient expression in mammalian
cell culture. Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No.
6,197,553B1). Plasmid isolation can be performed using a variety of
commercially available kits including, but not limited to Plasmid
Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and PureYield
MaxiPrep (Promega) kits as well as with commercially available
reagents.
[0033] Various exemplary embodiments of the cationic lipids of the
present invention, lipid nanoparticles and compositions comprising
the same, and their use to deliver active (e.g., therapeutic
agents), such as nucleic acids, to modulate gene and protein
expression, are described in further detail below.
[0034] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0035] Unless the context requires otherwise, throughout the
present specification and claims, the word "comprise" and
variations thereof, such as, "comprises" and "comprising" are to be
construed in an open and inclusive sense, that is, as "including,
but not limited to".
[0036] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
the appearances of the phrases "in one embodiment" or "in an
embodiment" in various places throughout this specification are not
necessarily all referring to the same embodiment. Furthermore, the
particular features, structures, or characteristics may be combined
in any suitable manner in one or more embodiments.
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. As used in the
specification and claims, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise.
[0038] The phrase "induce expression of a desired protein" refers
to the ability of a nucleic acid to increase expression of the
desired protein. To examine the extent of protein expression, a
test sample (e.g., a sample of cells in culture expressing the
desired protein) or a test mammal (e.g., a mammal such as a human
or an animal model such as a rodent (e.g., mouse) or a non-human
primate (e.g., monkey) model) is contacted with a nucleic acid
(e.g., nucleic acid in combination with a lipid of the present
invention). Expression of the desired protein in the test sample or
test animal is compared to expression of the desired protein in a
control sample (e.g., a sample of cells in culture expressing the
desired protein) or a control mammal (e.g., a mammal such as a
human or an animal model such as a rodent (e.g., mouse) or
non-human primate (e.g., monkey) model) that is not contacted with
or administered the nucleic acid. When the desired protein is
present in a control sample or a control mammal, the expression of
a desired protein in a control sample or a control mammal may be
assigned a value of 1.0. In particular embodiments, inducing
expression of a desired protein is achieved when the ratio of
desired protein expression in the test sample or the test mammal to
the level of desired protein expression in the control sample or
the control mammal is greater than 1, for example, about 1.1, 1.5,
2.0. 5.0 or 10.0. When a desired protein is not present in a
control sample or a control mammal, inducing expression of a
desired protein is achieved when any measurable level of the
desired protein in the test sample or the test mammal is detected.
One of ordinary skill in the art will understand appropriate assays
to determine the level of protein expression in a sample, for
example dot blots, northern blots, in situ hybridization, ELISA,
immunoprecipitation, enzyme function, and phenotypic assays, or
assays based on reporter proteins that can produce fluorescence or
luminescence under appropriate conditions.
[0039] The phrase "inhibiting expression of a target gene" refers
to the ability of a nucleic acid to silence, reduce, or inhibit the
expression of a target gene. To examine the extent of gene
silencing, a test sample (e.g., a sample of cells in culture
expressing the target gene) or a test mammal (e.g., a mammal such
as a human or an animal model such as a rodent (e.g., mouse) or a
non-human primate (e.g., monkey) model) is contacted with a nucleic
acid that silences, reduces, or inhibits expression of the target
gene. Expression of the target gene in the test sample or test
animal is compared to expression of the target gene in a control
sample (e.g., a sample of cells in culture expressing the target
gene) or a control mammal (e.g., a mammal such as a human or an
animal model such as a rodent (e.g., mouse) or non-human primate
(e.g., monkey) model) that is not contacted with or administered
the nucleic acid. The expression of the target gene in a control
sample or a control mammal may be assigned a value of 100%. In
particular embodiments, silencing, inhibition, or reduction of
expression of a target gene is achieved when the level of target
gene expression in the test sample or the test mammal relative to
the level of target gene expression in the control sample or the
control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In
other words, the nucleic acids are capable of silencing, reducing,
or inhibiting the expression of a target gene by at least about 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal
relative to the level of target gene expression in a control sample
or a control mammal not contacted with or administered the nucleic
acid. Suitable assays for determining the level of target gene
expression include, without limitation, examination of protein or
mRNA levels using techniques known to those of skill in the art,
such as, e.g., dot blots, northern blots, in situ hybridization,
ELISA, immunoprecipitation, enzyme function, as well as phenotypic
assays known to those of skill in the art.
[0040] An "effective amount" or "therapeutically effective amount"
of an active agent or therapeutic agent such as a therapeutic
nucleic acid is an amount sufficient to produce the desired effect,
e.g., an increase or inhibition of expression of a target sequence
in comparison to the normal expression level detected in the
absence of the nucleic acid. An increase in expression of a target
sequence is achieved when any measurable level is detected in the
case of an expression product that is not present in the absence of
the nucleic acid. In the case where the expression product is
present at some level prior to contact with the nucleic acid, an in
increase in expression is achieved when the fold increase in value
obtained with a nucleic acid such as mRNA relative to control is
about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,
5000, 10000 or greater. Inhibition of expression of a target gene
or target sequence is achieved when the value obtained with a
nucleic acid such as antisense oligonucleotide relative to the
control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,
45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays
for measuring expression of a target gene or target sequence
include, e.g., examination of protein or RNA levels using
techniques known to those of skill in the art such as dot blots,
northern blots, in situ hybridization, ELISA, immunoprecipitation,
enzyme function, fluorescence or luminescence of suitable reporter
proteins, as well as phenotypic assays known to those of skill in
the art.
[0041] The term "nucleic acid" as used herein refers to a polymer
containing at least two deoxyribonucleotides or ribonucleotides in
either single- or double-stranded form and includes DNA, RNA, and
hybrids thereof. DNA may be in the form of antisense molecules,
plasmid DNA, cDNA, PCR products, or vectors. RNA may be in the form
of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA,
miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA
(vRNA), and combinations thereof. Nucleic acids include nucleic
acids containing known nucleotide analogs or modified backbone
residues or linkages, which are synthetic, naturally occurring, and
non-naturally occurring, and which have similar binding properties
as the reference nucleic acid. Examples of such analogs include,
without limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2'-O-methyl
ribonucleotides, and peptide-nucleic acids (PNAs). Unless
specifically limited, the term "nucleic acid" encompasses nucleic
acids containing known analogues of natural nucleotides that have
similar binding properties as the reference nucleic acid. Unless
otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions), alleles, orthologs, single
nucleotide polymorphisms, and complementary sequences as well as
the sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes,
8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA) or
ribose (RNA), a base, and a phosphate group. Nucleotides are linked
together through the phosphate groups. "Bases" include purines and
pyrimidines, which further include natural compounds adenine,
thymine, guanine, cytosine, uracil, inosine, and natural analogs,
and synthetic derivatives of purines and pyrimidines, which
include, but are not limited to, modifications which place new
reactive groups such as, but not limited to, amines, alcohols,
thiols, carboxylates, and alkylhalides.
[0042] The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises partial length or entire length coding
sequences necessary for the production of a polypeptide or
precursor polypeptide.
[0043] "Gene product," as used herein, refers to a product of a
gene such as an RNA transcript or a polypeptide.
[0044] The term "lipid" refers to a group of organic compounds that
include, but are not limited to, esters of fatty acids and are
generally characterized by being poorly soluble in water, but
soluble in many organic solvents. Lipids are usually divided into
at least three classes: (1) "simple lipids," which include fats and
oils as well as waxes; (2) "compound lipids," which include
phospholipids and glycolipids; and (3) "derived lipids" such as
steroids.
[0045] A "steroid" is a compound comprising the following carbon
skeleton:
##STR00003##
Non-limiting examples of steroids include cholesterol, and the
like.
[0046] A "cationic lipid" refers to a lipid capable of being
positively charged. Exemplary cationic lipids include one or more
amine group(s) which bear the positive charge. Exemplary cationic
lipids are ionizable such that they can exist in a positively
charged or neutral form depending on pH. The ionization of the
cationic lipid affects the surface charge of the lipid nanoparticle
under different pH conditions. This charge state can influence
plasma protein absorption, blood clearance and tissue distribution
(Semple, S. C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well
as the ability to form endosomolytic non-bilayer structures (Hafez,
I. M., et al., Gene Ther 8:1188-1196 (2001)) critical to the
intracellular delivery of nucleic acids.
[0047] The term "lipid nanoparticle" refers to particles having at
least one dimension on the order of nanometers (e.g., 1-1,000 nm)
which include one or more of the compounds of formula (I) or other
specified cationic lipids. In some embodiments, lipid nanoparticles
are included in a formulation that can be used to deliver an active
agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to
a target site of interest (e.g., cell, tissue, organ, tumor, and
the like). In some embodiments, the lipid nanoparticles of the
invention comprise a nucleic acid. Such lipid nanoparticles
typically comprise a compound of Formula (I) and one or more
excipient selected from neutral lipids, charged lipids, steroids
and polymer conjugated lipids. In some embodiments, the active
agent or therapeutic agent, such as a nucleic acid, may be
encapsulated in the lipid portion of the lipid nanoparticle or an
aqueous space enveloped by some or all of the lipid portion of the
lipid nanoparticle, thereby protecting it from enzymatic
degradation or other undesirable effects induced by the mechanisms
of the host organism or cells e.g. an adverse immune response.
[0048] In various embodiments, the lipid nanoparticles have a mean
diameter of from about 30 nm to about 150 nm, from about 40 nm to
about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to
about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to
about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to
about 100 nm, from about 70 to about 90 nm, from about 80 nm to
about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35
nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm,
85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125
nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are
substantially non-toxic. In certain embodiments, nucleic acids,
when present in the lipid nanoparticles, are resistant in aqueous
solution to degradation with a nuclease. Lipid nanoparticles
comprising nucleic acids and their method of preparation are
disclosed in, e.g., U.S. Patent Publication Nos. 2004/0142025,
2007/0042031 and PCT Pub. Nos. WO 2013/016058 and WO 2013/086373,
the full disclosures of which are herein incorporated by reference
in their entirety for all purposes.
[0049] As used herein, "lipid encapsulated" refers to a lipid
nanoparticle that provides an active agent or therapeutic agent,
such as a nucleic acid (e.g., mRNA), with full encapsulation,
partial encapsulation, or both. In an embodiment, the nucleic acid
(e.g., mRNA) is fully encapsulated in the lipid nanoparticle.
[0050] The term "polymer conjugated lipid" refers to a molecule
comprising both a lipid portion and a polymer portion. An example
of a polymer conjugated lipid is a pegylated lipid. The term
"pegylated lipid" refers to a molecule comprising both a lipid
portion and a polyethylene glycol portion. Pegylated lipids are
known in the art and include
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-DMG) and the like.
[0051] The term "neutral lipid" refers to any of a number of lipid
species that exist either in an uncharged or neutral zwitterionic
form at a selected pH. At physiological pH, such lipids include,
but are not limited to, phosphotidylcholines such as
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
phophatidylethanolamines such as
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
sphingomyelins (SM), ceramides, steroids such as sterols and their
derivatives. Neutral lipids may be synthetic or naturally
derived.
[0052] The term "charged lipid" refers to any of a number of lipid
species that exist in either a positively charged or negatively
charged form independent of the pH within a useful physiological
range e.g. pH .about.3 to pH .about.9. Charged lipids may be
synthetic or naturally derived. Examples of charged lipids include
phosphatidylserines, phosphatidic acids, phosphatidylglycerols,
phosphatidylinositols, sterol hemisuccinates, dialkyl
trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyl
dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane
carbamoyl sterols (e.g. DC-Chol).
[0053] As used herein, the term "aqueous solution" refers to a
composition comprising water.
[0054] "Serum-stable" in relation to nucleic acid-lipid
nanoparticles means that the nucleotide is not significantly
degraded after exposure to a serum or nuclease assay that would
significantly degrade free DNA or RNA. Suitable assays include, for
example, a standard serum assay, a DNAse assay, or an RNAse
assay.
[0055] "Systemic delivery," as used herein, refers to delivery of a
therapeutic product that can result in a broad exposure of an
active agent within an organism. Some techniques of administration
can lead to the systemic delivery of certain agents, but not
others. Systemic delivery means that a useful, preferably
therapeutic, amount of an agent is exposed to most parts of the
body. Systemic delivery of lipid nanoparticles can be by any means
known in the art including, for example, intravenous,
intraarterial, subcutaneous, and intraperitoneal delivery. In some
embodiments, systemic delivery of lipid nanoparticles is by
intravenous delivery.
[0056] "Local delivery," as used herein, refers to delivery of an
active agent directly to a target site within an organism. For
example, an agent can be locally delivered by direct injection into
a disease site such as a tumor, other target site such as a site of
inflammation, or a target organ such as the liver, heart, pancreas,
kidney, and the like. Local delivery can also include topical
applications or localized injection techniques such as
intramuscular, subcutaneous or intradermal injection. Local
delivery does not preclude a systemic pharmacological effect.
[0057] "Alkyl" refers to a straight or branched hydrocarbon chain
radical consisting solely of carbon and hydrogen atoms, which is
saturated or unsaturated (i.e., contains one or more double and/or
triple bonds), having, for example, from one to twenty-four carbon
atoms (C.sub.1-C.sub.24 alkyl), four to twenty carbon atoms
(C.sub.4-C.sub.20 alkyl), six to sixteen carbon atoms
(C.sub.6-C.sub.16 alkyl), six to nine carbon atoms (C.sub.6-C.sub.9
alkyl), one to fifteen carbon atoms (C.sub.1-C.sub.15 alkyl),one to
twelve carbon atoms (C.sub.1-C.sub.12 alkyl), one to eight carbon
atoms (C.sub.1-C.sub.8 alkyl) or one to six carbon atoms
(C.sub.1-C.sub.6 alkyl) and which is attached to the rest of the
molecule by a single bond, e.g., methyl, ethyl, n propyl, 1
methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t
butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but 1
enyl, pent 1 enyl, penta 1,4 dienyl, ethynyl, propynyl, butynyl,
pentynyl, hexynyl, and the like. Unless stated otherwise
specifically in the specification, an alkyl group is optionally
substituted.
[0058] "Alkylene" or "alkylene chain" refers to a straight or
branched divalent hydrocarbon chain linking the rest of the
molecule to a radical group, consisting solely of carbon and
hydrogen, which is saturated or unsaturated (i.e., contains one or
more double and/or triple bonds), and having, for example, from one
to twenty-four carbon atoms (C.sub.1-C.sub.24 alkylene), one to
fifteen carbon atoms (C.sub.1-C.sub.15 alkylene),one to twelve
carbon atoms (C.sub.1-C.sub.12 alkylene), one to eight carbon atoms
(C.sub.1-C.sub.8 alkylene), one to six carbon atoms
(C.sub.1-C.sub.6 alkylene), two to four carbon atoms
(C.sub.2-C.sub.4 alkylene), one to two carbon atoms
(C.sub.1-C.sub.2 alkylene), e.g., methylene, ethylene, propylene,
n-butylene, ethenylene, propenylene, n-butenylene, propynylene,
n-butynylene, and the like. The alkylene chain is attached to the
rest of the molecule through a single or double bond and to the
radical group through a single or double bond. The points of
attachment of the alkylene chain to the rest of the molecule and to
the radical group can be through one carbon or any two carbons
within the chain. Unless stated otherwise specifically in the
specification, an alkylene chain may be optionally substituted.
[0059] "Heterocyclyl" or "heterocyclic ring" refers to a stable 3-
to 18-membered (e.g., 5, 6 or 7-membered) non-aromatic ring radical
having one to twelve ring carbon atoms (e.g., two to twelve) and
from one to six ring heteroatoms selected from the group consisting
of nitrogen, oxygen and sulfur. Unless stated otherwise
specifically in the specification, the heterocyclyl radical may be
a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which
may include fused or bridged ring systems; and the nitrogen, carbon
or sulfur atoms in the heterocyclyl radical may be optionally
oxidized; the nitrogen atom may be optionally quaternized; and the
heterocyclyl radical may be partially or fully saturated. Examples
of such heterocyclyl radicals include, but are not limited to,
dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl,
imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, octahydroindolyl, octahydroisoindolyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl,
pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl,
tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl,
thiamorpholinyl, 1-oxo-thiomorpholinyl, and
1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in
the specification, a heterocyclyl group may be optionally
substituted.
[0060] The term "substituted" used herein means any of the above
groups (e.g., alkyl, alkylene or heterocyclyl) wherein at least one
hydrogen atom (e.g., 1, 2, 3 or all hydrogen atoms) is replaced by
a bond to a non-hydrogen atom such as, but not limited to: a
halogen atom such as F, Cl, Br, or I; oxo groups (.dbd.O); hydroxyl
groups (--OH); C.sub.1-C.sub.12 alkyl groups; cycloalkyl groups;
--(C.dbd.O)OR'; --O(C.dbd.O)R'; --C(.dbd.O)R'; --OR';
--S(O).sub.xR'; --S--SR'; --C(.dbd.O)SR'; --SC(.dbd.O)R'; --NR'R';
--NR'C(.dbd.O)R'; --C(.dbd.O)NR'R'; --NR'C(.dbd.O)NR'R';
--OC(.dbd.O)NR'R'; --NR'C(.dbd.O)OR'; --NR'S(O).sub.xNR'R';
--NR'S(O).sub.xR'; and --S(O).sub.xNR'R', wherein: R' is, at each
occurrence, independently H, C.sub.1-C.sub.15 alkyl or cycloalkyl,
and x is 0, 1 or 2. In some embodiments the substituent is a
C.sub.1-C.sub.12 alkyl group. In other embodiments, the substituent
is a cycloalkyl group. In other embodiments, the substituent is a
halo group, such as fluoro. In other embodiments, the substituent
is a oxo group. In other embodiments, the substituent is a hydroxyl
group. In other embodiments, the substituent is an alkoxy group. In
other embodiments, the substituent is a carboxyl group. In other
embodiments, the substituent is an amine group.
[0061] "Optional" or "optionally" (e.g., optionally substituted)
means that the subsequently described event of circumstances may or
may not occur, and that the description includes instances where
said event or circumstance occurs and instances in which it does
not. For example, "optionally substituted alkyl" means that the
alkyl radical may or may not be substituted and that the
description includes both substituted alkyl radicals and alkyl
radicals having no substitution.
[0062] "Prodrug" is meant to indicate a compound that may be
converted under physiological conditions or by solvolysis to a
biologically active compound of the invention. Thus, the term
"prodrug" refers to a metabolic precursor of a compound of the
invention that is pharmaceutically acceptable. A prodrug may be
inactive when administered to a subject in need thereof, but is
converted in vivo to an active compound of the invention. Prodrugs
are typically rapidly transformed in vivo to yield the parent
compound of the invention, for example, by hydrolysis in blood. The
prodrug compound often offers advantages of solubility, tissue
compatibility or delayed release in a mammalian organism (see,
Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,
Amsterdam)). A discussion of prodrugs is provided in Higuchi, T.,
et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible
Carriers in Drug Design, Ed. Edward B. Roche, American
Pharmaceutical Association and Pergamon Press, 1987.
[0063] The term "prodrug" is also meant to include any covalently
bonded carriers, which release the active compound of the invention
in vivo when such prodrug is administered to a mammalian subject.
Prodrugs of a compound of the invention (e.g., compound of formula
(I)) may be prepared by modifying functional groups present in the
compound of the invention in such a way that the modifications are
cleaved, either in routine manipulation or in vivo, to the parent
compound of the invention. Prodrugs include compounds of the
invention wherein a hydroxy, amino or mercapto group is bonded to
any group that, when the prodrug of the compound of the invention
is administered to a mammalian subject, cleaves to form a free
hydroxy, free amino or free mercapto group, respectively. Examples
of prodrugs include, but are not limited to, acetate, formate and
benzoate derivatives of alcohol or amide derivatives of amine
functional groups in the compounds of the invention and the
like.
[0064] Embodiments of the invention disclosed herein are also meant
to encompass all pharmaceutically acceptable compounds of the
compound of Formula (I) being isotopically-labelled by having one
or more atoms replaced by an atom having a different atomic mass or
mass number. Examples of isotopes that can be incorporated into the
disclosed compounds include isotopes of hydrogen, carbon, nitrogen,
oxygen, phosphorous, fluorine, chlorine, and iodine, such as
.sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.13N, .sup.15N,
.sup.15O, .sup.17O, .sup.18O, .sup.31P, .sup.32P, .sup.35S,
.sup.18F, .sup.36Cl, .sup.123I, and .sup.125I, respectively. These
radiolabeled compounds can be useful to help determine or measure
the effectiveness of the compounds, by characterizing, for example,
the site or mode of action, or binding affinity to
pharmacologically important site of action. Certain
isotopically-labelled compounds having a structure of Formula (I)
or (II), for example, those incorporating a radioactive isotope,
are useful in drug and/or substrate tissue distribution studies.
The radioactive isotopes tritium, i.e., .sup.3H, and carbon-14,
i.e., .sup.14C, are particularly useful for this purpose in view of
their ease of incorporation and ready means of detection.
[0065] Substitution with heavier isotopes such as deuterium, i.e.,
.sup.2H, may afford certain therapeutic advantages resulting from
greater metabolic stability, for example, increased in vivo
half-life or reduced dosage requirements, and hence may be
preferred in some circumstances.
[0066] Substitution with positron emitting isotopes, such as
.sup.11C, .sup.18F, .sup.15O and .sup.13N, can be useful in
Positron Emission Topography (PET) studies for examining substrate
receptor occupancy. Isotopically-labeled compounds of Formula (I)
of (II) can generally be prepared by conventional techniques known
to those skilled in the art or by processes analogous to those
described in the Preparations and Examples as set out below using
an appropriate isotopically-labeled reagent in place of the
non-labeled reagent previously employed.
[0067] Embodiments of the invention disclosed herein are also meant
to encompass the in vivo metabolic products of the disclosed
compounds. Such products may result from, for example, the
oxidation, reduction, hydrolysis, amidation, esterification, and
the like of the administered compound, primarily due to enzymatic
processes. Accordingly, embodiments of the invention include
compounds produced by a process comprising administering a compound
of this invention to a mammal for a period of time sufficient to
yield a metabolic product thereof. Such products are typically
identified by administering a radiolabelled compound of the
invention in a detectable dose to an animal, such as rat, mouse,
guinea pig, monkey, or to human, allowing sufficient time for
metabolism to occur, and isolating its conversion products from the
urine, blood or other biological samples.
[0068] "Stable compound" and "stable structure" are meant to
indicate a compound that is sufficiently robust to survive
isolation to a useful degree of purity from a reaction mixture, and
formulation into an efficacious therapeutic agent.
[0069] "Mammal" includes humans and both domestic animals such as
laboratory animals and household pets (e.g., cats, dogs, swine,
cattle, sheep, goats, horses, rabbits), and non-domestic animals
such as wildlife and the like.
[0070] "Pharmaceutically acceptable carrier, diluent or excipient"
includes without limitation any adjuvant, carrier, excipient,
glidant, sweetening agent, diluent, preservative, dye/colorant,
flavor enhancer, surfactant, wetting agent, dispersing agent,
suspending agent, stabilizer, isotonic agent, solvent, or
emulsifier which has been approved by the United States Food and
Drug Administration as being acceptable for use in humans or
domestic animals.
[0071] "Pharmaceutically acceptable salt" includes both acid and
base addition salts.
[0072] "Pharmaceutically acceptable acid addition salt" refers to
those salts which retain the biological effectiveness and
properties of the free bases, which are not biologically or
otherwise undesirable, and which are formed with inorganic acids
such as, but are not limited to, hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as, but not limited to, acetic acid,
2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,
aspartic acid, benzenesulfonic acid, benzoic acid,
4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,
capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic
acid, citric acid, cyclamic acid, dodecylsulfuric acid,
ethane-1,2-disulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric
acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic
acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,
glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric
acid, lactic acid, lactobionic acid, lauric acid, maleic acid,
malic acid, malonic acid, mandelic acid, methanesulfonic acid,
mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic
acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid,
orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic
acid, pyroglutamic acid, pyruvic acid, salicylic acid,
4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,
tartaric acid, thiocyanic acid, p-toluenesulfonic acid,
trifluoroacetic acid, undecylenic acid, and the like.
[0073] "Pharmaceutically acceptable base addition salt" refers to
those salts which retain the biological effectiveness and
properties of the free acids, which are not biologically or
otherwise undesirable. These salts are prepared from addition of an
inorganic base or an organic base to the free acid. Salts derived
from inorganic bases include, but are not limited to, the sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc,
copper, manganese, aluminum salts and the like. Preferred inorganic
salts are the ammonium, sodium, potassium, calcium, and magnesium
salts. Salts derived from organic bases include, but are not
limited to, salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
ammonia, isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, diethanolamine, ethanolamine,
deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol,
dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,
hydrabamine, choline, betaine, benethamine, benzathine,
ethylenediamine, glucosamine, methylglucamine, theobromine,
triethanolamine, tromethamine, purines, piperazine, piperidine,
N-ethylpiperidine, polyamine resins and the like. Particularly
preferred organic bases are isopropylamine, diethylamine,
ethanolamine, trimethylamine, dicyclohexylamine, choline and
caffeine.
[0074] Often crystallizations produce a solvate of the compound of
the invention. As used herein, the term "solvate" refers to an
aggregate that comprises one or more molecules of a compound of the
invention with one or more molecules of solvent. The solvent may be
water, in which case the solvate may be a hydrate. Alternatively,
the solvent may be an organic solvent. Thus, the compounds of the
present invention may exist as a hydrate, including a monohydrate,
dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and
the like, as well as the corresponding solvated forms. The compound
of the invention may be true solvates, while in other cases, the
compound of the invention may merely retain adventitious water or
be a mixture of water plus some adventitious solvent.
[0075] A "pharmaceutical composition" refers to a formulation of a
compound of the invention and a medium generally accepted in the
art for the delivery of the biologically active compound to
mammals, e.g., humans. Such a medium includes all pharmaceutically
acceptable carriers, diluents or excipients therefor.
[0076] "Effective amount" or "therapeutically effective amount"
refers to that amount of a compound of the invention, or a lipid
nanoparticle comprising the same, which, when administered to a
mammal, preferably a human, is sufficient to effect treatment in
the mammal, preferably a human. The amount of a lipid nanoparticle
of the invention which constitutes a "therapeutically effective
amount" will vary depending on the compound, the condition and its
severity, the manner of administration, and the age of the mammal
to be treated, but can be determined routinely by one of ordinary
skill in the art having regard to his own knowledge and to this
disclosure.
[0077] "Treating" or "treatment" as used herein covers the
treatment of the disease or condition of interest in a mammal,
preferably a human, having the disease or condition of interest,
and includes:
[0078] (i) preventing the disease or condition from occurring in a
mammal, in particular, when such mammal is predisposed to the
condition but has not yet been diagnosed as having it;
[0079] (ii) inhibiting the disease or condition, i.e., arresting
its development;
[0080] (iii) relieving the disease or condition, i.e., causing
regression of the disease or condition; or
[0081] (iv) relieving the symptoms resulting from the disease or
condition, i.e., relieving pain without addressing the underlying
disease or condition. As used herein, the terms "disease" and
"condition" may be used interchangeably or may be different in that
the particular malady or condition may not have a known causative
agent (so that etiology has not yet been worked out) and it is
therefore not yet recognized as a disease but only as an
undesirable condition or syndrome, wherein a more or less specific
set of symptoms have been identified by clinicians.
[0082] The compounds of the invention, or their pharmaceutically
acceptable salts may contain one or more asymmetric centers and may
thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms that may be defined, in terms of absolute
stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino
acids. Embodiments of the present invention are meant to include
all such possible isomers, as well as their racemic and optically
pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)-
and (L)-isomers may be prepared using chiral synthons or chiral
reagents, or resolved using conventional techniques, for example,
chromatography and fractional crystallization. Conventional
techniques for the preparation/isolation of individual enantiomers
include chiral synthesis from a suitable optically pure precursor
or resolution of the racemate (or the racemate of a salt or
derivative) using, for example, chiral high pressure liquid
chromatography (HPLC). When the compounds described herein contain
olefinic double bonds or other centers of geometric asymmetry, and
unless specified otherwise, it is intended that the compounds
include both E and Z geometric isomers. Likewise, all tautomeric
forms are also intended to be included.
[0083] A "stereoisomer" refers to a compound made up of the same
atoms bonded by the same bonds but having different
three-dimensional structures, which are not interchangeable.
Embodiments of the present invention contemplates various
stereoisomers and mixtures thereof and includes "enantiomers",
which refers to two stereoisomers whose molecules are
nonsuperimposeable mirror images of one another.
[0084] A "tautomer" refers to a proton shift from one atom of a
molecule to another atom of the same molecule. Embodiments of the
present invention include tautomers of any said compounds.
[0085] Compounds
[0086] In an aspect, the invention provides novel lipid compounds
which are capable of combining with other lipid components such as
neutral lipids, charged lipids, steroids and/or polymer
conjugated-lipids to form lipid nanoparticles with
oligonucleotides. Without wishing to be bound by theory, it is
thought that these lipid nanoparticles shield oligonucleotides from
degradation in the serum and provide for effective delivery of
oligonucleotides to cells in vitro and in vivo.
[0087] In one embodiment, the lipid compounds have the structure of
Formula (I):
##STR00004##
or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein:
[0088] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--,
--C(.dbd.O)S--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)NR.sup.a--,
--OC(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)O-- or a direct
bond;
[0089] G.sup.1 is C.sub.1-C.sub.2 alkylene, --(C.dbd.O)--,
--O(C.dbd.O)--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)-- or a direct
bond;
[0090] G.sup.2 is --C(.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)S--,
--C(.dbd.O)NR.sup.a-- or a direct bond;
[0091] G.sup.3 is C.sub.1-C.sub.6 alkylene;
[0092] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0093] R.sup.1a and R.sup.1b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0094] R.sup.2a and R.sup.2b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.2b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0095] R.sup.3a and R.sup.3b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0096] R.sup.4a and R.sup.4b are, at each occurrence, independently
either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond;
[0097] R.sup.5 and R.sup.6 are each independently H or methyl;
[0098] R.sup.7 is C.sub.4-C.sub.20 alkyl;
[0099] R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12
alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to
which they are attached, form a 5, 6 or 7-membered heterocyclic
ring;
[0100] a, b, c and d are each independently an integer from 1 to
24; and
[0101] x is 0, 1 or 2.
[0102] In some embodiments, L.sup.1 and L.sup.2 are each
independently --O(C.dbd.O)--, --(C.dbd.O)O-- or a direct bond. In
other embodiments, G.sup.1 and G.sup.2 are each independently
--(C.dbd.O)-- or a direct bond. In some different embodiments,
L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)--,
--(C.dbd.O)O-- or a direct bond; and G.sup.1 and G.sup.2 are each
independently --(C.dbd.O)-- or a direct bond.
[0103] In some different embodiments, L.sup.1 and L.sup.2 are each
independently --C(.dbd.O)--, --O--, --S(O).sub.x--, --S--S--,
--C(.dbd.O)S--, --SC(.dbd.O)--, --NR.sup.a--,
--NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
--NR.sup.aC(.dbd.O)NR.sup.a, --OC(.dbd.O)NR.sup.a--,
--NR.sup.aC(.dbd.O)O--, --NR.sup.aS(O).sub.xNR.sup.a--,
--NR.sup.aS(O).sub.x-- or --S(O).sub.xNR.sup.a--.
[0104] In other of the foregoing embodiments, the compound has one
of the following structures (IA) or (IB):
##STR00005##
[0105] In some embodiments, the compound has structure (IA). In
other embodiments, the compound has structure (IB).
[0106] In any of the foregoing embodiments, one of L.sup.1 or
L.sup.2 is --O(C.dbd.O)--. For example, in some embodiments each of
L.sup.1 and L.sup.2 are --O(C.dbd.O)--.
[0107] In some different embodiments of any of the foregoing, one
of L.sup.1 or L.sup.2 is --(C.dbd.O)O--. For example, in some
embodiments each of L.sup.1 and L.sup.2 is --(C.dbd.O)O--.
[0108] In different embodiments, one of L.sup.1 or L.sup.2 is a
direct bond. As used herein, a "direct bond" means the group (e.g.,
L.sup.1 or L.sup.2) is absent. For example, in some embodiments
each of L.sup.1 and L.sup.2 is a direct bond.
[0109] In other different embodiments of the foregoing, for at
least one occurrence of R.sup.1a and R.sup.1b, R.sup.1a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.1b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
[0110] In still other different embodiments, for at least one
occurrence of R.sup.4a and R.sup.4b, R.sup.4a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.4b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
[0111] In more embodiments, for at least one occurrence of R.sup.2a
and R.sup.2b, R.sup.2a is H or C.sub.1-C.sub.12 alkyl, and R.sup.2b
together with the carbon atom to which it is bound is taken
together with an adjacent R.sup.2b and the carbon atom to which it
is bound to form a carbon-carbon double bond.
[0112] In other different embodiments of any of the foregoing, for
at least one occurrence of R.sup.3a and R.sup.3b, R.sup.3a is H or
C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom
to which it is bound is taken together with an adjacent R.sup.3b
and the carbon atom to which it is bound to form a carbon-carbon
double bond.
[0113] It is understood that "carbon-carbon" double bond refers to
one of the following structures:
##STR00006##
wherein R.sup.c and R.sup.d are, at each occurrence, independently
H or a substituent. For example, in some embodiments R.sup.c and
R.sup.d are, at each occurrence, independently H, C.sub.1-C.sub.12
alkyl or cycloalkyl, for example H or C.sub.1-C.sub.12 alkyl.
[0114] In various other embodiments, the compound has one of the
following structures (IC) or (ID):
##STR00007##
wherein e, f, g and h are each independently an integer from 1 to
12.
[0115] In some embodiments, the compound has structure (IC). In
other embodiments, the compound has structure (ID).
[0116] In various embodiments of the compounds of structures (IC)
or (ID), e, f, g and h are each independently an integer from 4 to
10.
[0117] In certain embodiments of the foregoing, a, b, c and d are
each independently an integer from 2 to 12 or an integer from 4 to
12. In other embodiments, a, b, c and d are each independently an
integer from 8 to 12 or 5 to 9. In some certain embodiments, a is
0. In some embodiments, a is 1. In other embodiments, a is 2. In
more embodiments, a is 3. In yet other embodiments, a is 4. In some
embodiments, a is 5. In other embodiments, a is 6. In more
embodiments, a is 7. In yet other embodiments, a is 8. In some
embodiments, a is 9. In other embodiments, a is 10. In more
embodiments, a is 11. In yet other embodiments, a is 12. In some
embodiments, a is 13. In other embodiments, a is 14. In more
embodiments, a is 15. In yet other embodiments, a is 16.
[0118] In some embodiments, b is 1. In other embodiments, b is 2.
In more embodiments, b is 3. In yet other embodiments, b is 4. In
some embodiments, b is 5. In other embodiments, b is 6. In more
embodiments, b is 7. In yet other embodiments, b is 8. In some
embodiments, b is 9. In other embodiments, b is 10. In more
embodiments, b is 11. In yet other embodiments, b is 12. In some
embodiments, b is 13. In other embodiments, b is 14. In more
embodiments, b is 15. In yet other embodiments, b is 16.
[0119] In some embodiments, c is 1. In other embodiments, c is 2.
In more embodiments, c is 3. In yet other embodiments, c is 4. In
some embodiments, c is 5. In other embodiments, c is 6. In more
embodiments, c is 7. In yet other embodiments, c is 8. In some
embodiments, c is 9. In other embodiments, c is 10. In more
embodiments, c is 11. In yet other embodiments, c is 12. In some
embodiments, c is 13. In other embodiments, c is 14. In more
embodiments, c is 15. In yet other embodiments, c is 16.
[0120] In some certain embodiments, d is 0. In some embodiments, d
is 1. In other embodiments, d is 2. In more embodiments, d is 3. In
yet other embodiments, d is 4. In some embodiments, d is 5. In
other embodiments, d is 6. In more embodiments, d is 7. In yet
other embodiments, d is 8. In some embodiments, d is 9. In other
embodiments, d is 10. In more embodiments, d is 11. In yet other
embodiments, d is 12. In some embodiments, d is 13. In other
embodiments, d is 14. In more embodiments, d is 15. In yet other
embodiments, d is 16.
[0121] In some embodiments, e is 1. In other embodiments, e is 2.
In more embodiments, e is 3. In yet other embodiments, e is 4. In
some embodiments, e is 5. In other embodiments, e is 6. In more
embodiments, e is 7. In yet other embodiments, e is 8. In some
embodiments, e is 9. In other embodiments, e is 10. In more
embodiments, e is 11. In yet other embodiments, e is 12.
[0122] In some embodiments, f is 1. In other embodiments, f is 2.
In more embodiments, f is 3. In yet other embodiments, f is 4. In
some embodiments, f is 5. In other embodiments, f is 6. In more
embodiments, f is 7. In yet other embodiments, f is 8. In some
embodiments, f is 9. In other embodiments, f is 10. In more
embodiments, f is 11. In yet other embodiments, f is 12.
[0123] In some embodiments, g is 1. In other embodiments, g is 2.
In more embodiments, g is 3. In yet other embodiments, g is 4. In
some embodiments, g is 5. In other embodiments, g is 6. In more
embodiments, g is 7. In yet other embodiments, g is 8. In some
embodiments, g is 9. In other embodiments, g is 10. In more
embodiments, g is 11. In yet other embodiments, g is 12.
[0124] In some embodiments, h is 1. In other embodiments, e is 2.
In more embodiments, h is 3. In yet other embodiments, h is 4. In
some embodiments, e is 5. In other embodiments, h is 6. In more
embodiments, h is 7. In yet other embodiments, h is 8. In some
embodiments, h is 9. In other embodiments, h is 10. In more
embodiments, h is 11. In yet other embodiments, h is 12.
[0125] In some other various embodiments, a and d are the same. In
some other embodiments, b and c are the same. In some other
specific embodiments and a and d are the same and b and c are the
same.
[0126] The sum of a and b and the sum of c and d are factors which
may be varied to obtain a lipid having the desired properties. In
one embodiment, a and b are chosen such that their sum is an
integer ranging from 14 to 24. In other embodiments, c and d are
chosen such that their sum is an integer ranging from 14 to 24. In
further embodiment, the sum of a and b and the sum of c and d are
the same. For example, in some embodiments the sum of a and b and
the sum of c and d are both the same integer which may range from
14 to 24. In still more embodiments, a. b, c and d are selected
such that the sum of a and b and the sum of c and d is 12 or
greater.
[0127] The substituents at R.sup.1a, R.sup.2a, R.sup.3a and
R.sup.4a are not particularly limited. In some embodiments, at
least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is H. In
certain embodiments R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a are H
at each occurrence. In certain other embodiments at least one of
R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.12
alkyl. In certain other embodiments at least one of R.sup.1a,
R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.8 alkyl. In
certain other embodiments at least one of R.sup.1a, R.sup.2a,
R.sup.3a and R.sup.4a is C.sub.1-C.sub.6 alkyl. In some of the
foregoing embodiments, the C.sub.1-C.sub.8 alkyl is methyl, ethyl,
n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or
n-octyl.
[0128] In certain embodiments of the foregoing, R.sup.1a, R.sup.1b,
R.sup.4a and R.sup.4b are C.sub.1-C.sub.12 alkyl at each
occurrence.
[0129] In further embodiments of the foregoing, at least one of
R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b is H or R.sup.1b,
R.sup.2b, R.sup.3b and R.sup.4b are H at each occurrence.
[0130] In certain embodiments of the foregoing, R.sup.1b together
with the carbon atom to which it is bound is taken together with an
adjacent R.sup.1b and the carbon atom to which it is bound to form
a carbon-carbon double bond. In other embodiments of the foregoing
R.sup.4b together with the carbon atom to which it is bound is
taken together with an adjacent R.sup.4b and the carbon atom to
which it is bound to form a carbon-carbon double bond.
[0131] The substituents at R.sup.5 and R.sup.6 are not particularly
limited in the foregoing embodiments. In certain embodiments one of
R.sup.5 or R.sup.6 is methyl. In other embodiments each of R.sup.5
or R.sup.6 is methyl.
[0132] The substituents at R.sup.7 are not particularly limited in
the foregoing embodiments. In certain embodiments R.sup.7 is
C.sub.6-C.sub.16 alkyl. In some other embodiments, R.sup.7 is
C.sub.6-C.sub.9 alkyl. In some of these embodiments, R.sup.7 is
substituted with --(C.dbd.O)OR.sup.b, --O(C.dbd.O)R.sup.b,
--C(.dbd.O)R.sup.b, --OR.sup.b, --S(O)R.sup.b, --S--SR.sup.b,
--C(.dbd.O)SR, --SC(.dbd.O)R.sup.b, --NR.sup.aR.sup.b,
--NR.sup.aC(.dbd.O)R.sup.b, --C(.dbd.O)NR.sup.aR.sup.b,
--NR.sup.aC(.dbd.O)NR.sup.aR.sup.b, --OC(.dbd.O)NR.sup.aR.sup.b,
--NR.sup.aC(.dbd.O)OR.sup.b, --NR.sup.aS(O).sub.xNR.sup.aR.sup.b,
--NR.sup.aS(O).sub.xR.sup.b or --S(O).sub.xNR.sup.aR.sup.b,
wherein: R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.b is
C.sub.1-C.sub.15 alkyl; and x is 0, 1 or 2. For example, in some
embodiments R.sup.7 is substituted with --(C.dbd.O)OR.sup.b or
--O(C.dbd.O)R.sup.b.
[0133] In various of the foregoing embodiments, R.sup.b is branched
C.sub.1-C.sub.15 alkyl. For example, in some embodiments R.sup.b
has one of the following structures:
##STR00008##
[0134] In certain other of the foregoing embodiments, one of
R.sup.8 or R.sup.9 is methyl. In other embodiments, both R.sup.8
and R.sup.9 are methyl.
[0135] In some different embodiments, R.sup.8 and R.sup.9, together
with the nitrogen atom to which they are attached, form a 5, 6 or
7-membered heterocyclic ring. In some embodiments of the foregoing,
R.sup.8 and R.sup.9, together with the nitrogen atom to which they
are attached, form a 5-membered heterocyclic ring, for example a
pyrrolidinyl ring. In some different embodiments of the foregoing,
R.sup.8 and R.sup.9, together with the nitrogen atom to which they
are attached, form a 6-membered heterocyclic ring, for example a
piperazinyl ring.
[0136] In still other embodiments of the foregoing compounds,
G.sup.3 is C.sub.2-C.sub.4 alkylene, for example C.sub.3
alkylene.
[0137] In various different embodiments, the compound has one of
the structures set forth in Table 1 below.
TABLE-US-00001 TABLE 1 Representative Compounds Preparation No.
Structure Method 1 ##STR00009## A 2 ##STR00010## A 3 ##STR00011## A
4 ##STR00012## B 5 ##STR00013## A 6 ##STR00014## A 7 ##STR00015## A
8 ##STR00016## A 9 ##STR00017## A 10 ##STR00018## A 11 ##STR00019##
A 12 ##STR00020## A 13 ##STR00021## A 14 ##STR00022## A 15
##STR00023## A 16 ##STR00024## B 17 ##STR00025## C 18 ##STR00026##
A 19 ##STR00027## A 20 ##STR00028## A 21 ##STR00029## A 22
##STR00030## A 23 ##STR00031## A 24 ##STR00032## A 25 ##STR00033##
B 26 ##STR00034## B 27 ##STR00035## B 28 ##STR00036## B 29
##STR00037## B 30 ##STR00038## B 31 ##STR00039## B 32 ##STR00040##
B 33 ##STR00041## B 34 ##STR00042## B 35 ##STR00043## A 36
##STR00044## C 37 ##STR00045## A 38 ##STR00046## A 39 ##STR00047##
A 40 ##STR00048## A 41 ##STR00049## A 42 ##STR00050## A 43
##STR00051## C 44 ##STR00052## A 45 ##STR00053## A 46 ##STR00054##
A
[0138] It is understood that any embodiment of the compounds of
Formula (I), as set forth above, and any specific substituent
and/or variable in the compound Formula (I), as set forth above,
may be independently combined with other embodiments and/or
substituents and/or variables of compounds of Formula (I) to form
embodiments of the inventions not specifically set forth above. In
addition, in the event that a list of substituents and/or variables
is listed for any particular R group, L group, G group, or
variables a-h, or x in a particular embodiment and/or claim, it is
understood that each individual substituent and/or variable may be
deleted from the particular embodiment and/or claim and that the
remaining list of substituents and/or variables will be considered
to be within the scope of the invention.
[0139] It is understood that in the present description,
combinations of substituents and/or variables of the depicted
formulae are permissible only if such contributions result in
stable compounds.
[0140] In some embodiments, compositions comprising any one or more
of the compounds of Formula (I) and a therapeutic agent are
provided. For example, in some embodiments, the compositions
comprise any of the compounds of Formula (I) and a therapeutic
agent and one or more excipient selected from neutral lipids,
steroids and polymer conjugated lipids. Other pharmaceutically
acceptable excipients and/or carriers are also included in various
embodiments of the compositions.
[0141] In some embodiments, the neutral lipid is selected from
DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the
neutral lipid is DSPC. In various embodiments, the molar ratio of
the compound to the neutral lipid ranges from about 2:1 to about
8:1.
[0142] In various embodiments, the compositions further comprise a
steroid or steroid analogue. In certain embodiments, the steroid or
steroid analogue is cholesterol. In some of these embodiments, the
molar ratio of the compound to cholesterol ranges from about 2:1 to
1:1.
[0143] In various embodiments, the polymer conjugated lipid is a
pegylated lipid. For example, some embodiments include a pegylated
diacylglycerol (PEG-DAG) such as
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG
succinate diacylglycerol (PEG-S-DAG) such as
4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(.omega.-methoxy(polyethoxy)eth-
yl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a
PEG dialkoxypropylcarbamate such as
w-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate
or
2,3-di(tetradecanoxy)propyl-N-(.omega.-methoxy(polyethoxy)ethyl)carbamate-
. In various embodiments, the molar ratio of the compound to the
pegylated lipid ranges from about 100:1 to about 25:1.
[0144] In some embodiments, the composition comprises a pegylated
lipid having the following structure (II):
##STR00055##
or a pharmaceutically acceptable salt, tautomer or stereoisomer
thereof, wherein:
[0145] R.sup.10 and R.sup.11 are each independently a straight or
branched, saturated or unsaturated alkyl chain containing from 10
to 30 carbon atoms, wherein the alkyl chain is optionally
interrupted by one or more ester bonds; and
[0146] z has a mean value ranging from 30 to 60.
[0147] In some embodiments, R.sup.10 and R.sup.11 are each
independently straight, saturated alkyl chains containing from 12
to 16 carbon atoms. In other embodiments, the average z is about
45.
[0148] In some embodiments of the foregoing composition, the
therapeutic agent comprises a nucleic acid. For example, in some
embodiments, the nucleic acid is selected from antisense, plasmid
DNA and messenger RNA.
[0149] In other different embodiments, the invention is directed to
a method for administering a therapeutic agent to a patient in need
thereof, the method comprising preparing or providing any of the
foregoing compositions and administering the composition to the
patient
[0150] For the purposes of administration, the compounds of the
present invention (typically in the form of lipid nanoparticles in
combination with a therapeutic agent) may be administered as a raw
chemical or may be formulated as pharmaceutical compositions.
Pharmaceutical compositions of the present invention comprise a
compound of Formula (I) and one or more pharmaceutically acceptable
carrier, diluent or excipient. The compound of Formula (I) is
present in the composition in an amount which is effective to form
a lipid nanoparticle and deliver the therapeutic agent, e.g., for
treating a particular disease or condition of interest. Appropriate
concentrations and dosages can be readily determined by one skilled
in the art.
[0151] Administration of the compositions of the invention can be
carried out via any of the accepted modes of administration of
agents for serving similar utilities. The pharmaceutical
compositions of the invention may be formulated into preparations
in solid, semi-solid, liquid or gaseous forms, such as tablets,
capsules, powders, granules, ointments, solutions, suspensions,
suppositories, injections, inhalants, gels, microspheres, and
aerosols. Typical routes of administering such pharmaceutical
compositions include, without limitation, oral, topical,
transdermal, inhalation, parenteral, sublingual, buccal, rectal,
vaginal, and intranasal. The term parenteral as used herein
includes subcutaneous injections, intravenous, intramuscular,
intradermal, intrasternal injection or infusion techniques.
Pharmaceutical compositions of the invention are formulated so as
to allow the active ingredients contained therein to be
bioavailable upon administration of the composition to a patient.
Compositions that will be administered to a subject or patient take
the form of one or more dosage units, where for example, a tablet
may be a single dosage unit, and a container of a compound of the
invention in aerosol form may hold a plurality of dosage units.
Actual methods of preparing such dosage forms are known, or will be
apparent, to those skilled in this art; for example, see Remington:
The Science and Practice of Pharmacy, 20th Edition (Philadelphia
College of Pharmacy and Science, 2000). The composition to be
administered will, in any event, contain a therapeutically
effective amount of a compound of the invention, or a
pharmaceutically acceptable salt thereof, for treatment of a
disease or condition of interest in accordance with the teachings
of this invention.
[0152] A pharmaceutical composition of the invention may be in the
form of a solid or liquid. In one aspect, the carrier(s) are
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) may be liquid, with the compositions
being, for example, an oral syrup, injectable liquid or an aerosol,
which is useful in, for example, inhalatory administration.
[0153] When intended for oral administration, the pharmaceutical
composition is preferably in either solid or liquid form, where
semi-solid, semi-liquid, suspension and gel forms are included
within the forms considered herein as either solid or liquid.
[0154] As a solid composition for oral administration, the
pharmaceutical composition may be formulated into a powder,
granule, compressed tablet, pill, capsule, chewing gum, wafer or
the like form. Such a solid composition will typically contain one
or more inert diluents or edible carriers. In addition, one or more
of the following may be present: binders such as
carboxymethylcellulose, ethyl cellulose, microcrystalline
cellulose, gum tragacanth or gelatin; excipients such as starch,
lactose or dextrins, disintegrating agents such as alginic acid,
sodium alginate, Primogel, corn starch and the like; lubricants
such as magnesium stearate or Sterotex; glidants such as colloidal
silicon dioxide; sweetening agents such as sucrose or saccharin; a
flavoring agent such as peppermint, methyl salicylate or orange
flavoring; and a coloring agent.
[0155] When the pharmaceutical composition is in the form of a
capsule, for example, a gelatin capsule, it may contain, in
addition to materials of the above type, a liquid carrier such as
polyethylene glycol or oil.
[0156] The pharmaceutical composition may be in the form of a
liquid, for example, an elixir, syrup, solution, emulsion or
suspension. The liquid may be for oral administration or for
delivery by injection, as two examples. When intended for oral
administration, preferred composition contain, in addition to the
present compounds, one or more of a sweetening agent,
preservatives, dye/colorant and flavor enhancer. In a composition
intended to be administered by injection, one or more of a
surfactant, preservative, wetting agent, dispersing agent,
suspending agent, buffer, stabilizer and isotonic agent may be
included.
[0157] The liquid pharmaceutical compositions of the invention,
whether they be solutions, suspensions or other like form, may
include one or more of the following adjuvants: sterile diluents
such as water for injection, saline solution, preferably
physiological saline, Ringer's solution, isotonic sodium chloride,
fixed oils such as synthetic mono or diglycerides which may serve
as the solvent or suspending medium, polyethylene glycols,
glycerin, propylene glycol or other solvents; antibacterial agents
such as benzyl alcohol or methyl paraben; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose; agents to act as cryoprotectants such
as sucrose or trehalose. The parenteral preparation can be enclosed
in ampoules, disposable syringes or multiple dose vials made of
glass or plastic. Physiological saline is a preferred adjuvant. An
injectable pharmaceutical composition is preferably sterile.
[0158] A liquid pharmaceutical composition of the invention
intended for either parenteral or oral administration should
contain an amount of a compound of the invention such that a
suitable dosage will be obtained.
[0159] The pharmaceutical composition of the invention may be
intended for topical administration, in which case the carrier may
suitably comprise a solution, emulsion, ointment or gel base. The
base, for example, may comprise one or more of the following:
petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil,
diluents such as water and alcohol, and emulsifiers and
stabilizers. Thickening agents may be present in a pharmaceutical
composition for topical administration. If intended for transdermal
administration, the composition may include a transdermal patch or
iontophoresis device.
[0160] The pharmaceutical composition of the invention may be
intended for rectal administration, in the form, for example, of a
suppository, which will melt in the rectum and release the drug.
The composition for rectal administration may contain an oleaginous
base as a suitable nonirritating excipient. Such bases include,
without limitation, lanolin, cocoa butter and polyethylene
glycol.
[0161] The pharmaceutical composition of the invention may include
various materials, which modify the physical form of a solid or
liquid dosage unit. For example, the composition may include
materials that form a coating shell around the active ingredients.
The materials that form the coating shell are typically inert, and
may be selected from, for example, sugar, shellac, and other
enteric coating agents. Alternatively, the active ingredients may
be encased in a gelatin capsule.
[0162] The pharmaceutical composition of the invention in solid or
liquid form may include an agent that binds to the compound of the
invention and thereby assists in the delivery of the compound.
Suitable agents that may act in this capacity include a monoclonal
or polyclonal antibody, or a protein.
[0163] The pharmaceutical composition of the invention may consist
of dosage units that can be administered as an aerosol. The term
aerosol is used to denote a variety of systems ranging from those
of colloidal nature to systems consisting of pressurized packages.
Delivery may be by a liquefied or compressed gas or by a suitable
pump system that dispenses the active ingredients. Aerosols of
compounds of the invention may be delivered in single phase,
bi-phasic, or tri-phasic systems in order to deliver the active
ingredient(s). Delivery of the aerosol includes the necessary
container, activators, valves, subcontainers, and the like, which
together may form a kit. One skilled in the art, without undue
experimentation may determine preferred aerosols.
[0164] The pharmaceutical compositions of the invention may be
prepared by methodology well known in the pharmaceutical art. For
example, a pharmaceutical composition intended to be administered
by injection can be prepared by combining the lipid nanoparticles
of the invention with sterile, distilled water or other carrier so
as to form a solution. A surfactant may be added to facilitate the
formation of a homogeneous solution or suspension. Surfactants are
compounds that non-covalently interact with the compound of the
invention so as to facilitate dissolution or homogeneous suspension
of the compound in the aqueous delivery system.
[0165] The compositions of the invention, or their pharmaceutically
acceptable salts, are administered in a therapeutically effective
amount, which will vary depending upon a variety of factors
including the activity of the specific therapeutic agent employed;
the metabolic stability and length of action of the therapeutic
agent; the age, body weight, general health, sex, and diet of the
patient; the mode and time of administration; the rate of
excretion; the drug combination; the severity of the particular
disorder or condition; and the subject undergoing therapy.
[0166] Compositions of the invention may also be administered
simultaneously with, prior to, or after administration of one or
more other therapeutic agents. Such combination therapy includes
administration of a single pharmaceutical dosage formulation of a
composition of the invention and one or more additional active
agents, as well as administration of the composition of the
invention and each active agent in its own separate pharmaceutical
dosage formulation. For example, a composition of the invention and
the other active agent can be administered to the patient together
in a single oral dosage composition such as a tablet or capsule, or
each agent administered in separate oral dosage formulations. Where
separate dosage formulations are used, the compounds of the
invention and one or more additional active agents can be
administered at essentially the same time, i.e., concurrently, or
at separately staggered times, i.e., sequentially; combination
therapy is understood to include all these regimens.
[0167] Preparation methods for the above compounds and compositions
are described herein below and/or known in the art.
[0168] It will be appreciated by those skilled in the art that in
the process described herein the functional groups of intermediate
compounds may need to be protected by suitable protecting groups.
Such functional groups include hydroxy, amino, mercapto and
carboxylic acid. Suitable protecting groups for hydroxy include
trialkylsilyl or diarylalkylsilyl (for example,
t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),
tetrahydropyranyl, benzyl, and the like. Suitable protecting groups
for amino, amidino and guanidino include t-butoxycarbonyl,
benzyloxycarbonyl, and the like. Suitable protecting groups for
mercapto include --C(O)--R'' (where R'' is alkyl, aryl or
arylalkyl), p-methoxybenzyl, trityl and the like. Suitable
protecting groups for carboxylic acid include alkyl, aryl or
arylalkyl esters. Protecting groups may be added or removed in
accordance with standard techniques, which are known to one skilled
in the art and as described herein. The use of protecting groups is
described in detail in Green, T. W. and P. G. M. Wutz, Protective
Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill
in the art would appreciate, the protecting group may also be a
polymer resin such as a Wang resin, Rink resin or a
2-chlorotrityl-chloride resin.
[0169] It will also be appreciated by those skilled in the art,
although such protected derivatives of compounds of this invention
may not possess pharmacological activity as such, they may be
administered to a mammal and thereafter metabolized in the body to
form compounds of the invention which are pharmacologically active.
Such derivatives may therefore be described as "prodrugs". All
prodrugs of compounds of this invention are included within the
scope of the invention.
[0170] Furthermore, all compounds of the invention which exist in
free base or acid form can be converted to their pharmaceutically
acceptable salts by treatment with the appropriate inorganic or
organic base or acid by methods known to one skilled in the art.
Salts of the compounds of the invention can be converted to their
free base or acid form by standard techniques.
[0171] The following Reaction Scheme illustrates methods to make
compounds of this invention, i.e., compounds of formula (I):
##STR00056##
or a pharmaceutically acceptable salt, tautomer or stereoisomer
thereof, wherein R.sup.1a, R.sup.1b, R.sup.2a, R.sup.2b, R.sup.3a,
R.sup.3b, R.sup.4a, R.sup.4b, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, L.sup.1, L.sup.2, G.sup.1, G.sup.2, G.sup.3, a, b, c and d
are as defined herein. It is understood that one skilled in the art
may be able to make these compounds by similar methods or by
combining other methods known to one skilled in the art. It is also
understood that one skilled in the art would be able to make, in a
similar manner as described below, other compounds of Formula (I)
not specifically illustrated below by using the appropriate
starting components and modifying the parameters of the synthesis
as needed. In general, starting components may be obtained from
sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,
Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or
synthesized according to sources known to those skilled in the art
(see, for example, Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or
prepared as described in this invention.
##STR00057##
[0172] Embodiments of the compound of structure (I) (e.g.,
compounds A-5 and A-7) can be prepared according to General
Reaction Scheme 1 ("Method A"), wherein R.sup.1a, R.sup.1b,
R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b, R.sup.4a, R.sup.4b,
R.sup.5, R.sup.6, R.sup.8, R.sup.9, L.sup.1, L.sup.2, G.sup.1,
G.sup.2, G.sup.3, a, b, c and d are as defined herein, and R.sup.7'
represents R.sup.7 or a C.sub.3-C.sub.19 alkyl. Referring to
General Reaction Scheme 1, compounds of structure A-1 and A2 can be
purchased from commercial sources or prepared according to methods
familiar to one of ordinary skill in the art. A solution of A-1 and
A-2 is treated with a reducing agent (e.g., sodium
triacetoxyborohydride) to obtain A-3 after any necessary work up. A
solution of A-3 and a base (e.g. trimethylamine, DMAP) is treated
with acyl chloride A-4 (or carboxylic acid and DCC) to obtain A-5
after any necessary work up and/or purification. A-5 can be reduced
with LiAlH4 A-6 to give A-7 after any necessary work up and/or
purification.
##STR00058##
[0173] Embodiments of the compound of structure (I) (e.g., compound
B-5) can be prepared according to General Reaction Scheme 2
("Method B"), wherein R.sup.1a, R.sup.1b, R.sup.2a, R.sup.2b,
R.sup.3a, R.sup.3b, R.sup.4a, R.sup.4b, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, L.sup.1, L.sup.2, G.sup.3, a, b, c and d are as
defined herein. Referring to General Reaction Scheme 2, compounds
of structure B-1 and B-2 can be purchased from commercial sources
or prepared according to methods familiar to one of ordinary skill
in the art. A mixture of B-1 (in excess), B-2 and a base (e.g.,
potassium carbonate) is heated to obtain B-3 after any necessary
work up. A solution of B-3 and a base (e.g. trimethylamine, DMAP)
is treated with acyl chloride B4 (or carboxylic acid and DCC) to
obtain B-5 after any necessary work up and/or purification.
##STR00059##
[0174] Other embodiments of the compound of Formula (I) (e.g., C-9)
are prepared according to General Reaction Scheme 3. As illustrated
in General Reaction Scheme 3, an appropriately protected ketone
(C-1) is reacted under reductive amination conditions with amine
C-2 to yield C-3. Acylation of C-3 with acid chloride C-4 yields
acylated product C-5. Removal of the alcohol protecting group on
C-5 followed by reaction with C-7 and/or C-8 and appropriate
activating reagent (e.g., DCC) yields the desired compound C-9.
[0175] The following examples are provided for purpose of
illustration and not limitation.
Example 1
Synthesis of Compound 1
[0176] Compound 1 was prepared according to method A from compound
5 to yield 240 mg of colorless oil, 0.32 mmol, 61%). 1HNMR (400
MHz, CDCl3) .delta.: 5.43-5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H),
2.39-2.25 (m, 7H), 2.22 (s, 6H), 2.06 (q, 6.8 Hz, 8H), 1.53
(quintet, 7.3 Hz, 2H), 1.41-1.11 (54H), 0.92-0.87 (m, 9H).
Example 2
Synthesis of Compound 2
[0177] Compound 2 was prepared according to method A as follows:
Compound 7 (0.84 g, 0.96 mmol) was dissolved in THF (15 mL) and LAH
(2 eq. 1.92 mmol, 73 mg, MW37.95) was added in portions at RT.
After the reaction mixture was heated at 60 C overnight, sodium
sulfate hydrate was added. The mixture was stirred for 2 h,
filtered through a layer of silica gel. The filtrate was
concentrated to give a slightly yellow oil (0.86 g). The crude
product was purified by gravity column chromatography on silica gel
(0 to 4% MeOH in chloroform). This gave the desired product as a
colorless oil (420 mg, 0.49 mmol, 51%). 1HNMR (400 MHz, CDCl3)
.delta.: 5.43-5.30 (m, 12H), 2.78 (t, 6.4 Hz, 6H), 2.40-2.25 (m,
7H), 2.22 (s, 6H), 2.06 (q, 6.8 Hz, 12H), 1.53 (quintet, 7.3 Hz,
2H), 1.41-1.10 (58H), 0.90 (t, 6.8 Hz, 9H).
Example 3
Synthesis of Compound 3
[0178] Compound 3 was prepared according to method A from compound
8 to yield 123 mg of colorless oil, 0.15 mmol, 41%). 1HNMR (400
MHz, CDCl3) .delta.: 5.43-5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H),
2.35-2.24 (m, 5H), 2.22 (s, 6H), 2.15 (d, 5.5 Hz, 2H), 2.06 (q, 6.8
Hz, 8H), 1.52 (quintet, 7.3 Hz, 2H), 1.40-1.09 (65H), 0.92-0.87 (m,
12H).
Example 4
Synthesis of Compound 5
##STR00060##
[0180] Compound 5 was prepared according to method A as
follows:
Step 1
[0181] 3-dimethylamine-1-propylamine (6 mmol, 612 mg) and the
ketone 5a (3.16 g, 6 mmol) were mixed in DCE (25 mL) and then
treated with sodium triacetoxyborohydride (8.49 mmol, 1.8 g) and
AcOH (6 mmol, 0.36 g, 0.340 mL). The mixture was stirred at rt
under a Ar atmosphere for 2 days. The reaction mixture was quenched
by adding 1 N NaOH (ca 20 mL), and the product was extracted with a
mixture of hexane and ethyl acetate (ca 5%). The organic extract
was washed with water/brine (1:1), brine and dried (Na2SO4).
Concentrated to give the desired product 5b as a yellow oil (3.55
g). The crude product was used for the next step without any
further purification.
Step 2
[0182] A solution of nonanoyl chloride (212 mg, 1.2 mmol) in
benzene (10 mL) was added via syringe to a solution of compound 5b
(600 mg, 0.978 mmol) and triethylamine (5 mmol, 0.7 mL, 5 eq) and
DMAP (20 mg) in benzene (10 mL) at RT in 10 min. After addition,
the mixture was then diluted with a mixture of hexane and ethyl
acetate (ca 5%), washed with water, washed with brine, dried over
sodium sulfate, filtered and concentrated. The crude product (0.77
g) was purified by gravity column chromatography on silica gel
(230-400 mesh silica gel, 40 g, MeOH in chloroform, 0 to 4%). This
gave the desired product 5 as a colorless oil (563 mg, 0.75 mmol,
76%). 1HNMR (400 MHz, CDCl3) .delta.: 5.43-5.30 (m, 8H), 4.56-4.36
(br., 0.3H, due to slow isomerization about amide bond), 3.64
(quintet, 7 Hz, 0.7H), 3.12-3.09 (m, 2H), 2.78 (t, 6.4 Hz, 4H),
2.33-2.25 (m, 4H), 2.23, 2.22 (two sets of singlet, 6H), 2.06
(q-like, 6.8 Hz, 8H), 1.76-1.66 (m, 4H), 1.50-1.40 (m, 4H),
1.40-1.15 (46H), 0.90 (t, 6.7 Hz, 6H), 0.88 (t, 6.8 Hz, 3H).
Example 5
Synthesis of Compound 6
[0183] Compound 6 was prepared according to the general procedure A
to yield 0.98 g of slightly yellow oil, 1.13 mmol, 58%. 1HNMR (400
MHz, CDCl3) .delta.: 5.43-5.30 (m, 12H), 4.55-4.32 (br., 0.3H, due
to slow isomerization about amide bond), 3.63 (quintet-like, 7 Hz,
0.7H), 3.15-3.09 (m, 2H), 2.78 (t, 6.4 Hz, 6H), 2.33-2.25 (m, 4H),
2.22, 2.23 (two sets of singlet, 6H), 2.06 (q-like, 6.8 Hz, 12H),
1.76-1.60 (m, 4H), 1.49-1.16 (54H), 0.90 (t-like, 6.8 Hz, 9H).
Example 6
Synthesis of Compound 7
[0184] Compound 7 was prepared according to method A as
follows:
[0185] To a solution of 2-ethylheptanoic acid (1.5 eq. 0.83 mmol,
130 mg) in benzene (6 mL) and DMF (5-10 uL) was added oxalyl
chloride (5 eq, 2.8 mmol, 349 mg, 0.24 mL) at RT. The mixture was
stirred at RT for 30 min and then heated at 60 C for 2 h under Ar.
The mixture was concentrated. The residue was taken up in benzene
(6 mL) and concentrated again to remove any oxalyl chloride. The
residual oil (light yellow) was taken in 4 mL of benzene and added
via syringe to a solution of compound 5b (1 eq., 0.55 mmol, 337 mg)
and triethylamine (5 eq, 2.8 mmol, 283 mg, 390 uL) and DMAP (10 mg)
in benzene (6 mL) at RT in 10 min. After addition, the resulting
mixture was stirred at RT overnight. TLC showed that there was not
much reaction. The reaction was concentrated and dried well and
used in the following. The residue was taken up in DCM (20 mL).
DMAP (200 mg, 1.64 mmol) was added, followed by addition of DCC
(1.64 mmol, 338 mg). The mixture was stirred for 11 days and
filtered. The filtrate was washed with 5% NaOH (100 mL). The
organic phase was washed with brine, dried over sodium sulfate.
Filtration and Concentration gave light brown oil (0.89 g). The
crude product (0.89 g) was purified by column chromatography on
silica gel (0 to 4% MeOH in chloroform). This gave the desired
product as a colorless oil (122 mg, 0.16 mmol, 29%). .sup.1HNMR
(400 MHz, CDCl3) .delta.: 5.43-5.30 (m, 8H), 4.69-4.51 (very br.,
estimated 0.4H, due to slow isomerization about amide bond), 3.72
(quintet-like, 6.9 Hz, 0.6H), 3.19-3.09 (m, 2H), 2.78 (t, 6.4 Hz,
4H), 2.55 (quintet-like, 6.5 Hz, 0.5H), 2.42 (quintet-like, 6.5 Hz,
0.5H), 2.29 (q-like, but could be two overlap triplets, 6.9 Hz,
2H), 2.24, 2.23 (two sets of singlet, integration ratio is about
1:1, 6H), 2.09-2.02 (m, 8H), 1.77-1.58 (m, 4H), 1.55-1.15 (48H),
0.93-0.85 (m, 12H).
Example 7
Synthesis of Compound 8
[0186] Compound 8 was prepared according to the general procedure A
to yield 0.39 g of colorless oil, 0.46 mmol, 56%. 1HNMR (400 MHz,
CDCl3) .delta.: 5.43-5.30 (m, 8H), 4.55-4.32 (very br., estimated
0.3H, due to slow isomerization about amide bond), 3.71
(quintet-like, 7 Hz, 0.7H), 3.17-3.08 (m, 2H), 2.78 (t, 6.4 Hz,
4H), 2.59 (quintet-like, 6.5 Hz, 0.5H), 2.46 (quintet-like, 6.5 Hz,
0.5H), 2.40 (t, 7 Hz, 1H), 2.31 (t, 7 Hz, 1H), 2.28, 2.25 (two sets
of singlet, integration ratio is about 1:1, 6H), 2.09-2.02 (m, 8H),
1.79-1.69 (m, 2H), 1.66-1.57 (m, 2H), 1.55-1.16 (62H), 0.92-0.86
(m, 12H).
Example 8
Synthesis of Compound 9
##STR00061##
[0188] Compound 9 was prepared according to method A as
follows:
Step 1
[0189] 3-dimethylamine-1-propylamine (1 eq. 1.3 mmol, 133 mg, 163
uL; MW102.18, d 0.812) and the ketone 9a (1 eq., 0.885 g, 1.3 mmol)
were mixed in DCE (8 mL) and then treated with sodium
triacetoxyborohydride (1.4 eq., 1.82 mmol, 386 mg; MW211.94) and
AcOH (1 eq., 1.3 mmol, 78 mg, 74 uL, MW 60.05, d 1.06). The mixture
was stirred at RT under an Ar atmosphere for 2 days. The reaction
mixture was diluted with hexanes-EtOAc (9:1) and quenched by adding
0.1 N NaOH (20 mL). The organic phase was separated, washed with
sat NaHCO.sub.3, brine, dried over sodium sulfate, decanted and
concentrated to give the desired product 9b as a slightly yellow
cloudy oil (1.07 g, 1.398 mmol).
Step 2
[0190] A solution of nonanoyl chloride (1.3 eq., 1.27 mmol, 225 mg)
in benzene (10 mL) was added via syringe to a solution of the
compound 9b from step 1 (0.75 g, 0.98 mmol) and triethylamine (5
eq, 4.90 mmol, 0.68 mL) and DMAP (20 mg) in benzene (10 mL) at RT
in 10 min. After addition, the mixture was stirred at RT overnight.
Methanol (5.5 mL) was added to remove excess acyl chloride. After 3
h, the mixture was filtered through a pad of silica gel (1.2 cm).
Concentration gave a colorless oil (0.70 g).
[0191] The crude product (0.70 g) was purified by flash dry column
chromatography on silica gel (0 to 4% MeOH in chloroform). This
yielded 457 mg of colorless oil, 0.50 mmol, 51%. 1HNMR (400 MHz,
CDCl3) .delta.: 4.54-4.36 (very br., estimated 0.3H, due to slow
isomerization about amide bond), 3.977, 3.973 (two sets of
doublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09
(m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two sets of singlet, 6H),
1.76-1.56 (m, 10H), 1.49-1.39 (m, 4H), 1.37-1.11 (62H), 0.92-0.86
(m, 15H).
Example 9
Synthesis of Compound 10
[0192] Compound 10 was prepared according to the general procedure
A to yield 245 mg of colorless oil, 0.27 mmol, total yield 53% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.87 (quintet-like,
6.3 Hz, 2H), 4.54-4.36 (very br., estimated 0.3H, due to slow
isomerization about amide bond), 3.63 (quintet-like, 6.8 Hz, 0.7H),
3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two sets of
singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m, 12H), 1.37-1.11
(60H), 0.92-0.86 (m, 15H).
Example 10
Synthesis of Compound 11
[0193] Compound 11 was prepared according to the general procedure
A to yield 239 mg of colorless oil, 0.26 mmol, total yield 52% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.87 (quintet-like,
6.3 Hz, 2H), 4.54-4.36 (very br., estimated 0.3H, due to slow
isomerization about amide bond), 3.63 (quintet-like, 6.8 Hz, 0.7H),
3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two sets of
singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m, 12H), 1.37-1.11
(62H), 0.92-0.86 (m, 15H).
Example 11
Synthesis of Compound 12
[0194] Compound 12 was prepared according to the general procedure
A to yield 198 mg of colorless oil, 0.20 mmol, total yield 46% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.54-4.36 (very br.,
estimated 0.3H, due to slow isomerization about amide bond), 3.974,
3.971 (two sets of doublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8
Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two
sets of singlet, 6H), 1.76-1.56 (m, 10H), 1.49-1.39 (m, 4H),
1.37-1.11 (76H), 0.92-0.86 (m, 15H).
Example 12
Synthesis of Compound 13
[0195] Compound 13 was prepared according to the general procedure
A to yield 217 mg of colorless oil, 0.21 mmol, total yield 49% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.54-4.36 (very br.,
estimated 0.3H, due to slow isomerization about amide bond), 3.973,
3.970 (two sets of doublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8
Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two
sets of singlet, 6H), 1.76-1.56 (m, 10H), 1.49-1.39 (m, 4H),
1.37-1.11 (78H), 0.92-0.86 (m, 15H).
Example 13
Synthesis of Compound 14
[0196] Compound 14 was prepared according to the general procedure
A to yield 263 mg of colorless oil, 0.29 mmol, total yield 39% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.54-4.36 (br.,
estimated 0.3H, due to slow isomerization about amide bond), 3.977,
3.973 (two sets of doublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8
Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43 (m, 6H), 2.34-2.26 (m, 6H),
1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H), 1.49-1.38 (m, 4H), 1.37-1.11
(60H), 0.92-0.86 (m, 15H).
Example 14
Synthesis of Compound 15
[0197] Compound 15 was prepared according to the general procedure
A to yield 234 mg of colorless oil, 0.25 mmol, total yield 34% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.54-4.36 (br.,
estimated 0.3H, due to slow isomerization about amide bond), 3.977,
3.973 (two sets of doublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8
Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43 (m, 6H), 2.34-2.26 (m, 6H),
1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H), 1.49-1.38 (m, 4H), 1.37-1.11
(62H), 0.92-0.86 (m, 15H).
Example 15
Synthesis of Compound 16
##STR00062##
[0199] Compound 16 was prepared according to method B as
follows:
[0200] To a solution of the acid 018-19 (0.5 g, 0.90 mmol),
N-hydroxysuccinimide (1.2 eq, 1.08 mmol, 124 mg) and DMAP (0.3 eq,
0.27 mmol, 33 mg) in DCM (20 mL) was added DCC (2 eq, 1.8 mmol, 371
mg). The resulting mixture was stirred at RT for 16 h. The reaction
mixture was then filtered and added into a solution of the amine
021-24 (1.26 mmol, 288 mg) in DCM (10 mL) and triethylamine (5
mmol, 696 uL). After 15 days, the mixture was concentrated. The
residue was taken up in hexane/ethyl acetate/Et3N (ca 9:1:0.3) and
was filtered through a small pad of silica gel, washed with a
mixture of hexane/ethyl acetate/Et3N (ca 9:1:0.3). The filtrate was
concentrated and a yellow oil was obtained (580 mg). The yellow oil
was purified by column chromatography on silica gel (eluted with a
gradient mixture of MeOH in Chloroform, 0 to 4.2%). This gave the
desired product as a colorless oil (102 mg, 0.13 mmol, 14%).
.sup.1HNMR (400 MHz, CDCl3) .delta.: 5.43-5.30 (m, 8H), 3.38-3.29
(m, 3H), 3.28-3.23 (m, 1H), 2.78 (t, 6.4 Hz, 4H), 2.56-2.47 (m,
1H), 2.30-2.24 (m, 2H), 2.23, 2.22 (two sets of singlet, 6H),
2.09-2.02 (m, 8H), 1.71 (quintet-like, 7.4 Hz, 2H), 1.66-1.48
(overlapped with water; estimated 4H), 1.47-1.18 (m, 50H),
0.92-0.86 (m, 9H).
Example 16
Synthesis of Compound 24
[0201] Compound 24 was prepared according to the general procedure
A to yield 279 mg of slightly yellow oil, 0.29 mmol, total yield
44% for 2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.88
(quintet-like, 6.3 Hz, 3H), 3.62 (quintet-like, 6.8 Hz, 1H),
3.14-3.08 (m, 2H), 2.33-2.25 (m, 10H), 2.23, 2.22 (two sets of
singlet, 6H), 1.76-1.58 (m, 10H), 1.52 (q-like, 6.7 Hz, 12H),
1.49-1.39 (m, 4H), 1.38-1.14 (50H), 0.89 (t-like, 18H).
Example 17
Synthesis of Compound 35
[0202] Compound 35 was prepared according to the general procedure
A to yield 260 mg of slightly yellow oil, 0.29 mmol, total yield
33% for 2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.66-4.52
(very br., estimated 0.3H, due to slow isomerization about amide
bond), 3.977, 3.973 (two sets of doublets, 5.8 Hz, 4H), 3.71
(quintet-like, 6.8 Hz, 0.7H), 3.19-3.09 (m, 2H), 2.54, 2.42 (two
sets of quintet-like, 6.8 Hz, integration ratio is about 1:1.2,
1H), 2.33-2.25 (m, 6H), 2.24, 2.22 (two sets of singlet, 6H),
1.77-1.11 (74H), 0.93-0.85 (m, 18H).
Example 18
Synthesis of Compound 17
##STR00063##
[0204] Compound 17 was prepared according to method C as
follows:
Step 1
[0205] 3-dimethylamino-1-propylamine (1 eq. 4.14 mmol, 423 mg, 521
uL) and ketone 17a (1 eq., 2.0 g, 4.14 mmol) were mixed in DCE (30
mL) and then treated with sodium triacetoxyborohydride (1.4 eq.,
5.80 mmol, 1.229 g) and AcOH (1 eq., 4.14 mmol, 249 mg, 235 uL).
The mixture was stirred at RT under Ar atmosphere for 2 days.
[0206] The reaction mixture was diluted with a mixture of hexanes
and EtOAc (9:1, 200 mL) and quenched by adding dilute NaOH solution
(0.1 N, 270 mL). The two phases were separated. The organic phase
was washed with sat NaHCO.sub.3, brine, dried over sodium sulfate
and filtered through a pad of silica gel. The pad was washed with
200 mL of a mixture of hexane and EtOAc (9:1). Then the pad was
washed 200 mL of a mixture of DCM/MeOH/Et3N (85:15:1). The
DCM/MeOH/Et3N washing was concentrated to give the desired product
(17b) as a colorless oil (1.749 g, 3.07 mmol, 74%).
Step 2
[0207] A solution of nonanoyl chloride (0.333 mL) in benzene (10
mL) was added to a solution of compound 17b (0.75 g) and
triethylamine (0.92 mL) and DMAP (20 mg) in benzene (20 mL) at RT.
The mixture was stirred at RT overnight. MeOH (1 mL) was added and
the mixture continued to stir for 2 h. The reaction mixture was
filtered through a pad of silica gel. Concentration of the filtrate
gave the desired product (17c) as a yellow oil (0.945 g).
Step 3
[0208] To a flask containing 17c (0.945 g, 1.33 mmol and EtOH (25
mL) was added p-toluenesulfonic acid hydrate (1.33 mmol, 253 mg) at
room temperature. The resulting mixture was stirred overnight at
RT. The reaction mixture was heated at 85 C for 2 h. More PTSA (160
mg) was added and the reaction mixture continued to heat at 75 C
overnight. The mixture was concentrated. The residue was taken up
in DCM and washed with dilute NH4OH solution. The organic phase was
washed with a mixture of sat sodium bicarbonate and brine; dried
over sodium sulfate. Concentration gave the desired product (17 d)
as a slightly yellow viscous oil (0.799 g, 1.47 mmol). The crude
product was purified by silica gel column chromatography (0 to 15%
methanol in DCM with trace of triethlyamine). This gave 17 d as a
colorless oil (647 mg, 1.20 mmol, 90%).
Step 4
[0209] To a solution of 17 d (216 mg, 0.40 mmol), 2-butyloctanoic
acid (5 eq, 2 mmol, 401 mg), and 4-dimethylaminopyridine (DMAP)
(5.5 eq. 2.2 mmol, 269 mg) in dichloromethane (20 mL) was added DCC
(5.5 eq, 2.2 mmol, 454 mg). After being stirred over for 4 days, 3
mL of MeOH was added. The mixture continued to stir for another 16
h. The mixture was filtered and the filtrate was concentrated to
dryness. The crude product was purified by gravity column
chromatography on silica gel (MeOH in DCM, 0 to 6%). This gave the
desired compound (17) as a slightly yellow oil (colorless oil, 175
mg, 0.19 mmol, 48%). .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.07,
4.06 (two sets of triplets, 6.7 Hz, 4H), 3.64 (quintet-like, 6.8
Hz, 1H), 3.21-3.09 (two sets of multiplets, 2H), 3.00-2.37 (br.
6H), 2.36-2.20 (m, 6H), 2.05-1.85 (m, 2H), 1.79-1.53 (m, 10H),
1.52-1.39 (m, 8H), 1.37-1.03 (58H), 0.91-0.86 (m, 15H).
Example 19
Synthesis of Compound 36
[0210] Compound 36 was prepared according to the general procedure
C to yield 156 mg of colorless oil, 0.15 mmol, 38% for the last
step. .sup.1HNMR (400 MHz, CDCl3) S: 4.07 (triplets, 6.7 Hz, 4H),
3.65 (quintet-like, 6.8 Hz, 1H), 3.21 (t-like, 6.8 Hz, 2H),
3.10-3.03 (br. 2H), 2.79, 2.78 (two sets of singlet, 6H), 2.35-2.28
(m, 4H), 2.09 (quintet-like, 7.5 Hz, 2H), 1.67-1.54 (m, 10H),
1.54-1.38 (m, 8H), 1.38-1.03 (74H), 0.91-0.86 (m, 15H).
Example 20
Synthesis of Compound 37
[0211] Compound 37 was prepared according to the general procedure
A to yield 397 mg of colorless oil, 0.49 mmol, total yield 60% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 5.43-5.30 (m, 8H),
4.13 (q, 7.1 Hz, 2H), 4.56-4.34 (br. 0.3H), 3.63 (quintet-like, 6.9
Hz, 0.7H), 3.15-3.08 (m, 2H), 2.78 (t-like, 6.4 Hz, 4H), 2.39-2.21
(m, 12H), 2.06 (q-like, 6.9 Hz, 8H), 1.79-1.55 (m, 6H), 1.50-1.40
(m, 4H), 1.40-1.15 (m, 45H), 0.90 (t-like, 6.8 Hz, 6H).
Example 21
Synthesis of Compound 38
[0212] Compound 38 was prepared according to method A as
follows:
##STR00064##
Step 1
[0213] To a solution of 38a (1 eq., 1.266 g, 1.79 mmol) in DCE (15
mL) was added 3-dimethylamino-1-propylamine (1 eq. 1.79 mmol, 183
mg, 225 uL), followed by addition of sodium triacetoxyborohydride
(1.4 eq., 2.51 mmol, 531 mg) and AcOH (1 eq., 1.79 mmol, 107 mg,
101 uL). The mixture was stirred at RT under Ar atmosphere for 3
days.
[0214] The residue was diluted with hexanes-EtOAc (9:1, 150 mL) and
washed with dilute NaOH solution (0.12 N, 100 mL), sat NaHCO.sub.3,
brine and dried over sodium sulfate. The organic phase was filtered
through a pad of silica gel. The pad was washed with 200 mL of a
mixture of hexane and EtOAc (9:1). Then the pad was washed with 200
mL of a mixture of DCM/MeOH/Et3N (85:15:1). The DCM/MeOH/Et3N
washing was concentrated and dried on high vacuum line to give the
desired product (38b) as a colorless oil (1.1 g, 1.38 mmol,
77%).
Step 2
[0215] A solution of nonanoyl chloride (1.5 eq., 0.68 mmol, 120 mg)
in benzene (5 mL) was added to a solution of 38b (0.45 mmol, 360
mg) and triethylamine (5 eq, 2.25 mmol, 228 mg, 314 uL) and DMAP
(10 mg) in benzene (10 mL) at RT in 2 min under Ar. After addition,
the mixture was stirred at RT overnight. MeOH (1 mL) was added and
the mixture continued to stir 2 h. The crude was filtered through a
pad of silica gel. The filtrate was concentrated. The residue (457
mg) was purified by flash column chromatography on silica gel
(230-400 mesh silica gel, 40 g, MeOH in chloroform, 0 to 4.6%).
This gave the desired product (38) as a colorless oil (410 mg, 0.44
mmol, 98%). .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.61-4.35 (br.,
estimated 0.4H, due to slow isomerization about amide bond), 3.974,
3.964 (two sets of doublets, 5.7 Hz, 4H), 3.64 (quintet-like, 7.0
Hz, 0.6H), 3.14-3.08 (m, 2H), 2.34-2.25 (m, 8H), 2.23 (broad s,
6H), 1.77-1.58 (m, 10H), 1.53-1.39 (m, 4H), 1.37-1.15 (66H),
0.92-0.86 (m, 15H).
Example 22
Synthesis of Compound 39
[0216] Compound 39 was prepared according to the general procedure
A to yield 370 mg of colorless oil, 0.40 mmol, total yield 69% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.61-4.35 (br.,
estimated 0.4H, due to slow isomerization about amide bond), 3.974,
3.964 (two sets of doublets, 5.7 Hz, 4H), 3.64 (quintet-like, 7.0
Hz, 0.6H), 3.14-3.08 (m, 2H), 2.34-2.25 (m, 8H), 2.230, 2.221 (two
sets of singlet, 6H), 1.75-1.58 (m, 10H), 1.51-1.39 (m, 4H),
1.37-1.15 (64H), 0.92-0.86 (m, 15H).
Example 23
Synthesis of Compound 40
[0217] Compound 40 was prepared according to the general procedure
A to yield 382 mg of colorless oil, 0.39 mmol, total yield 68% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.60-4.35 (br.,
estimated 0.3H, due to slow isomerization about amide bond), 4.13
(q, 7.2 Hz, 2H), 3.973, 3.964 (two sets of doublets, 5.7 Hz, 4H),
3.63 (quintet-like, 7.0 Hz, 0.7H), 3.14-3.08 (m, 2H), 2.34-2.25 (m,
10H), 2.229, 2.220 (two sets of singlet, 6H), 1.75-1.58 (m, 12H),
1.51-1.39 (m, 4H), 1.37-1.15 (64H), 0.89 (t-like, 7.8 Hz, 12H).
Example 24
Synthesis of Compound 41
[0218] Compound 41 was prepared according to the general procedure
A to yield 309 mg of colorless oil, 0.30 mmol, total yield 73% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.60-4.35 (br.,
estimated 0.3H, due to slow isomerization about amide bond), 3.972,
3.962 (two sets of doublets, 5.7 Hz, 4H), 3.64 (quintet-like, 7.1
Hz, 0.7H), 3.14-3.08 (m, 2H), 2.34-2.25 (m, 8H), 2.23, 2.22 (two
sets of singlet, 6H), 1.75-1.58 (m, 10H), 1.51-1.39 (m, 4H),
1.35-1.21 (82H), 0.92-0.86 (m, 15H).
Example 25
Synthesis of Compound 42
[0219] Compound 42 was prepared according to the general procedure
A to yield 235 mg of colorless oil, 0.23 mmol, total yield 56% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.75-4.49 (br.,
estimated 0.4H, due to slow isomerization about amide bond), 3.97,
3.96 (two sets of doublets, 5.3 Hz, 4H), 3.72 (quintet-like, 7 Hz,
0.6H), 3.21-3.05 (m, 2H), 2.53, 2.42 (two sets of quintet-like, 6.6
Hz, integration ratio is about 1:1.7, 1H), 2.32-2.25 (m, 6H), 2.24,
2.22 (two sets of singlet, 6H), 1.78-1.56 (m, 10H), 1.53-1.39 (m,
6H), 1.38-1.17 (76H), 0.93-0.85 (m, 18H).
Example 26
Synthesis of Compound 43
[0220] Compound 43 was prepared according to the general procedure
C to yield 187 mg of colorless oil, 0.23 mmol, 57% for the last
step. .sup.1HNMR (400 MHz, CDCl3) S: 4.077, 4.071 (two sets of
triplets, 6.7 Hz, 4H), 4.56-4.34 (br. 0.3H), 3.64 (quintet-like,
6.9 Hz, 0.7H), 3.15-3.09 (m, 2H), 2.34-2.24 (m, 6H), 2.234-2.224
(two sets of singlet, 6H), 1.76-1.58 (m, 10H), 1.55-1.39 (m, 8H),
1.39-1.10 (48H), 0.92-0.86 (m, 15H).
Example 27
Synthesis of Compound 44
[0221] Compound 44 was prepared according to the general procedure
A to yield 260 mg of colorless oil, 0.22 mmol, total yield 53% for
2 steps. .sup.1HNMR (400 MHz, CDCl3) .delta.: 4.59-4.35 (br.,
estimated 0.3H, due to slow isomerization about amide bond),
4.03-3.95 (m, 6H), 3.63 (quintet-like, 6.9 Hz, 0.7H), 3.14-3.08 (m,
2H), 2.33-2.24 (m, 10H), 2.229, 2.221 (two sets of singlet,
6H),1.75-1.57 (m, 12H), 1.51-1.40 (m, 4H), 1.40-1.08 (87H),
0.92-0.86 (m, 18H).
Example 28
Luciferase mRNA In Vivo Evaluation Using the Lipid Nanoparticle
Compositions
[0222] Cationic lipid (MC3), DSPC, cholesterol and PEG-lipid were
solubilized in ethanol at a molar ratio of 50:10:38.5:1.5. Lipid
nanoparticles (LNP) were prepared at a total lipid to mRNA weight
ratio of approximately 10:1 to 30:1. Briefly, the mRNA was diluted
to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps
were used to mix the ethanolic lipid solution with the mRNA aqueous
solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow
rates above 15 ml/min. The ethanol was then removed and the
external buffer replaced with PBS by dialysis. Finally, the lipid
nanoparticles were filtered through a 0.2 .mu.m pore sterile
filter. Lipid nanoparticle particle size was 70-90 nm diameter as
determined by quasi-elastic light scattering using a Nicomp 370
submicron particle sizer (Santa Barbara, Calif.).
[0223] Studies were performed in 6-8 week old female C57BL/6 mice
(Charles River) according to guidelines established by an
institutional animal care committee (ACC) and the Canadian Council
on Animal Care (CCAC). Varying doses of mRNA-lipid nanoparticle
were systemically administered by tail vein injection and animals
euthanized at specific time points (1, 2, 4, 8 and 24 hrs)
post-administration. Liver and spleen were collected in
pre-weighted tubes, weights determined, immediately snap frozen in
liquid nitrogen and stored at -80.degree. C. until processing for
analysis.
[0224] For liver, approximately 50 mg was dissected for analyses in
a 2 mL FastPrep tubes (MP Biomedicals, Solon Ohio). 1/4'' ceramic
sphere (MP Biomedicals) was added to each tube and 500 .mu.L of Glo
Lysis Buffer--GLB (Promega, Madison Wis.) equilibrated to room
temperature was added to liver tissue. Liver tissues were
homogenized with the FastPrep24 instrument (MP Biomedicals) at
2.times.6.0 m/s for 15 seconds. Homogenate was incubated at room
temperature for 5 minutes prior to a 1:4 dilution in GLB and
assessed using SteadyGlo Luciferase assay system (Promega).
Specifically, 50 .mu.L of diluted tissue homogenate was reacted
with 50 .mu.L of SteadyGlo substrate, shaken for 10 seconds
followed by 5 minute incubation and then quantitated using a
CentroXS.sup.3 LB 960 luminometer (Berthold Technologies, Germany).
The amount of protein assayed was determined by using the BCA
protein assay kit (Pierce, Rockford Ill.). Relative luminescence
units (RLU) were then normalized to total ug protein assayed. To
convert RLU to ng luciferase a standard curve was generated with
QuantiLum Recombinant Luciferase (Promega). Based in the data
provided in FIG. 1, the four-hour time point was chosen for
efficacy evaluation of the lipid formulations (see Example 29).
[0225] The FLuc mRNA (L-6107) from Trilink Biotechnologies will
express a luciferase protein, originally isolated from the firefly,
Photinus pyralis. FLuc is commonly used in mammalian cell culture
to measure both gene expression and cell viability. It emits
bioluminescence in the presence of the substrate, luciferin. This
capped and polyadenylated mRNA is fully substituted with
5-methylcytidine and pseudouridine.
Example 29
Determination of Efficacy of Lipid Nanoparticle Formulations
Containing Various Cationic Lipids Using an In Vivo Luciferase mRNA
Expression Rodent Model
[0226] The cationic lipids shown in Table 2 have previously been
tested with nucleic acids. For comparative purposes, these lipids
were also used to formulate lipid nanoparticles containing the FLuc
mRNA (L-6107) using an in line mixing method, as described in
example 28 and in PCT/US10/22614, which is hereby incorporated by
reference in its entirety. Lipid nanoparticles were formulated
using the following molar ratio: 50% Cationic lipid/10%
distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG
lipid ("PEG-DMG", i.e.,
(1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with
an average PEG molecular weight of 2000). Relative activity was
determined by measuring luciferase expression in the liver 4 hours
following administration via tail vein injection as described in
example 28. The activity was compared at a dose of 0.3 and 1.0 mg
mRNA/kg and expressed as ng luciferase/g liver measured 4 hours
after administration, as described in Example 28.
TABLE-US-00002 TABLE 2 Lipids showing activity with mRNA Liver Luc
@ Liver Luc @ 0.3 mg/kg 1.0 mg/kg Compound dose dose Structure MC2
4 .+-. 1 N/D ##STR00065## DLinDMA 13 .+-. 3 67 .+-. 20 ##STR00066##
MC4 41 .+-. 10 N/D ##STR00067## XTC2 80 .+-. 28 237 .+-. 99
##STR00068## MC3 198 .+-. 126 757 .+-. 528 ##STR00069## 319 (2%
PEG) 258 .+-. 67 681 .+-. 203 ##STR00070## 137 281 .+-. 203 588
.+-. 303 ##STR00071##
[0227] The novel lipids of the invention and selected comparator
lipids shown in Table 3 were formulated using the following molar
ratio: 50%/cationic lipid/10%/distearoylphosphatidylcholine
(DSPC)/38.5% Cholesterol/1.5% PEG lipid ("PEG-DMA"
2-[2-(w-methoxy(polyethyleneglycol.sub.2000)ethoxy]-N,N-ditetradecylaceta-
mide). Relative activity was determined by measuring luciferase
expression in the liver 4 hours following administration via tail
vein injection as described in Example 28. The activity was
compared at a dose of 0.3 and 1.0 mg mRNA/kg and expressed as ng
luciferase/g liver measured 4 hours after administration, as
described in Example 28. A plot of selected data is given in FIG. 3
(from top to bottom: circle=compound 10; triangle=compound 6;
square=MC3).
TABLE-US-00003 TABLE 3 Exemplary Cationic lipids and Comparator
Lipids Liver Luc @ Liver Luc @ 0.3 mg/kg (ng 1.0 mg/kg (ng No.
pK.sub.a luc/g liver) luc/g liver) Structure MC3 6.09 603 .+-. 150
2876 .+-. 622 ##STR00072## A 7.05 * * ##STR00073## B 6.17 95 .+-.
41 1131 .+-. 384 ##STR00074## C 6.36 24 .+-. 4 77 .+-. 19
##STR00075## 1 5.64 54 .+-. 8 226 .+-. 20 ##STR00076## 5 6.27 603
.+-. 167 3640 .+-. 601 ##STR00077## 6 6.14 19 .+-. 4 211 .+-. 119
##STR00078## 7 5.93 833 .+-. 401 8859 .+-. 780 ##STR00079## 8 5.35
105 .+-. 98 1238 .+-. 153 ##STR00080## 9 6.27 2381 .+-. 1162 17157
.+-. 2470 ##STR00081## 10 6.16 2379 .+-. 93 26181 .+-. 2900
##STR00082## 11 6.13 2273 .+-. 294 16502 .+-. 4301 ##STR00083## 12
6.21 3336 .+-. 1394 13577 .+-. 1948 ##STR00084## 13 6.22 1537 .+-.
777 10907 .+-. 2032 ##STR00085## 14 6.33 2851 .+-. 438 15445 .+-.
3693 ##STR00086## 15 6.32 2708 .+-. 924 15930 .+-. 4711
##STR00087## 16 6.37 231 .+-. 100 1185 .+-. 838 ##STR00088## 17
6.29 837 .+-. 260 6703 .+-. 689 ##STR00089## 24 6.14 1120 .+-. 376
7425 .+-. 2810 ##STR00090## 35 5.97 1083 .+-. 350 8554 .+-. 4587
##STR00091## 36 6.13 541 .+-. 91 4736 .+-. 980 ##STR00092## 37 5.61
* * ##STR00093## 38 6.45 905 .+-. 443 5353 .+-. 2082 ##STR00094##
39 6.45 779 .+-. 82 5180 .+-. 2116 ##STR00095## 40 6.57 753 .+-.
156 2203 .+-. 1555 ##STR00096## 41 ND.sup..dagger. 832 .+-. 298
7437 .+-. 1612 ##STR00097## *not tested; pKa out of range
.sup..dagger.not determined
Example 30
Determination of pk.sub.a of Formulated Lipids
[0228] As described elsewhere, the pKa of formulated cationic
lipids is correlated with the effectiveness of LNPs for delivery of
nucleic acids (see Jayaraman et al, Angewandte Chemie,
International Edition (2012), 51(34), 8529-8533; Semple et al,
Nature Biotechnology 28, 172-176 (2010)). The preferred range of
pKa is .about.5 to .about.7. The pKa of each cationic lipid was
determined in lipid nanoparticles using an assay based on
fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid (TNS).
Lipid nanoparticles comprising of cationic
lipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) in PBS at a
concentration of 0.4 mM total lipid are prepared using the in-line
process as described in Example 28. TNS was prepared as a 100 .mu.M
stock solution in distilled water. Vesicles were diluted to 24
.mu.M lipid in 2 mL of buffered solutions containing, 10 mM HEPES,
10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranged
from 2.5 to 11. An aliquot of the TNS solution was added to give a
final concentration of 1 .mu.M and following vortex mixing
fluorescence intensity was measured at room temperature in a SLM
Aminco Series 2 Luminescence Spectrophotometer using excitation and
emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit
analysis was applied to the fluorescence data and the pKa was
measured as the pH giving rise to half-maximal fluorescence
intensity (see FIG. 2).
[0229] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent
application publications, U.S. patent applications, foreign
patents, foreign patent applications and non-patent publications
referred to in this specification and/or listed in the Application
Data Sheet, including U.S. Provisional Patent Application Ser. No.
62/186,210, filed Jun. 29, 2015, are incorporated herein by
reference, in their entirety. Aspects of the embodiments can be
modified, if necessary to employ concepts of the various patents,
applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of
the above-detailed description. In general, in the following
claims, the terms used should not be construed to limit the claims
to the specific embodiments disclosed in the specification and the
claims, but should be construed to include all possible embodiments
along with the full scope of equivalents to which such claims are
entitled. Accordingly, the claims are not limited by the
disclosure.
* * * * *