U.S. patent application number 17/517111 was filed with the patent office on 2022-02-24 for supramolecular modification of proteins.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Daniel Griffith Anderson, Eric Andrew Appel, Robert S. Langer, Matthew J. Webber.
Application Number | 20220054642 17/517111 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-24 |
United States Patent
Application |
20220054642 |
Kind Code |
A1 |
Webber; Matthew J. ; et
al. |
February 24, 2022 |
SUPRAMOLECULAR MODIFICATION OF PROTEINS
Abstract
The modification of biomolecules, small molecules, and other
agents of via conjugation of excipients, tags, or labels is of
great importance. For example, the modification of therapeutic
agents can confer improved stability, solubility, duration of
action, or pharmacological properties. Supramolecular chemistry
utilizes specific, directional, reversible, non-covalent molecular
recognition motifs in order to achieve organization of molecules,
and can be used to complex tags to agents of interest (e.g.,
insulin, glucagon, antibodies). The present invention provides
useful supramolecular complexes wherein an agent of interest is
specifically bound to a host via non-covalent interactions, and
wherein the host is conjugated to a tag. The present invention also
provides methods and compounds useful in preparing supramolecular
complexes, and methods of treating diseases using the
supramolecular complexes.
Inventors: |
Webber; Matthew J.;
(Cambridge, MA) ; Appel; Eric Andrew; (Palo Alto,
CA) ; Langer; Robert S.; (Newton, MA) ;
Anderson; Daniel Griffith; (Framingham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Appl. No.: |
17/517111 |
Filed: |
November 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15765585 |
Apr 3, 2018 |
11191841 |
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PCT/US2016/055755 |
Oct 6, 2016 |
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17517111 |
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62334910 |
May 11, 2016 |
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62237836 |
Oct 6, 2015 |
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International
Class: |
A61K 47/54 20060101
A61K047/54; A61K 47/60 20060101 A61K047/60; A61K 47/34 20060101
A61K047/34; C07K 14/62 20060101 C07K014/62; C07D 519/00 20060101
C07D519/00; C07K 14/605 20060101 C07K014/605; A61K 47/68 20060101
A61K047/68; A61K 38/26 20060101 A61K038/26; A61K 38/28 20060101
A61K038/28; C07K 16/28 20060101 C07K016/28 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under
EB000244 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. An agent associated with a tag, wherein: the agent is
specifically bound to a host via non-covalent interactions; and the
host is conjugated to the tag.
2. The agent associated with a tag of claim 1, wherein the agent is
selected from the group consisting of proteins, peptides, nucleic
acids, polysaccharides, lipids, small molecules, diagnostic agents,
and imaging agents.
3. The agent of claim 2, wherein the agent is selected from the
group consisting of proteins, peptides, nucleic acids, and small
molecules.
4. The agent of claim 3, wherein the agent is a protein.
5. The agent of claim 4, wherein the protein is a therapeutic
protein.
6. The agent of claim 5, wherein the protein is insulin.
7. The agent of claim 4, wherein the protein is glucagon.
8. The agent of any of claims 1-7, wherein the host is a
macrocycle.
9. The agent of claim 8, wherein the macrocyclic host is selected
from the group consisting of cucurbiturils, cyclodextrins,
calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers,
cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands.
10. The agent of claim 9, wherein the macrocyclic host is an
optionally substituted cucurbituril.
11. The agent of claim 10, wherein the cucurbituril is an
optionally substituted cucurbit[n]uril (CB[n]), wherein n is an
integer between 5-10, inclusive.
12. The agent of claim 11, wherein n is 7; and the cucurbituril is
optionally substituted cucurbit[7]uril (CB[7]).
13. The agent of claim 1, wherein the tag is selected from the
group consisting of polymers, therapeutic agents, targeting agents,
radionuclides, fluorophores, chromophores, phosphorescent agents,
dyes, chemiluminescent agents, particles, colorimetric labels,
magnetic labels, haptens, biomolecules, fatty acids, hydrocarbon
chains, small molecules, excipients, and diagnostic agents.
14. The agent of claim 13, wherein the host is conjugated to the
tag.
15. The agent of claim 14, wherein the tag is a polymer.
16. The agent of claim 15, wherein the polymer is a polyether.
17. The agent of claim 16, wherein the polyether is polyethylene
glycol (PEG).
18. The agent of any of claims 1-17, wherein the host is conjugated
to the tag via a linker.
19. The agent of claim 18, wherein the linker is of Formula (L-1):
##STR00024## wherein: each of L.sup.1 and L.sup.2 is independently
a bond, optionally substituted alkylene, or optionally substituted
heteroalkylene; and A is a bond, optionally substituted
heterocyclyl, or optionally substituted heteroaryl.
20. The agent of claim 19, wherein A is optionally substituted
heteroaryl.
21. The agent of claim 20, wherein A is an optionally substituted
triazole moiety.
22. The agent of claim 18, wherein the linker is of Formula (L-2a)
or (L-2b): ##STR00025## wherein: each of L.sup.1 and L.sup.2 is
independently a bond, optionally substituted alkylene or optionally
substituted heteroalkylene; and Y is CR.sup.a or N; m is an integer
from 0-10, inclusive; each instance of R.sup.a is independently
hydrogen, halogen, optionally substituted alkyl, optionally
substituted heteroalkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted acyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, --OR.sup.aa, --SR.sup.aa,
--N(R.sup.aa).sub.2, --NO.sub.2, or --CN, or two R.sup.a attached
to adjacent atoms are joined to form an optionally substituted
carbocyclyl, optionally substituted heterocyclyl, optionally
substituted aryl, optionally substituted heteroaryl ring; each
occurrence of R.sup.aa is independently hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted acyl, optionally
substituted sulfonyl, an oxygen protecting group, or a nitrogen
protecting group, or two R.sup.aa are joined to form an optionally
substituted heterocyclic or optionally substituted heteroaryl
ring.
23. The agent of claim 22, wherein the linker is of Formula (L-3a)
or (L-3b): ##STR00026##
24. The agent of claim 23, wherein L.sup.1 is optionally
substituted alkylene.
25. The agent of claim 23, wherein L.sup.2 is optionally
substituted heteroalkylene.
26. The agent of any one of claims 22-25, wherein the linker is of
one of the following formulae: ##STR00027##
27. The agent of any one of claims 1-26, wherein the non-covalent
interaction is a guest-host interaction.
28. The agent of any one of claims 1-26, wherein the agent is a
protein, and the host binds an amino acid residue on the
protein.
29. The agent of claim 28, wherein the host binds an N-terminal
amino acid on the protein.
30. The agent of claim 28 or 29, wherein the host binds an amino
acid selected from the group consisting of phenylalanine,
tryptophan, and tyrosine.
31. The agent of claim 28, wherein the amino acid is
phenylalanine.
32. The agent of any one of claims 28-31, wherein the protein is
insulin; and the host binds to the B1 phenylalanine of insulin.
33. A compound comprising an optionally substituted cucurbituril
conjugated to a tag via a linker, or a salt thereof; wherein: the
tag is selected from the group consisting of polymers, therapeutic
agents, targeting agents, radionuclides, fluorophores,
chromophores, phosphorescent agents, dyes, chemiluminescent agents,
particles, colorimetric labels, magnetic labels, haptens,
biomolecules, small molecules, fatty acids, hydrocarbon chains,
excipients, and diagnostic agents.
34. The compound of claim 33, wherein the compound is of Formula
(CB-1): CB-L.sup.1-A-L.sup.2-TAG (CB-1), or a salt thereof,
wherein: CB is an optionally substituted cucurbituril; TAG is a tag
selected from the group consisting of polymers, therapeutic agents,
targeting agents, radionuclides, fluorophores, chromophores,
phosphorescent agents, dyes, chemiluminescent agents, particles,
colorimetric labels, magnetic labels, haptens, biomolecules, small
molecules, fatty acids, hydrocarbon chains, excipients, and
diagnostic agents; each of L.sup.1 and L.sup.2 is independently a
bond, optionally substituted alkylene, or optionally substituted
heteroalkylene; and A is a bond, optionally substituted
heterocyclyl, or optionally substituted heteroaryl.
35. The compound of claim 34, wherein A is optionally substituted
heteroaryl.
36. The compound of claim 35, wherein A is an optionally
substituted triazole moiety.
37. The compound of claim 33, wherein the compound is of Formula
(CB-2a) or Formula (CB-2b): ##STR00028## wherein: CB is an
optionally substituted cucurbituril; TAG is a tag selected from the
group consisting of polymers, therapeutic agents, targeting agents,
radionuclides, fluorophores, chromophores, phosphorescent agents,
dyes, chemiluminescent agents, particles, colorimetric labels,
magnetic labels, haptens, biomolecules, small molecules, fatty
acids, hydrocarbon chains, excipients, and diagnostic agents; each
of L.sup.1 and L.sup.2 is independently a bond, optionally
substituted alkylene, or optionally substituted heteroalkylene; and
Y is CR.sup.a or N; each instance of R.sup.a is independently
hydrogen, halogen, optionally substituted alkyl, optionally
substituted heteroalkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted acyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, --OR.sup.aa, --SR.sup.aa,
--N(R.sup.aa).sub.2, --NO.sub.2, or --CN, or two R.sup.a attached
to adjacent atoms are joined to form an optionally substituted
carbocyclyl, optionally substituted heterocyclyl, optionally
substituted aryl, optionally substituted heteroaryl ring; each
occurrence of R.sup.aa is independently hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted acyl, optionally
substituted sulfonyl, an oxygen protecting group, or a nitrogen
protecting group, or two R.sup.aa are joined to form an optionally
substituted heterocyclic or optionally substituted heteroaryl
ring.
38. The compound of claim 37, wherein the compound is of Formula
(CB-3a) or (CB-3b): ##STR00029##
39. The compound of claim 38, wherein L.sup.1 is optionally
substituted alkylene.
40. The compound of claim 38, wherein L.sup.2 is optionally
substituted heteroalkylene.
41. The compound of any one of claims 37-40, wherein the compound
is of one of the following formulae: ##STR00030##
42. The compound of any of claims 33-41, wherein the cucurbituril
is an optionally substituted cucurbit[n]uril (CB[n]), wherein n is
an integer between 5-10, inclusive.
43. The compound of claim 42, wherein the cucurbituril is
optionally substituted cucurbit[7]uril (CB[7]).
44. The compound of any one of claims 33-43, wherein the tag is a
polymer.
45. The compound of claim 44, wherein the polymer is a
polyether.
46. The compound of claim 45, wherein the polyether is polyethylene
glycol (PEG).
47. The compound of any one of claims 33-46, wherein the compound
is of Formula (PEG-T-CB[7]): ##STR00031##
48. A method of preparing the agent associated with a tag of any
one of claims 1-31, the method comprising: contacting an agent with
a host; wherein the host is conjugated to the tag; and wherein the
tag is selected from the group consisting of polymers, therapeutic
agents, targeting agents, radionuclides, fluorophores,
chromophores, phosphorescent agents, dyes, chemiluminescent agents,
particles, colorimetric labels, magnetic labels, haptens,
biomolecules, small molecules, fatty acids, hydrocarbon chains,
excipients, and diagnostic agents.
49. The method of claim 48, wherein the host is a compound of any
one of claims 31-47.
50. The method of claim 48 or 49, wherein the step of contacting is
performed in vitro.
51. The method of claim 48 or 49, wherein the step of contacting is
performed in vivo.
52. A method of preparing a compound of any one of claims 31-47,
the method comprising contacting a cucurbituril derivative
comprising a first click chemistry handle with a tag comprising a
second click chemistry handle.
53. The method of claim 52, the method comprising contacting a
cucurbituril derivative comprising an azide moiety with a tag
comprising an alkyne moiety.
54. The method of claim 53, the method comprising coupling a
compound of Formula (Az-1): ##STR00032## or salt thereof, with a
compound of Formula (Ak-1): ##STR00033## or salt thereof, to yield
a compound of Formula (CB-2a) or (CB-2b): ##STR00034## or salt
thereof, wherein: CB is an optionally substituted cucurbituril; TAG
is a tag selected from the group consisting of polymers,
therapeutic agents, targeting agents, radionuclides, fluorophores,
dyes, chemiluminescent agents, particles, colorimetric labels,
magnetic labels, haptens, biomolecules, small molecules, fatty
acids, hydrocarbon chains, excipients, and diagnostic agents; each
of L.sup.1 and L.sup.2 is independently a bond, optionally
substituted alkylene or heteroalkylene; m is an integer from 0-10,
inclusive; each instance of R.sup.a is independently hydrogen,
halogen, optionally substituted alkyl, optionally substituted
heteroalkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted acyl, optionally substituted
carbocyclyl, optionally substituted heterocyclyl, optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted acyl, --OR.sup.aa, --SR.sup.aa, --N(R.sup.aa).sub.2,
--NO.sub.2, or --CN, or two R.sup.a attached to adjacent atoms are
joined to form an optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, optionally
substituted heteroaryl ring; and each occurrence of R.sup.aa is
independently hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, optionally substituted sulfonyl, an
oxygen protecting group, or a nitrogen protecting group, or two
R.sup.aa are joined to form an optionally substituted heterocyclic
or optionally substituted heteroaryl ring.
55. The method of claim 54, comprising contacting a compound of
Formula (Az-1), or salt thereof, with a compound of one of the
following formulae: ##STR00035## or a salt thereof.
56. The method of any one of claim 54 or 55, wherein the compound
of Formula (Az-1) is of Formula (Az-2): ##STR00036##
57. The method of claim 56, wherein CB is optionally substituted
cucurbit[7]uril (CB[7]).
58. The method of any one of claims 52-57, wherein the tag
comprising a click chemistry handle is of Formula (PEG-DBCO):
##STR00037##
59. The method of any one of claims 52-58, wherein the step of
coupling is performed in the presence of copper.
60. The method of any one of claims 52-59, wherein the step of
coupling is a copper-free reaction.
61. A pharmaceutical composition comprising an agent of any one of
claims 1-32, or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable excipient.
62. A method of treating a disease in a subject in need thereof
comprising administering to the subject a therapeutically effective
amount of an agent of any one of claims 1-32, or a pharmaceutically
acceptable salt thereof, or a pharmaceutical composition of claim
61.
63. The method of claim 62, wherein the disease is a proliferative
disease, genetic disorder, a disease associated with angiogenesis,
an inflammatory disease, cardiovascular disease, hepatic disease,
spleen disease, pulmonary disease, painful condition, hematological
disease, neurological disease, psychiatric disorder, autoimmune
disease, infectious disease, metabolic disease, or endocrine
disease.
64. The method of claim 62, wherein the disease is a diabetic
condition.
65. The method of claim 64, wherein the disease is Type I
diabetes.
66. A kit comprising an agent of any one of claims 1-32, or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition of claim 61, and instructions for administering the
compound or composition to a subject.
67. A kit comprising a compound comprising a cucurbituril and a
click chemistry handle, or salt thereof; instructions for preparing
a compound of any one of claims 33-47; and optionally a second
compound comprising a tag and a click chemistry handle, or salt
thereof.
68. The agent of claim 4, wherein the agent is an antibody.
69. The agent of claim 68, wherein the agent is a monoclonal
antibody
70. The agent of claim 68, wherein the agent is an antibody
directed against human CD20.
71. The method of claim 63, wherein the disease is a proliferative
disease.
72. The method of claim 71, wherein the proliferative disease is
cancer.
73. The method of claim 72, wherein the cancer is a hematological
cancer.
74. The method of claim 72, wherein the cancer is a lymphoma,
leukemia, or multiple myeloma.
75. The method of claim 72, wherein the cancer is leukemia.
76. An agent according to any one of claims 1-32, or a
pharmaceutical composition of claim 61, for use in treating a
disease or condition in a subject in need thereof.
77. Use of an agent according to any one of claims 1-32, or a
pharmaceutical composition of claim 61, for the preparation of a
medicament.
78. Use of an agent according to any one of claims 1-32, or a
pharmaceutical composition of claim 61, for use in treating a
disease or condition in a subject in need thereof.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. provisional patent applications, U.S. Ser. No.
62/237,836, filed Oct. 6, 2015, and U.S. Ser. No. 62/334,910, filed
May 11, 2016, both of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] The practice of medicine has been transformed by the use of
biopharmaceuticals, with many drugs coming to market in recent
years in the form of peptides and proteins (e.g., insulin,
antibodies, cytokines). See, e.g., Carter P J. Nat Rev Immunol
2006, 6(5): 343-357; Aggarwal R S. Nat Biotechnol 2014, 32(1):
32-39. These new classes of drugs have an array of attendant
complications that arise from poor chemical and/or structural
stability that can lead to the active drug being converted into an
inactive and/or potentially immunogenic form. See, e.g., Shire et
al. J Pharm Sci 2004, 93(6): 1390-1402; Hermeling et al.
Pharmaceutical research 2004, 21(6): 897-903. Extensive efforts
have been devoted to the development of excipients for use in the
formulation of biomolecules (e.g., proteins, antibodies) to confer
improved stability. See, e.g., Wang et al. Int J Pharm 1999,
185(2): 129-188. For example, excipients commonly used in the
formulation of biomolecules include salts, sugars, amino acids,
non-ionic surfactants, chelators, anti-microbial preservatives,
carrier proteins, and polymers. See, e.g., Kamerzell et al. Adv
Drug Deliv Rev 2011, 63(13): 1118-1159. The mechanisms by which
these excipients act are varied and can include direct interaction
with the protein via electrostatic, cation-n, or hydrogen bonding
interactions, preferential protein hydration, enhancement of
dispersive forces, cryo-stabilization during lyophilization,
coating of exposed hydrophobic patches on protein surfaces, or
coating of air-liquid and solid-liquid interfaces to prevent
nucleation of protein unfolding and aggregation. An alternate
approach to promote stability or modify pharmacological activity of
drugs or other agents is through direct covalent modification. One
of the most common modification strategies has been covalent
attachment of poly(ethylene glycol) (PEG), which is intended to
increase protein solubility, limit exposure to proteolytic enzymes
or opsonins, increase the effective hydrodynamic size of the
protein to reduce glomerular filtration, and/or inhibit protein
aggregation. See, e.g., Veronese F M. Biomaterials 2001, 22(5):
405-417; Roberts et al. Adv Drug Deliv Rev 2002, 54(4): 459-476;
Harris et al. Nat Rev Drug Discov 2003, 2(3): 214-221; Jevsevar et
al. Biotechnology Journal 2010, 5(1): 113-128; Veronese et al. Drug
Discov Today 2005, 10(21): 1451-1458. An alternative approach has
relied on direct conjugation of zwitterionic polymers to fulfill
similar objectives. See, e.g., Keefe et al. Nature Chemistry 2012,
4(1): 59-63. PEGylated biopharmaceuticals have been used clinically
for a range of diseases, for example, with interferon treatment of
hepatitis B, hepatitis C, and multiple sclerosis. An important
issue with direct conjugation is ensuring site-specific
modification away from the active site of the therapeutic in order
to limit deleterious effects of the modification on protein
function.
[0004] Supramolecular chemistry, defined as "chemistry beyond the
molecule," utilizes specific, directional, reversible, non-covalent
molecular recognition motifs in order to achieve organization of
molecules. See, e.g., Lehn et al. Angewandte Chemie International
Edition in English 1988, 27(1): 89-112; Lehn et al. Proc Natl Acad
Sci USA 2002, 99(8): 4763-4768; Supramolecular host-guest motifs
typically comprise a discrete macrocyclic host with a cavity that
is selective for complimentary binding to certain guest ligands.
See, e.g., Schneider et al. Angew Chem Int Edit 1991, 30(11):
1417-1436. In aqueous environments, several families of cyclic
hosts, including cyclodextrins and cucurbiturils, select for
inclusion of hydrophobic guests within their cavity. See, e.g., Liu
et al. Journal of the American Chemical Society 2005, 127(45):
15959-15967; Del Valle et al. Process Biochem 2004, 39(9):
1033-1046; Biedermann et al. Journal of the American Chemical
Society 2012, 134(37): 15318-15323. The affinity of a number of
hydrophobic small molecule drugs with these hosts has been
leveraged as a strategy to solubilize a wide variety of
pharmaceutical compounds. See, e.g., Davis et al. Nat Rev Drug
Discov 2004, 3(12): 1023-1035; Uekama et al. Crit Rev Ther Drug
1987, 3(1): 1-40. Cucurbit[7]uril (CB[7]) has also been
demonstrated to bind proteins with N-terminal aromatic amino acids
(e.g., tryptophan and phenylalanine) through a combination of
R-group inclusion within the cavity and electrostatic interactions
between the protonated N-terminal amine and the carbonyl
functionality on the CB[7] portal. For example, CB [7] can bind to
insulin via its B1 phenylalanine residue. See, e.g., Bush et al.
Journal of the American Chemical Society 2005, 127(41):
14511-14517; Heitmann et al. Journal of the American Chemical
Society 2006, 128(38): 12574-12581; Chinai et al. Journal of the
American Chemical Society 2011, 133(23): 8810-8813.
[0005] New strategies for the modification and formulation of
compounds are needed, and supramolecular guest-host chemistry has
great utility in this regard. In particular, supramolecular
conjugates of therapeutic agents can help achieve improved
stability and duration of action of the therapeutic agent.
SUMMARY OF THE INVENTION
[0006] Alternative means of modifying biomolecules that do not rely
on covalent bonding are desired; in particular, site-specific
non-covalent complexation is an attractive option. Moreover, this
concept can be extended beyond biomolecules to aid in the
modification of small molecules, diagnostic agents, imaging agents,
etc. Supramolecular chemistry utilizes specific, directional,
reversible, non-covalent molecular recognition motifs in order to
achieve organization of molecules. In particular, supramolecular
chemistry can be used to bind agents, such as biomolecules (e.g.,
peptides, proteins, nucleic acids), small molecule drugs, and
imaging agents, and diagnostic agents to excipients and other tags.
The present invention provides useful supramolecular complexes
comprising an agent associated with a tag, wherein the agent is
specifically bound to a host via non-covalent guest-host
interactions, and wherein the host is conjugated to the tag. The
present invention also provides methods and compounds useful in
preparing the supramolecular complexes described herein, methods of
treating diseases using the supramolecular complexes, as well as
compositions and kits comprising the supramolecular complexes.
[0007] In one aspect, the present invention provides an agent
associated with a tag (also referred to herein as a "supramolecular
complex"), wherein the agent is specifically bound to a host via
non-covalent guest-host interactions, and wherein the host is
conjugated to the tag. In certain embodiments of the invention, the
agent that is associated with a tag is a biomolecule (e.g.,
peptide, protein, nucleic acid), small molecule drug, excipient,
diagnostic agent, imaging agent, etc. In some embodiments, the host
is a macrocyclic host, such as a cucurbituril, cyclodextrin,
calixarene, pillararene, porphyrin, metallacrown, crown ether,
cyclotriveratrylene, cryptophane, or carcerand. Examples of tags
that are associated with the agent include excipients, labels,
therapeutic agents, diagnostic agents, etc.
[0008] In another aspect, the present invention describes methods
for preparing supramolecular complexes comprising an agent
associated with a tag. In some embodiments, these methods comprise
contacting an agent with a host conjugated to a tag (also referred
to herein as a "host-tag conjugate").
[0009] In another aspect, the present invention provides a host
conjugated to a tag ("host-tag conjugate"). These compounds are
useful in the preparation of the supramolecular complexes described
herein. In some embodiments, the host is a macrocyclic host. In
some embodiments, the host conjugated to a tag is a cucurbituril;
and the tag is optionally selected from the group consisting of
polymers, targeting agents, radionuclides, fluorophores,
chromophores, phosphorescent agents, dyes, chemiluminescent agents,
particles, colorimetric labels, magnetic labels, haptens,
biomolecules, fatty acids, hydrocarbon chains, excipients, and
small molecules. In certain embodiments, the tag is another
biomolecule, such as a peptide, protein (e.g., antibody), nucleic
acid, etc.
[0010] In another aspect, the present invention describes methods
for preparing the host-tag conjugates described herein. In certain
embodiments of the invention, these compounds can be prepared by
linking a host molecule to a tag using covalent bond-forming
reactions. For example, in some embodiments, "click chemistry" is
used to conjugate the host to the tag.
[0011] In another aspect, the present invention provides
pharmaceutical compositions including a supramolecular complex
described herein, and optionally a pharmaceutically acceptable
excipient. In certain embodiments, the pharmaceutical composition
comprises an effective amount (e.g., therapeutically or
prophylactically effective amount) of the supramolecular
complex).
[0012] The present invention also provides methods for treating
diseases in a subject in need thereof by administering to a subject
in need thereof an effective amount of a supramolecular complex
described herein, or a pharmaceutical composition thereof. In
addition, the present invention provides uses of the supramolecular
complexes provided herein for the treatment of diseases described
herein. The present invention also provides uses of the
supramolecular complexes provided herein for the preparation of
medicaments (i.e., medicaments for the treatment of diseases
described herein). Further, the present invention provides
supramolecular complexes, and pharmaceutical compositions thereof,
for use in the treatment of diseases described herein.
[0013] In another aspect, the present disclosure relates to kits
comprising a supramolecular complex described herein, or
pharmaceutical composition thereof, as described herein. The kits
may be useful in a method of the disclosure. In certain
embodiments, the kit further includes instructions for using the
complex or pharmaceutical composition. In yet another aspect, the
present invention relates to kits comprising compounds that are
useful in the preparation of supramolecular complexes or
intermediates thereto as described herein.
[0014] The details of certain embodiments of the invention are set
forth in the Detailed Description of Certain Embodiments. Other
features, objects, and advantages of the invention will be apparent
from the Definitions, Examples, Figures, and Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
[0016] FIGS. 1A-1B. Supramolecular PEGylation of native insulin.
(FIG. 1A) A copper free "click" reaction between a cucurbit[7]uril
(CB[7]) supramolecular host molecule bearing a single azide moiety
and a dibenzocyclooctyne-functionalized poly(ethylene glycol)
polymer (PEG-DBCO; M.sub.n=5 kDa, 10 kDa, or 30 kDa) yields
PEG-T-CB[7] conjugate upon triazole formation (n.b. only one of two
possible regioisomers for triazole formation is shown). (FIG. 1B)
Schematic representation of supramolecular PEGylation of native
insulin with PEG-T-CB[7] via strong non-covalent binding of the
CB[7] moiety to the B1 phenylalanine residue.
[0017] FIGS. 2A-2D. Characterization of PEG-T-CB[7] conjugates.
(FIG. 2A) UV-visible absorbance of PEG.sub.30k-DBCO and
PEG.sub.30k-T-CB[7] indicates a change in the .lamda..sub.max of
the DBCO moiety upon conjugation of the CB[7]-N.sub.3 and
corresponding formation of a triazole. (FIG. 2B) Overlay of
refractive index and absorbance (triazole .lamda..sub.max=245 nm)
traces from gel permeation chromatography demonstrates co-elution
of the triazole and the polymeric species. (FIG. 2C) MALDI-MS
characterization indicates an increase in mass of the PEG species
corresponding to conjugation of the CB[7] moiety. (FIG. 2D)
Acridine orange (AO) competitive binding assay with native insulin
indicates no significant alteration of the CB[7]-Insulin binding
occurs following PEG conjugation (CB[7], K.sub.D=2.3.+-.0.2 .mu.M;
PEG.sub.5k-T-CB[7], K.sub.D=1.4.+-.0.1 .mu.M; PEG.sub.10k-T-CB[7],
K.sub.D=1.3.+-.0.1 .mu.M; PEG.sub.30k-T-CB[7], K.sub.D=2.1.+-.0.3
.mu.M), using AO (8 .mu.M), CB[7] or PEG-T-CB[7] (6 .mu.M), and
insulin (varied from 0-15 .mu.M), with binding studies performed in
water. The intensity at 510 nm was used in fitting the decay of
signal.
[0018] FIGS. 3A-3B. Stabilization of native insulin with
PEG-T-CB[7] conjugates. (FIG. 3A) Kinetic studies of the
aggregation of various insulin formulations at pH 7.4, 37.degree.
C., in physiological buffer with continuous agitation over the
course of 100 hours. (FIG. 3B) Quantification of the aggregation
time of various insulin formulations (where CB[7].sub.5k denotes
addition of PEG.sub.5k-T-CB[7]). Rapid aggregation of native
insulin with and without CB[7] is observed, while supramolecular
PEGylation of insulin with PEG-T-CB[7] promotes dramatically
increased stability under these conditions. At the 50-day endpoint
of the study, none of the PEG-T-CB[7] formulations had
aggregated.
[0019] FIG. 4A. In vivo performance of CB[7] conjugates. In vivo
assessment of blood glucose levels in STZ diabetic mice following
administration of native insulin (1 IU/kg) injected alone or
formulated with CB[7] or the PEG-T-CB[7] conjugates. Insulin was
injected at t=0, and blood glucose was monitored for 6 hours
following insulin administration (n=5 mice/group). The dotted gray
line shows the standard criteria for normoglycemic in this strain
of mice (<200 mg/dL). FIG. 4B. Insulin aged under physiologic
conditions with continuous agitation for 100 days in formulation
with CB[7]-PEG conjugates retains its activity when assaying for
phosphorylation of Ser.sup.473 on AKT (formulation potency: Aged
Insulin, LogEC.sub.50=-4.8.+-.0.06; CB[7],
LogEC.sub.50=-4.9.+-.0.07; CB[7]-PEG.sub.5k,
LogEC.sub.50=-6.8.+-.0.04; CB[7]-PEG.sub.10k,
LogEC.sub.50=-6.7.+-.0.04; CB[7]-PEG.sub.30k,
LogEC.sub.50=-6.7.+-.0.04 Fresh Insulin,
LogEC.sub.50=-6.8.+-.0.04).
[0020] FIG. 5. Matrix assisted laser desorption ionization mass
spectroscopy (MALDI-MS) of CB[7]-PEG conjugates following
click-addition of CB[7]-N.sub.3 to PEG-DBCO.
[0021] FIG. 6. .sup.1H-NMR (400 MHz) of PEG.sub.5k-T-CB[7] in
D.sub.2O.
[0022] FIG. 7. Near-ultraviolet circular dichroism spectra of
insulin or insulin formulated with CB[7]-PEG conjugates. Spectra
demonstrate no changes in insulin circular dichroism signal, with a
characteristic .alpha.-helical signature.
[0023] FIG. 8. Kinetic studies of the aggregation of various
formulations of native insulin (1 mg/ml) at pH 7.4 and 37.degree.
C. in PBS with continuous agitation for insulin alone, or
formulated with PEG (1 equivalent; M.sub.n=10 kDa or 30 kDa), with
and without unmodified CB[7] (1 equivalent).
[0024] FIGS. 9A-9C. Validation of stability with other
biopharmaceuticals. (FIG. 9) Anti-CD20 antibody, aged in
formulation with CB[7] or CB[7]-PEG was evaluated for reactivity
against CD20.sup.+ Raji cells, analyzed by flow cytometry after
incubation with cells and subsequent labeling with a fluorescent
anti-IgG secondary antibody. Median fluorescent intensity (MFI) is
shown for each group. Antibody reactivity of Raji cells is
preserved only in the case where antibody is aged in the presence
of CB[7]-PEG. (FIG. 9B) Glucagon, when formulated with
CB[7]-PEG.sub.10k, remains soluble for at least 24 hours, whereas
it readily precipitates from solution when dissolved alone. (FIG.
9C) Near ultraviolet circular dichroism demonstrates glucagon aged
for 24 hours in the presence of CB[7]-PEG.sub.10k remains in an
active .alpha.-helix conformation, and there is no appreciable
glucagon in solution when aged.
Definitions
Chemical Definitions
[0025] Definitions of specific functional groups and chemical terms
are described in more detail below. The chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed.,
inside cover, and specific functional groups are generally defined
as described therein. Additionally, general principles of organic
chemistry, as well as specific functional moieties and reactivity,
are described in Organic Chemistry, Thomas Sorrell, University
Science Books, Sausalito, 1999; Smith and March, March's Advanced
Organic Chemistry, 5.sup.th Edition, John Wiley & Sons, Inc.,
New York, 2001; Larock, Comprehensive organic Transformations, VCH
Publishers, Inc., New York, 1989; and Carruthers, Some Modern
Methods of Organic Synthesis, 3.sup.rd Edition, Cambridge
University Press, Cambridge, 1987.
[0026] Compounds described herein can comprise one or more
asymmetric centers, and thus can exist in various stereoisomeric
forms, e.g., enantiomers and/or diastereomers. For example, the
compounds described herein can be in the form of an individual
enantiomer, diastereomer or geometric isomer, or can be in the form
of a mixture of stereoisomers, including racemic mixtures and
mixtures enriched in one or more stereoisomer. Isomers can be
isolated from mixtures by methods known to those skilled in the
art, including chiral high pressure liquid chromatography (HPLC)
and the formation and crystallization of chiral salts; or preferred
isomers can be prepared by asymmetric syntheses. See, for example,
Jacques et al., Enantiomers, Racemates and Resolutions (Wiley
Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725
(1977); Eliel, E. L. Stereochemistry of Carbon Compounds
(McGraw-Hill, N Y, 1962); and Wilen, S. H., Tables of Resolving
Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of
Notre Dame Press, Notre Dame, Ind. 1972). The invention
additionally encompasses compounds as individual isomers
substantially free of other isomers, and alternatively, as mixtures
of various isomers.
[0027] Unless otherwise stated, structures depicted herein are also
meant to include compounds that differ only in the presence of one
or more isotopically enriched atoms. For example, compounds having
the present structures except for the replacement of hydrogen by
deuterium or tritium, replacement of .sup.19F with .sup.18F, or the
replacement of .sup.12C with .sup.13C or .sup.14C are within the
scope of the disclosure. Such compounds are useful, for example, as
analytical tools or probes in biological assays.
[0028] When a range of values is listed, it is intended to
encompass each value and sub-range within the range. For example
"C.sub.1-6 alkyl" is intended to encompass, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.1-6, C.sub.1-5,
C.sub.1-4, C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-5, C.sub.2-4,
C.sub.2-3, C.sub.3-6, C.sub.3-5, C.sub.3-4, C.sub.4-6, C.sub.4-5,
and C.sub.5-6 alkyl.
[0029] The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and
carbocyclic groups. Likewise, the term "heteroaliphatic" refers to
heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic
groups.
[0030] The term "alkyl" refers to a radical of a straight-chain or
branched saturated hydrocarbon group having from 1 to 10 carbon
atoms ("C.sub.1-10 alkyl"). In some embodiments, an alkyl group has
1 to 9 carbon atoms ("C.sub.1-9 alkyl"). In some embodiments, an
alkyl group has 1 to 8 carbon atoms ("C.sub.1-8 alkyl"). In some
embodiments, an alkyl group has 1 to 7 carbon atoms ("C.sub.1-7
alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon
atoms ("C.sub.1-6 alkyl"). In some embodiments, an alkyl group has
1 to 5 carbon atoms ("C.sub.1-5 alkyl"). In some embodiments, an
alkyl group has 1 to 4 carbon atoms ("C.sub.1-4 alkyl"). In some
embodiments, an alkyl group has 1 to 3 carbon atoms ("C.sub.1-3
alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon
atoms ("C.sub.1-2 alkyl"). In some embodiments, an alkyl group has
1 carbon atom ("C.sub.1 alkyl"). In some embodiments, an alkyl
group has 2 to 6 carbon atoms ("C.sub.2-6 alkyl"). Examples of
C.sub.1-6 alkyl groups include methyl (C.sub.1), ethyl (C.sub.2),
propyl (C.sub.3) (e.g., n-propyl, isopropyl), butyl (C.sub.4)
(e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C.sub.5)
(e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl,
tertiary amyl), and hexyl (C.sub.6) (e.g., n-hexyl). Additional
examples of alkyl groups include n-heptyl (C.sub.7), n-octyl
(C.sub.8), and the like. Unless otherwise specified, each instance
of an alkyl group is independently unsubstituted (an "unsubstituted
alkyl") or substituted (a "substituted alkyl") with one or more
substituents (e.g., halogen, such as F). In certain embodiments,
the alkyl group is an unsubstituted C.sub.1-10 alkyl (such as
unsubstituted C.sub.1-6 alkyl, e.g., --CH.sub.3 (Me), unsubstituted
ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl
(n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu,
e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl
(tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted
isobutyl (i-Bu)). In certain embodiments, the alkyl group is a
substituted C.sub.1-10 alkyl (such as substituted C.sub.1-6 alkyl,
e.g., --CF.sub.3, Bn).
[0031] The term "heteroalkyl" refers to an alkyl group, which
further includes at least one heteroatom (e.g., 1, 2, 3, or 4
heteroatoms) selected from oxygen, nitrogen, or sulfur within
(i.e., inserted between adjacent carbon atoms of) and/or placed at
one or more terminal position(s) of the parent chain. In certain
embodiments, a heteroalkyl group refers to a saturated group having
from 1 to 10 carbon atoms and 1 or more heteroatoms within the
parent chain ("heteroC.sub.1-10 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 9 carbon atoms
and 1 or more heteroatoms within the parent chain ("heteroC.sub.1-9
alkyl"). In some embodiments, a heteroalkyl group is a saturated
group having 1 to 8 carbon atoms and 1 or more heteroatoms within
the parent chain ("heteroC.sub.1-8 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 7 carbon atoms
and 1 or more heteroatoms within the parent chain ("heteroC.sub.1-7
alkyl"). In some embodiments, a heteroalkyl group is a saturated
group having 1 to 6 carbon atoms and 1 or more heteroatoms within
the parent chain ("heteroC.sub.1-6 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 5 carbon atoms
and 1 or 2 heteroatoms within the parent chain ("heteroC.sub.1-5
alkyl"). In some embodiments, a heteroalkyl group is a saturated
group having 1 to 4 carbon atoms and for 2 heteroatoms within the
parent chain ("heteroC.sub.1-4 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 3 carbon atoms
and 1 heteroatom within the parent chain ("heteroC.sub.1-3 alkyl").
In some embodiments, a heteroalkyl group is a saturated group
having 1 to 2 carbon atoms and 1 heteroatom within the parent chain
("heteroC.sub.1-2 alkyl"). In some embodiments, a heteroalkyl group
is a saturated group having 1 carbon atom and 1 heteroatom
("heteroC.sub.1 alkyl"). In some embodiments, a heteroalkyl group
is a saturated group having 2 to 6 carbon atoms and 1 or 2
heteroatoms within the parent chain ("heteroC.sub.2-6 alkyl").
Unless otherwise specified, each instance of a heteroalkyl group is
independently unsubstituted (an "unsubstituted heteroalkyl") or
substituted (a "substituted heteroalkyl") with one or more
substituents. In certain embodiments, the heteroalkyl group is an
unsubstituted heteroC.sub.1-10 alkyl. In certain embodiments, the
heteroalkyl group is a substituted heteroC.sub.1-10 alkyl.
[0032] A "hydrocarbon chain" refers to a substituted or
unsubstituted divalent alkyl, alkenyl, or alkynyl group. A
hydrocarbon chain includes (1) one or more chains of carbon atoms
immediately between the two radicals of the hydrocarbon chain; (2)
optionally one or more hydrogen atoms on the chain(s) of carbon
atoms; and (3) optionally one or more substituents ("non-chain
substituents," which are not hydrogen) on the chain(s) of carbon
atoms. A chain of carbon atoms consists of consecutively connected
carbon atoms ("chain atoms") and does not include hydrogen atoms or
heteroatoms. However, a non-chain substituent of a hydrocarbon
chain may include any atoms, including hydrogen atoms, carbon
atoms, and heteroatoms. For example, hydrocarbon chain
--C.sup.AH(C.sup.BH.sub.2C.sup.CH.sub.3-- includes one chain atom
C.sup.A, one hydrogen atom on C.sup.A, and non-chain substituent
--(C.sup.BH.sub.2C.sup.CH.sub.3). The term "C.sub.x hydrocarbon
chain," wherein x is a positive integer, refers to a hydrocarbon
chain that includes x number of chain atom(s) between the two
radicals of the hydrocarbon chain. If there is more than one
possible value of x, the smallest possible value of x is used for
the definition of the hydrocarbon chain. For example,
--CH(C.sub.2H.sub.5)-- is a C.sub.1 hydrocarbon chain, and
##STR00001##
is a C.sub.3 hydrocarbon chain. When a range of values is used, the
meaning of the range is as described herein. For example, a
C.sub.3-10 hydrocarbon chain refers to a hydrocarbon chain where
the number of chain atoms of the shortest chain of carbon atoms
immediately between the two radicals of the hydrocarbon chain is 3,
4, 5, 6, 7, 8, 9, or 10. A hydrocarbon chain may be saturated
(e.g., --(CH.sub.2).sub.4--). A hydrocarbon chain may also be
unsaturated and include one or more C.dbd.C and/or C.ident.C bonds
anywhere in the hydrocarbon chain. For instance,
--CH.dbd.CH--(CH.sub.2).sub.2--, --CH.sub.2--C.ident.C--CH.sub.2--,
and --C.ident.C--CH.dbd.CH-- are all examples of a unsubstituted
and unsaturated hydrocarbon chain. In certain embodiments, the
hydrocarbon chain is unsubstituted (e.g., --C.ident.C-- or
--(CH.sub.2).sub.4--). In certain embodiments, the hydrocarbon
chain is substituted (e.g., --CH(C.sub.2H.sub.5)-- and
--CF.sub.2--). Any two substituents on the hydrocarbon chain may be
joined to form an optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, or
optionally substituted heteroaryl ring. For instance,
##STR00002##
are all examples of a hydrocarbon chain. In contrast, in certain
embodiments,
##STR00003##
are not within the scope of the hydrocarbon chains described
herein. When a chain atom of a C.sub.x hydrocarbon chain is
replaced with a heteroatom, the resulting group is referred to as a
C.sub.x hydrocarbon chain wherein a chain atom is replaced with a
heteroatom, as opposed to a C.sub.x-1 hydrocarbon chain. For
example,
##STR00004##
is a C.sub.3 hydrocarbon chain wherein one chain atom is replaced
with an oxygen atom.
[0033] The term "alkenyl" refers to a radical of a straight-chain
or branched hydrocarbon group having from 2 to 10 carbon atoms and
one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double
bonds). In some embodiments, an alkenyl group has 2 to 9 carbon
atoms ("C.sub.2-9 alkenyl"). In some embodiments, an alkenyl group
has 2 to 8 carbon atoms ("C.sub.2-8 alkenyl"). In some embodiments,
an alkenyl group has 2 to 7 carbon atoms ("C.sub.2-7 alkenyl"). In
some embodiments, an alkenyl group has 2 to 6 carbon atoms
("C.sub.2-6 alkenyl"). In some embodiments, an alkenyl group has 2
to 5 carbon atoms ("C.sub.2-5 alkenyl"). In some embodiments, an
alkenyl group has 2 to 4 carbon atoms ("C.sub.2-4 alkenyl"). In
some embodiments, an alkenyl group has 2 to 3 carbon atoms
("C.sub.2-3 alkenyl"). In some embodiments, an alkenyl group has 2
carbon atoms ("C.sub.2 alkenyl"). The one or more carbon-carbon
double bonds can be internal (such as in 2-butenyl) or terminal
(such as in 1-butenyl). Examples of C.sub.2-4 alkenyl groups
include ethenyl (C.sub.2), 1-propenyl (C.sub.3), 2-propenyl
(C.sub.3), 1-butenyl (C.sub.4), 2-butenyl (C.sub.4), butadienyl
(C.sub.4), and the like. Examples of C.sub.2-6 alkenyl groups
include the aforementioned C.sub.2-4 alkenyl groups as well as
pentenyl (C.sub.5), pentadienyl (C.sub.5), hexenyl (C.sub.6), and
the like. Additional examples of alkenyl include heptenyl
(C.sub.7), octenyl (C.sub.8), octatrienyl (C.sub.8), and the like.
Unless otherwise specified, each instance of an alkenyl group is
independently unsubstituted (an "unsubstituted alkenyl") or
substituted (a "substituted alkenyl") with one or more
substituents. In certain embodiments, the alkenyl group is an
unsubstituted C.sub.2-10 alkenyl. In certain embodiments, the
alkenyl group is a substituted C.sub.2-10 alkenyl. In an alkenyl
group, a C.dbd.C double bond for which the stereochemistry is not
specified
##STR00005##
may be an (E)- or (Z)-double bond.
[0034] The term "heteroalkenyl" refers to an alkenyl group, which
further includes at least one heteroatom (e.g., 1, 2, 3, or 4
heteroatoms) selected from oxygen, nitrogen, or sulfur within
(i.e., inserted between adjacent carbon atoms of) and/or placed at
one or more terminal position(s) of the parent chain. In certain
embodiments, a heteroalkenyl group refers to a group having from 2
to 10 carbon atoms, at least one double bond, and 1 or more
heteroatoms within the parent chain ("heteroC.sub.2-10 alkenyl").
In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms
at least one double bond, and 1 or more heteroatoms within the
parent chain ("heteroC.sub.2-9 alkenyl"). In some embodiments, a
heteroalkenyl group has 2 to 8 carbon atoms, at least one double
bond, and 1 or more heteroatoms within the parent chain
("heteroC.sub.2-8 alkenyl"). In some embodiments, a heteroalkenyl
group has 2 to 7 carbon atoms, at least one double bond, and 1 or
more heteroatoms within the parent chain ("heteroC.sub.2-7
alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 6
carbon atoms, at least one double bond, and 1 or more heteroatoms
within the parent chain ("heteroC.sub.2-6 alkenyl"). In some
embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at
least one double bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-5 alkenyl"). In some embodiments, a
heteroalkenyl group has 2 to 4 carbon atoms, at least one double
bond, and 1 or 2 heteroatoms within the parent chain
("heteroC.sub.2-4 alkenyl"). In some embodiments, a heteroalkenyl
group has 2 to 3 carbon atoms, at least one double bond, and 1
heteroatom within the parent chain ("heteroC.sub.2-3 alkenyl"). In
some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at
least one double bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-6 alkenyl"). Unless otherwise specified, each
instance of a heteroalkenyl group is independently unsubstituted
(an "unsubstituted heteroalkenyl") or substituted (a "substituted
heteroalkenyl") with one or more substituents. In certain
embodiments, the heteroalkenyl group is an unsubstituted
heteroC.sub.2-10 alkenyl. In certain embodiments, the heteroalkenyl
group is a substituted heteroC.sub.2-10 alkenyl.
[0035] The term "alkynyl" refers to a radical of a straight-chain
or branched hydrocarbon group having from 2 to 10 carbon atoms and
one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple
bonds) ("C.sub.2-10 alkynyl"). In some embodiments, an alkynyl
group has 2 to 9 carbon atoms ("C.sub.2-9 alkynyl"). In some
embodiments, an alkynyl group has 2 to 8 carbon atoms ("C.sub.2-8
alkynyl"). In some embodiments, an alkynyl group has 2 to 7 carbon
atoms ("C.sub.2-7 alkynyl"). In some embodiments, an alkynyl group
has 2 to 6 carbon atoms ("C.sub.2-6 alkynyl"). In some embodiments,
an alkynyl group has 2 to 5 carbon atoms ("C.sub.2-5 alkynyl"). In
some embodiments, an alkynyl group has 2 to 4 carbon atoms
("C.sub.2-4 alkynyl"). In some embodiments, an alkynyl group has 2
to 3 carbon atoms ("C.sub.2-3 alkynyl"). In some embodiments, an
alkynyl group has 2 carbon atoms ("C.sub.2 alkynyl"). The one or
more carbon-carbon triple bonds can be internal (such as in
2-butynyl) or terminal (such as in 1-butynyl). Examples of
C.sub.2-4 alkynyl groups include, without limitation, ethynyl
(C.sub.2), 1-propynyl (C.sub.3), 2-propynyl (C.sub.3), 1-butynyl
(C.sub.4), 2-butynyl (C.sub.4), and the like. Examples of C.sub.2-6
alkenyl groups include the aforementioned C.sub.2-4 alkynyl groups
as well as pentynyl (C.sub.5), hexynyl (C.sub.6), and the like.
Additional examples of alkynyl include heptynyl (C.sub.7), octynyl
(C.sub.8), and the like. Unless otherwise specified, each instance
of an alkynyl group is independently unsubstituted (an
"unsubstituted alkynyl") or substituted (a "substituted alkynyl")
with one or more substituents. In certain embodiments, the alkynyl
group is an unsubstituted C.sub.2-10 alkynyl. In certain
embodiments, the alkynyl group is a substituted C.sub.2-10
alkynyl.
[0036] The term "heteroalkynyl" refers to an alkynyl group, which
further includes at least one heteroatom (e.g., 1, 2, 3, or 4
heteroatoms) selected from oxygen, nitrogen, or sulfur within
(i.e., inserted between adjacent carbon atoms of) and/or placed at
one or more terminal position(s) of the parent chain. In certain
embodiments, a heteroalkynyl group refers to a group having from 2
to 10 carbon atoms, at least one triple bond, and 1 or more
heteroatoms within the parent chain ("heteroC.sub.2-10 alkynyl").
In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms,
at least one triple bond, and 1 or more heteroatoms within the
parent chain ("heteroC.sub.2-9 alkynyl"). In some embodiments, a
heteroalkynyl group has 2 to 8 carbon atoms, at least one triple
bond, and 1 or more heteroatoms within the parent chain
("heteroC.sub.2-8 alkynyl"). In some embodiments, a heteroalkynyl
group has 2 to 7 carbon atoms, at least one triple bond, and 1 or
more heteroatoms within the parent chain ("heteroC.sub.2-7
alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 6
carbon atoms, at least one triple bond, and 1 or more heteroatoms
within the parent chain ("heteroC.sub.2-6 alkynyl"). In some
embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at
least one triple bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-5 alkynyl"). In some embodiments, a
heteroalkynyl group has 2 to 4 carbon atoms, at least one triple
bond, and 1 or 2 heteroatoms within the parent chain
("heteroC.sub.2-4 alkynyl"). In some embodiments, a heteroalkynyl
group has 2 to 3 carbon atoms, at least one triple bond, and 1
heteroatom within the parent chain ("heteroC.sub.2-3 alkynyl"). In
some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at
least one triple bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-6 alkynyl"). Unless otherwise specified, each
instance of a heteroalkynyl group is independently unsubstituted
(an "unsubstituted heteroalkynyl") or substituted (a "substituted
heteroalkynyl") with one or more substituents. In certain
embodiments, the heteroalkynyl group is an unsubstituted
heteroC.sub.2-10 alkynyl. In certain embodiments, the heteroalkynyl
group is a substituted heteroC.sub.2-10 alkynyl.
[0037] The term "carbocyclyl" or "carbocyclic" refers to a radical
of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring
carbon atoms ("C.sub.3-14 carbocyclyl") and zero heteroatoms in the
non-aromatic ring system. In some embodiments, a carbocyclyl group
has 3 to 10 ring carbon atoms ("C.sub.3-10 carbocyclyl"). In some
embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms
("C.sub.3-8 carbocyclyl"). In some embodiments, a carbocyclyl group
has 3 to 7 ring carbon atoms ("C.sub.3-7 carbocyclyl"). In some
embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms
("C.sub.3-6 carbocyclyl"). In some embodiments, a carbocyclyl group
has 4 to 6 ring carbon atoms ("C.sub.4-6 carbocyclyl"). In some
embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms
("C.sub.5-6 carbocyclyl"). In some embodiments, a carbocyclyl group
has 5 to 10 ring carbon atoms ("C.sub.5-10 carbocyclyl"). Exemplary
C.sub.3-6 carbocyclyl groups include, without limitation,
cyclopropyl (C.sub.3), cyclopropenyl (C.sub.3), cyclobutyl
(C.sub.4), cyclobutenyl (C.sub.4), cyclopentyl (C.sub.5),
cyclopentenyl (C.sub.5), cyclohexyl (C.sub.6), cyclohexenyl
(C.sub.6), cyclohexadienyl (C.sub.6), and the like. Exemplary
C.sub.3-8 carbocyclyl groups include, without limitation, the
aforementioned C.sub.3-6 carbocyclyl groups as well as cycloheptyl
(C.sub.7), cycloheptenyl (C.sub.7), cycloheptadienyl (C.sub.7),
cycloheptatrienyl (C.sub.7), cyclooctyl (C.sub.8), cyclooctenyl
(C.sub.8), bicyclo[2.2.1]heptanyl (C.sub.7), bicyclo[2.2.2]octanyl
(C.sub.8), and the like. Exemplary C.sub.3-10 carbocyclyl groups
include, without limitation, the aforementioned C.sub.3-8
carbocyclyl groups as well as cyclononyl (C.sub.9), cyclononenyl
(C.sub.9), cyclodecyl (C.sub.10), cyclodecenyl (C.sub.10),
octahydro-1H-indenyl (C.sub.9), decahydronaphthalenyl (C.sub.10),
spiro[4.5]decanyl (C.sub.10), and the like. As the foregoing
examples illustrate, in certain embodiments, the carbocyclyl group
is either monocyclic ("monocyclic carbocyclyl") or polycyclic
(e.g., containing a fused, bridged or spiro ring system such as a
bicyclic system ("bicyclic carbocyclyl") or tricyclic system
("tricyclic carbocyclyl")) and can be saturated or can contain one
or more carbon-carbon double or triple bonds. "Carbocyclyl" also
includes ring systems wherein the carbocyclyl ring, as defined
above, is fused with one or more aryl or heteroaryl groups wherein
the point of attachment is on the carbocyclyl ring, and in such
instances, the number of carbons continue to designate the number
of carbons in the carbocyclic ring system. Unless otherwise
specified, each instance of a carbocyclyl group is independently
unsubstituted (an "unsubstituted carbocyclyl") or substituted (a
"substituted carbocyclyl") with one or more substituents. In
certain embodiments, the carbocyclyl group is an unsubstituted
C.sub.3-14 carbocyclyl. In certain embodiments, the carbocyclyl
group is a substituted C.sub.3-14 carbocyclyl.
[0038] In some embodiments, "carbocyclyl" is a monocyclic,
saturated carbocyclyl group having from 3 to 14 ring carbon atoms
("C.sub.3-14 cycloalkyl"). In some embodiments, a cycloalkyl group
has 3 to 10 ring carbon atoms ("C.sub.3-10 cycloalkyl"). In some
embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms
("C.sub.3-8 cycloalkyl"). In some embodiments, a cycloalkyl group
has 3 to 6 ring carbon atoms ("C.sub.3-6 cycloalkyl"). In some
embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms
("C.sub.4-6 cycloalkyl"). In some embodiments, a cycloalkyl group
has 5 to 6 ring carbon atoms ("C.sub.5-6 cycloalkyl"). In some
embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms
("C.sub.5-10 cycloalkyl"). Examples of C.sub.5-6 cycloalkyl groups
include cyclopentyl (C.sub.5) and cyclohexyl (C.sub.5). Examples of
C.sub.3-6 cycloalkyl groups include the aforementioned C.sub.5-6
cycloalkyl groups as well as cyclopropyl (C.sub.3) and cyclobutyl
(C.sub.4). Examples of C.sub.3-8 cycloalkyl groups include the
aforementioned C.sub.3-6 cycloalkyl groups as well as cycloheptyl
(C.sub.7) and cyclooctyl (C.sub.8). Unless otherwise specified,
each instance of a cycloalkyl group is independently unsubstituted
(an "unsubstituted cycloalkyl") or substituted (a "substituted
cycloalkyl") with one or more substituents. In certain embodiments,
the cycloalkyl group is an unsubstituted C.sub.3-14 cycloalkyl. In
certain embodiments, the cycloalkyl group is a substituted
C.sub.3-14 cycloalkyl.
[0039] The term "heterocyclyl" or "heterocyclic" refers to a
radical of a 3- to 14-membered non-aromatic ring system having ring
carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom
is independently selected from nitrogen, oxygen, and sulfur ("3-14
membered heterocyclyl"). In heterocyclyl groups that contain one or
more nitrogen atoms, the point of attachment can be a carbon or
nitrogen atom, as valency permits. A heterocyclyl group can either
be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a
fused, bridged or spiro ring system such as a bicyclic system
("bicyclic heterocyclyl") or tricyclic system ("tricyclic
heterocyclyl")), and can be saturated or can contain one or more
carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring
systems can include one or more heteroatoms in one or both rings.
"Heterocyclyl" also includes ring systems wherein the heterocyclyl
ring, as defined above, is fused with one or more carbocyclyl
groups wherein the point of attachment is either on the carbocyclyl
or heterocyclyl ring, or ring systems wherein the heterocyclyl
ring, as defined above, is fused with one or more aryl or
heteroaryl groups, wherein the point of attachment is on the
heterocyclyl ring, and in such instances, the number of ring
members continue to designate the number of ring members in the
heterocyclyl ring system. Unless otherwise specified, each instance
of heterocyclyl is independently unsubstituted (an "unsubstituted
heterocyclyl") or substituted (a "substituted heterocyclyl") with
one or more substituents. In certain embodiments, the heterocyclyl
group is an unsubstituted 3-14 membered heterocyclyl. In certain
embodiments, the heterocyclyl group is a substituted 3-14 membered
heterocyclyl.
[0040] In some embodiments, a heterocyclyl group is a 5-10 membered
non-aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-10 membered heterocyclyl"). In
some embodiments, a heterocyclyl group is a 5-8 membered
non-aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-8 membered heterocyclyl"). In some
embodiments, a heterocyclyl group is a 5-6 membered non-aromatic
ring system having ring carbon atoms and 1-4 ring heteroatoms,
wherein each heteroatom is independently selected from nitrogen,
oxygen, and sulfur ("5-6 membered heterocyclyl"). In some
embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms
selected from nitrogen, oxygen, and sulfur. In some embodiments,
the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected
from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6
membered heterocyclyl has 1 ring heteroatom selected from nitrogen,
oxygen, and sulfur.
[0041] Exemplary 3-membered heterocyclyl groups containing 1
heteroatom include, without limitation, azirdinyl, oxiranyl, and
thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1
heteroatom include, without limitation, azetidinyl, oxetanyl, and
thietanyl. Exemplary 5-membered heterocyclyl groups containing 1
heteroatom include, without limitation, tetrahydrofuranyl,
dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl,
pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary
5-membered heterocyclyl groups containing 2 heteroatoms include,
without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms
include, without limitation, triazolinyl, oxadiazolinyl, and
thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing
1 heteroatom include, without limitation, piperidinyl,
tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary
6-membered heterocyclyl groups containing 2 heteroatoms include,
without limitation, piperazinyl, morpholinyl, dithianyl, and
dioxanyl. Exemplary 6-membered heterocyclyl groups containing 2
heteroatoms include, without limitation, triazinanyl. Exemplary
7-membered heterocyclyl groups containing 1 heteroatom include,
without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary
8-membered heterocyclyl groups containing 1 heteroatom include,
without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary
bicyclic heterocyclyl groups include, without limitation,
indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,
tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,
decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl,
decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl,
octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl,
naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl,
1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,
5,6-dihydro-4H-furo[3,2-b]pyrrolyl,
6,7-dihydro-5H-furo[3,2-b]pyranyl,
5,7-dihydro-4H-thieno[2,3-c]pyranyl,
2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl,
2,3-dihydrofuro[2,3-b]pyridinyl,
4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,
4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,
4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,
1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.
[0042] The term "aryl" refers to a radical of a monocyclic or
polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system
(e.g., having 6, 10, or 14 .pi. electrons shared in a cyclic array)
having 6-14 ring carbon atoms and zero heteroatoms provided in the
aromatic ring system ("C.sub.6-14 aryl"). In some embodiments, an
aryl group has 6 ring carbon atoms ("C.sub.6 aryl"; e.g., phenyl).
In some embodiments, an aryl group has 10 ring carbon atoms
("C.sub.10 aryl"; e.g., naphthyl such as 1-naphthyl and
2-naphthyl). In some embodiments, an aryl group has 14 ring carbon
atoms ("C.sub.14 aryl"; e.g., anthracyl). "Aryl" also includes ring
systems wherein the aryl ring, as defined above, is fused with one
or more carbocyclyl or heterocyclyl groups wherein the radical or
point of attachment is on the aryl ring, and in such instances, the
number of carbon atoms continue to designate the number of carbon
atoms in the aryl ring system. Unless otherwise specified, each
instance of an aryl group is independently unsubstituted (an
"unsubstituted aryl") or substituted (a "substituted aryl") with
one or more substituents. In certain embodiments, the aryl group is
an unsubstituted C.sub.6-14 aryl. In certain embodiments, the aryl
group is a substituted C.sub.6-14 aryl.
[0043] "Aralkyl" is a subset of "alkyl" and refers to an alkyl
group substituted by an aryl group, wherein the point of attachment
is on the alkyl moiety.
[0044] The term "heteroaryl" refers to a radical of a 5-14 membered
monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic
ring system (e.g., having 6, 10, or 14 .pi. electrons shared in a
cyclic array) having ring carbon atoms and 1-4 ring heteroatoms
provided in the aromatic ring system, wherein each heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("5-14
membered heteroaryl"). In heteroaryl groups that contain one or
more nitrogen atoms, the point of attachment can be a carbon or
nitrogen atom, as valency permits. Heteroaryl polycyclic ring
systems can include one or more heteroatoms in one or both rings.
"Heteroaryl" includes ring systems wherein the heteroaryl ring, as
defined above, is fused with one or more carbocyclyl or
heterocyclyl groups wherein the point of attachment is on the
heteroaryl ring, and in such instances, the number of ring members
continue to designate the number of ring members in the heteroaryl
ring system. "Heteroaryl" also includes ring systems wherein the
heteroaryl ring, as defined above, is fused with one or more aryl
groups wherein the point of attachment is either on the aryl or
heteroaryl ring, and in such instances, the number of ring members
designates the number of ring members in the fused polycyclic
(aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein
one ring does not contain a heteroatom (e.g., indolyl, quinolinyl,
carbazolyl, and the like) the point of attachment can be on either
ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl)
or the ring that does not contain a heteroatom (e.g.,
5-indolyl).
[0045] In some embodiments, a heteroaryl group is a 5-10 membered
aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms provided in the aromatic ring system, wherein each
heteroatom is independently selected from nitrogen, oxygen, and
sulfur ("5-10 membered heteroaryl"). In some embodiments, a
heteroaryl group is a 5-8 membered aromatic ring system having ring
carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring
system, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some
embodiments, a heteroaryl group is a 5-6 membered aromatic ring
system having ring carbon atoms and 1-4 ring heteroatoms provided
in the aromatic ring system, wherein each heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("5-6
membered heteroaryl"). In some embodiments, the 5-6 membered
heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen,
and sulfur. In some embodiments, the 5-6 membered heteroaryl has
1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In
some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom
selected from nitrogen, oxygen, and sulfur. Unless otherwise
specified, each instance of a heteroaryl group is independently
unsubstituted (an "unsubstituted heteroaryl") or substituted (a
"substituted heteroaryl") with one or more substituents. In certain
embodiments, the heteroaryl group is an unsubstituted 5-14 membered
heteroaryl. In certain embodiments, the heteroaryl group is a
substituted 5-14 membered heteroaryl.
[0046] Exemplary 5-membered heteroaryl groups containing 1
heteroatom include, without limitation, pyrrolyl, furanyl, and
thiophenyl. Exemplary 5-membered heteroaryl groups containing 2
heteroatoms include, without limitation, imidazolyl, pyrazolyl,
oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary
5-membered heteroaryl groups containing 3 heteroatoms include,
without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
Exemplary 5-membered heteroaryl groups containing 4 heteroatoms
include, without limitation, tetrazolyl. Exemplary 6-membered
heteroaryl groups containing 1 heteroatom include, without
limitation, pyridinyl. Exemplary 6-membered heteroaryl groups
containing 2 heteroatoms include, without limitation, pyridazinyl,
pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups
containing 3 or 4 heteroatoms include, without limitation,
triazinyl and tetrazinyl, respectively. Exemplary 7-membered
heteroaryl groups containing 1 heteroatom include, without
limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary
5,6-bicyclic heteroaryl groups include, without limitation,
indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl,
isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl,
benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,
benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
Exemplary 6,6-bicyclic heteroaryl groups include, without
limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,
cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary
tricyclic heteroaryl groups include, without limitation,
phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl,
phenothiazinyl, phenoxazinyl and phenazinyl.
[0047] "Heteroaralkyl" is a subset of "alkyl" and refers to an
alkyl group substituted by a heteroaryl group, wherein the point of
attachment is on the alkyl moiety.
[0048] Affixing the suffix "-ene" to a group indicates the group is
a divalent moiety, e.g., alkylene is the divalent moiety of alkyl,
alkenylene is the divalent moiety of alkenyl, alkynylene is the
divalent moiety of alkynyl, heteroalkylene is the divalent moiety
of heteroalkyl, heteroalkenylene is the divalent moiety of
heteroalkenyl, heteroalkynylene is the divalent moiety of
heteroalkynyl, carbocyclylene is the divalent moiety of
carbocyclyl, heterocyclylene is the divalent moiety of
heterocyclyl, arylene is the divalent moiety of aryl, and
heteroarylene is the divalent moiety of heteroaryl.
[0049] A group is optionally substituted unless expressly provided
otherwise. The term "optionally substituted" refers to being
substituted or unsubstituted. In certain embodiments, alkyl,
alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are
optionally substituted. "Optionally substituted" refers to a group
which may be substituted or unsubstituted (e.g., "substituted" or
"unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl,
"substituted" or "unsubstituted" alkynyl, "substituted" or
"unsubstituted" heteroalkyl, "substituted" or "unsubstituted"
heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl,
"substituted" or "unsubstituted" carbocyclyl, "substituted" or
"unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl
or "substituted" or "unsubstituted" heteroaryl group). In general,
the term "substituted" means that at least one hydrogen present on
a group is replaced with a permissible substituent, e.g., a
substituent which upon substitution results in a stable compound,
e.g., a compound which does not spontaneously undergo
transformation such as by rearrangement, cyclization, elimination,
or other reaction. Unless otherwise indicated, a "substituted"
group has a substituent at one or more substitutable positions of
the group, and when more than one position in any given structure
is substituted, the substituent is either the same or different at
each position. The term "substituted" is contemplated to include
substitution with all permissible substituents of organic
compounds, and includes any of the substituents described herein
that results in the formation of a stable compound. The present
invention contemplates any and all such combinations in order to
arrive at a stable compound. For purposes of this invention,
heteroatoms such as nitrogen may have hydrogen substituents and/or
any suitable substituent as described herein which satisfy the
valencies of the heteroatoms and results in the formation of a
stable moiety. The invention is not intended to be limited in any
manner by the exemplary substituents described herein.
[0050] Exemplary carbon atom substituents include, but are not
limited to, halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H,
--SO.sub.3H, --OH, --OR.sup.aa, --ON(R.sup.bb).sub.2,
--N(R.sup.bb).sub.2, --N(R.sup.bb).sub.3.sup.+X.sup.-,
--N(OR.sup.cc)R.sup.bb, --SH, --SR.sup.aa, --SSR.sup.cc,
--C(.dbd.O)R.sup.aa, --CO.sub.2H, --CHO, --C(OR.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --OC(.dbd.O)R.sup.aa, --OCO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --OC(.dbd.O)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.O)R.sup.aa, --NR.sup.bbCO.sub.2R.sup.aa,
--NR.sup.bbC(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --OC(.dbd.NR.sup.bb)R.sup.aa,
--OC(.dbd.NR.sup.bb)OR.sup.aa,
--C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--OC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--C(.dbd.O)NR.sup.bbSO.sub.2R.sup.aa, --NR.sup.bbSO.sub.2R.sup.aa,
--SO.sub.2N(R.sup.bb).sub.2, --SO.sub.2R.sup.aa,
--SO.sub.2OR.sup.aa, --OSO.sub.2R.sup.aa, --S(.dbd.O)R.sup.aa,
--OS(.dbd.O)R.sup.aa, --Si(R.sup.aa).sub.3,
--OSi(R.sup.aa).sub.3--C(.dbd.S)N(R.sup.bb).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.S)SR.sup.aa, --SC(.dbd.S)SR.sup.aa,
--SC(.dbd.O)SR.sup.aa, --OC(.dbd.O)SR.sup.aa,
--SC(.dbd.O)OR.sup.aa, --SC(.dbd.O)R.sup.aa,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2,
--OP(.dbd.O)(R.sup.aa).sub.2, --OP(.dbd.O)(OR.sup.cc).sub.2,
--P(.dbd.O)(N(R.sup.bb).sub.2).sub.2,
--OP(.dbd.O)(N(R.sup.bb).sub.2).sub.2,
--NR.sup.bbP(.dbd.O)(R.sup.aa).sub.2,
--NR.sup.bbP(.dbd.O)(OR.sup.cc).sub.2,
--NR.sup.bbP(.dbd.O)(N(R.sup.bb).sub.2).sub.2, --P(R.sup.cc).sub.2,
--P(OR.sup.cc).sub.2, --P(R.sup.cc).sub.3.sup.+X.sup.-,
--P(OR.sup.cc).sub.3.sup.+X.sup.-, --P(R.sup.cc).sub.4,
--P(OR.sup.cc).sub.4, --OP(R.sup.cc).sub.2,
--OP(R.sup.cc).sub.3.sup.+X.sup.-, --OP(OR.sup.cc).sub.2,
--OP(OR.sup.cc).sub.3.sup.+X.sup.-, --OP(R.sup.cc).sub.4,
--OP(OR.sup.cc).sub.4, --B(R.sup.aa).sub.2, --B(OR.sup.cc).sub.2,
--BR.sup.aa(OR.sup.cc), C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl,
C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,
heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,
heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; wherein X.sup.- is a
counterion;
[0051] or two geminal hydrogens on a carbon atom are replaced with
the group .dbd.O, .dbd.S, .dbd.NN(R.sup.bb).sub.2,
.dbd.NNR.sup.bbC(.dbd.O)R.sup.aa,
.dbd.NNR.sup.bbC(.dbd.O)OR.sup.aa,
.dbd.NNR.sup.bbS(.dbd.O).sub.2R.sup.aa, .dbd.NR.sup.bb, or
.dbd.NOR.sup.cc;
[0052] each instance of R.sup.aa is, independently, selected from
C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl,
C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10alkenyl, heteroC.sub.2-10alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, or two R.sup.aa groups are joined to form a
3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0053] each instance of R.sup.bb is, independently, selected from
hydrogen, --OH, --OR.sup.aa, --N(R.sup.cc).sub.2, --CN,
--C(.dbd.O)R.sup.aa, --C(.dbd.O)N(R.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --SO.sub.2R.sup.aa,
--C(.dbd.NR.sup.cc)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2,
--P(.dbd.O)(N(R.sup.cc).sub.2).sub.2, C.sub.1-10 alkyl, C.sub.1-10
perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
heteroC.sub.1-10alkyl, heteroC.sub.2-10alkenyl,
heteroC.sub.2-10alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered
heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two
R.sup.bb groups are joined to form a 3-14 membered heterocyclyl or
5-14 membered heteroaryl ring, wherein each alkyl, alkenyl,
alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,
heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; wherein X.sup.- is a
counterion;
[0054] each instance of R.sup.cc is, independently, selected from
hydrogen, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10
alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, or two R.sup.cc groups are joined to form a
3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0055] each instance of R.sup.dd is, independently, selected from
halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H,
--OH, --OR.sup.ee, --ON(R.sup.ff).sub.2, --N(R.sup.ff).sub.2,
--N(R.sup.ff).sub.3.sup.+X.sup.-, --N(OR.sup.ee)R.sup.ff, --SH,
--SR.sup.ee, --SSR.sup.ee, --C(.dbd.O)R.sup.ee, --CO.sub.2H,
--CO.sub.2R.sup.ee, --OC(.dbd.O)R.sup.ee, --OCO.sub.2R.sup.ee,
--C(.dbd.O)N(R.sup.ff).sub.2, --OC(.dbd.O)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.O)R.sup.ee, --NR.sup.ffCO.sub.2R.sup.ee,
--NR.sup.ffC(.dbd.O)N(R.sup.ff).sub.2,
--C(.dbd.NR.sup.ff)OR.sup.ee, --OC(.dbd.NR.sup.ff)R.sup.ee,
--OC(.dbd.NR.sup.ff)OR.sup.ee,
--C(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--OC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffSO.sub.2R.sup.ee, --SO.sub.2N(R.sup.ff).sub.2,
--SO.sub.2R.sup.ee, --SO.sub.2OR.sup.ee, --OSO.sub.2R.sup.ee,
--S(.dbd.O)R.sup.ee, --Si(R.sup.ee).sub.3, --OSi(R.sup.ee).sub.3,
--C(.dbd.S)N(R.sup.ff).sub.2, --C(.dbd.O)SR.sup.ee,
--C(.dbd.S)SR.sup.ee, --SC(.dbd.S)SR.sup.ee,
--P(.dbd.O)(OR.sup.ee).sub.2, --P(.dbd.O)(R.sup.ee).sub.2,
--OP(.dbd.O)(R.sup.ee).sub.2, --OP(.dbd.O)(OR.sup.ee).sub.2,
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, 3-10 membered
heterocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups,
or two geminal R.sup.dd substituents can be joined to form .dbd.O
or .dbd.S; wherein X.sup.- is a counterion;
[0056] each instance of R.sup.ee is, independently, selected from
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, heteroC.sub.1-6 alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6 alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg
groups;
[0057] each instance of R.sup.ff is, independently, selected from
hydrogen, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6alkyl,
heteroC.sub.2-6alkenyl, heteroC.sub.2-6alkynyl, C.sub.3-10
carbocyclyl, 3-10 membered heterocyclyl, C.sub.6-10 aryl and 5-10
membered heteroaryl, or two R.sup.ff groups are joined to form a
3-10 membered heterocyclyl or 5-10 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups;
and each instance of R.sup.gg is, independently, halogen, --CN,
--NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H, --OH, --OC.sub.1-6
alkyl, --ON(C.sub.1-6 alkyl).sub.2, --N(C.sub.1-6 alkyl).sub.2,
--N(C.sub.1-6 alkyl).sub.3.sup.+X.sup.-, --NH(C.sub.1-6
alkyl).sub.2.sup.+X.sup.-, --NH.sub.2(C.sub.1-6
alkyl).sup.+X.sup.-, --NH.sub.3.sup.+X.sup.-, --N(OC.sub.1-6
alkyl)(C.sub.1-6 alkyl), --N(OH)(C.sub.1-6 alkyl), --NH(OH), --SH,
--SC.sub.1-6 alkyl, --SS(C.sub.1-6 alkyl), --C(.dbd.O)(C.sub.1-6
alkyl), --CO.sub.2H, --CO.sub.2(C.sub.1-6 alkyl),
--OC(.dbd.O)(C.sub.1-6 alkyl), --OCO.sub.2(C.sub.1-6 alkyl),
--C(.dbd.O)NH.sub.2, --C(.dbd.O)N(C.sub.1-6 alkyl).sub.2,
--OC(.dbd.O)NH(C.sub.1-6 alkyl), --NHC(.dbd.O)(C.sub.1-6 alkyl),
--N(C.sub.1-6 alkyl)C(.dbd.O)(C.sub.1-6 alkyl),
--NHCO.sub.2(C.sub.1-6 alkyl), --NHC(.dbd.O)N(C.sub.1-6
alkyl).sub.2, --NHC(.dbd.O)NH(C.sub.1-6 alkyl),
--NHC(.dbd.O)NH.sub.2, --C(.dbd.NH)O(C.sub.1-6 alkyl),
--OC(.dbd.NH)(C.sub.1-6 alkyl), --OC(.dbd.NH)OC.sub.1-6 alkyl,
--C(.dbd.NH)N(C.sub.1-6 alkyl).sub.2, --C(.dbd.NH)NH(C.sub.1-6
alkyl), --C(.dbd.NH)NH.sub.2, --OC(.dbd.NH)N(C.sub.1-6
alkyl).sub.2, --OC(NH)NH(C.sub.1-6 alkyl), --OC(NH)NH.sub.2,
--NHC(NH)N(C.sub.1-6 alkyl).sub.2, --NHC(.dbd.NH)NH.sub.2,
--NHSO.sub.2(C.sub.1-6 alkyl), --SO.sub.2N(C.sub.1-6 alkyl).sub.2,
--SO.sub.2NH(C.sub.1-6 alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2C.sub.1-6 alkyl, --SO.sub.2OC.sub.1-6 alkyl,
--OSO.sub.2C.sub.1-6 alkyl, --SOC.sub.1-6 alkyl, --Si(C.sub.1-6
alkyl).sub.3, --OSi(C.sub.1-6 alkyl).sub.3 --C(.dbd.S)N(C.sub.1-6
alkyl).sub.2, C(.dbd.S)NH(C.sub.1-6 alkyl), C(.dbd.S)NH.sub.2,
--C(.dbd.O)S(C.sub.1-6 alkyl), --C(.dbd.S)SC.sub.1-6 alkyl,
--SC(.dbd.S)SC.sub.1-6 alkyl, --P(.dbd.O)(OC.sub.1-6 alkyl).sub.2,
--P(.dbd.O)(C.sub.1-6 alkyl).sub.2, --OP(.dbd.O)(C.sub.1-6
alkyl).sub.2, --OP(.dbd.O)(OC.sub.1-6 alkyl).sub.2, C.sub.1-6
alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two
geminal R.sup.gg substituents can be joined to form .dbd.O or
.dbd.S; wherein X.sup.- is a counterion.
[0058] The term "halo" or "halogen" refers to fluorine (fluoro,
--F), chlorine (chloro, --Cl), bromine (bromo, --Br), or iodine
(iodo, --I).
[0059] Nitrogen atoms can be substituted or unsubstituted as
valency permits, and include primary, secondary, tertiary, and
quaternary nitrogen atoms. Exemplary nitrogen atom substituents
include, but are not limited to, hydrogen, --OH, --OR.sup.aa,
--N(R.sup.cc).sub.2, --CN, --C(.dbd.O)R.sup.aa,
--C(.dbd.O)N(R.sup.cc).sub.2, --CO.sub.2R.sup.aa,
--SO.sub.2R.sup.aa, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.cc)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc,
--P(.dbd.O)(OR.sup.cc).sub.2, --P(.dbd.O)(R.sup.aa).sub.2,
--P(.dbd.O)(N(R.sup.cc).sub.2).sub.2, C.sub.1-10 alkyl, C.sub.1-10
perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
heteroC.sub.1-10alkyl, heteroC.sub.2-10alkenyl,
heteroC.sub.2-10alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered
heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two
R.sup.cc groups attached to an N atom are joined to form a 3-14
membered heterocyclyl or 5-14 membered heteroaryl ring, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups,
and wherein R.sup.aa, R.sup.bb, R.sup.cc and R.sup.dd are as
defined above.
[0060] In certain embodiments, the substituent present on the
nitrogen atom is an nitrogen protecting group (also referred to
herein as an "amino protecting group"). Nitrogen protecting groups
include, but are not limited to, --OH, --OR.sup.aa,
--N(R.sup.cc).sub.2, --C(.dbd.O)R.sup.aa,
--C(.dbd.O)N(R.sup.cc).sub.2, --CO.sub.2R.sup.aa,
--SO.sub.2R.sup.aa, --C(.dbd.NR.sup.cc)R.sup.aa,
--C(.dbd.NR.sup.cc)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc, C.sub.MO alkyl (e.g.,
aralkyl, heteroaralkyl), C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
heteroC.sub.1-10 alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10
alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl,
C.sub.6-14 aryl, and 5-14 membered heteroaryl groups, wherein each
alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups,
and wherein R.sup.aa, R.sup.bb, R.sup.cc and R.sup.dd are as
defined herein. Nitrogen protecting groups are well known in the
art and include those described in detail in Protecting Groups in
Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3.sup.rd
edition, John Wiley & Sons, 1999, incorporated herein by
reference.
[0061] For example, nitrogen protecting groups such as amide groups
(e.g., --C(.dbd.O)R.sup.aa) include, but are not limited to,
formamide, acetamide, chloroacetamide, trichloroacetamide,
trifluoroacetamide, phenylacetamide, 3-phenylpropanamide,
picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl
derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide,
o-nitrophenoxyacetamide, acetoacetamide,
(N'-dithiobenzyloxyacylamino)acetamide,
3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,
2-methyl-2-(o-nitrophenoxy)propanamide,
2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,
3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine
derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.
[0062] Nitrogen protecting groups such as carbamate groups (e.g.,
--C(.dbd.O)OR.sup.aa) include, but are not limited to, methyl
carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc),
9-(2-sulfo)fluorenylmethyl carbamate,
9-(2,7-dibromo)fluoroenylmethyl carbamate,
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl
carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),
2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl
carbamate (Teoc), 2-phenylethyl carbamate (hZ),
1-(1-adamantyl)-1-methylethyl carbamate (Adpoc),
1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl
carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),
1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'-
and 4'-pyridyl)ethyl carbamate (Pyoc),
2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate
(BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc),
allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc),
cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),
8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio
carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),
p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl
carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl
carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl
carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl
carbamate, 2-(p-toluenesulfonyl)ethyl carbamate,
[2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl
carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc),
2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl
carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate,
m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl
carbamate, 5-benzisoxazolylmethyl carbamate,
2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc),
m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate,
o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate,
phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl
thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate,
cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl
carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl
carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate,
1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,
1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,
2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl
carbamate, isobutyl carbamate, isonicotinyl carbamate,
p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl
carbamate, 1-methylcyclohexyl carbamate,
1-methyl-1-cyclopropylmethyl carbamate,
1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,
1-methyl-1-(p-phenylazophenyl)ethyl carbamate,
1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl
carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate,
2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl
carbamate, and 2,4,6-trimethylbenzyl carbamate.
[0063] Nitrogen protecting groups such as sulfonamide groups (e.g.,
--S(.dbd.O).sub.2R.sup.aa) include, but are not limited to,
p-toluenesulfonamide (Ts), benzenesulfonamide,
2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr),
2,4,6-trimethoxybenzenesulfonamide (Mtb),
2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),
2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),
4-methoxybenzenesulfonamide (Mbs),
2,4,6-trimethylbenzenesulfonamide (Mts),
2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),
2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc),
methanesulfonamide (Ms), .beta.-trimethylsilylethanesulfonamide
(SES), 9-anthracenesulfonamide,
4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),
benzylsulfonamide, trifluoromethylsulfonamide, and
phenacylsulfonamide.
[0064] Other nitrogen protecting groups include, but are not
limited to, phenothiazinyl-(10)-acyl derivative,
N'-p-toluenesulfonylaminoacyl derivative, N'-phenylaminothioacyl
derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine
derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide,
N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide,
N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane
adduct (STABASE), 5-substituted
1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted
1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted
3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,
N-[2-(trimethylsilyl)ethoxy]methylamine (SEM),
N-3-acetoxypropylamine,
N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary
ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,
N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),
N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),
N-9-phenylfluorenylamine (PhF),
N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino
(Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine,
N-benzylideneamine, N-p-methoxybenzylideneamine,
N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,
N--(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine,
N-p-nitrobenzylideneamine, N-salicylideneamine,
N-5-chlorosalicylideneamine,
N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,
N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,
N-borane derivative, N-diphenylborinic acid derivative,
N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper
chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine
N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide
(Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates,
dibenzyl phosphoramidate, diphenyl phosphoramidate,
benzenesulfenamide, o-nitrobenzenesulfenamide (Nps),
2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide,
2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide,
and 3-nitropyridinesulfenamide (Npys).
[0065] In certain embodiments, the substituent present on an oxygen
atom is an oxygen protecting group (also referred to herein as an
"hydroxyl protecting group"). Oxygen protecting groups include, but
are not limited to, --R.sup.aa, --N(R.sup.bb).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.O)R.sup.aa, --CO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--S(.dbd.O)R.sup.aa, --SO.sub.2R.sup.aa, --Si(R.sup.aa).sub.3,
--P(R.sup.cc).sub.2, --P(R.sup.cc).sub.3.sup.+X.sup.-,
--P(OR.sup.cc).sub.2, --P(OR.sup.cc).sub.3.sup.+X.sup.-,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2, and
--P(.dbd.O)(N(R.sup.bb).sub.2).sub.2, wherein X.sup.-, R.sup.aa,
R.sup.bb, and R.sup.cc are as defined herein. Oxygen protecting
groups are well known in the art and include those described in
detail in Protecting Groups in Organic Synthesis, T. W. Greene and
P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999,
incorporated herein by reference.
[0066] Exemplary oxygen protecting groups include, but are not
limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM),
t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),
benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM),
(4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM),
t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl,
2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,
bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),
tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,
tetrahydrothiopyranyl, 1-methoxycyclohexyl,
4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl,
4-methoxytetrahydrothiopyranyl S,S-dioxide,
1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP),
1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,
1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl,
2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl,
p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl,
4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,
p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,
.alpha.-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,
di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxyphenyl)methyl,
3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,
1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido,
trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl
(TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl
(DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS),
t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl,
triphenylsilyl, diphenylmethylsilyl (DPMS),
t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate,
acetate, chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,
phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,
4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate
(levulinoyldithioacetal), pivaloate, adamantoate, crotonate,
4-methoxycrotonate, benzoate, p-phenylbenzoate,
2,4,6-trimethylbenzoate (mesitoate), methyl carbonate,
9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate,
2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl
carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),
2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate,
vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc),
p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl
carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,
p-nitrobenzyl carbonate, S-benzyl thiocarbonate,
4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate,
2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate,
o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate,
2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate,
2-(methylthiomethoxymethyl)benzoate,
2,6-dichloro-4-methylphenoxyacetate,
2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,
isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,
o-(methoxyacyl)benzoate, .alpha.-naphthoate, nitrate, alkyl
N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,
borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,
sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate
(Ts).
[0067] In certain embodiments, the substituent present on a sulfur
atom is a sulfur protecting group (also referred to as a "thiol
protecting group"). Sulfur protecting groups include, but are not
limited to, --R.sup.aa, --N(R.sup.bb).sub.2, --C(.dbd.O)SR.sup.aa,
--C(.dbd.O)R.sup.aa, --CO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--S(.dbd.O)R.sup.aa, --SO.sub.2R.sup.aa, --Si(R.sup.aa).sub.3,
--P(R.sup.cc).sub.2, --P(R.sup.cc).sub.3.sup.+X.sup.-,
--P(OR.sup.cc).sub.2, --P(OR.sup.cc).sub.3.sup.+X.sup.-,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2, and
--P(.dbd.O)(N(R.sup.bb).sub.2).sub.2, wherein R.sup.aa, R.sup.bb,
and R.sup.cc are as defined herein. Sulfur protecting groups are
well known in the art and include those described in detail in
Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.
Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, incorporated
herein by reference.
[0068] A "counterion" or "anionic counterion" is a negatively
charged group associated with a positively charged group in order
to maintain electronic neutrality. An anionic counterion may be
monovalent (i.e., including one formal negative charge). An anionic
counterion may also be multivalent (i.e., including more than one
formal negative charge), such as divalent or trivalent. Exemplary
counterions include halide ions (e.g., F.sup.-, Cl.sup.-, Br.sup.-,
I.sup.-), NO.sub.3.sup.-, ClO.sub.4.sup.-, OH.sup.-,
H.sub.2PO.sub.4.sup.-, HCO.sub.3.sup.-, HSO.sub.4.sup.-, sulfonate
ions (e.g., methansulfonate, trifluoromethanesulfonate,
p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate,
naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate,
ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions
(e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate,
glycolate, gluconate, and the like), BF.sub.4.sup.-,
PF.sub.4.sup.-, PF.sub.6.sup.-, AsF.sub.6.sup.-, SbF.sub.6.sup.-,
B[3,5-(CF.sub.3).sub.2C.sub.6H.sub.3].sub.4].sup.-,
B(C.sub.6F.sub.5).sub.4.sup.-, BPh.sub.4.sup.-,
Al(OC(CF.sub.3).sub.3).sub.4.sup.-, and carborane anions (e.g.,
CB.sub.11H.sub.12.sup.- or (HCB.sub.11Me.sub.5Br.sub.6).sup.-).
Exemplary counterions which may be multivalent include
CO.sub.3.sup.2-, HPO.sub.4.sup.2-, PO.sub.4.sup.3-,
B.sub.4O.sub.7.sup.2-, SO.sub.4.sup.2-, S.sub.2O.sub.3.sup.2-,
carboxylate anions (e.g., tartrate, citrate, fumarate, maleate,
malate, malonate, gluconate, succinate, glutarate, adipate,
pimelate, suberate, azelate, sebacate, salicylate, phthalates,
aspartate, glutamate, and the like), and carboranes.
[0069] As used herein, use of the phrase "at least one instance"
refers to 1, 2, 3, 4, or more instances, but also encompasses a
range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2,
from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.
[0070] The term "heteroatom" refers to an atom that is not hydrogen
or carbon. In certain embodiments, the heteroatom is nitrogen. In
certain embodiments, the heteroatom is oxygen. In certain
embodiments, the heteroatom is sulfur.
[0071] A "counterion" or "anionic counterion" is a negatively
charged group associated with a positively charged group in order
to maintain electronic neutrality. An anionic counterion may be
monovalent (i.e., including one formal negative charge). An anionic
counterion may also be multivalent (i.e., including more than one
formal negative charge), such as divalent or trivalent. Exemplary
counterions include halide ions (e.g., F.sup.-, Cl.sup.-, Br.sup.-,
I.sup.-), NO.sub.3.sup.-, ClO.sub.4.sup.-, OH.sup.-,
H.sub.2PO.sub.4.sup.-, HCO.sub.3.sup.-, HSO.sub.4.sup.-, sulfonate
ions (e.g., methansulfonate, trifluoromethanesulfonate,
p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate,
naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate,
ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions
(e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate,
glycolate, gluconate, and the like), BF.sub.4.sup.-,
PF.sub.4.sup.-, PF.sub.6.sup.-, AsF.sub.6.sup.-, SbF.sub.6.sup.-,
B[3,5-(CF.sub.3).sub.2C.sub.6H.sub.3].sub.4].sup.-,
B(C.sub.6F.sub.5).sub.4.sup.-, BPh.sub.4.sup.-,
Al(OC(CF.sub.3).sub.3).sub.4.sup.-, and carborane anions (e.g.,
CB.sub.11H.sub.12.sup.- or (HCB.sub.11Me.sub.5Br.sub.6).sup.-).
Exemplary counterions which may be multivalent include
CO.sub.3.sup.2-, HPO.sub.4.sup.2-, PO.sub.4.sup.3-,
B.sub.4O.sub.7.sup.2-, SO.sub.4.sup.2-, S.sub.2O.sub.3.sup.2-,
carboxylate anions (e.g., tartrate, citrate, fumarate, maleate,
malate, malonate, gluconate, succinate, glutarate, adipate,
pimelate, suberate, azelate, sebacate, salicylate, phthalates,
aspartate, glutamate, and the like), and carboranes.
[0072] The term "small molecule" refers to molecules, whether
naturally-occurring or artificially created (e.g., via chemical
synthesis) that have a relatively low molecular weight. Typically,
a small molecule is an organic compound (i.e., it contains carbon).
The small molecule may contain multiple carbon-carbon bonds,
stereocenters, and other functional groups (e.g., amines, hydroxyl,
carbonyls, and heterocyclic rings, etc.). In certain embodiments,
the molecular weight of a small molecule is not more than about
1,000 g/mol, not more than about 900 g/mol, not more than about 800
g/mol, not more than about 700 g/mol, not more than about 600
g/mol, not more than about 500 g/mol, not more than about 400
g/mol, not more than about 300 g/mol, not more than about 200
g/mol, or not more than about 100 g/mol. In certain embodiments,
the molecular weight of a small molecule is at least about 100
g/mol, at least about 200 g/mol, at least about 300 g/mol, at least
about 400 g/mol, at least about 500 g/mol, at least about 600
g/mol, at least about 700 g/mol, at least about 800 g/mol, or at
least about 900 g/mol, or at least about 1,000 g/mol. Combinations
of the above ranges (e.g., at least about 200 g/mol and not more
than about 500 g/mol) are also possible. In certain embodiments,
the small molecule is a therapeutically active agent such as a drug
(e.g., a molecule approved by the U.S. Food and Drug Administration
as provided in the Code of Federal Regulations (C.F.R.)). The small
molecule may also be complexed with one or more metal atoms and/or
metal ions. In this instance, the small molecule is also referred
to as a "small organometallic molecule." Preferred small molecules
are biologically active in that they produce a biological effect in
animals, preferably mammals, more preferably humans. Small
molecules include, but are not limited to, radionuclides and
imaging agents. In certain embodiments, the small molecule is a
drug. Preferably, though not necessarily, the drug is one that has
already been deemed safe and effective for use in humans or animals
by the appropriate governmental agency or regulatory body. For
example, drugs approved for human use are listed by the FDA under
21 C.F.R. .sctn..sctn. 330.5, 331 through 361, and 440 through 460,
incorporated herein by reference; drugs for veterinary use are
listed by the FDA under 21 C.F.R. .sctn..sctn. 500 through 589,
incorporated herein by reference. All listed drugs are considered
acceptable for use in accordance with the present invention.
[0073] The "molecular weight" of a monovalent moiety --R is
calculated by subtracting 1 from the molecular weight of the
compound R--H. The "molecular weight" of a divalent moiety -L- is
calculated by subtracting 2 from the molecular weight of the
compound H-L-H.
[0074] The term "lipophilic" or "hydrophobic" refers to the ability
of a compound to dissolve, or the ability of a moiety of a compound
to assist the compound in dissolving in fats, oils, lipids, and/or
non-polar solvents (e.g., hexane or toluene). Lipophilic moieties
include, but are not limited to, substituted or unsubstituted,
branched or unbranched alkyl groups having 1 to 50 carbon atoms. In
certain embodiments, the lipophilic moiety is an alkyl group
including at least 1, at least 6, at least 12, at least 18, at
least 24, at least 36, or at least 50 carbon atoms. In certain
embodiments, the lipophilic moiety is an alkyl group including at
most 50, at most 36, at most 24, at most 18, at most 12, or at most
6 carbon atoms. Combinations of the above-referenced ranges (e.g.,
at least about 1 and at most about 24 carbon atoms) are also within
the scope of the invention. In certain embodiments, the lipophilic
moiety is unsubstituted alkyl. In certain embodiments, the
lipophilic moiety is unsubstituted and unbranched alkyl. In certain
embodiments, the lipophilic moiety is unsubstituted and unbranched
C.sub.1-24 alkyl. In certain embodiments, the lipophilic moiety is
unsubstituted and unbranched C.sub.6-24 alkyl. In certain
embodiments, the lipophilic moiety is unsubstituted and unbranched
C.sub.12-24 alkyl.
Other Definitions
[0075] The following definitions are more general terms used
throughout the present application.
[0076] As generally defined herein, "biomolecule" refers to any
compound or biological material which may be found in a living
organism. Examples of classes of biomolecules include, but are not
limited to, proteins, peptides, nucleic acids, lipids,
polysaccharides, small molecules, primary metabolites, secondary
metabolites, natural products, etc.
[0077] As used herein, the term "polymer" refers to any substance
comprising at least two repeating structural units (i.e.,
"monomers") which are associated with one another. In some
embodiments, monomers are covalently associated with one another.
In some embodiments, monomers are non-covalently associated with
one another. Polymers may be homopolymers or copolymers comprising
two or more monomers. In terms of sequence, copolymers may be
random, block, graft, or comprise a combination of random, block,
and/or graft sequences. In some embodiments, block copolymers are
diblock copolymers. In some embodiments, block copolymers are
triblock copolymers. In some embodiments, polymers can be linear or
branched polymers. In some embodiments, polymers in accordance with
the invention comprise blends, mixtures, and/or adducts of any of
the polymers described herein. Typically, polymers in accordance
with the present invention are organic polymers. In some
embodiments, polymers are hydrophilic. In some embodiments,
polymers are hydrophobic. In some embodiments, polymers modified
with one or more moieties and/or functional groups.
[0078] A "protein," "peptide," or "polypeptide" comprises a polymer
of amino acid residues linked together by peptide bonds. The term
refers to proteins, polypeptides, and peptides of any size,
structure, or function. Typically, a protein will be at least three
amino acids long. A protein may refer to an individual protein or a
collection of proteins. Inventive proteins preferably contain only
natural amino acids, although non-natural amino acids (i.e.,
compounds that do not occur in nature but that can be incorporated
into a polypeptide chain) and/or amino acid analogs as are known in
the art may alternatively be employed. Also, one or more of the
amino acids in a protein may be modified, for example, by the
addition of a chemical agent such as a carbohydrate group, a
hydroxyl group, a phosphate group, a farnesyl group, an isofamesyl
group, a fatty acid group, a linker for conjugation or
functionalization, or other modification. A protein may also be a
single molecule or may be a multi-molecular complex. A protein may
be a fragment of a naturally occurring protein or peptide. A
protein may be naturally occurring, recombinant, synthetic, or any
combination of these. A "native" or "wild type" protein or peptide
refers to the protein or peptide as it is found in nature (i.e.,
without further modification). In certain embodiments, a protein
useful in the present invention is a therapeutic protein. Examples
of therapeutic proteins are provided below and elsewhere
herein.
[0079] As generally defined herein, "therapeutic protein" refers to
any protein or protein-based therapy that may be administered to a
subject and have a therapeutic effect. Such therapies include
protein replacement and protein supplementation therapies. Such
therapies also include the administration of exogenous or foreign
protein, antibody therapies, and cell or cell-based therapies.
Therapeutic proteins include infusible therapeutic proteins,
enzymes, enzyme cofactors, hormones, blood clotting factors,
cytokines, growth factors, monoclonal antibodies, and polyclonal
antibodies.
[0080] Examples of enzymes contemplated in the present invention
include, but are not limited to, oxidoreductases, transferases,
hydrolases, lyases, isomerases, and ligases.
[0081] Examples of hormones contemplated in the present invention
include, but are not limited to, Melatonin
(N-acetyl-5-methoxytryptamine), Serotonin, Thyroxine (or
tetraiodothyronine) (a thyroid hormone), Triiodothyronine (a
thyroid hormone), Epinephrine (or adrenaline), Norepinephrine (or
noradrenaline), Dopamine (or prolactin inhibiting hormone),
Antimullerian hormone (or mullerian inhibiting factor or hormone),
adipokines (e.g., Leptin and Adiponectin), Adrenocorticotropic
hormone (or corticotropin), Angiotensinogen and angiotensin,
Antidiuretic hormone (or vasopressin, arginine vasopressin),
Atrial-natriuretic peptide (or atriopeptin), Calcitonin,
Cholecystokinin, Corticotropin-releasing hormone, Erythropoietin,
Follicle-stimulating hormone, Gastrin, Ghrelin, Glucagon,
Glucagon-like peptide (GLP-1), GIP, Gonadotropin-releasing hormone,
Growth hormone-releasing hormone, Human chorionic gonadotropin,
Human placental lactogen, Growth hormone, Inhibin, Insulin,
Insulin-like growth factor (or somatomedin), Luteinizing hormone,
Melanocyte stimulating hormone, Orexin, Oxytocin, Parathyroid
hormone, Prolactin, Relaxin, Secretin, Somatostatin,
Thrombopoietin, Thyroid-stimulating hormone (or thyrotropin),
Thyrotropin-releasing hormone, Cortisol, Aldosterone, Testosterone,
Dehydroepiandrosterone, Androstenedione, Dihydrotestosterone,
Estradiol, Estrone, Estriol, Progesterone, Calcitriol
(1,25-dihydroxyvitamin D3), Calcidiol (25-hydroxyvitamin D3),
Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane, Prolactin
releasing hormone, Lipotropin, Brain natriuretic peptide,
Neuropeptide Y, Histamine, Endothelin, Pancreatic polypeptide,
Renin, and Enkephalin.
[0082] Examples of blood and blood coagulation factors contemplated
in the present invention include, but are not limited to, Factor I
(fibrinogen), Factor II (prothrombin), tissue factor, Factor V
(proaccelerin, labile factor), Factor VII (stable factor,
proconvertin), Factor VIII (antihemophilic globulin), Factor IX
(Christmas factor or plasma thromboplastin component), Factor X
(Stuart-Prower factor), Factor Xa, Factor XI, Factor XII (Hageman
factor), Factor XIII (fibrin-stabilizing factor), von Willebrand
factor, prekallikrein (Fletcher factor), high-molecular weight
kininogen (HMWK) (Fitzgerald factor), fibronectin, fibrin,
thrombin, antithrombin, antithrombin III, heparin cofactor II,
protein C, protein S, protein Z, protein Z-related protease
inhibitot (ZPI), plasminogen, alpha 2-antiplasmin, tissue
plasminogen activator (tPA), urokinase, plasminogen activator
inhibitor-1 (PAI1), von Heldebrant factor, plasminogen activator
inhibitor-2 (PAI2), cancer procoagulant, and epoetin alfa (Epogen,
Procrit).
[0083] Examples of cytokines include lymphokines, interleukins, and
chemokines, type 1 cytokines, such as IFN-.gamma., TGF-.beta., and
type 2 cytokines, such as IF-4, IF-10, and IF-13.
[0084] Examples of growth factors include Adrenomedullin (AM),
Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic
proteins (BMPs), Brain-derived neurotrophic factor (BDNF),
Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast
growth factor (FGF), Glial cell line-derived neurotrophic factor
(GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte
macrophage colony-stimulating factor (GM-CSF), Growth
differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF),
Hepatoma-derived growth factor (HDGF), Insulin-like growth factor
(IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve
growth factor (NGF) and other neurotrophins, Platelet-derived
growth factor (PDGF), Thrombopoietin (TPO), Transforming growth
factor alpha (TGF-.alpha.), Transforming growth factor beta
(TGF-.beta.), Tumour_necrosis_factor-alpha (TNF-.alpha.), Vascular
endothelial growth factor (VEGF), Wnt Signaling Pathway, placental
growth factor (PlGF), [(Foetal Bovine Somatotrophin)] (FBS), IF-1,
IF-2, IF-3, IF-4, IF-5, IF-6, and IF-7.
[0085] Examples of monoclonal antibodies contemplated in the
present invention include, but are not limited to, Abagovomab,
Abciximab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab,
Alacizumab pegol, AFD, Alemtuzumab, Altumomab pentetate, Anatumomab
mafenatox, Anrukinzumab, Anti-thymocyte globin, Apolizumab,
Arcitumomab, Aselizumab, Atlizumab (tocilizumab), Atorolimumab,
Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab,
Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Biciromab,
Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin,
Briakinumab, Canakinumab, Cantuzumab mertansine, Capromab
pendetide, Catumaxomab, Cedelizumab, Certolizumab pegol, Cetuximab,
Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumab
tetraxetan, Conatumumab, Dacetuzumab, Daclizumab, Daratumumab,
Denosumab, Detumomab, Dorlimomab aritox, Dorlixizumab, Ecromeximab,
Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab,
Elotuzumab, Elsilimomab, Enlimomab pegol, Epitumomab cituxetan,
Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab,
Fanolesomab, Faralimomab, Farletuzumab, Felvizumab, Fezakinumab,
Figitumumab, Fontolizumab, Foravirumab, Fresolimumab, Galiximab,
Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, GC1008,
Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab,
Ibalizumab, Ibritumomab tiuxetan, Igovomab, Imciromab, Infliximab,
Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab,
Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Lemalesomab,
Lerdelimumab, Lexatumumab, Libivirumab, Lintuzumab, Lorvotuzumab
mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab,
Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab,
Mitumomab, Morolimumab, Motavizumab, Muromonab-CD3, Nacolomab
tafenatox, Naptumomab estafenatox, Natalizumab, Nebacumab,
Necitumumab, Nerelimomab, Nimotuzumab, Nofetumomab merpentan,
Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Omalizumab,
Oportuzumab monatox, Oregovomab, Otelixizumab, Pagibaximab,
Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pemtumomab,
Pertuzumab, Pexelizumab, Pintumomab, Priliximab, Pritumumab,
Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab
Reslizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab,
Rovelizumab, Ruplizumab, Satumomab pendetide, Sevirumab,
Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Solanezumab,
Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab
tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox,
Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab,
Teplizumab, Ticilimumab (tremelimumab), Tigatuzumab, Tocilizumab
(atlizumab), Toralizumab, Tositumomab, Trastuzumab, Tremelimumab,
Tucotuzumab celmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab,
Vapaliximab, Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab,
Volociximab, Votumumab, Zalutumumab, Zanolimumab, Ziralimumab, and
Zolimomab aritox.
[0086] Examples of infusion therapy or injectable therapeutic
proteins contemplated in the present invention include, but are not
limited to, Tocilizumab (Roche/Actemra.RTM.), alpha-1 antitrypsin
(Kamada/AAT), Hematide.RTM. (Affymax and Takeda, synthetic
peptide), albinterferon alfa-2b (Novartis/Zalbin.TM.), Rhucin.RTM.
(Pharming Group, Cl inhibitor replacement therapy), tesamorelin
(Theratechnologies/Egrifta, synthetic growth hormone-releasing
factor), ocrelizumab (Genentech, Roche and Biogen), belimumab
(GlaxoSmithKline/Benlysta.RTM.), pegloticase (Savient
Pharmaceuticals/Krystexxa.TM.), taliglucerase alfa
(Protalix/Uplyso), agalsidase alfa (Shire/Replagal.RTM.),
velaglucerase alfa (Shire).
[0087] Other examples of therapeutic proteins contemplated in the
present invention include, but are not limited to, imiglucerase for
the treatment of Gaucher's disease (e.g., CEREZYME.TM.),
a-galactosidase A (a-gal A) for the treatment of Fabry disease
(e.g., agalsidase beta, FABRYZYME.TM.), acid a-glucosidase (GAA)
for the treatment of Pompe disease (e.g., alglucosidase alfa, rhGAA
(e.g., LUMIZYME.TM., MYOZYME.TM.), arylsulfatase B for the
treatment of Mucopolysaccharidoses (e.g., laronidase,
ALDURAZYME.TM., idursulfase, ELAPRASE.TM., arylsulfatase B,
NAGLAZYME.TM.).
[0088] As used herein, the terms "associated with," "conjugated,"
"linked," "attached," "tethered," and "complex" when used with
respect to two or more moieties, means that the moieties are
physically associated or connected with one another, either
directly or via one or more additional moieties that serves as a
linking agent, to form a structure that is sufficiently stable so
that the moieties remain physically associated under the conditions
in which the structure is used, e.g., physiological conditions. The
moieties can be connected via covalent or non-covalent
interactions. In some instances, moieties are connected by covalent
bonds. In some instances, moieties are connected by non-covalent
interactions. In some embodiments, a sufficient number of weaker
interactions can provide sufficient stability for moieties to
remain physically associated under a variety of different
conditions.
[0089] The terms "polynucleotide", "nucleotide sequence", "nucleic
acid", "nucleic acid molecule", "nucleic acid sequence", and
"oligonucleotide" refer to a series of nucleotide bases (also
called "nucleotides") in DNA and RNA, and mean any chain of two or
more nucleotides. The polynucleotides can be chimeric mixtures or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, its hybridization parameters,
etc. The antisense oligonuculeotide may comprise a modified base
moiety which is selected from the group including, but not limited
to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,
2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, a thio-guanine, and
2,6-diaminopurine. A nucleotide sequence typically carries genetic
information, including the information used by cellular machinery
to make proteins and enzymes. These terms include double- or
single-stranded genomic and cDNA, RNA, any synthetic and
genetically manipulated polynucleotide, and both sense and
antisense polynucleotides. This includes single- and
double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA
hybrids, as well as "protein nucleic acids" (PNAs) formed by
conjugating bases to an amino acid backbone. This also includes
nucleic acids containing carbohydrate or lipids. Exemplary DNAs
include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA),
plasmid DNA (pDNA), genomic DNA (gDNA), complementary DNA (cDNA),
antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite
DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA),
provirus, lysogen, repetitive DNA, satellite DNA, and viral DNA.
Exemplary RNAs include single-stranded RNA (ssRNA), double-stranded
RNA (dsRNA), small interfering RNA (siRNA), messenger RNA (mRNA),
precursor messenger RNA (pre-mRNA), small hairpin RNA or short
hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer
RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA
(hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA
(long ncRNA or IncRNA), satellite RNA, viral satellite RNA, signal
recognition particle RNA, small cytoplasmic RNA, small nuclear RNA
(snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA),
polyinosinic acid, ribozyme, flexizyme, small nucleolar RNA
(snoRNA), spliced leader RNA, viral RNA, and viral satellite
RNA.
[0090] The terms "nucleic acid" or "nucleic acid sequence",
"nucleic acid molecule", "nucleic acid fragment" or
"polynucleotide" may be used interchangeably with "gene", "mRNA
encoded by a gene" and "cDNA".
[0091] The term "mRNA" or "mRNA molecule" refers to messenger RNA,
or the RNA that serves as a template for protein synthesis in a
cell. The sequence of a strand of mRNA is based on the sequence of
a complementary strand of DNA comprising a sequence coding for the
protein to be synthesized.
[0092] The term "siRNA" or "siRNA molecule" refers to small
inhibitory RNA duplexes that induce the RNA interference (RNAi)
pathway, where the siRNA interferes with the expression of specific
genes with a complementary nucleotide sequence. siRNA molecules can
vary in length (e.g., between 18-30 or 20-25 basepairs) and contain
varying degrees of complementarity to their target mRNA in the
antisense strand. Some siRNA have unpaired overhanging bases on the
5' or 3' end of the sense strand and/or the antisense strand. The
term siRNA includes duplexes of two separate strands, as well as
single strands that can form hairpin structures comprising a duplex
region.
[0093] The term "amino acid" refers to a molecule containing both
an amino group and a carboxyl group. Amino acids include
alpha-amino acids and beta-amino acids, the structures of which are
depicted below. In certain embodiments, an amino acid is an alpha
amino acid.
##STR00006##
[0094] Suitable amino acids include, without limitation, natural
alpha-amino acids such as D- and L-isomers of the 20 common
naturally occurring alpha-amino acids found in peptides (e.g., A,
R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as
provided below), unnatural alpha-amino acids natural beta-amino
acids (e.g., beta-alanine), and unnnatural beta-amino acids.
Exemplary natural alpha-amino acids include L-alanine (A),
L-arginine (R), L-asparagine (N), L-aspartic acid (D), L-cysteine
(C), L-glutamic acid (E), L-glutamine (Q), glycine (G), L-histidine
(H), L-isoleucine (I), L-leucine (L), L-lysine (K), L-methionine
(M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine
(T), L-tryptophan (W), L-tyrosine (Y), and L-valine (V). Exemplary
unnatural alpha-amino acids include D-arginine, D-asparagine,
D-aspartic acid, D-cysteine, D-glutamic acid, D-glutamine,
D-histidine, D-isoleucine, D-leucine, D-lysine, D-methionine,
D-phenylalanine, D-proline, D-serine, D-threonine, D-tryptophan,
D-tyrosine, D-valine, Di-vinyl, .alpha.-methyl-alanine (Aib),
.alpha.-methyl-arginine, .alpha.-methyl-asparagine,
.alpha.-methyl-aspartic acid, .alpha.-methyl-cysteine,
.alpha.-methyl-glutamic acid, .alpha.-methyl-glutamine,
.alpha.-methyl-histidine, .alpha.-methyl-isoleucine,
.alpha.-methyl-leucine, .alpha.-methyl-lysine,
.alpha.-methyl-methionine, .alpha.-methyl-phenylalanine,
.alpha.-methyl-proline, .alpha.-methyl-serine,
.alpha.-methyl-threonine, .alpha.-methyl-tryptophan,
.alpha.-methyl-tyrosine, .alpha.-methyl-valine, norleucine,
terminally unsaturated alpha-amino acids and bis alpha-amino acids
(e.g., modified cysteine, modified lysine, modified tryptophan,
modified serine, modified threonine, modified proline, modified
histidine, modified alanine, and the like). There are many known
unnatural amino acids any of which may be included in the peptides
of the present invention. See for example, S. Hunt, The Non-Protein
Amino Acids: In Chemistry and Biochemistry of the Amino Acids,
edited by G. C. Barrett, Chapman and Hall, 1985.
[0095] As generally used herein, "radionuclide" refers to a
radioactive nuclide (also referred to as "radioactive isotope" or
"radioisotope"). Radioactive nuclides contain excess nuclear energy
and are capable of undergoing radioactive decay to emit radiation
(e.g., gamma radiation, alpha particles, beta particles) or
internal conversion to eject an electron. Radionuclides can be used
in radiolabeling and radioactive tracing.
[0096] As used herein, "Fluorophores" are fluorescent chemical
compounds that emit light upon light excitation. Fluorophores
contemplated in the present invention include, but are not limited
to, dyes. Commonly recognized fluorophores include, but are not
limited to, xanthene derivatives (e.g., fluorescein, rhodamine,
Oregon green, eosin, Texas red), cyanine derivatives (e.g.,
cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine,
merocyanine), squaraine derivatives and ring-substituted squaraines
(e.g., Seta, SeTau, Square dyes), naphthalene derivatives (e.g.,
dansyl and prodan derivatives), coumarin derivatives, oxadiazole
derivatives: (e.g., pyridyloxazole, nitrobenzoxadiazole,
benzoxadiazole), anthracene derivatives (e.g., anthraquinones,
including DRAQ5, DRAQ7, CyTRAK Orange), pyrene derivatives (e.g.,
cascade blue), oxazine derivatives (e.g., Nile red, Nile blue,
cresyl violet, oxazine 170), acridine derivatives (e.g., proflavin,
acridine orange, acridine yellow), arylmethine derivatives (e.g.,
auramine, crystal violet, malachite green), and tetrapyrrole
derivatives (e.g., porphin, phthalocyanine, bilirubin). Examples of
more recent fluorphores and their vendors include, but are not
limited to, CF dye (Biotium), DRAQ and CyTRAK probes (BioStatus),
BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor
(Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich),
FluoProbes (Interchim), Abberior Dyes (Abberior), DY and MegaStokes
Dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec),
Seta, SeTau and Square Dyes (SETA BioMedicals), Quasar and Cal
Fluor dyes (Biosearch Technologies), SureLight Dyes (APC, RPEPerCP,
Phycobilisomes)(Columbia Biosciences), APC, APCXL, RPE, and BPE
(Phyco-Biotech, Greensea, Prozyme, Flogen). Other examples of
fluorophores contemplated in the present invention include, but are
not limited to, hydroxycoumarin, aminocoumarin, methoxycoumarin,
Cascade Blue, Pacific Blue, Pacific Orange, Lucifer yellow, NBD,
R-Phycoerythrin (PE), PE-Cy5 conjugates (e.g., Cychrome, R670,
Tri-Color, Quantum Red), PE-Cy7 conjugates, Red 613 (PE-Texas Red),
PerCP (Peridinin chlorophyll protein), TruRed (PerCP-Cy5.5
conjugate), FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5,
Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B,
Texas Red, Allophycocyanin (APC), and APC-Cy7 conjugates. Other
examples of fluorophores include, but are not limited to, hoechst,
DAPI, SYTOX Blue, Chromomycin A3, Mithramycin, YOYO-1, Ethidium
Bromide, Acridine Orange, SYTOX Green, TOTO-1, TO-PRO-1 TOTO:
Cyanine Dimer, TO-PRO: Cyanine Monomer, Thiazole Orange, CyTRAK
Orange 5, Propidium Iodide (PI), LDS, 7-AAD (7-aminoactinomycin D),
SYTOX Orange, TOTO-3, TO-PRO-3, DRAQ5, DRAQ7, Indo-1, Fluo-3,
Fluo-4, DCFH (2'7'Dichorodihydrofluorescein), DHR
(Dihydrorhodamine), and SNARF. Fluorophores also include
fluorescent proteins, exampels of which include, but are not
limited to, green fluorescent protein (e.g., wild-type, Y66H
mutation, Y66F mutation, Y66W mutation, S65A mutation, S65C
mutation, S65L mutation, TurboGFP, TagGFP, S65T mutation, GFPuv,
EGFP), EBFP, EBFP2, Azurite, T-Sapphire, Cerulean, mCFP,
mTurquoise2, ECFP, CyPet, mKeima-Red, TagCFP, AmCyan1, mTFPl,
Midoriishi Cyan, Emerald, Azami Green, ZsGreenl, TagYFP, EYFP,
Topaz, Venus, mCitrine, Ypet, TurboYFP, ZsYellowl, Kusabira Orange,
mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP,
DsRed monomer, DsRed2, mStrawberry, TurboFP602, AsRed2, mRFPl,
J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry,
HcRedl, Katusha, P3 (phycobilisome complex[6]), Peridinin
Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and
mRaspberry.
[0097] As used herein, "chemiluminescent agents" refers to
molecules that release energy in the form of heat or light as a
result of a chemical reaction.
[0098] As used herein, "hapten" refers to a molecule (e.g., small
molecule) that triggers an immune response (e.g., production of
antibodies) when bound to a carrier molecule such as a protein.
[0099] As used herein, "diagnostic agent" refers to molecule that
can be used to visualize or characterize a certain biological
process and/or to diagnose a certain condition in a subject.
[0100] As generally defined herein, "particles" include any organic
or inorganic particles. Specific examples of particles include, but
are not limited to, organic nanoparticles such as lipid
nanoparticles, liposomes, and micelles. Other examples of particles
include, but are not limited to, metallic nanoparticles.
[0101] As used herein, "colorimetric labels" refers to any molecule
that can be observed or quantified by colorimetry. "Colorimetry"
refers to the detection (e.g., quantification) of a molecule or
compound by measuring its absorbance of a specific wavelength of
light.
[0102] As used herein, "fatty acid" refers to a carboxylic acid
with a saturated or unsaturated, substituted or unsubstituted,
branched or unbranched aliphatic chain. In certain embodiments, the
aliphatic chain comprises 4 to 28 carbon atoms. In certain
embodiments, the aliphatic chain of the fatty acid comprises more
than 28 carbon atoms. Derivatives of fatty acid are also
contemplated in the invention (e.g., ester and amide derivatives).
As defined herein, "short-chain fatty acids" are fatty acids with
aliphatic tails of fewer than six carbons (e.g., butyric acid). As
defined herein, "medium-chain fatty acids" are fatty acids with
aliphatic tails of 6-12 carbons. As defined herein, "long-chain
fatty acids" are fatty acids with aliphatic tails 13 to 21 carbons.
As also defined herein, "very long chain fatty acids" are fatty
acids with aliphatic tails longer than 22 carbons. Examples of
unsaturated fatty acids include, but are not limited to,
myristoleic acid, palmitoleic acid, sapienic, oleic acid, elaidic
acid, vaccenic acid, linoleic acid, linoelaidic acid,
.alpha.-linolenic acid, arachidonic acid, eicosapentaenoic acid,
and erucic acid, docosahexaenoic acid. Examples of saturated fatty
acids include, but are not limited to, caprylic acid, capric acid,
lauric acid, myristic acid, palmitic acid, stearic acid, arachidic
acid, behenic acid, lignoceric acid, and cerotic acid.
[0103] As used herein, the term "salt" refers to any and all salts,
and encompasses pharmaceutically acceptable salts.
[0104] The term "pharmaceutically acceptable salt" refers to those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response, and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, Berge et al. describe pharmaceutically acceptable
salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19,
incorporated herein by reference. Pharmaceutically acceptable salts
of the compounds of this invention include those derived from
suitable inorganic and organic acids and bases. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids, such as hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid, and
perchloric acid or with organic acids, such as acetic acid, oxalic
acid, maleic acid, tartaric acid, citric acid, succinic acid, or
malonic acid or by using other methods known in the art such as ion
exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium, and N.sup.+(C.sub.1-4 alkyl).sub.4.sup.-
salts. Representative alkali or alkaline earth metal salts include
sodium, lithium, potassium, calcium, magnesium, and the like.
Further pharmaceutically acceptable salts include, when
appropriate, nontoxic ammonium, quaternary ammonium, and amine
cations formed using counterions such as halide, hydroxide,
carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate,
and aryl sulfonate.
[0105] The term "solvate" refers to forms of the compound, or a
salt thereof, that are associated with a solvent, usually by a
solvolysis reaction. This physical association may include hydrogen
bonding. Conventional solvents include water, methanol, ethanol,
acetic acid, DMSO, THF, diethyl ether, and the like. The compounds
described herein may be prepared, e.g., in crystalline form, and
may be solvated. Suitable solvates include pharmaceutically
acceptable solvates and further include both stoichiometric
solvates and non-stoichiometric solvates. In certain instances, the
solvate will be capable of isolation, for example, when one or more
solvent molecules are incorporated in the crystal lattice of a
crystalline solid. "Solvate" encompasses both solution-phase and
isolatable solvates. Representative solvates include hydrates,
ethanolates, and methanolates.
[0106] The term "hydrate" refers to a compound that is associated
with water. Typically, the number of the water molecules contained
in a hydrate of a compound is in a definite ratio to the number of
the compound molecules in the hydrate. Therefore, a hydrate of a
compound may be represented, for example, by the general formula Rx
H.sub.2O, wherein R is the compound, and x is a number greater than
0. A given compound may form more than one type of hydrate,
including, e.g., monohydrates (x is 1), lower hydrates (x is a
number greater than 0 and smaller than 1, e.g., hemihydrates
(R.0.5H.sub.2O)), and polyhydrates (x is a number greater than 1,
e.g., dihydrates (R.2H.sub.2O) and hexahydrates (R.6H.sub.2O)).
[0107] The term "tautomers" or "tautomeric" refers to two or more
interconvertible compounds resulting from at least one formal
migration of a hydrogen atom and at least one change in valency
(e.g., a single bond to a double bond, a triple bond to a single
bond, or vice versa). The exact ratio of the tautomers depends on
several factors, including temperature, solvent, and pH.
Tautomerizations (i.e., the reaction providing a tautomeric pair)
may catalyzed by acid or base. Exemplary tautomerizations include
keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine,
and enamine-to-(a different enamine) tautomerizations. It is also
to be understood that compounds that have the same molecular
formula but differ in the nature or sequence of bonding of their
atoms or the arrangement of their atoms in space are termed
"isomers". Isomers that differ in the arrangement of their atoms in
space are termed "stereoisomers".
[0108] Stereoisomers that are not mirror images of one another are
termed "diastereomers" and those that are non-superimposable mirror
images of each other are termed "enantiomers". When a compound has
an asymmetric center, for example, it is bonded to four different
groups, a pair of enantiomers is possible. An enantiomer can be
characterized by the absolute configuration of its asymmetric
center and is described by the R- and S-sequencing rules of Cahn
and Prelog, or by the manner in which the molecule rotates the
plane of polarized light and designated as dextrorotatory or
levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral
compound can exist as either individual enantiomer or as a mixture
thereof. A mixture containing equal proportions of the enantiomers
is called a "racemic mixture".
[0109] The term "prodrugs" refers to compounds that have cleavable
groups and become by solvolysis or under physiological conditions
the compounds described herein, which are pharmaceutically active
in vivo. Such examples include, but are not limited to, choline
ester derivatives and the like, N-alkylmorpholine esters and the
like. Other derivatives of the compounds described herein have
activity in both their acid and acid derivative forms, but in the
acid sensitive form often offer advantages of solubility, tissue
compatibility, or delayed release in the mammalian organism (see,
Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier,
Amsterdam 1985). Prodrugs include acid derivatives well known to
practitioners of the art, such as, for example, esters prepared by
reaction of the parent acid with a suitable alcohol, or amides
prepared by reaction of the parent acid compound with a substituted
or unsubstituted amine, or acid anhydrides, or mixed anhydrides.
Simple aliphatic or aromatic esters, amides, and anhydrides derived
from acidic groups pendant on the compounds described herein are
particular prodrugs. In some cases it is desirable to prepare
double ester type prodrugs such as (acyloxy)alkyl esters or
((alkoxycarbonyl)oxy)alkylesters. C1-C8 alkyl, C2-C8 alkenyl, C2-C8
alkynyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters
of the compounds described herein may be preferred.
[0110] The terms "composition" and "formulation" are used
interchangeably.
[0111] A "subject" to which administration is contemplated refers
to a human (i.e., male or female of any age group, e.g., pediatric
subject (e.g., infant, child, or adolescent) or adult subject
(e.g., young adult, middle-aged adult, or senior adult)) or
non-human animal. In certain embodiments, the non-human animal is a
mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey),
commercially relevant mammal (e.g., cattle, pig, horse, sheep,
goat, cat, or dog), or bird (e.g., commercially relevant bird, such
as chicken, duck, goose, or turkey)). In certain embodiments, the
non-human animal is a fish, reptile, or amphibian. The non-human
animal may be a male or female at any stage of development. The
non-human animal may be a transgenic animal or genetically
engineered animal. The term "patient" refers to a human subject in
need of treatment of a disease. The subject may also be a plant. In
certain embodiments, the plant is a land plant. In certain
embodiments, the plant is a non-vascular land plant. In certain
embodiments, the plant is a vascular land plant. In certain
embodiments, the plant is a seed plant. In certain embodiments, the
plant is a cultivated plant. In certain embodiments, the plant is a
dicot. In certain embodiments, the plant is a monocot. In certain
embodiments, the plant is a flowering plant. In some embodiments,
the plant is a cereal plant, e.g., maize, corn, wheat, rice, oat,
barley, rye, or millet. In some embodiments, the plant is a legume,
e.g., a bean plant, e.g., soybean plant. In some embodiments, the
plant is a tree or shrub.
[0112] The term "administer," "administering," or "administration"
refers to implanting, absorbing, ingesting, injecting, inhaling, or
otherwise introducing a compound described herein, or a composition
thereof, in or on a subject.
[0113] The terms "treatment," "treat," and "treating" refer to
reversing, alleviating, delaying the onset of, or inhibiting the
progress of a disease described herein. In some embodiments,
treatment may be administered after one or more signs or symptoms
of the disease have developed or have been observed. In other
embodiments, treatment may be administered in the absence of signs
or symptoms of the disease. For example, treatment may be
administered to a susceptible subject prior to the onset of
symptoms (e.g., in light of a history of symptoms and/or in light
of exposure to a pathogen). Treatment may also be continued after
symptoms have resolved, for example, to delay or prevent
recurrence.
[0114] The terms "condition," "disease," and "disorder" are used
interchangeably.
[0115] An "effective amount" of a compound described herein refers
to an amount sufficient to elicit the desired biological response.
An effective amount of a compound described herein may vary
depending on such factors as the desired biological endpoint, the
pharmacokinetics of the compound, the condition being treated, the
mode of administration, and the age and health of the subject. In
certain embodiments, an effective amount is a therapeutically
effective amount. In certain embodiments, an effective amount is a
prophylactic treatment. In certain embodiments, an effective amount
is the amount of a compound described herein in a single dose. In
certain embodiments, an effective amount is the combined amounts of
a compound described herein in multiple doses.
[0116] A "therapeutically effective amount" of a compound described
herein is an amount sufficient to provide a therapeutic benefit in
the treatment of a condition or to delay or minimize one or more
symptoms associated with the condition. A therapeutically effective
amount of a compound means an amount of therapeutic agent, alone or
in combination with other therapies, which provides a therapeutic
benefit in the treatment of the condition. The term
"therapeutically effective amount" can encompass an amount that
improves overall therapy, reduces or avoids symptoms, signs, or
causes of the condition, and/or enhances the therapeutic efficacy
of another therapeutic agent.
Medical Conditions
[0117] The term "metabolic disorder" refers to any disorder that
involves an alteration in the normal metabolism of carbohydrates,
lipids, proteins, nucleic acids, or a combination thereof. A
metabolic disorder is associated with either a deficiency or excess
in a metabolic pathway resulting in an imbalance in metabolism of
nucleic acids, proteins, lipids, and/or carbohydrates. Factors
affecting metabolism include, and are not limited to, the endocrine
(hormonal) control system (e.g., the insulin pathway, the
enteroendocrine hormones including GLP-1, PYY or the like), the
neural control system (e.g., GLP-1 in the brain), or the like.
Examples of metabolic disorders include, but are not limited to,
diabetes (e.g., Type I diabetes, Type II diabetes, gestational
diabetes), hyperglycemia, hyperinsulinemia, insulin resistance, and
obesity.
[0118] A "diabetic condition" (also referred to as "diabetes")
refers to diabetes and pre-diabetes. Diabetes refers to a group of
metabolic diseases in which a person has high blood sugar, either
because the body does not produce enough insulin, or because cells
do not respond to the insulin that is produced. This high blood
sugar produces the classical symptoms of polyuria (frequent
urination), polydipsia (increased thirst) and polyphagia (increased
hunger). There are several types of diabetes. Type I diabetes
results from the body's failure to produce insulin, and presently
requires the person to inject insulin or wear an insulin pump. Type
II diabetes results from insulin resistance a condition in which
cells fail to use insulin properly, sometimes combined with an
absolute insulin deficiency. Gestational diabetes occurs when
pregnant women without a previous diagnosis of diabetes develop a
high blood glucose level. Other forms of diabetes include
congenital diabetes, which is due to genetic defects of insulin
secretion, cystic fibrosis-related diabetes, steroid diabetes
induced by high doses of glucocorticoids, and several forms of
monogenic diabetes, e.g., mature onset diabetes of the young (e.g.,
MODY 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Pre-diabetes indicates a
condition that occurs when a person's blood glucose levels are
higher than normal but not high enough for a diagnosis of diabetes.
All forms of diabetes increase the risk of long-term complications.
These typically develop after many years, but may be the first
symptom in those who have otherwise not received a diagnosis before
that time. The major long-term complications relate to damage to
blood vessels. Diabetes doubles the risk of cardiovascular disease
and macrovascular diseases such as ischemic heart disease (angina,
myocardial infarction), stroke, and peripheral vascular disease.
Diabetes also causes microvascular complications, e.g., damage to
the small blood vessels. Diabetic retinopathy, which affects blood
vessel formation in the retina of the eye, can lead to visual
symptoms, reduced vision, and potentially blindness. Diabetic
nephropathy, the impact of diabetes on the kidneys, can lead to
scarring changes in the kidney tissue, loss of small or
progressively larger amounts of protein in the urine, and
eventually chronic kidney disease requiring dialysis. Diabetic
neuropathy is the impact of diabetes on the nervous system, most
commonly causing numbness, tingling and pain in the feet and also
increasing the risk of skin damage due to altered sensation.
Together with vascular disease in the legs, neuropathy contributes
to the risk of diabetes-related foot problems, e.g., diabetic foot
ulcers, that can be difficult to treat and occasionally require
amputation.
[0119] The term "genetic disease" refers to a disease caused by one
or more abnormalities in the genome of a subject, such as a disease
that is present from birth of the subject. Genetic diseases may be
heritable and may be passed down from the parents' genes. A genetic
disease may also be caused by mutations or changes of the DNAs
and/or RNAs of the subject. In such cases, the genetic disease will
be heritable if it occurs in the germline. Exemplary genetic
diseases include, but are not limited to, Aarskog-Scott syndrome,
Aase syndrome, achondroplasia, acrodysostosis, addiction,
adreno-leukodystrophy, albinism, ablepharon-macrostomia syndrome,
alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency,
Alport's syndrome, Alzheimer's disease, asthma, autoimmune
polyglandular syndrome, androgen insensitivity syndrome, Angelman
syndrome, ataxia, ataxia telangiectasia, atherosclerosis, attention
deficit hyperactivity disorder (ADHD), autism, baldness, Batten
disease, Beckwith-Wiedemann syndrome, Best disease, bipolar
disorder, brachydactyl), breast cancer, Burkitt lymphoma, chronic
myeloid leukemia, Charcot-Marie-Tooth disease, Crohn's disease,
cleft lip, Cockayne syndrome, Coffin Lowry syndrome, colon cancer,
congenital adrenal hyperplasia, Cornelia de Lange syndrome,
Costello syndrome, Cowden syndrome, craniofrontonasal dysplasia,
Crigler-Najjar syndrome, Creutzfeldt-Jakob disease, cystic
fibrosis, deafness, depression, diabetes, diastrophic dysplasia,
DiGeorge syndrome, Down's syndrome, dyslexia, Duchenne muscular
dystrophy, Dubowitz syndrome, ectodermal dysplasia Ellis-van
Creveld syndrome, Ehlers-Danlos, epidermolysis bullosa, epilepsy,
essential tremor, familial hypercholesterolemia, familial
Mediterranean fever, fragile X syndrome, Lriedreich's ataxia,
Gaucher disease, glaucoma, glucose galactose malabsorption,
glutaricaciduria, gyrate atrophy, Goldberg Shprintzen syndrome
(velocardiofacial syndrome), Gorlin syndrome, Hailey-Hailey
disease, hemihypertrophy, hemochromatosis, hemophilia, hereditary
motor and sensory neuropathy (HMSN), hereditary non polyposis
colorectal cancer (HNPCC), Huntington's disease, immunodeficiency
with hyper-IgM, juvenile onset diabetes, Klinefelter's syndrome,
Kabuki syndrome, Leigh's disease, long QT syndrome, lung cancer,
malignant melanoma, manic depression, Marfan syndrome, Menkes
syndrome, miscarriage, mucopolysaccharide disease, multiple
endocrine neoplasia, multiple sclerosis, muscular dystrophy,
myotrophic lateral sclerosis, myotonic dystrophy,
neurofibromatosis, Niemann-Pick disease, Noonan syndrome, obesity,
ovarian cancer, pancreatic cancer, Parkinson's disease, paroxysmal
nocturnal hemoglobinuria, Pendred syndrome, peroneal muscular
atrophy, phenylketonuria (PKU), polycystic kidney disease,
Prader-Willi syndrome, primary biliary cirrhosis, prostate cancer,
REAR syndrome, Refsum disease, retinitis pigmentosa,
retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia,
severe combined immunodeficiency, sickle cell anemia, spina bifida,
spinal muscular atrophy, spinocerebellar atrophy, sudden adult
death syndrome, Tangier disease, Tay-Sachs disease,
thrombocytopenia absent radius syndrome, Townes-Brocks syndrome,
tuberous sclerosis, Turner syndrome, Usher syndrome, von
Hippel-Lindau syndrome, Waardenburg syndrome, Weaver syndrome,
Wemer syndrome, Williams syndrome, Wilson's disease, xeroderma
piginentosum, and Zellweger syndrome.
[0120] A "proliferative disease" refers to a disease that occurs
due to abnormal growth or extension by the multiplication of cells
(Walker, Cambridge Dictionary of Biology; Cambridge University
Press: Cambridge, UK, 1990). A proliferative disease may be
associated with: 1) the pathological proliferation of normally
quiescent cells; 2) the pathological migration of cells from their
normal location (e.g., metastasis of neoplastic cells); 3) the
pathological expression of proteolytic enzymes such as the matrix
metalloproteinases (e.g., collagenases, gelatinases, and
elastases); or 4) the pathological angiogenesis as in proliferative
retinopathy and tumor metastasis. Exemplary proliferative diseases
include cancers (i.e., "malignant neoplasms"), benign neoplasms,
angiogenesis, inflammatory diseases, and autoimmune diseases.
[0121] The term "angiogenesis" refers to the physiological process
through which new blood vessels form from pre-existing vessels.
Angiogenesis is distinct from vasculogenesis, which is the de novo
formation of endothelial cells from mesoderm cell precursors. The
first vessels in a developing embryo form through vasculogenesis,
after which angiogenesis is responsible for most blood vessel
growth during normal or abnormal development. Angiogenesis is a
vital process in growth and development, as well as in wound
healing and in the formation of granulation tissue. However,
angiogenesis is also a fundamental step in the transition of tumors
from a benign state to a malignant one, leading to the use of
angiogenesis inhibitors in the treatment of cancer. Angiogenesis
may be chemically stimulated by angiogenic proteins, such as growth
factors (e.g., FGF). "Pathological angiogenesis" refers to abnormal
(e.g., excessive or insufficient) angiogenesis that amounts to
and/or is associated with a disease.
[0122] The terms "neoplasm" and "tumor" are used herein
interchangeably and refer to an abnormal mass of tissue wherein the
growth of the mass surpasses and is not coordinated with the growth
of a normal tissue. A neoplasm or tumor may be "benign" or
"malignant," depending on the following characteristics: degree of
cellular differentiation (including morphology and functionality),
rate of growth, local invasion, and metastasis. A "benign neoplasm"
is generally well differentiated, has characteristically slower
growth than a malignant neoplasm, and remains localized to the site
of origin. In addition, a benign neoplasm does not have the
capacity to infiltrate, invade, or metastasize to distant sites.
Exemplary benign neoplasms include, but are not limited to, lipoma,
chondroma, adenomas, acrochordon, senile angiomas, seborrheic
keratoses, lentigos, and sebaceous hyperplasias. In some cases,
certain "benign" tumors may later give rise to malignant neoplasms,
which may result from additional genetic changes in a subpopulation
of the tumor's neoplastic cells, and these tumors are referred to
as "pre-malignant neoplasms." An exemplary pre-malignant neoplasm
is a teratoma. In contrast, a "malignant neoplasm" is generally
poorly differentiated (anaplasia) and has characteristically rapid
growth accompanied by progressive infiltration, invasion, and
destruction of the surrounding tissue. Furthermore, a malignant
neoplasm generally has the capacity to metastasize to distant
sites. The term "metastasis," "metastatic," or "metastasize" refers
to the spread or migration of cancerous cells from a primary or
original tumor to another organ or tissue and is typically
identifiable by the presence of a "secondary tumor" or "secondary
cell mass" of the tissue type of the primary or original tumor and
not of that of the organ or tissue in which the secondary
(metastatic) tumor is located. For example, a prostate cancer that
has migrated to bone is said to be metastasized prostate cancer and
includes cancerous prostate cancer cells growing in bone
tissue.
[0123] The term "cancer" refers to a class of diseases
characterized by the development of abnormal cells that proliferate
uncontrollably and have the ability to infiltrate and destroy
normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th
ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990.
Exemplary cancers include, but are not limited to, acoustic
neuroma; adenocarcinoma; adrenal gland cancer; anal cancer;
angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma,
hemangiosarcoma); appendix cancer; benign monoclonal gammopathy;
biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast
cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of
the breast, mammary cancer, medullary carcinoma of the breast);
brain cancer (e.g., meningioma, glioblastomas, glioma (e.g.,
astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer;
carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma);
choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer
(e.g., colon cancer, rectal cancer, colorectal adenocarcinoma);
connective tissue cancer; epithelial carcinoma; ependymoma;
endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic
hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer,
uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the
esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular
cancer (e.g., intraocular melanoma, retinoblastoma); familiar
hypereosinophilia; gall bladder cancer; gastric cancer (e.g.,
stomach adenocarcinoma); gastrointestinal stromal tumor (GIST);
germ cell cancer; head and neck cancer (e.g., head and neck
squamous cell carcinoma, oral cancer (e.g., oral squamous cell
carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal
cancer, nasopharyngeal cancer, oropharyngeal cancer));
hematopoietic cancers (e.g., leukemia such as acute lymphocytic
leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic
leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic
leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic
lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL));
lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL)
and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse
large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma),
follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic
lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone
B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT)
lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal
zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt
lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's
macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large
cell lymphoma, precursor B-lymphoblastic lymphoma and primary
central nervous system (CNS) lymphoma; and T-cell NHL such as
precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell
lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,
mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell
lymphoma, extranodal natural killer T-cell lymphoma, enteropathy
type T-cell lymphoma, subcutaneous panniculitis-like T-cell
lymphoma, and anaplastic large cell lymphoma); a mixture of one or
more leukemia/lymphoma as described above; and multiple myeloma
(MM)), heavy chain disease (e.g., alpha chain disease, gamma chain
disease, mu chain disease); hemangioblastoma; hypopharynx cancer;
inflammatory myofibroblastic tumors; immunocytic amyloidosis;
kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell
carcinoma); liver cancer (e.g., hepatocellular cancer (HCC),
malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma,
small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC),
adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis
(e.g., systemic mastocytosis); muscle cancer; myelodysplastic
syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD)
(e.g., polycythemia vera (PV), essential thrombocytosis (ET),
agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF),
chronic idiopathic myelofibrosis, chronic myelocytic leukemia
(CML), chronic neutrophilic leukemia (CNL), hypereosinophilic
syndrome (HES)); neuroblastoma; neurofibroma (e.g.,
neurofibromatosis (NF) type 1 or type 2, schwannomatosis);
neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine
tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone
cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian
embryonal carcinoma, ovarian adenocarcinoma); papillary
adenocarcinoma; pancreatic cancer (e.g., pancreatic
andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN),
Islet cell tumors); penile cancer (e.g., Paget's disease of the
penis and scrotum); pinealoma; primitive neuroectodermal tumor
(PNT); plasma cell neoplasia; paraneoplastic syndromes;
intraepithelial neoplasms; prostate cancer (e.g., prostate
adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland
cancer; skin cancer (e.g., squamous cell carcinoma (SCC),
keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small
bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g.,
malignant fibrous histiocytoma (MFH), liposarcoma, malignant
peripheral nerve sheath tumor (MPNST), chondrosarcoma,
fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small
intestine cancer; sweat gland carcinoma; synovioma; testicular
cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid
cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid
carcinoma (PTC), medullary thyroid cancer); urethral cancer;
vaginal cancer; and vulvar cancer (e.g., Paget's disease of the
vulva).
[0124] The term "inflammatory disease" refers to a disease caused
by, resulting from, or resulting in inflammation. The term
"inflammatory disease" may also refer to a dysregulated
inflammatory reaction that causes an exaggerated response by
macrophages, granulocytes, and/or T-lymphocytes leading to abnormal
tissue damage and/or cell death. An inflammatory disease can be
either an acute or chronic inflammatory condition and can result
from infections or non-infectious causes. Inflammatory diseases
include, without limitation, atherosclerosis, arteriosclerosis,
autoimmune disorders, multiple sclerosis, systemic lupus
erythematosus, polymyalgia rheumatica (PMR), gouty arthritis,
degenerative arthritis, tendonitis, bursitis, psoriasis, cystic
fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory
arthritis, Sjogren's syndrome, giant cell arteritis, progressive
systemic sclerosis (scleroderma), ankylosing spondylitis,
polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes
(e.g., Type I), myasthenia gravis, Hashimoto's thyroiditis, Graves'
disease, Goodpasture's disease, mixed connective tissue disease,
sclerosing cholangitis, inflammatory bowel disease, Crohn's
disease, ulcerative colitis, pernicious anemia, inflammatory
dermatoses, usual interstitial pneumonitis (UIP), asbestosis,
silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis,
sarcoidosis, desquamative interstitial pneumonia, lymphoid
interstitial pneumonia, giant cell interstitial pneumonia, cellular
interstitial pneumonia, extrinsic allergic alveolitis, Wegener's
granulomatosis and related forms of angiitis (temporal arteritis
and polyarteritis nodosa), inflammatory dermatoses, hepatitis,
delayed-type hypersensitivity reactions (e.g., poison ivy
dermatitis), pneumonia, respiratory tract inflammation, Adult
Respiratory Distress Syndrome (ARDS), encephalitis, immediate
hypersensitivity reactions, asthma, hayfever, allergies, acute
anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis,
cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic
injury), reperfusion injury, allograft rejection, host-versus-graft
rejection, appendicitis, arteritis, blepharitis, bronchiolitis,
bronchitis, cervicitis, cholangitis, chorioamnionitis,
conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis,
endometritis, enteritis, enterocolitis, epicondylitis,
epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis,
gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis,
nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis,
pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis,
phlebitis, pneumonitis, proctitis, prostatitis, rhinitis,
salpingitis, sinusitis, stomatitis, synovitis, testitis,
tonsillitis, urethritis, urocystitis, uveitis, vaginitis,
vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis,
osteomyelitis, optic neuritis, temporal arteritis, transverse
myelitis, necrotizing fasciitis, and necrotizing enterocolitis. An
ocular inflammatory disease includes, but is not limited to,
post-surgical inflammation.
[0125] An "autoimmune disease" refers to a disease arising from an
inappropriate immune response of the body of a subject against
substances and tissues normally present in the body. In other
words, the immune system mistakes some part of the body as a
pathogen and attacks its own cells. This may be restricted to
certain organs (e.g., in autoimmune thyroiditis) or involve a
particular tissue in different places (e.g., Goodpasture's disease
which may affect the basement membrane in both the lung and
kidney). The treatment of autoimmune diseases is typically with
immunosuppression, e.g., medications which decrease the immune
response. Exemplary autoimmune diseases include, but are not
limited to, glomerulonephritis, Goodpasture's syndrome, necrotizing
vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus
erythematosis, rheumatoid arthritis, psoriatic arthritis, systemic
lupus erythematosis, psoriasis, ulcerative colitis, systemic
sclerosis, dermatomyositis/polymyositis, anti-phospholipid antibody
syndrome, scleroderma, pemphigus vulgaris, ANCA-associated
vasculitis (e.g., Wegener's granulomatosis, microscopic
polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease,
Reiter's syndrome, ankylosing spondylitis, Lyme disease,
Guillain-Barre syndrome, Hashimoto's thyroiditis, and
cardiomyopathy.
[0126] The term "liver disease" or "hepatic disease" refers to
damage to or a disease of the liver. Non-limiting examples of liver
disease include intrahepatic cholestasis (e.g., alagille syndrome,
biliary liver cirrhosis), fatty liver (e.g., alcoholic fatty liver,
Reye's syndrome), hepatic vein thrombosis, hepatolenticular
degeneration (i.e., Wilson's disease), hepatomegaly, liver abscess
(e.g., amebic liver abscess), liver cirrhosis (e.g., alcoholic,
biliary, and experimental liver cirrhosis), alcoholic liver
diseases (e.g., fatty liver, hepatitis, cirrhosis), parasitic liver
disease (e.g., hepatic echinococcosis, fascioliasis, amebic liver
abscess), jaundice (e.g., hemolytic, hepatocellular, cholestatic
jaundice), cholestasis, portal hypertension, liver enlargement,
ascites, hepatitis (e.g., alcoholic hepatitis, animal hepatitis,
chronic hepatitis (e.g., autoimmune, hepatitis B, hepatitis C,
hepatitis D, drug induced chronic hepatitis), toxic hepatitis,
viral human hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C,
hepatitis D, hepatitis E), granulomatous hepatitis, secondary
biliary cirrhosis, hepatic encephalopathy, varices, primary biliary
cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma,
hemangiomas, bile stones, liver failure (e.g., hepatic
encephalopathy, acute liver failure), angiomyolipoma, calcified
liver metastases, cystic liver metastases, fibrolamellar
hepatocarcinoma, hepatic adenoma, hepatoma, hepatic cysts (e.g.,
Simple cysts, Polycystic liver disease, hepatobiliary cystadenoma,
choledochal cyst), mesenchymal tumors (mesenchymal hamartoma,
infantile hemangioendothelioma, hemangioma, peliosis hepatis,
lipomas, inflammatory pseudotumor), epithelial tumors (e.g., bile
duct hamartoma, bile duct adenoma), focal nodular hyperplasia,
nodular regenerative hyperplasia, hepatoblastoma, hepatocellular
carcinoma, cholangiocarcinoma, cystadenocarcinoma, tumors of blood
vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma,
embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma,
carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary
lymphoma, peliosis hepatis, erythrohepatic porphyria, hepatic
porphyria (e.g., acute intermittent porphyria, porphyria cutanea
tarda), and Zellweger syndrome.
[0127] The term "spleen disease" refers to a disease of the spleen.
Example of spleen diseases include, but are not limited to,
splenomegaly, spleen cancer, asplenia, spleen trauma, idiopathic
purpura, Felty's syndrome, Hodgkin's disease, and immune-mediated
destruction of the spleen.
[0128] The term "lung disease" or "pulmonary disease" refers to a
disease of the lung. Examples of lung diseases include, but are not
limited to, bronchiectasis, bronchitis, bronchopulmonary dysplasia,
interstitial lung disease, occupational lung disease, emphysema,
cystic fibrosis, acute respiratory distress syndrome (ARDS), severe
acute respiratory syndrome (SARS), asthma (e.g., intermittent
asthma, mild persistent asthma, moderate persistent asthma, severe
persistent asthma), chronic bronchitis, chronic obstructive
pulmonary disease (COPD), emphysema, interstitial lung disease,
sarcoidosis, asbestosis, aspergilloma, aspergillosis, pneumonia
(e.g., lobar pneumonia, multilobar pneumonia, bronchial pneumonia,
interstitial pneumonia), pulmonary fibrosis, pulmonary
tuberculosis, rheumatoid lung disease, pulmonary embolism, and lung
cancer (e.g., non-small-cell lung carcinoma (e.g., adenocarcinoma,
squamous-cell lung carcinoma, large-cell lung carcinoma),
small-cell lung carcinoma).
[0129] A "hematological disease" includes a disease which affects a
hematopoietic cell or tissue. Hematological diseases include
diseases associated with aberrant hematological content and/or
function. Examples of hematological diseases include diseases
resulting from bone marrow irradiation or chemotherapy treatments
for cancer, diseases such as pernicious anemia, hemorrhagic anemia,
hemolytic anemia, aplastic anemia, sickle cell anemia,
sideroblastic anemia, anemia associated with chronic infections
such as malaria, trypanosomiasis, HTV, hepatitis vims or other
viruses, myelophthisic anemias caused by marrow deficiencies, renal
failure resulting from anemia, anemia, polycythemia, infectious
mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), acute
myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute
myelomonocytic leukemia (AMMoL), polycythemia vera, lymphoma, acute
lymphocytic leukemia (ALL), chronic lymphocytic leukemia, Wilm's
tumor, Ewing's sarcoma, retinoblastoma, hemophilia, disorders
associated with an increased risk of thrombosis, herpes,
thalassemia, antibody-mediated disorders such as transfusion
reactions and erythroblastosis, mechanical trauma to red blood
cells such as micro-angiopathic hemolytic anemias, thrombotic
thrombocytopenic purpura and disseminated intravascular
coagulation, infections by parasites such as Plasmodium, chemical
injuries from, e.g., lead poisoning, and hypersplenism.
[0130] The term "neurological disease" refers to any disease of the
nervous system, including diseases that involve the central nervous
system (brain, brainstem and cerebellum), the peripheral nervous
system (including cranial nerves), and the autonomic nervous system
(parts of which are located in both central and peripheral nervous
system). Neurodegenerative diseases refer to a type of neurological
disease marked by the loss of nerve cells, including, but not
limited to, Alzheimer's disease, Parkinson's disease, amyotrophic
lateral sclerosis, tauopathies (including frontotemporal dementia),
and Huntington's disease. Examples of neurological diseases
include, but are not limited to, headache, stupor and coma,
dementia, seizure, sleep disorders, trauma, infections, neoplasms,
neuro-ophthalmology, movement disorders, demyelinating diseases,
spinal cord disorders, and disorders of peripheral nerves, muscle
and neuromuscular junctions. Addiction and mental illness, include,
but are not limited to, bipolar disorder and schizophrenia, are
also included in the definition of neurological diseases. Further
examples of neurological diseases include acquired epileptiform
aphasia; acute disseminated encephalomyelitis;
adrenoleukodystrophy; agenesis of the corpus callosum; agnosia;
Aicardi syndrome; Alexander disease; Alpers' disease; alternating
hemiplegia; Alzheimer's disease; amyotrophic lateral sclerosis;
anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia;
apraxia; arachnoid cysts; arachnoiditis; Amold-Chiari malformation;
arteriovenous malformation; Asperger syndrome; ataxia
telangiectasia; attention deficit hyperactivity disorder; autism;
autonomic dysfunction; back pain; Batten disease; Behcet's disease;
Bell's palsy; benign essential blepharospasm; benign focal;
amyotrophy; benign intracranial hypertension; Binswanger's disease;
blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury;
brain abscess; bbrain injury; brain tumors (including glioblastoma
multiforme); spinal tumor; Brown-Sequard syndrome; Canavan disease;
carpal tunnel syndrome (CTS); causalgia; central pain syndrome;
central pontine myelinolysis; cephalic disorder; cerebral aneurysm;
cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism;
cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced
neuropathy and neuropathic pain; Chiari malformation; chorea;
chronic inflammatory demyelinating polyneuropathy (CIDP); chronic
pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma,
including persistent vegetative state; congenital facial diplegia;
corticobasal degeneration; cranial arteritis; craniosynostosis;
Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's
syndrome; cytomegalic inclusion body disease (CIBD);
cytomegalovirus infection; dancing eyes-dancing feet syndrome;
Dandy-Walker syndrome; Dawson disease; De Morsier's syndrome;
Dejerine-Klumpke palsy; dementia; dermatomyositis; diabetic
neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia;
dystonias; early infantile epileptic encephalopathy; empty sella
syndrome; encephalitis; encephaloceles; encephalotrigeminal
angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's
disease; Fahr's syndrome; fainting; familial spastic paralysis;
febrile seizures; Fisher syndrome; Friedreich's ataxia;
frontotemporal dementia and other "tauopathies"; Gaucher's disease;
Gerstmann's syndrome; giant cell arteritis; giant cell inclusion
disease; globoid cell leukodystrophy; Guillain-Barre syndrome;
HTFV-1 associated myelopathy; Hallervorden-Spatz disease; head
injury; headache; hemifacial spasm; hereditary spastic paraplegia;
heredopathia atactica polyneuritiformis; herpes zoster oticus;
herpes zoster; Hirayama syndrome; HIV-associated dementia and
neuropathy (see also neurological manifestations of AIDS);
holoprosencephaly; Huntington's disease and other polyglutamine
repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism;
hypoxia; immune-mediated encephalomyelitis; inclusion body
myositis; incontinentia pigmenti; infantile; phytanic acid storage
disease; Infantile Refsum disease; infantile spasms; inflammatory
myopathy; intracranial cyst; intracranial hypertension; Joubert
syndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsbourne
syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander
disease; kuru; Fafora disease; Fambert-Eaton myasthenic syndrome;
Fandau-Kleffner syndrome; lateral medullary (Wallenberg) syndrome;
learning disabilities; Feigh's disease; Fennox-Gastaut syndrome;
Fesch-Nyhan syndrome; leukodystrophy; Fewy body dementia;
lissencephaly; locked-in syndrome; Fou Gehrig's disease (aka motor
neuron disease or amyotrophic lateral sclerosis); lumbar disc
disease; lyme disease-neurological sequelae; Machado-Joseph
disease; macrencephaly; megalencephaly; Melkersson-Rosenthal
syndrome; Menieres disease; meningitis; Menkes disease;
metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher
syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome;
monomelic amyotrophy; motor neurone disease; moyamoya disease;
mucopolysaccharidoses; multi-infarct dementia; multifocal motor
neuropathy; multiple sclerosis and other demyelinating disorders;
multiple system atrophy with postural hypotension; muscular
dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis;
myoclonic encephalopathy of infants; myoclonus; myopathy; myotonia
congenital; narcolepsy; neurofibromatosis; neuroleptic malignant
syndrome; neurological manifestations of AIDS; neurological
sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis;
neuronal migration disorders; Niemann-Pick disease;
O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal
dysraphism sequence; Ohtahara syndrome; olivopontocerebellar
atrophy; opsoclonus myoclonus; optic neuritis; orthostatic
hypotension; overuse syndrome; paresthesia; Parkinson's disease;
paramyotonia congenita; paraneoplastic diseases; paroxysmal
attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease;
periodic paralyses; peripheral neuropathy; painful neuropathy and
neuropathic pain; persistent vegetative state; pervasive
developmental disorders; photic sneeze reflex; phytanic acid
storage disease; Pick's disease; pinched nerve; pituitary tumors;
polymyositis; porencephaly; Post-Polio syndrome; postherpetic
neuralgia (PHN); postinfectious encephalomyelitis; postural
hypotension; Prader-Willi syndrome; primary lateral sclerosis;
prion diseases; progressive; hemifacial atrophy; progressive
multifocal leukoencephalopathy; progressive sclerosing
poliodystrophy; progressive supranuclear palsy; pseudotumor
cerebri; Ramsay-Hunt syndrome (Type I and Type II); Rasmussen's
Encephalitis; reflex sympathetic dystrophy syndrome; Refsum
disease; repetitive motion disorders; repetitive stress injuries;
restless legs syndrome; retrovirus-associated myelopathy; Rett
syndrome; Reye's syndrome; Saint Vitus Dance; Sandhoff disease;
Schilder's disease; schizencephaly; septo-optic dysplasia; shaken
baby syndrome; shingles; Shy-Drager syndrome; Sjogren's syndrome;
sleep apnea; Soto's syndrome; spasticity; spina bifida; spinal cord
injury; spinal cord tumors; spinal muscular atrophy; stiff-person
syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing
panencephalitis; subarachnoid hemorrhage; subcortical
arteriosclerotic encephalopathy; sydenham chorea; syncope;
syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal
arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic
outlet syndrome; tic douloureux; Todd's paralysis; Tourette
syndrome; transient ischemic attack; transmissible spongiform
encephalopathies; transverse myelitis; traumatic brain injury;
tremor; trigeminal neuralgia; tropical spastic paraparesis;
tuberous sclerosis; vascular dementia (multi-infarct dementia);
vasculitis including temporal arteritis; Von Hippel-Lindau Disease
(VHL); Wallenberg's syndrome; Werdnig-Hoffman disease; West
syndrome; whiplash; Williams syndrome; Wilson's disease; and
Zellweger syndrome.
[0131] A "painful condition" includes, but is not limited to,
neuropathic pain (e.g., peripheral neuropathic pain), central pain,
deafferentiation pain, chronic pain (e.g., chronic nociceptive
pain, and other forms of chronic pain such as post-operative pain,
e.g., pain arising after hip, knee, or other replacement surgery),
pre-operative pain, stimulus of nociceptive receptors (nociceptive
pain), acute pain (e.g., phantom and transient acute pain),
noninflammatory pain, inflammatory pain, pain associated with
cancer, wound pain, burn pain, postoperative pain, pain associated
with medical procedures, pain resulting from pruritus, painful
bladder syndrome, pain associated with premenstrual dysphoric
disorder and/or premenstrual syndrome, pain associated with chronic
fatigue syndrome, pain associated with pre-term labor, pain
associated with withdrawal symptoms from drug addiction, joint
pain, arthritic pain (e.g., pain associated with crystalline
arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis,
reactive arthritis, rheumatoid arthritis or Reiter's arthritis),
lumbosacral pain, musculo-skeletal pain, headache, migraine, muscle
ache, lower back pain, neck pain, toothache, dental/maxillofacial
pain, visceral pain and the like. One or more of the painful
conditions contemplated herein can comprise mixtures of various
types of pain provided above and herein (e.g. nociceptive pain,
inflammatory pain, neuropathic pain, etc.). In some embodiments, a
particular pain can dominate. In other embodiments, the painful
condition comprises two or more types of pains without one
dominating. A skilled clinician can determine the dosage to achieve
a therapeutically effective amount for a particular subject based
on the painful condition.
[0132] The term "psychiatric disorder" refers to a disease of the
mind and includes diseases and disorders listed in the Diagnostic
and Statistical Manual of Mental Disorders--Fourth Edition
(DSM-IV), published by the American Psychiatric Association,
Washington D. C. (1994). Psychiatric disorders include, but are not
limited to, anxiety disorders (e.g., acute stress disorder
agoraphobia, generalized anxiety disorder, obsessive-compulsive
disorder, panic disorder, posttraumatic stress disorder, separation
anxiety disorder, social phobia, and specific phobia), childhood
disorders, (e.g., attention-deficit/hyperactivity disorder, conduct
disorder, and oppositional defiant disorder), eating disorders
(e.g., anorexia nervosa and bulimia nervosa), mood disorders (e.g.,
depression, bipolar disorder, cyclothymic disorder, dysthymic
disorder, and major depressive disorder), personality disorders
(e.g., antisocial personality disorder, avoidant personality
disorder, borderline personality disorder, dependent personality
disorder, histrionic personality disorder, narcissistic personality
disorder, obsessive-compulsive personality disorder, paranoid
personality disorder, schizoid personality disorder, and
schizotypal personality disorder), psychotic disorders (e.g., brief
psychotic disorder, delusional disorder, schizoaffective disorder,
schizophreniform disorder, schizophrenia, and shared psychotic
disorder), substance-related disorders (e.g., alcohol dependence,
amphetamine dependence, cannabis dependence, cocaine dependence,
hallucinogen dependence, inhalant dependence, nicotine dependence,
opioid dependence, phencyclidine dependence, and sedative
dependence), adjustment disorder, autism, delirium, dementia,
multi-infarct dementia, learning and memory disorders (e.g.,
amnesia and age-related memory loss), and Tourette's disorder.
[0133] A "cardiovascular disease" (CVD) refers to a disease that
involves the heart or blood vessels. Cardiovascular disease
includes coronary artery diseases (e.g., angina and myocardial
infarction (i.e., heart attack). Other types of cardiovascular
disease include, but are not limited to, stroke, hypertensive heart
disease, rheumatic heart disease, cardiomyopathy, atrial
fibrillation, congenital heart disease, endocarditis, aortic
aneurysms, peripheral artery disease and venous thrombosis. CVDs
that affect the blood vessels (i.e., "vascular diseases") include,
but are not limited to, coronary artery disease (i.e., coronary
heart disease or ischemic heart disease), peripheral arterial
disease, cerebrovascular disease (e.g., stroke), renal artery
stenosis, and aortic aneurysm. CVDs that affect the heart include,
but are not limited to, cardiomyopathy, hypertensive heart disease,
heart failure, pulmonary heart disease, cardiac dysrhythmias,
inflammatory heart disease (e.g., endocarditis, inflammatory
cardiomegaly, myocarditis), valvular heart disease, congenital
heart disease, and rheumatic heart disease.
[0134] An "infectious disease" refers to any disease caused by a
pathogen (i.e., pathogenic microorganisms). An infectious disease
may be caused by bacteria, viruses, parasites, or fungi. In certain
embodiments, the infectious disease is a bacterial infection. In
certain embodiments, the infectious disease is a viral
infection.
[0135] An "endocrine disease" refers to a disease involving the
endrocrine system. Examples of endrocrine diseases include, but are
not limited to, adrenal disorders (e.g., adrenal insufficiency
(e.g., Addison's disease, mineralocorticoid deficiency), adrenal
hormone excess (e.g., Conn's syndrome, Cushing's syndrome,
glucocorticoid remediable aldosteronism (GRA), pheochromocytoma),
congenital adrenal hyperplasia (i.e., adrenogenital syndrome),
adrenocortical carcinoma)), glucose homeostasis disorders (e.g.,
diabetes mellitus, hypoglycemia, glucagonoma), thyroid disorders
(e.g., goiter, hyperthyroidism (e.g., Graves-Basedow disease, toxic
multinodular goiter), hypothyroidism, thyroiditis (e.g.,
Hashimoto's thyroiditis), thyroid cancer, thyroid hormone
resistance), calcium homeostasis disorders and Metabolic bone
diseases (e.g., parathyroid gland disorders (e.g., primary
hyperparathyroidism, secondary hyperparathyroidism, tertiary
hyperparathyroidism, hypoparathyroidism (e.g.,
pseudohypoparathyroidism)), osteoporosis, psteitis deformans (i.e.,
Paget's disease of bone), rickets, osteomalacia), pituitary gland
disorders (e.g., diabetes insipidus, hypopituitarism (or
panhypopituitarism), pituitary tumors (e.g., pituitary adenomas,
prolactinoma (or hyperprolactinemia), acromegaly, gigantism,
Cushing's disease))), sex hormone disorders (e.g., disorders of sex
development or intersex disorders (e.g., hermaphroditism, gonadal
dysgenesis, androgen insensitivity syndromes), hypogonadism
(gonadotropin deficiency) (e.g., Kallmann syndrome, Klinefelter
syndrome, Turner syndrome, ovarian failure (i.e., premature
menopause), testicular failure), disorders of puberty (e.g.,
delayed puberty, precocious puberty), menstrual function or
fertility disorders (e.g., amenorrhea, polycystic ovary syndrome)),
and tumours of the endocrine glands.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0136] Therapeutic agents, diagnostic agents, and imaging agents
have an array of attendant complications that arise from poor
chemical and/or structural stability that can lead to the agent
being converted into an inactive and/or potentially immunogenic
form. Extensive efforts have been devoted to the development of
excipients, labels, and tags for use in the formulation of agents
to confer improved stability, solubility, duration of action, or
pharmacological properties. More generally, the modification of
biomolecules, small molecules, and other agents of via conjugation
of tags or labels is of great importance. One approach to tagging
drugs or other agents is through direct covalent modification.
Alternative means of modifying biomolecules that do not rely on
covalent bonding are desired; in particular, site-specific
non-covalent complexation. Supramolecular chemistry utilizes
specific, directional, reversible, non-covalent molecular
recognition motifs in order to achieve organization of molecules.
As described in the present invention, supramolecular chemistry can
be used to bind agents to excipients, labels, and other tags.
[0137] The present invention provides useful supramolecular
complexes comprising an agent associated with a tag, wherein the
agent is specifically bound to a host via non-covalent guest-host
interactions, and wherein the host is conjugated to the tag. The
present invention also provides methods and compounds useful in
preparing the supramolecular complexes described herein, and
methods of treating diseases using the supramolecular
complexes.
Supramolecular Complexes
[0138] The present invention provides supramolecular complexes
comprising an agent associated with a tag, wherein:
[0139] (1) the agent is specifically bound to a host via
non-covalent interactions; and
[0140] (2) the host is conjugated to the tag.
[0141] It is to be understood that the agent that is associated
with a tag can be anything. In certain embodiments, the agent that
is associated with a tag is selected from the group consisting of
biomolecules, small molecule drugs, imaging agents, and diagnostic
agents. In certain embodiments, the agent is a small molecule. In
certain embodiments, the agent is a therapeutic small molecule. In
certain embodiments, the agent is a biomolecule. In certain
embodiments, the agent is a therapeutic biomolecule. In certain
embodiments, the agent is a biomolecule selected from the group
consisting of proteins, peptides, nucleic acids, polysaccharides,
and lipids. In certain embodiments, the agent is a peptide. In
certain embodiments, the agent is a protein. In certain
embodiments, the agent is an enzyme. In certain embodiments, the
agent is an antibody or antibody fragment. In certain embodiments,
the agent is a monoclonal antibody. In certain embodiments, the
agent is a human antibody or a humanized antibody. In certain
embodiments, the agent is an antibody directed against human CD20.
Examples of anti-CD20 antibodies include, but are not limited to,
rituximab, obinutuzumab, ibritumomab tiuxetan, tositumomab,
ofatumumab (Genmab), obinutuzumab (Gazyva), ocaratuzumab,
ocrelizumab, and IMMU-106 (veltuzumab). In certain emnodiments, the
agent is an antibody useful in the treatment of cancer (e.g.,
trastuzumab, rituximab). In certain embodiments, the agent is a
polysaccharide. In certain embodiments, the agent is a lipid. In
certain embodiments, the agent is a nucleic acid, (e.g., DNA, RNA,
and hybrids thereof). In certain embodiments, the agent is a
protein. In certain embodiments, the agent is a therapeutic
protein. In certain embodiments, the agent is a modified protein.
In certain embodiments, the agent is a native or wild-type protein.
In certain embodiments, the agent is GLP-1 or an analogue thereof.
In certain embodiments, the agent is an interferon. In certain
embodiments, the agent is a growth factor (e.g., BMP-2, VEGF,
FGF-2, PDGF). In certain embodiments, the agent is a growth
hormone. In certain embodiments, the agent is glucagon. In certain
embodiments, the agent is insulin. In certain embodiments, the
agent is a derivative of insulin. In certain embodiments, the agent
is native or wild-type insulin.
[0142] As described herein, the agent is specifically bound to a
host via non-covalent interactions. It is to be understood that
when the agent binds to the host, a "supramolecular complex"
results. In certain embodiments, the host is a macrocycle. In
certain embodiments, the macrocyclic host is selected from the
group consisting of cucurbiturils, cyclodextrins, calixarenes,
pillararenes, porphyrins, metallacrowns, crown ethers,
cyclotriveratrylenes, cryptophanes, cyclophanes, and carcerands. In
certain embodiments, the macrocyclic host is a cyclophane. In
certain embodiments, the macrocyclic host is a cyclophane
comprising paraquat moieties. In certain embodiments, the host is
cyclobis(paraquat-p-phenylene). In certain embodiments, the
macrocyclic host is a cucurbituril, wherein cucurbituril is as
defined herein. In certain embodiments, the host is an optionally
substituted acyclic cucurbituril. See, e.g., Isaacs el at. Nat.
Chem. 2012, 4, 503-510. In certain embodiments, the macrocyclic
host is an optionally substituted cucurbituril. In certain
embodiments, the macrocyclic host is a substituted cucurbituril. In
certain embodiments, the host is an optionally substituted
cucurbit[n]uril (CB[n]), wherein n is an integer between 5-10,
inclusive. In certain embodiments, n is 5. In certain embodiments,
n is 6. In certain embodiments, n is 7. In certain embodiments, n
is 8. In certain embodiments, n is 9. In certain embodiments, n is
10. In certain embodiments, n is 7, and the host is optionally
substituted cucurbit[7]uril (CB[7]).
[0143] As generally defined herein, "host" or "host molecule"
refers to a moeity which forms a complex with a second moiety
(generally defined herein as "guest" or "guest moiety") via
non-covalent interactions. See, e.g., Lehn et al. Angewandte Chemie
International Edition in English 1988, 27(1): 89-112; Lehn et al.
Proc Natl Acad Sci USA 2002, 99(8): 4763-4768; Schneider et al.
Angew Chem Int Edit 1991, 30(11): 1417-1436. See, e.g., Liu et al.
Journal of the American Chemical Society 2005, 127(45):
15959-15967; Del Valle et al. Process Biochem 2004, 39(9):
1033-1046; Biedermann et al. Journal of the American Chemical
Society 2012, 134(37): 15318-15323. See, e.g., Davis et al. Nat Rev
Drug Discov 2004, 3(12): 1023-1035; Uekama et al. Grit Rev Ther
Drug 1987, 3(1): 1-40. It is to be understood that when a host
binds to a guest, a supramolecular complex is formed. A host may
bind to a guest via any combination of non-covalent interactions.
Examples of non-covalent interactions include, but are not limited
to, electrostatic interactions, .pi.-interactions, hydrogen bonds,
ionic bonds, van der Waals forces, and hydrophobic interactions. In
some instances, the host is acyclic. In some instances, a host is a
macrocycle (also referred to as a "inclusion compound"), and the
guest binds inside the cavity of the macrocycle. Examples of
macrocyclic hosts include, but are not limited to, cucurbiturils,
cyclodextrins, calixarenes, pillararenes, porphyrins,
metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes,
cyclophanes and carcerands.
[0144] As generally defined herein, "cucurbituril" refers to a
macrocyclic polymer comprising glycouril monomers linked by
methylene (--CH.sub.2--) bridges. A cucurbituril may be referred to
as "cucurbit[n]uril" or "CB[n]", wherein n is the number of
glycouril monomers present in the macrocycle. In certain
embodiments, the cucurbituril In certain embodiments, the
cucurbituril comprises 5 glycouril units (cucurbit[5]uril or
CB[5]). In certain embodiments, the cucurbituril comprises 6
glycouril units (cucurbit[6]uril or CB[6]). In certain embodiments,
the cucurbituril comprises 7 glycouril units (cucurbit[7]uril or
CB[7]). In certain embodiments, the cucurbituril comprises 8
glycouril units (cucurbit[8]uril or CB[8]). In certain embodiments,
the cucurbituril comprises 9 glycouril units (cucurbit[9]uril or
CB[9]). In certain embodiments, the cucurbituril comprises 10
glycouril units (cucurbit[10]uril or CB[10]). In certain
embodiments, the cucurbituril is substituted. In certain
embodiments, the cucurbituril is unsubstituted. Cucurbiturils, as
generally defined herein, are of the following formula:
##STR00007##
wherein a.sup.1 indicates the point of attachment to b.sup.1;
a.sup.2 indicates the point of attachment to b.sup.2; and n is an
integer between 2-20, inclusive. The formula above may be
optionally substituted. "Optionally substituted" refers to a
hydrogen atom being replaced with a substituent described herein.
For example, an unsubstituted cucurbituril comprising 7 glycouril
units (cucurbit[7]uril or CB[7]) is of the following formula:
##STR00008##
[0145] As generally defined herein, "cyclodextrin" refers to a
macrocyclic polymer comprising sugar monomers (often referred to as
"cyclic oligosaccharide" or "cycloamylose"). In some instances, a
cyclodextrin comprises 5 or more .alpha.-D-glucopyranoside units.
Typical cyclodextrins are made up of 6 glucose monomers
(alpha-cyclodextrin), 7 glucose monomers (beta-cyclodextrin), or 8
glucose monomers (gamma-cyclodextrin).
[0146] The supramolecular complexes described herein comprise an
agent associated with a tag. In certain embodiments, the tag is
selected from the group consisting of polymers, therapeutic agents,
targeting agents, radionuclides, fluorophores, chromophores,
phosphorescent agents, dyes, chemiluminescent agents, particles,
colorimetric labels, magnetic labels, haptens, biomolecules, small
molecules, fatty acids, hydrocarbon chains, excipients, and
diagnostic agents. The tag may be useful for stabilizing,
solubilizing, modulating the duration of action of the agent, or
improving the pharmacological properties of the agent. In certain
embodiments, the tag is a an excipient (e.g., pharmaceutically
acceptable excipient). Examines of pharmaceutically acceptable
excipients are provided elsewhere herein. In certain embodiments,
the tag is a polymer. In certain embodiments, the tag is a
polyboronic acid or a polyboronic ester. In certain embodiments,
the tag is a polyether. In certain embodiments, the tag is a
polyglycol. In certain embodiments, the tag is polyethylene glycol
(PEG). In certain embodiments, the tag is a copolymer of PEG. In
certain embodiments, the tag is PEG, and the PEG has a molecular
weight <1 kDa. In certain embodiments, the PEG has a molecular
weight between 1-10 kDa, inclusive. In certain embodiments, the PEG
has a molecular weight of approximately 5 kDa. In certain
embodiments, the PEG has a molecular weight between 5-10 kDa,
inclusive. In certain embodiments, the PEG has a molecular weight
of approximately 10 kDa. In certain embodiments, In certain
embodiments, the PEG has a molecular weight between 10-30 kDa,
inclusive. In certain embodiments, the PEG has a molecular weight
of approximately 30 kDa. In certain embodiments, the PEG has a
molecular weight >30 kDa. In certain embodiments, the PEG has a
molecular weight under 100 kDa.
[0147] The supramolecular complexes described herein comprise an
agent bound to a host via non-covalent interactions. In certain
embodiments, the non-covalent interactions are guest-host
interactions. In certain embodiments, the non-covalent interactions
involve electrostatic interactions, .pi.-interactions, van der
Waals forces, or hydrophobic interactions. In certain embodiments,
the non-covalent interactions involve hydrogen bonding. In certain
embodiments, the non-covalent interactions are site-specific. In
certain embodiments, the non-covalent interactions are
reversible.
[0148] It is to be understood that the supramolecular complexes
described herein comprise an agent which comprises one or more
guest moieties capable of binding to a host. In certain
embodiments, the guest moiety is an amino acid. In certain
embodiments, the agent is a biomolecule (e.g., peptide or protein)
comprising one or more amino acids, and the host binds to an amino
acid of the biomolecule. In certain embodiments, the agent is a
protein, and the host binds to an amino acid of the protein. In
certain embodiments, the host binds an amino acid selected from the
group consisting of phenylalanine, tryptophan, and tyrosine. In
certain embodiments, the host binds to phenylalanine. In certain
embodiments, the host binds an N-terminal amino acid of the
protein. In certain embodiments, the host binds to an N-terminal
phenylalanine, tryptophan, or tyrosine of the protein. In certain
embodiments, the host binds to an N-terminal phenylalanine of the
protein. In certain embodiments, the protein is insulin, and the
host binds to a phenylalanine of insulin. In certain embodiments,
the protein is insulin and the host binds to the B1 phenylalanine
of insulin. In certain embodiments, the host that binds to an amino
acid of insulin is an optionally substituted cucurbituril. In
certain embodiments, the host that binds to an amino acid of
insulin is an optionally substituted cucurbit[7]uril.
[0149] In particular embodiments of the invention, the agent
associated with a tag is a biomolecule, the host is a macrocycle,
and the tag is a polymer. In certain embodiments, the agent
associated with a tag is a protein, the host is an optionally
substituted cucurbituril, and the tag conjugated to the host is a
polymer. In certain embodiments, the agent associated with a tag is
a protein, the host is an optionally substituted cucurbituril, and
the tag conjugated to the host is an excipient (e.g.,
pharmaceutically acceptable excipient). In certain embodiments, the
agent associated with a tag is a protein, the host is an optionally
substituted cucurbituril, and the tag conjugated to the host is
PEG. In certain embodiments, the agent associated with a tag is a
protein, the host is an optionally substituted cucurbituril, and
the tag conjugated to the host is an excipient. In certain
embodiments, the agent associated with a tag is a protein, the host
is an optionally substituted cucurbituril, and the tag conjugated
to the host is a polymer. In certain embodiments, the protein is
insulin, the host is an optionally substituted cucurbituril, and
the tag is a polymer. In certain embodiments, the protein is
insulin, the host is an optionally substituted cucurbituril, and
the tag is an excipient. In certain embodiments, the protein is
insulin, the host is an optionally substituted cucurbituril, and
the tag is PEG. In certain embodiments, the protein is insulin, the
host is an optionally substituted cucurbit[7]uril, and the tag is a
polymer. In certain embodiments, the protein is insulin, the host
is an optionally substituted cucurbit[7]uril, and the tag is an
excipient. In certain embodiments, the protein is insulin, the host
is an optionally substituted cucurbit[7]uril, and the tag is PEG.
In certain embodiments, the protein is insulin, the host is
macrocycle, and the tag is polyboronic acid or polyboronic ester.
In certain embodiments, the protein is insulin, the host is an
optionally substituted cucurbituril, and the tag is polyboronic
acid or polyboronic ester.
[0150] In certain embodiments, the protein associated with a tag is
glucagon, the host is a macrocycle, and the tag is a polymer. In
certain embodiments, the protein associated with a tag is glucagon,
the host is a macrocycle, and the tag is an excipient. In certain
embodiments, the protein is glucagon, the host is a macrocycle, and
the tag is PEG. In certain embodiments, the protein is glucagon,
the host is an optionally substituted cucurbituril, and the tag is
a polymer. In certain embodiments, the protein is glucagon, the
host is an optionally substituted cucurbituril, and the tag is an
excipient. In certain embodiments, the protein is glucagon, the
host is an optionally substituted cucurbituril, and the tag is PEG.
In certain embodiments, the protein is glucagon, the host is an
optionally substituted cucurbit[7]uril, and the tag is a polymer.
In certain embodiments, the protein is glucagon, the host is an
optionally substituted cucurbit[7]uril, and the tag is an
excipient. In certain embodiments, the protein is glucagon, the
host is an optionally substituted cucurbit[7]uril, and the tag is
PEG. In certain embodiments, the protein is glucagon, the host is
macrocycle, and the tag is polyboronic acid or polyboronic ester.
In certain embodiments, the protein is glucagon, the host is an
optionally substituted cucurbituril, and the tag is polyboronic
acid or polyboronic ester.
[0151] In certain embodiments, the agent associated with a tag is
an antibody, the host is a macrocycle, and the tag is a polymer. In
certain embodiments, the agent is an anti-CD20 antibody, the host
is a macrocycle, and the tag is PEG. In certain embodiments, the
agent associated with a tag is an anti-CD20 protein, the host is an
optionally substituted cucurbituril, and the tag conjugated to the
host is a polymer. In certain embodiments, the agent associated
with a tag is an anti-CD20 protein, the host is an optionally
substituted cucurbituril, and the tag conjugated to the host is an
excipient. In certain embodiments, the agent associated with a tag
is an anti-CD20 protein, the host is an optionally substituted
cucurbituril, and the tag conjugated to the host is PEG. In certain
embodiments, the agent is an anti-CD20 antibody, the host is an
optionally substituted cucurbit[7]uril, and the tag is PEG. In
certain embodiments, the agent is an anti-CD20 antibody, the host
is an optionally substituted cucurbit[7]uril, and the tag is a
polymer. In certain embodiments, the agent is Rituximab, the host
is cucurbit[7]uril, and the tag is a polymer. In certain
embodiments, the agent is Rituximab, the host is cucurbit[7]uril,
and the tag is PEG.
[0152] The supramolecular complexes described herein comprise a
host conjugated to a tag. In certain embodiments, the host is
directly conjugated to the tag (i.e., via a covalent bond). In
certain embodiments, the host is conjugated to the tag via a
linker.
[0153] In certain embodiments, the linker is a hydrocarbon chain.
In certain embodiments, the linker is a polymer (e.g.,
polyethylene). In certain embodiments, the linker is an amino acid
or peptide linker. In certain embodiments, the linker comprises a
triazole. In certain embodiments, the linker comprises an amide. In
certain embodiments, the linker comprises a disulfide. In certain
embodiments, the linker comprises an ester. In certain embodiments,
the linker comprises a thioether. In certain embodiments, the
linker comprises a carbonate. In certain embodiments, the linker
comprises a carbamate. In certain embodiments, the linker comprises
a thiourea.
[0154] In certain embodiments, the linker is selected from the
group consisting of optionally substituted alkylene, optionally
substituted heteroalkylene, optionally substituted alkenylene,
optionally substituted heteroalkenylene, optionally substituted
alkynylene, optionally substituted heteroalkynylene, optionally
substituted carbocyclylene, optionally substituted heterocyclylene,
optionally substituted arylene, optionally substituted
heteroarylene, optionally substituted acylene, and combinations
thereof.
[0155] In certain embodiments, the host is conjugated to a tag via
a linker of Formula (L-1):
##STR00009##
wherein:
[0156] each of L.sup.1 and L.sup.2 is independently a bond,
optionally substituted alkylene, or optionally substituted
heteroalkylene; and
[0157] A is a bond, optionally substituted heterocyclyl, or
optionally substituted heteroaryl.
[0158] As generally defined herein, each of L.sup.1 and L.sup.2 is
independently a bond, optionally substituted alkylene, or
optionally substituted heteroalkylene. In certain embodiments, at
least one of L.sup.1 and L.sup.2 is a bond. In certain embodiments,
both L.sup.1 and L.sup.2 are bonds. In certain embodiments, at
least one of L.sup.1 and L.sup.2 is optionally substituted
alkylene. In certain embodiments, both L.sup.1 and L.sup.2 are
optionally substituted alkylene. In certain embodiments, at least
one of L.sup.1 and L.sup.2 is optionally substituted
heteroalkylene. In certain embodiments, both L.sup.1 and L.sup.2
are optionally substituted heteroalkylene. In certain embodiments,
L.sup.1 is optionally substituted alkylene and L.sup.2 is
optionally substituted heteroalkylene.
[0159] As generally defined herein, A is a bond, optionally
substituted heterocyclyl, or optionally substituted heteroaryl. In
certain embodiments, A is a bond. In certain embodiments, A is
optionally substituted heterocyclyl. In certain embodiments, A is
optionally substituted heteroaryl. In certain embodiments, A is
optionally substituted five-membered heteroaryl. In certain
embodiments, A is optionally substituted triazole. In certain
embodiments, A is a polycyclic ring system. In certain embodiments,
A is a tetracyclic ring system. In certain embodiments, A is a
tetracyclic ring system comprising a triazole moiety.
[0160] In certain embodiments, L.sup.1 is optionally substituted
alkylene, L.sup.2 is optionally substituted heteroalkylene, and A
comprises a triazole moiety.
[0161] In certain embodiments, the host is conjugated to the tag
via linker of Formula (L-2a) or (L-2b):
##STR00010##
wherein:
[0162] each of L.sup.1 and L.sup.2 is independently a bond,
optionally substituted alkylene or optionally substituted
heteroalkylene; and
[0163] Y is CR.sup.a or N;
[0164] m is an integer from 0-10, inclusive;
[0165] each instance of R.sup.a is independently hydrogen, halogen,
optionally substituted alkyl, optionally substituted heteroalkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted acyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl,
--OR.sup.aa, --SR.sup.aa, --N(R.sup.aa).sub.2, --NO.sub.2, or --CN,
or two R.sup.a attached to adjacent atoms are joined to form an
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl ring;
[0166] each occurrence of R.sup.aa is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl,
optionally substituted sulfonyl, an oxygen protecting group, or a
nitrogen protecting group, or two R.sup.aa are joined to form an
optionally substituted heterocyclic or optionally substituted
heteroaryl ring.
[0167] In certain embodiments, the linker is of Formula the linker
is of Formula (L-3a) or (L-3b):
##STR00011##
[0168] In certain specific embodiments, the linker is of one of the
following formulae:
##STR00012##
[0169] The present invention provides supramolecular complexes
comprising an agent associated with a tag. In certain embodiments,
the tag improves the stability of the agent. In certain
embodiments, the tag improves the solubility of the agent. In
certain embodiments, the agent is a therapeutic agent, and the tag
modulates the duration of action of the therapeutic agent. In
certain embodiments, the tag improves the pharmacological
properties of the agent. In particular embodiments of the present
invention, the agent is a biomolecule, and the tag helps to
stabilize the biomolecule. In certain embodiments, the agent is a
therapeutic biomolecule, and the tag modulates the duration of
action of the biomolecule. In certain embodiments, the agent is a
therapeutic biomolecule, and the tag improves the pharmacological
properties of the biomolecule. In certain embodiments, the agent is
a biomolecule, and the tag is a polymer. In certain embodiments,
the agent is a biomolecule, and the tag is an excipient. In certain
embodiments, the agent is a biomolecule, and the tag is
polyethylene glycol (PEG). In certain embodiments, the PEGylated
biomolecule exhibits greater stability and/or improved duration of
action compared with the biomolecule in its wild type or unmodified
form. In certain embodiments, the biomolecule is a protein, and the
tag is a polymer. In certain embodiments, the protein is a
therapeutic protein and the tag is a polymer. In certain
embodiments, the agent is a protein and the tag is a polymer. In
certain embodiments, the agent is a protein and the tag is an
excipient. In certain embodiments, the agent is a protein and the
tag is a PEG. In certain embodiments, the protein is insulin and
the tag is a polymer. In certain embodiments, the protein is
insulin and the tag is an excipient. In certain embodiments, the
protein is insulin and the tag is PEG. In certain embodiments, the
PEGylated insulin exhibits greater stability and/or improved or
sustained duration of action compared with insulin in its native or
unmodified form. In certain embodiments, the protein is glucagon
and the tag is a polymer. In certain embodiments, the protein is
glucagon and the tag is an excipient. In certain embodiments, the
protein is glucagon and the tag is PEG. In certain embodiments, the
PEGylated glucagon exhibits greater stability and/or improved or
sustained duration of action compared with glucagon in its native
or unmodified form. In certain embodiments, the agent is an
antibody and the tag is a polymer. In certain embodiments, the
agent is an antibody and the tag is an excipient. In certain
embodiments, the agent is an antibody and the tag is PEG. In
certain embodiments, the PEGylated antibody exhibits greater
stability and/or improved or sustained duration of action compared
with the antibody in its native or unmodified form. In certain
embodiments, the agent is an anti-CD20 antibody and the tag is an
excipient. In certain embodiments, the agent is an anti-CD20
antibody and the tag is PEG. In certain embodiments, the PEGylated
anti-CD20 antibody exhibits greater stability and/or improved or
sustained duration of action compared with the antibody in its
native or unmodified form.
Host-Tag Conjugates
[0170] The present invention also provides compounds which are
useful in preparing the supramolecular complexes described herein.
In general, these compounds comprise a host conjugated to a tag
(referred to herein as "host-tag conjugates"). In certain
embodiments, the host is a macrocycle. In certain embodiments, the
macrocyclic host is selected from the group consisting of
cucurbiturils, cyclodextrins, calixarenes, pillararenes,
porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes,
cryptophanes, and carcerands. In certain embodiments, the
macrocyclic host is a cucurbituril, wherein cucurbituril is as
defined herein. In certain embodiments, the macrocyclic host is an
optionally substituted cucurbituril. In certain embodiments, the
macrocyclic host is a substituted cucurbituril. In certain
embodiments, the host is an optionally substituted cucurbit[n]uril
(CB[n]), wherein n is an integer between 5-10, inclusive. In
certain embodiments, n is 5. In certain embodiments, n is 6. In
certain embodiments, n is 7. In certain embodiments, n is 8. In
certain embodiments, n is 9. In certain embodiments, n is 10. In
certain embodiments, n is 7, and the host is optionally substituted
optionally substituted cucurbit[7]uril (CB[7]).
[0171] In certain embodiments, the tag is selected from the group
consisting of polymers, therapeutic agents, targeting agents,
radionuclides, fluorophores, chromophores, phosphorescent agents,
dyes, chemiluminescent agents, particles, colorimetric labels,
magnetic labels, haptens, biomolecules, fatty acids, hydrocarbon
chains, small molecules, and diagnostic agents. In certain
embodiments, the tag is a polymer. In certain embodiments, the tag
is a polyboronic acid or a polyboronic ester. In certain
embodiments, the tag is a polyether. In certain embodiments, the
tag is a polyglycol. In certain embodiments, the tag is
polyethylene glycol (PEG). The PEG can be of any molecular
weight.
[0172] In certain embodiments, the host is conjugated to the tag
via a linker. In certain embodiments, the linker is selected from
the group consisting of optionally substituted alkylene, optionally
substituted heteroalkylene, optionally substituted alkenylene,
optionally substituted heteroalkenylene, optionally substituted
alkynylene, optionally substituted heteroalkynylene, optionally
substituted carbocyclylene, optionally substituted heterocyclylene,
optionally substituted arylene, optionally substituted
heteroarylene, optionally substituted acylene, and combinations
thereof.
[0173] In certain embodiments, the linker is a bond or optionally
substituted aliphatic. In certain embodiments, the linker is a
bond. The linker is optional, and the tag may be directly attached
to the host. In certain embodiments, the linker is a hydrocarbon
chain. In certain embodiments, the linker is a polymer (e.g.,
polyethylene). In certain embodiments, the linker is an amino acid
or peptide linker. In certain embodiments, the linker comprises a
triazole. In certain embodiments, the linker comprises an amide. In
certain embodiments, the linker comprises a disulfide. In certain
embodiments, the linker comprises an ester. In certain embodiments,
the linker comprises a thioether. In certain embodiments, the
linker comprises a carbonate. In certain embodiments, the linker
comprises a carbamate. In certain embodiments, the linker comprises
a thiourea.
[0174] In certain embodiments, the host-tag conjugate comprises a
cucurbituril conjugated to a polymer. In certain embodiments, the
cucurbituril is CB[7], and the polymer is PEG.
[0175] In certain embodiments, a compound comprising a host
conjugated to a tag via a linker is of Formula (CB-1):
CB-L.sup.1-A-L.sup.2-TAG (CB-1),
or a salt thereof, wherein:
[0176] CB is an optionally substituted cucurbituril;
[0177] TAG is a tag selected from the group consisting of polymers,
therapeutic agents, targeting agents, radionuclides, fluorophores,
chromophores, phosphorescent agents, dyes, chemiluminescent agents,
particles, colorimetric labels, magnetic labels, haptens,
biomolecules, small molecules, fatty acids, hydrocarbon chains, and
diagnostic agents;
[0178] each of L and L2 is independently a bond, optionally
substituted alkylene, or optionally substituted heteroalkylene;
and
[0179] A is a bond, optionally substituted heterocyclyl, or
optionally substituted heteroaryl.
[0180] In certain embodiments, A is optionally substituted
heteroaryl. In certain embodiments, A is optionally substituted
five-membered heteroaryl. In certain embodiments, A is optionally
substituted five-membered heteroaryl. A is an optionally
substituted triazole moiety.
[0181] As generally defined herein, "tag" or TAG is anything that
can be conjugated to the host. In certain embodiments, the tag is
selected from the group consisting of polymers, therapeutic agents,
targeting agents, radionuclides, fluorophores, chromophores,
phosphorescent agents, dyes, chemiluminescent agents, particles,
colorimetric labels, magnetic labels, haptens, biomolecules, small
molecules, fatty acids, hydrocarbon chains, and diagnostic agents.
In certain embodiments, the tag is a polymer. In certain
embodiments, the tag is a polyboronic acid or a polyboronic ester.
In certain embodiments, the tag is a polyether. In certain
embodiments, the tag is a polyglycol. In certain embodiments, the
tag is polyethylene glycol (PEG). In certain embodiments, the tag
is PEG, and the PEG has a molecular weight <1 kDa. In certain
embodiments, the PEG has a molecular weight between 1-10 kDa,
inclusive. In certain embodiments, the PEG has a molecular weight
of approximately 5 kDa. In certain embodiments, the PEG has a
molecular weight between 5-10 kDa, inclusive. In certain
embodiments, the PEG has a molecular weight of approximately 10
kDa. In certain embodiments, the PEG has a molecular weight between
10-30 kDa, inclusive. In certain embodiments, the PEG has a
molecular weight of approximately 30 kDa. In certain embodiments,
the PEG has a molecular weight >30 kDa. In certain embodiments,
the PEG has a molecular weight under 100 kDa.
[0182] In certain embodiments, a compound comprising a host
conjugated to a tag via a linker is of Formula (CB-2a) or
(CB-2b):
##STR00013##
wherein:
[0183] CB is an optionally substituted cucurbituril;
[0184] TAG is a tag selected from the group consisting of polymers,
therapeutic agents, targeting agents, radionuclides, fluorophores,
dyes, chemiluminescent agents, particles, colorimetric labels,
magnetic labels, haptens, biomolecules, small molecules, and
diagnostic agents;
[0185] each of L.sup.1 and L.sup.2 is independently a bond,
optionally substituted alkylene, or optionally substituted
heteroalkylene; and
[0186] Y is CR.sup.a or N;
[0187] each instance of R.sup.a is independently hydrogen, halogen,
optionally substituted alkyl, optionally substituted heteroalkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted acyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl,
--OR.sup.aa, --SR.sup.aa, --N(R.sup.aa).sub.2, --NO.sub.2, or --CN,
or two R.sup.a attached to adjacent atoms are joined to form an
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl ring;
[0188] each occurrence of R.sup.aa is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl,
optionally substituted sulfonyl, an oxygen protecting group, or a
nitrogen protecting group, or two R.sup.aa are joined to form an
optionally substituted heterocyclic or optionally substituted
heteroaryl ring.
[0189] In certain embodiments, the compounds of Formulae (CB-2a)
and (CB-2b) are of Formula (CB-3a) and (CB-3b), respectively:
##STR00014##
Or salt thereof.
[0190] In certain embodiments, L.sup.1 is optionally substituted
alkylene. In certain embodiments, L.sup.2 is optionally substituted
heteroalkylene. In certain embodiments, the compounds Formulae
(CB-3a) and (CB-3b) of one of the following formulae:
##STR00015##
or a salt thereof.
[0191] In certain embodiments, the host conjugated to a tag via a
linker is of the Formula (PEG-T-CB[7]):
##STR00016##
or a salt thereof.
[0192] In certain embodiments, the host conjugated to a tag via a
linker is of the Formula (CB-5):
##STR00017##
or salt thereof, wherein p is an integer between 0-20,
inclusive.
Methods
[0193] The present invention also provides methods of preparing
supramolecular complexes described herein, as well as compounds
that are useful in preparing the supramolecular complexes.
Methods for Preparing Supramolecular Complexes
[0194] Provided herein are methods for preparing agents associated
with tags, wherein the agent is specifically bound to a host via
non-covalent interactions, and wherein the host is conjugated to
the tag (referred to herein as "supramolecular complexes"). A
typical method for preparing a supramolecular conjugate of the
present invention comprises:
[0195] contacting an agent with a host;
[0196] wherein the host is conjugated to the tag; and
[0197] wherein the tag is selected from the group consisting of
polymers, therapeutic agents, targeting agents, radionuclides,
fluorophores, chromophores, phosphorescent agents, dyes,
chemiluminescent agents, particles, colorimetric labels, magnetic
labels, haptens, biomolecules, small molecules, and diagnostic
agents.
[0198] It is to be understood that the agent that is associated
with a tag can be anything. In certain embodiments, the agent that
is associated with a tag is selected from the group consisting of
biomolecules, small molecule drugs, imaging agents, and diagnostic
agents. In certain embodiments, the agent is a small molecule. In
certain embodiments, the agent is a therapeutic small molecule. In
certain embodiments, the agent is a biomolecule. In certain
embodiments, the agent is a therapeutic biomolecule. In certain
embodiments, the agent is a biomolecule selected from the group
consisting of proteins, peptides, nucleic acids, polysaccharides,
and lipids. In certain embodiments, the agent is a peptide. In
certain embodiments, the agent is a protein. In certain
embodiments, the agent is an enzyme. In certain embodiments, the
agent is an antibody or antibody fragment. In certain embodiments,
the agent is a monoclonal antibody. In certain embodiments, the
agent is a polyclonal antibody. In certain embodiments, the agent
is an antibody for human CD20. Examples of anti-CD20 antibodies
include, but are not limited to, rituximab, obinutuzumab,
Ibritumomab tiuxetan, tositumomab, Ofatumumab (Genmab),
obinutuzumab (Gazyva), Ocaratuzumab, Ocrelizumab, and IMMU-106
(veltuzumab). In certain emnodiments, the agent is an anti-cancer
antibody (e.g., trastuzumab, rituximab). In certain embodiments,
the agent is a polysaccharide. In certain embodiments, the agent is
a lipid. In certain embodiments, the agent is a nucleic acid.
(e.g., DNA, RNA, and hybrids thereof). In certain embodiments, the
agent is a protein. In certain embodiments, the agent is a
therapeutic protein. In certain embodiments, the agent is a
modified protein. In certain embodiments, the agent is a native or
wild-type protein. In certain embodiments, the agent is GLP-1 or an
analogue thereof. In certain embodiments, the agent is an
interferon. In certain embodiments, the agent is a growth factor
(e.g., BMP-2, VEGF, FGF-2, PDGF). In certain embodiments, the agent
is a growth hormone. In certain embodiments, the agent is glucagon.
In certain embodiments, the agent is insulin. In certain
embodiments, the agent is a derivative of insulin. In certain
embodiments, the agent is native or wild type insulin.
[0199] As described herein, method for preparing supramolecular
complexes of the present invention comprises contacting an agent
with a host. In certain embodiments, the host is a macrocycle. In
certain embodiments, the macrocyclic host is selected from the
group consisting of cucurbiturils, cyclodextrins, calixarenes,
pillararenes, porphyrins, metallacrowns, crown ethers,
cyclotriveratrylenes, cryptophanes, cyclophanes, and carcerands. In
certain embodiments, the macrocyclic host is a cyclophane. In
certain embodiments, the macrocyclic host is a cyclophane
comprising paraquat moieties. In certain embodiments, the host is
cyclobis(paraquat-p-phenylene). In certain embodiments, the
macrocyclic host is a cucurbituril, wherein cucurbituril is as
defined herein. In certain embodiments, the host is an optionally
substituted acyclic cucurbituril. See, e.g., Isaacs et al. Nat.
Chem. 2012, 4, 503-510. In certain embodiments, the macrocyclic
host is an optionally substituted cucurbituril. In certain
embodiments, the macrocyclic host is a substituted cucurbituril. In
certain embodiments, the host is an optionally substituted
cucurbit[n]uril (CB[n]), wherein n is an integer between 5-10,
inclusive. In certain embodiments, n is 5. In certain embodiments,
n is 6. In certain embodiments, n is 7. In certain embodiments, n
is 8. In certain embodiments, n is 9. In certain embodiments, n is
10. In certain embodiments, n is 7, and the host is optionally
substituted cucurbit[7]uril (CB[7]).
[0200] As described herein, the host is conjugated to a tag. In
certain embodiments, the tag is selected from the group consisting
of polymers, therapeutic agents, targeting agents, radionuclides,
fluorophores, chromophores, phosphorescent agents, dyes,
chemiluminescent agents, particles, colorimetric labels, magnetic
labels, haptens, biomolecules, small molecules, excipients, and
diagnostic agents. In certain embodiments, the tag is a polymer. In
certain embodiments, the tag is an excipient (e.g., a
pharmaceutically acceptable excipient). In certain embodiments, the
tag is a polyboronic acid or a polyboronic ester. In certain
embodiments, the tag is a polyether. In certain embodiments, the
tag is a polyglycol. In certain embodiments, the tag is
polyethylene glycol (PEG).
[0201] As described herein, the method for preparing supramolecular
complexes comprises contacting an agent (e.g., biomolecule, small
molecule, imaging agent, or diagnostic agent) with a host
conjugated to a tag. In certain embodiments, the method comprises
contacting a biomolecule with a host conjugated to a tag. In
certain embodiments, the method comprises contacting a protein with
a host conjugated to a tag. In certain embodiments, the method
comprises contacting a therapeutic protein with a host conjugated
to a tag. In certain embodiments, the method comprises contacting
insulin with a host conjugated to a tag.
[0202] In particular embodiments, the method comprises contacting a
biomolecule with a cucurbituril conjugated to a tag. In certain
embodiments, the method comprises contacting a biomolecule with an
optionally substituted CB[7] conjugated to a tag. In certain
embodiments, the method comprises contacting a biomolecule with a
host conjugated to a polymer. In certain embodiments, the method
comprises contacting a biomolecule with a host conjugated to
polyethylene glycol (PEG). In certain embodiments, the method
comprises contacting a biomolecule with a cucurbituril conjugated
to a polymer. In certain embodiments, the method comprises
contacting a biomolecule with CB[7] conjugated to PEG. In certain
embodiments, the method comprises contacting a biomolecule with a
compound of any one of Formulae (CB-1), (CB-2a), (CB-2b), (CB-3a),
(CB-3b), (CB-4a), (CB-4b), (CB-4c), (CB-4d), (CB-5) or
(PEG-T-CB[7]).
[0203] In particular embodiments, the method comprises contacting
insulin with an optionally substituted cucurbituril conjugated to a
polymer. In particular embodiments, the method comprises contacting
insulin with an optionally substituted CB[7] conjugated to PEG. In
particular embodiments, the method comprises contacting insulin
with a compound of any one of Formulae (CB-1), (CB-2a), (CB-2b),
(CB-3a), (CB-3b), (CB-4a), (CB-4b), (CB-4c), (CB-4d), (CB-5) or
(PEG-T-CB[7]).
[0204] In certain embodiments, the step of contacting an agent with
a host conjugated to a tag is performed in vitro. In certain
embodiments, the step of contacting an agent with a host conjugated
to a tag is performed in vivo. In certain embodiments, the step of
contacting is performed in a solvent. In certain embodiments, the
step of contacting is performed in an organic solvent. In certain
embodiments, the step of contacting is performed in an inorganic
solvent. In certain embodiments, the solvent is water. In certain
embodiments, the step of contacting is performed at ambient
temperature. In certain embodiments, the step of contacting is
performed at a temperature above ambient temperature. In certain
embodiments, an excess (i.e., superstoichiometric amount) of
host-tag conjugate relative to the agent is used. In certain
embodiments, an excess of the agent relative to the host-tag
conjugate is used.
Methods for Preparing Host-Tag Conjugates
[0205] As described herein, host molecules that are conjugated to
tags (also referred to herein as "host-tag conjugates") are
compounds that are useful in preparing supramolecular conjugates.
The present invention provides methods for preparing host-tag
conjugates.
[0206] It should be understood that any covalent bond-forming
reaction can be used to conjugate a tag to a host in order provide
a host-tag conjugate of the present invention. Exemplary reactions
include, but are not limited to, alkylation reactions, metathesis
reactions, addition reactions, substitution reactions,
cycloaddition reactions, etc. In certain embodiments, the reaction
used to conjugate the host to the tag is a cycloaddition reaction.
In certain embodiments, the cycloaddition is a [4+2] cycloaddition.
In certain embodiments, the cycloaddition is a 1,3-dipolar
cycloaddition. In certain embodiments, the reaction is an
azide-alkyne cycloaddition (i.e., Huisgen cycloaddition). In
certain embodiments, the cycloaddition is performed in the presence
of a catalyst. In certain embodiments, the catalyst is a copper
catalyst. In certain embodiments, the cycloaddition reaction is a
copper-free reaction. In certain embodiments, the azide-alkyne
cycloaddition involved a cyclic alkyne and is strain-promoted.
[0207] In certain embodiments, the reaction used to conjugate the
host to the tag is a "click chemistry" reaction (e.g., the Huisgen
alkyne-azide cycloaddition). It is to be understood that any "click
chemistry" reaction known in the art can be used to this end. Click
chemistry is a chemical approach introduced by Sharpless in 2001
and describes chemistry tailored to generate substances quickly and
reliably by joining small units together. See, e.g., Kolb, Finn and
Sharpless Angewandte Chemie International Edition (2001) 40:
2004-2021; Evans, Australian Journal of Chemistry (2007) 60:
384-395). Exemplary coupling reactions (some of which may be
classified as "click chemistry") include, but are not limited to,
formation of esters, thioesters, amides (e.g., such as peptide
coupling) from activated acids or acyl halides; nucleophilic
displacement reactions (e.g., such as nucleophilic displacement of
a halide or ring opening of strained ring systems); azide-alkyne
Huisgen cycloaddition; thiol-yne addition; imine formation; Michael
additions (e.g., maleimide addition); and Diels-Alder reactions
(e.g., tetrazine [4+2] cycloaddition).
[0208] In certain embodiments, the method of preparing a host-tag
conjugate comprises contacting a cucurbituril derivative comprising
a first click chemistry handle with a tag comprising a second click
chemistry handle. In certain embodiments, the method comprises
contacting a cucurbituril derivative comprising an azide moiety
with a tag comprising an alkyne moiety. In certain embodiments, the
alkyne is a strained alkyne. In certain embodiments, the method
comprises contacting a cucurbituril derivative comprising an alkyne
moiety with a tag comprising an azide moiety. In certain
embodiments, the alkyne moiety is a strained alkyne.
[0209] In certain embodiments, the method comprises coupling a
compound of Formula (Az-1):
##STR00018##
or salt thereof, with a compound of Formula (Ak-1):
##STR00019##
or salt thereof, to yield a compound of Formula (CB-2a) or
(CB-2b):
##STR00020##
or salt thereof, wherein:
[0210] CB is an optionally substituted cucurbituril;
[0211] TAG is a tag selected from the group consisting of polymers,
therapeutic agents, targeting agents, radionuclides, fluorophores,
dyes, chemiluminescent agents, particles, colorimetric labels,
magnetic labels, haptens, biomolecules, fatty acids, hydrocarbon
chains, small molecules, excipients, and diagnostic agents;
[0212] each of L.sup.1 and L.sup.2 is independently a bond,
optionally substituted alkylene or heteroalkylene;
[0213] m is an integer from 0-10, inclusive;
[0214] each instance of R.sup.a is independently hydrogen, halogen,
optionally substituted alkyl, optionally substituted heteroalkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted acyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl,
--OR.sup.aa, --SR.sup.aa, --N(R.sup.aa).sub.2, --NO.sub.2, or --CN,
or two R.sup.a attached to adjacent atoms are joined to form an
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl ring; and
[0215] each occurrence of R.sup.aa is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl,
optionally substituted sulfonyl, an oxygen protecting group, or a
nitrogen protecting group, or two R.sup.aa are joined to form an
optionally substituted heterocyclic or optionally substituted
heteroaryl ring.
[0216] In certain embodiments, CB is optionally substituted
cucurbit[7]uril (CB[7]). In certain embodiments, CB is substituted
cucurbit[7]uril (CB[7]). In certain embodiments, CB is
unsubstituted cucurbit[7]uril (CB[7]).
[0217] In certain embodiments, the method comprises contacting a
compound of Formula (Az-1), or salt thereof, with a compound of one
of the following formulae:
##STR00021##
or a salt thereof.
[0218] In particular embodiments, the compound of Formula (Az-1) is
of Formula (Az-2):
##STR00022##
or salt thereof.
[0219] In certain embodiments, the tag with a click chemistry
handle is of Formula (PEG-DBCO):
##STR00023##
or salt thereof.
Pharmaceutical Compositions, Kits, and Administration
[0220] The present disclosure provides pharmaceutical compositions
comprising a supramolecular complex or acceptable salt thereof, and
optionally a pharmaceutically acceptable excipient. In certain
embodiments, the pharmaceutical composition described herein
comprises any supramolecular complex described herein or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable excipient.
[0221] In certain embodiments, the supramolecular complex described
herein is provided in an effective amount in the pharmaceutical
composition. In certain embodiments, the effective amount is a
therapeutically effective amount. In certain embodiments, the
effective amount is a prophylactically effective amount. In certain
embodiments, the effective amount is an amount effective for
treating a metabolic disorder in a subject in need thereof. In
certain embodiments, the effective amount is an amount effective
for preventing a metabolic disorder in a subject in need thereof.
In certain embodiments, the effective amount is an amount effective
for treating a diabetic condition in a subject in need thereof. In
certain embodiments, the effective amount is an amount effective
for preventing a diabetic condition in a subject in need thereof.
In certain embodiments, the effective amount is an amount effective
for treating a proliferative disease in a subject in need thereof.
In certain embodiments, the effective amount is an amount effective
for preventing a proliferative disease in a subject in need
thereof. In certain embodiments, the effective amount is an amount
effective for treating cancer (e.g., a lymphoma or leukemia) in a
subject in need thereof. In certain embodiments, the effective
amount is an amount effective for preventing a recurrence of cancer
(e.g., a lymphoma or leukemia) in a subject in need thereof.
[0222] In certain embodiments, the subject is an animal. The animal
may be of either sex and may be at any stage of development. In
certain embodiments, the subject described herein is a human. In
certain embodiments, the subject is a non-human animal. In certain
embodiments, the subject is a mammal. In certain embodiments, the
subject is a non-human mammal. In certain embodiments, the subject
is a domesticated animal, such as a dog, cat, cow, pig, horse,
sheep, or goat. In certain embodiments, the subject is a companion
animal, such as a dog or cat. In certain embodiments, the subject
is a livestock animal, such as a cow, pig, horse, sheep, or goat.
In certain embodiments, the subject is a zoo animal. In another
embodiment, the subject is a research animal, such as a rodent
(e.g., mouse, rat), dog, pig, or non-human primate. In certain
embodiments, the animal is a genetically engineered animal. In
certain embodiments, the animal is a transgenic animal (e.g.,
transgenic mice and transgenic pigs). In certain embodiments, the
subject is a fish or reptile.
[0223] Pharmaceutical compositions described herein can be prepared
by any method known in the art of pharmacology. In general, such
preparatory methods include bringing the compound described herein
(i.e., the "active ingredient") into association with a carrier or
excipient, and/or one or more other accessory ingredients, and
then, if necessary and/or desirable, shaping, and/or packaging the
product into a desired single- or multi-dose unit.
[0224] Pharmaceutical compositions can be prepared, packaged,
and/or sold in bulk, as a single unit dose, and/or as a plurality
of single unit doses. A "unit dose" is a discrete amount of the
pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient is generally
equal to the dosage of the active ingredient which would be
administered to a subject and/or a convenient fraction of such a
dosage, such as one-half or one-third of such a dosage.
[0225] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition described herein will
vary, depending upon the idagent, size, and/or condition of the
subject treated and further depending upon the route by which the
composition is to be administered. The composition may comprise
between 0.1% and 100% (w/w) active ingredient.
[0226] Pharmaceutically acceptable excipients used in the
manufacture of provided pharmaceutical compositions include inert
diluents, dispersing and/or granulating agents, surface active
agents and/or emulsifiers, disintegrating agents, binding agents,
preservatives, buffering agents, lubricating agents, and/or oils.
Excipients such as cocoa butter and suppository waxes, coloring
agents, coating agents, sweetening, flavoring, and perfuming agents
may also be present in the composition.
[0227] Exemplary diluents include calcium carbonate, sodium
carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate,
calcium hydrogen phosphate, sodium phosphate lactose, sucrose,
cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,
inositol, sodium chloride, dry starch, cornstarch, powdered sugar,
and mixtures thereof.
[0228] Exemplary granulating and/or dispersing agents include
potato starch, corn starch, tapioca starch, sodium starch
glycolate, clays, alginic acid, guar gum, citrus pulp, agar,
bentonite, cellulose, and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and
mixtures thereof.
[0229] Exemplary surface active agents and/or emulsifiers include
natural emulsifiers (e.g., acacia, agar, alginic acid, sodium
alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,
gelatin, egg yolk, casein, wool fat, cholesterol, wax, and
lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and
Veegum (magnesium aluminum silicate)), long chain amino acid
derivatives, high molecular weight alcohols (e.g., stearyl alcohol,
cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene
glycol distearate, glyceryl monostearate, and propylene glycol
monostearate, polyvinyl alcohol), carbomers (e.g., carboxy
polymethylene, polyacrylic acid, acrylic acid polymer, and
carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene
sorbitan monolaurate (Tween.RTM. 20), polyoxyethylene sorbitan
(Tween.RTM. 60), polyoxyethylene sorbitan monooleate (Tween.RTM.
80), sorbitan monopalmitate (Span.RTM. 40), sorbitan monostearate
(Span.RTM. 60), sorbitan tristearate (Span.RTM. 65), glyceryl
monooleate, sorbitan monooleate (Span.RTM. 80), polyoxyethylene
esters (e.g., polyoxyethylene monostearate (Myrj.RTM. 45),
polyoxyethylene hydrogenated castor oil, polyethoxylated castor
oil, polyoxymethylene stearate, and Solutol.RTM.), sucrose fatty
acid esters, polyethylene glycol fatty acid esters (e.g.,
Cremophor.RTM.), polyoxyethylene ethers, (e.g., polyoxyethylene
lauryl ether (Brij.RTM. 30)), poly(vinyl-pyrrolidone), diethylene
glycol monolaurate, triethanolamine oleate, sodium oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium
lauryl sulfate, Pluronic.RTM. F-68, poloxamer P-188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, and/or mixtures thereof. In certain embodiments of the
invention, one or more of these surface active agents/emulsifiers
is the tag conjugated to the host in the supramolecular complexes
described herein. In certain embodiments of the invention, one or
more of these surface active agents/emulsifiers is the tag
conjugated to the host in the host-tag conjugates described
herein.
[0230] Exemplary binding agents include starch (e.g., cornstarch
and starch paste), gelatin, sugars (e.g., sucrose, glucose,
dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.),
natural and synthetic gums (e.g., acacia, sodium alginate, extract
of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks,
carboxymethylcellulose, methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, microcrystalline cellulose, cellulose acetate,
poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum.RTM.),
and larch arabogalactan), alginates, polyethylene oxide,
polyethylene glycol, inorganic calcium salts, silicic acid,
polymethacrylates, waxes, water, alcohol, and/or mixtures
thereof.
[0231] Exemplary preservatives include antioxidants, chelating
agents, antimicrobial preservatives, antifungal preservatives,
antiprotozoan preservatives, alcohol preservatives, acidic
preservatives, and other preservatives. In certain embodiments, the
preservative is an antioxidant. In other embodiments, the
preservative is a chelating agent.
[0232] Exemplary antioxidants include alpha tocopherol, ascorbic
acid, acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and sodium sulfite.
[0233] Exemplary chelating agents include
ethylenediaminetetraacetic acid (EDTA) and salts and hydrates
thereof (e.g., sodium edetate, disodium edetate, trisodium edetate,
calcium disodium edetate, dipotassium edetate, and the like),
citric acid and salts and hydrates thereof (e.g., citric acid
monohydrate), fumaric acid and salts and hydrates thereof, malic
acid and salts and hydrates thereof, phosphoric acid and salts and
hydrates thereof, and tartaric acid and salts and hydrates thereof.
Exemplary antimicrobial preservatives include benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and thimerosal.
[0234] Exemplary antifungal preservatives include butyl paraben,
methyl paraben, ethyl paraben, propyl paraben, benzoic acid,
hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium
benzoate, sodium propionate, and sorbic acid.
[0235] Exemplary alcohol preservatives include ethanol,
polyethylene glycol, phenol, phenolic compounds, bisphenol,
chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
[0236] Exemplary acidic preservatives include vitamin A, vitamin C,
vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic
acid, ascorbic acid, sorbic acid, and phytic acid. Other
preservatives include tocopherol, tocopherol acetate, deteroxime
mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated
hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate
(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium
metabisulfite, potassium sulfite, potassium metabisulfite,
Glydant.RTM. Plus, Phenonip.RTM., methylparaben, Germall.RTM. 115,
Germaben.RTM. II, Neolone.RTM., Kathon.RTM., and Euxyl.RTM..
[0237] Exemplary buffering agents include citrate buffer solutions,
acetate buffer solutions, phosphate buffer solutions, ammonium
chloride, calcium carbonate, calcium chloride, calcium citrate,
calcium glubionate, calcium gluceptate, calcium gluconate,
D-gluconic acid, calcium glycerophosphate, calcium lactate,
propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium
phosphate, phosphoric acid, tribasic calcium phosphate, calcium
hydroxide phosphate, potassium acetate, potassium chloride,
potassium gluconate, potassium mixtures, dibasic potassium
phosphate, monobasic potassium phosphate, potassium phosphate
mixtures, sodium acetate, sodium bicarbonate, sodium chloride,
sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic
sodium phosphate, sodium phosphate mixtures, tromethamine,
magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free
water, isotonic saline, Ringer's solution, ethyl alcohol, and
mixtures thereof.
[0238] Exemplary lubricating agents include magnesium stearate,
calcium stearate, stearic acid, silica, talc, malt, glyceryl
behanate, hydrogenated vegetable oils, polyethylene glycol, sodium
benzoate, sodium acetate, sodium chloride, leucine, magnesium
lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
[0239] Exemplary natural oils include almond, apricot kernel,
avocado, babassu, bergamot, black current seed, borage, cade,
camomile, canola, caraway, carnauba, castor, cinnamon, cocoa
butter, coconut, cod liver, coffee, corn, cotton seed, emu,
eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,
grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui
nut, lavandin, lavender, lemon, litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary synthetic oils include, but are not
limited to, butyl stearate, caprylic triglyceride, capric
triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,
isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,
silicone oil, and mixtures thereof.
[0240] Liquid dosage forms for oral and parenteral administration
include pharmaceutically acceptable emulsions, microemulsions,
solutions, suspensions, syrups and elixirs. In addition to the
active ingredients, the liquid dosage forms may comprise inert
diluents commonly used in the art such as, for example, water or
other solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ,
olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan,
and mixtures thereof. Besides inert diluents, the oral compositions
can include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents. In
certain embodiments for parenteral administration, the complexes
described herein are mixed with solubilizing agents such as
Cremophor.RTM., alcohols, oils, modified oils, glycols,
polysorbates, cyclodextrins, polymers, and mixtures thereof.
[0241] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0242] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0243] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This can be accomplished by the use of a
liquid suspension of crystalline or amorphous material with poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution, which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form may be
accomplished by dissolving or suspending the drug in an oil
vehicle.
[0244] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing the
complexes described herein with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol, or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active ingredient.
[0245] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active ingredient is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or (a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
(b) binders such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia,
(c) humectants such as glycerol, (d) disintegrating agents such as
agar, calcium carbonate, potato or tapioca starch, alginic acid,
certain silicates, and sodium carbonate, (e) solution retarding
agents such as paraffin, (f) absorption accelerators such as
quaternary ammonium compounds, (g) wetting agents such as, for
example, cetyl alcohol and glycerol monostearate, (h) absorbents
such as kaolin and bentonite clay, and (i) lubricants such as talc,
calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of
capsules, tablets, and pills, the dosage form may include a
buffering agent.
[0246] Solid compositions of a similar type can be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the art of pharmacology. They may optionally
comprise opacifying agents and can be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of encapsulating compositions which can be used
include polymeric substances and waxes. Solid compositions of a
similar type can be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as
well as high molecular weight polyethylene glycols and the
like.
[0247] The active ingredient can be in a micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings, and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active ingredient can be admixed with at least one inert diluent
such as sucrose, lactose, or starch. Such dosage forms may
comprise, as is normal practice, additional substances other than
inert diluents, e.g., tableting lubricants and other tableting aids
such a magnesium stearate and microcrystalline cellulose. In the
case of capsules, tablets and pills, the dosage forms may comprise
buffering agents. They may optionally comprise opacifying agents
and can be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
encapsulating agents which can be used include polymeric substances
and waxes.
[0248] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by the skilled artisan that such compositions
are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design
and/or perform such modification with ordinary experimentation.
[0249] Complexes provided herein are typically formulated in dosage
unit form for ease of administration and uniformity of dosage. It
will be understood, however, that the total daily usage of the
compositions described herein will be decided by a physician within
the scope of sound medical judgment. The specific therapeutically
effective dose level for any particular subject or organism will
depend upon a variety of factors including the disease being
treated and the severity of the disorder; the activity of the
specific active ingredient employed; the specific composition
employed; the age, body weight, general health, sex, and diet of
the subject; the time of administration, route of administration,
and rate of excretion of the specific active ingredient employed;
the duration of the treatment; drugs used in combination or
coincidental with the specific active ingredient employed; and like
factors well known in the medical arts.
[0250] The complexes and compositions provided herein can be
administered by any route, including enteral (e.g., oral),
parenteral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, subcutaneous, intraventricular,
transdermal, interdermal, rectal, intravaginal, intraperitoneal,
topical (as by powders, ointments, creams, and/or drops), mucosal,
nasal, bucal, sublingual; by intratracheal instillation, bronchial
instillation, and/or inhalation; and/or as an oral spray, nasal
spray, and/or aerosol. Specifically contemplated routes are oral
administration, intravenous administration (e.g., systemic
intravenous injection), regional administration via blood and/or
lymph supply, and/or direct administration to an affected site. In
general, the most appropriate route of administration will depend
upon a variety of factors including the nature of the agent (e.g.,
its stability in the environment of the gastrointestinal tract),
and/or the condition of the subject (e.g., whether the subject is
able to tolerate oral administration). In certain embodiments, the
compound or pharmaceutical composition described herein is suitable
for topical administration to the eye of a subject.
[0251] The exact amount of a complex required to achieve an
effective amount will vary from subject to subject, depending, for
example, on species, age, and general condition of a subject,
severity of the side effects or disorder, idagent of the particular
compound, mode of administration, and the like. An effective amount
may be included in a single dose (e.g., single oral dose) or
multiple doses (e.g., multiple oral doses). In certain embodiments,
when multiple doses are administered to a subject or applied to a
tissue or cell, any two doses of the multiple doses include
different or substantially the same amounts of a compound described
herein. In certain embodiments, when multiple doses are
administered to a subject or applied to a tissue or cell, the
frequency of administering the multiple doses to the subject or
applying the multiple doses to the tissue or cell is three doses a
day, two doses a day, one dose a day, one dose every other day, one
dose every third day, one dose every week, one dose every two
weeks, one dose every three weeks, or one dose every four weeks. In
certain embodiments, the frequency of administering the multiple
doses to the subject or applying the multiple doses to the tissue
or cell is one dose per day. In certain embodiments, the frequency
of administering the multiple doses to the subject or applying the
multiple doses to the tissue or cell is two doses per day. In
certain embodiments, the frequency of administering the multiple
doses to the subject or applying the multiple doses to the tissue
or cell is three doses per day. In certain embodiments, when
multiple doses are administered to a subject or applied to a tissue
or cell, the duration between the first dose and last dose of the
multiple doses is one day, two days, four days, one week, two
weeks, three weeks, one month, two months, three months, four
months, six months, nine months, one year, two years, three years,
four years, five years, seven years, ten years, fifteen years,
twenty years, or the lifetime of the subject, tissue, or cell. In
certain embodiments, the duration between the first dose and last
dose of the multiple doses is three months, six months, or one
year. In certain embodiments, the duration between the first dose
and last dose of the multiple doses is the lifetime of the subject,
tissue, or cell. In certain embodiments, a dose (e.g., a single
dose, or any dose of multiple doses) described herein includes
independently between 0.1 .mu.g and 1 .mu.g, between 0.001 mg and
0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg,
between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30
mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between
300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a
compound described herein. In certain embodiments, a dose described
herein includes independently between 1 mg and 3 mg, inclusive, of
a compound described herein. In certain embodiments, a dose
described herein includes independently between 3 mg and 10 mg,
inclusive, of a compound described herein. In certain embodiments,
a dose described herein includes independently between 10 mg and 30
mg, inclusive, of a compound described herein. In certain
embodiments, a dose described herein includes independently between
30 mg and 100 mg, inclusive, of a compound described herein.
[0252] Dose ranges as described herein provide guidance for the
administration of provided pharmaceutical compositions to an adult.
The amount to be administered to, for example, a child or an
adolescent can be determined by a medical practitioner or person
skilled in the art and can be lower or the same as that
administered to an adult.
[0253] A complex or composition, as described herein, can be
administered in combination with one or more additional
pharmaceutical agents (e.g., therapeutically and/or
prophylactically active agents). The compounds or compositions can
be administered in combination with additional pharmaceutical
agents that improve their activity (e.g., activity (e.g., potency
and/or efficacy) in treating a disease in a subject in need
thereof, in preventing a disease in a subject in need thereof, in
reducing the risk to develop a disease in a subject in need
thereof, and/or in inhibiting the activity of a protein kinase in a
subject or cell), improve bioavailability, improve safety, reduce
drug resistance, reduce and/or modify metabolism, inhibit
excretion, and/or modify distribution in a subject or cell. It will
also be appreciated that the therapy employed may achieve a desired
effect for the same disorder, and/or it may achieve different
effects. In certain embodiments, a pharmaceutical composition
described herein including a compound described herein and an
additional pharmaceutical agent shows a synergistic effect that is
absent in a pharmaceutical composition including one of the
compound and the additional pharmaceutical agent, but not both.
[0254] The supramolecular complex or composition can be
administered concurrently with, prior to, or subsequent to one or
more additional pharmaceutical agents, which may be useful as,
e.g., combination therapies. Pharmaceutical agents include
therapeutically active agents. Pharmaceutical agents also include
prophylactically active agents. Pharmaceutical agents include small
organic molecules such as drug compounds (e.g., compounds approved
for human or veterinary use by the U.S. Food and Drug
Administration as provided in the Code of Federal Regulations
(CFR)), peptides, proteins, carbohydrates, monosaccharides,
oligosaccharides, polysaccharides, nucleoproteins, mucoproteins,
lipoproteins, synthetic polypeptides or proteins, small molecules
linked to proteins, glycoproteins, steroids, nucleic acids, DNAs,
RNAs, nucleotides, nucleosides, oligonucleotides, antisense
oligonucleotides, lipids, hormones, vitamins, and cells. In certain
embodiments, the additional pharmaceutical agent is a
pharmaceutical agent useful for treating and/or preventing a
disease (e.g., proliferative disease, hematological disease,
neurological disease, painful condition, psychiatric disorder, or
metabolic disorder). Each additional pharmaceutical agent may be
administered at a dose and/or on a time schedule determined for
that pharmaceutical agent. The additional pharmaceutical agents may
also be administered together with each other and/or with the
compound or composition described herein in a single dose or
administered separately in different doses. The particular
combination to employ in a regimen will take into account
compatibility of the compound described herein with the additional
pharmaceutical agent(s) and/or the desired therapeutic and/or
prophylactic effect to be achieved. In general, it is expected that
the additional pharmaceutical agent(s) in combination be utilized
at levels that do not exceed the levels at which they are utilized
individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually.
[0255] Also encompassed by the disclosure are kits (e.g.,
pharmaceutical packs). The kits provided may comprise a
pharmaceutical composition or supramolecular complex described
herein and a container (e.g., a vial, ampule, bottle, syringe,
and/or dispenser package, or other suitable container). In some
embodiments, provided kits may optionally further include a second
container comprising a pharmaceutical excipient for dilution or
suspension of a pharmaceutical composition or compound described
herein. In some embodiments, the pharmaceutical composition or
compound described herein provided in the first container and the
second container are combined to form one unit dosage form.
[0256] Thus, in one aspect, provided are kits including a first
container comprising a supramolecular complex or pharmaceutical
composition described herein. In certain embodiments, the kits are
useful for treating a disease (e.g., proliferative disease,
hematological disease, neurological disease, painful condition,
psychiatric disorder, or metabolic disorder) in a subject in need
thereof. In certain embodiments, the kits are useful for preventing
a disease (e.g., proliferative disease, hematological disease,
neurological disease, painful condition, psychiatric disorder, or
metabolic disorder) in a subject in need thereof. In certain
embodiments, the kits are useful for reducing the risk of
developing a disease (e.g., proliferative disease, hematological
disease, neurological disease, painful condition, psychiatric
disorder, or metabolic disorder) in a subject in need thereof. In
certain embodiments, the kits are useful for inhibiting the
activity (e.g., aberrant activity, such as increased activity) of a
protein kinase in a subject or cell.
[0257] In certain embodiments, a kit described herein further
includes instructions for using the kit. A kit described herein may
also include information as required by a regulatory agency such as
the U.S. Food and Drug Administration (FDA). In certain
embodiments, the information included in the kits is prescribing
information. In certain embodiments, the kits and instructions
provide for treating a disease (e.g., proliferative disease,
hematological disease, neurological disease, painful condition,
psychiatric disorder, or metabolic disorder) in a subject in need
thereof. In certain embodiments, the kits and instructions provide
for preventing a disease (e.g., proliferative disease,
hematological disease, neurological disease, painful condition,
psychiatric disorder, or metabolic disorder) in a subject in need
thereof. In certain embodiments, the kits and instructions provide
for reducing the risk of developing a disease (e.g., proliferative
disease, hematological disease, neurological disease, painful
condition, psychiatric disorder, or metabolic disorder) in a
subject in need thereof. In certain embodiments, the kits and
instructions provide for inhibiting the activity (e.g., aberrant
activity, such as increased activity) of a protein kinase in a
subject or cell. A kit described herein may include one or more
additional pharmaceutical agents described herein as a separate
composition.
Methods of Treatment and Use
[0258] The present invention also provides methods for the
treatment or prevention of diseases including, but not limited to,
proliferative diseases, diseases associated with angiogenesis,
genetic disorders, inflammatory diseases, cardiovascular diseases,
hepatic diseases, splenic diseases, gastrointestinal diseases,
pulmonary diseases, painful conditions, hematological diseases,
neurological diseases, psychiatric disorders, autoimmune diseases,
infectious diseases, neurological diseases, metabolic disease, or
endocrine diseases. In certain embodiments, a method of treating a
disease in a subject in need thereof comprises administering to the
subject a therapeutically effective amount of a supramolecular
complex described herein, or a salt thereof.
[0259] In certain embodiments, the disease is a metabolic disease.
In certain embodiments, the disease is diabetes. In certain
embodiments, the disease is Type II diabetes.
[0260] In certain embodiments, the disease is a proliferative
disease. In certain embodiments, the disease is cancer. In certain
embodiments, the disease is a hematological cancer. In certain
embodiments, the disease is a lymphoma, leukemia, or myeloma. In
certain embodiments, the disease is lymphoma. In certain
embodiments, the disease is leukemia. In certain embodiments, the
disease is chronic lymphocytic leukemia.
[0261] All types of autoimmune and inflammatory diseases disclosed
herein or known in the art are contemplated as being treatable
using the inventive compositions and methods described herein.
[0262] In certain embodiments, the subject being treated is a
mammal. In certain embodiments, the subject is a human. In certain
embodiments, the subject is a domesticated animal, such as a dog,
cat, cow, pig, horse, sheep, or goat. In certain embodiments, the
subject is a companion animal such as a dog or cat. In certain
embodiments, the subject is a livestock animal such as a cow, pig,
horse, sheep, or goat. In certain embodiments, the subject is a zoo
animal. In another embodiment, the subject is a research animal
such as a rodent, dog, or non-human primate. In certain
embodiments, the subject is a non-human transgenic animal such as a
transgenic mouse or transgenic pig.
[0263] In certain embodiments, the methods described herein include
administering to a subject or contacting a biological sample with
an effective amount of a supramolecular complex described herein,
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, or a pharmaceutical composition
thereof. In certain embodiments, the methods described herein
include administering to a subject or contacting a biological
sample with an effective amount of a supramolecular complex
described herein, or a pharmaceutically acceptable salt thereof, or
a pharmaceutical composition thereof. In certain embodiments, the
supramolecular complex is contacted with a biological sample. In
certain embodiments, the supramolecular complex is administered to
a subject. In certain embodiments, the supramolecular is
administered in combination with one or more additional
pharmaceutical agents.
[0264] The present invention also provides uses of supramolecular
complexes described herein for the treatment of diseases described
herein. The present invention also provides uses of supramolecular
complexes described herein for the preparation of medicaments for
treating diseases described herein.
EXAMPLES
[0265] In order that the invention described herein may be more
fully understood, the following examples are set forth. The
examples described in this application are offered to illustrate
the supramolecular complexes and methods provided herein and are
not to be construed in any way as limiting their scope.
PEGylation of Insulin
[0266] Insulin is one of the most widely used biopharmaceuticals,
and is a key component in the treatment of both type-I and type-II
diabetes. See, e.g., Patterson et al. Lancet 2009, 373(9680):
2027-2033; Zimmet et al. Nature 2001, 414(6865): 782-787; Wild et
al. Diabetes Care 2004, 27(5): 1047-1053. The protein is known to
have limited solution stability, even in refrigerated storage, and
is prone to the formation of amyloid fibrils. See, e.g., Jimenez et
al. Proc Natl Acad Sci USA 2002, 99(14): 9196-9201; Sluzky et al.
Proc Natl Acad Sci USA 1991, 88(21): 9377-9381; Fisher et al. The
Journal of pharmacy and pharmacology 1981, 33(4): 203-206. Insulin
used clinically is formulated with a number of excipients to
maximize its shelf life; most available formulations are stable for
up to one month when refrigerated. See, e.g., Grajower et al.
Diabetes Care 2003, 26(9): 2665-2669. However, the incorporation of
insulin into pumps or implantable delivery devices, along with
variable handling from patients, introduces complications that
could reduce protein stability, leading to limited therapeutic
efficacy. See, e.g., Lougheed et al. Diabetologia 1980, 19(1): 1-9;
Renard et al. Current opinion in pharmacology 2002, 2(6): 708-716.
Extensive work has been done to achieve site-specific PEGylation of
insulin to control both its physical stability and pharmacological
properties. See, e.g., Hinds et al. Adv Drug Deliv Rev 2002, 54(4):
505-530; Hinds et al. Bioconjug Chem 2000, 11(2): 195-201; Uchio et
al. Adv Drug Deliv Rev 1999, 35(2-3): 289-306. Specifically,
PEGylation has been shown to reduce insulin aggregation and fibril
formation, reduce immunogenicity and allergenicity, and increase
serum circulation half-life. Alternatively, a number of insulin
variants have been developed to reduce aggregation or promote
long-lasting activity, as well as other approaches to improve
stability by supramolecular zinc-stapling, and approaches to
facilitate glucose-responsive bioavailability. See, e.g., Hirsch I
B. N Engl J Med 2005, 352(2): 174-183; Owens D R. Nat Rev Drug
Discov 2002, 1(7): 529-540; Phillips et al. J Biol Chem 2010,
285(16): 11755-11759; Chou et al. Proc Natl Acad Sci USA 2015,
112(8): 2401-2406.
[0267] We developed a strategy to utilize a CB[7] host-modified
with a single PEG chain for supramolecular PEGylation of native
insulin via host-guest complexation. Supramolecular PEGylation of
native insulin enhances stability of the protein by preventing
aggregation and also provides longer lasting pharmacological
activity in a diabetic mouse model. This strategy could be employed
as an excipient-only approach to provide both enhanced stability
and long-lasting activity of therapeutic proteins through
supramolecular complex formation.
[0268] Though CB[7] exhibits exceptional binding affinities for a
broad range of guest molecules it has been significantly more
difficult to functionalize or conjugate than other classes of
cyclic host molecules. See, e.g., Rekharsky et al. Proc Natl Acad
Sci USA 2007, 104(52): 20737-20742; Moghaddam et al. J Am Chem Soc
2011, 133: 3570-3581; Cao et al. Angewandte Chemie 2014, 53(4):
988-993; Kim et al. Chem Soc Rev 2007, 36(2): 267-279; Lee et al.
Acc Chem Res 2003, 36(8): 621-630. Two synthetic routes have
recently been described to prepare monofunctionalized CB[7]
derivatives with synthetically useful handles. See, e.g.,
Vinciguerra et al. Journal of the American Chemical Society 2012,
134(31): 13133-13140; Ahn et al. Angewandte Chemie 2013, 52(11):
3140-3144; Cao et al. Angewandte Chemie 2013, 52(46): 12033-12037.
Using the Isaacs group methods, CB[7]-N.sub.3 was synthesized to
prepare supramolecular PEG conjugates by of copper-free "click"
chemistry. Dibenzocyclooctyne (DBCO)-modified poly(ethylene glycol)
(PEG-DBCO) polymers of varying molecular weights (M.sub.n=5 kDa, 10
kDa, and 30 kDa) were reacted with CB[7]-N.sub.3 to yield
PEG-T-CB[7], connected via a triazole linkage (FIG. 1A). PEG chains
of three different molecular weights were selected to enable a
study of the effect of PEG chain length on the stability and
pharmacological activity of native insulin following supramolecular
PEGylation.
[0269] Gel permeation chromatography (GPC) with UV/visible
detection (FIGS. 2A and 2B) was performed to verify the reaction
between PEG-DBCO and CB[7]-N.sub.3 and characterize the
PEG-T-CB[7]. The UV/visible spectrum upon GPC elution demonstrates
a characteristic change in absorbance from the PEG-DBCO starting
material to PEG-T-CB[7] resulting from triazole formation, with a
blue-shift in .sub.max from 290 nm to 248 nm. Moreover, these DBCO
and triazole signatures appear concurrent with elution of the
polymeric backbone, as indicated by the refractive index trace.
Additionally, matrix-assisted laser desorption ionization (MALDI)
mass spectrometry demonstrates a characteristic increase in
molecular weight for PEG-T-CB[7] relative to the PEG-DBCO starting
material (FIG. 2C). The shift in the observed peak of the MALDI-MS
spectrum following reaction of the 30 kDa PEG-DBCO with
CB[7]-N.sub.3 corresponds to a mass increase of approximately 1200
Da, consistent with addition of a single CB[7]-N.sub.3 moiety.
[0270] As mentioned above, CB[7] has been shown to bind to the B1
phenylalanine residue of native insulin. To confirm binding of
PEG-T-CB[7] to native insulin, a competitive binding assay was
performed using acridine orange (AO) (FIG. 2D). In this assay, the
fluorescent emission spectrum of AO is collected. The fluorescence
intensity of AO increases when in complex with CB[7] relative to
its emission when free in solution. As insulin is added, the CB[7]
AO complex dissociates as a result of stronger binding affinity of
the B1 Phenylalanine residue to the CB[7], resulting in a reduction
to the measured fluorescence intensity of AO. When fitting this
data to a one-site competitive binding model, no significant
difference in the binding constant toward insulin was observed in
comparing unmodified authentic CB[7] to PEG-T-CB[7] (CB[7],
K.sub.D=2.3.+-.0.2 .mu.M vs. PEG.sub.30k-T-CB[7],
K.sub.D=2.1.+-.0.3 .mu.M). Furthermore, binding to insulin by
PEG-T-CB[7] does not alter the secondary structure of native
insulin, as measured by circular dichroism spectroscopy (FIG. 7),
suggesting no negative effect of supramolecular PEGylation on the
secondary structure of the protein.
[0271] Insulin is well known to form fibrillar aggregates in
physiologic conditions. To determine whether PEG-T-CB[7] can
stabilize insulin and prevent its aggregation, assays were
performed in physiologic salt concentrations at pH 7.4 and
37.degree. C. with continuous agitation to determine the kinetics
of insulin aggregation in a solution of 1 mg/mL protein (FIG. 3A).
Specifically, transmittance was measured at 540 nm, a wavelength
where both insulin and the PEG-T-CB[7] have negligible absorbance
in order to detect the formation of aggregates, which scatter
light, in solution over time. Defining aggregation time (t.sub.A)
as the time after which a 10% reduction in transmittance is
observed, native insulin aggregates after 13.6.+-.0.2 h of
agitation and insulin formulated with unmodified CB[7] displays an
aggregation time of 14.2.+-.0.4 h. Meanwhile, insulin formulated
with all molecular weights of PEG-T-CB[7] did not aggregate in 100
hours of kinetic study. After the initial 100 h study, data were
collected once daily to determine the duration of stability for
each PEG-T-CB[7]/insulin formulation (FIG. 3B). At the 50 day
endpoint of these studies, formulation of insulin with
PEG.sub.5k-T-CB[7], PEG.sub.10k-T-CB[7], and PEG.sub.30k-T-CB[7]
remained stable and were not aggregated. Serial monitoring for 100
days indicated no insulin aggregation in CB[7]-PEG formulated
groups, and furthermore, in vitro activity assays at the study
endpoint demonstrated preserved insulin signaling for CB[7]-PEG
formulations (FIG. 4B). As a control, the addition of PEG polymers
of comparable molecular weights alone and in combination with
unmodified CB[7] was not found to prolong insulin stability (FIG.
8), highlighting the importance of supramolecular PEGylation with
PEG-T-CB[7] in enhancing the stability of native insulin.
[0272] Here we have demonstrated that supramolecular PEGylation
using PEG-T-CB[7] conjugates significantly inhibits insulin
aggregation. Since the process of insulin aggregation is initiated
by a stochastic nucleation event and is dependent on the specific
materials and conditions used, it can be challenging to directly
compare these findings to others reported previously. Aggregation
of insulin is most commonly studied using thioflavin T (ThT). See,
e.g., Krebs et al. Journal of structural biology 2005, 149(1):
30-37. In order to avoid confounding effects on account of
competitive binding of ThT to CB[7], aggregation was monitored here
using transmittance. Nonetheless, the observed time for aggregation
of native insulin in our studies is consistent with those from a
number of published reports. See, e.g., Dutta Choudhury et al. J
Phys Chem B 2009, 113(7): 1891-1898; Lee et al. Angewandte Chemie
2014, 53(29): 7461-7465. The significant extension in the timeframe
of insulin stability afforded by supramolecular PEGylation with
PEG-T-CB[7] observed here is therefore noteworthy. Furthermore,
this was achieved using a non-covalent supramolecular modification
approach, demonstrating a viable alternative to methods that have
stabilized insulin through covalent PEGylation on the B1 N-terminal
amine. Previously published findings have suggested unmodified
CB[7] can inhibit amyloid formation as measured by the ThT assay,
whereas the studies performed here suggest covalent PEG conjugation
to the CB[7] is required to inhibit aggregation of insulin upon
supramolecular modification.
[0273] A mouse model of insulin-deficient diabetes, prepared using
streptozotocin to induce pancreatic .beta.-cell death, was used to
evaluate the function of PEG-T-CB[7] conjugates in vivo (FIG. 4A).
Following the onset of diabetes, mice were fasted overnight to
ensure an average starting blood glucose level of approximately 450
mg/dL. See, e.g., Like et al. Science 1976, 193(4251): 415-417.
Insulin was then administered subcutaneously at a dose of 1 IU/kg
in a saline vehicle. Alternatively, an identical dose of insulin
was administered along with CB[7], PEG.sub.5k-T-CB [7],
PEG.sub.10k-T-CB[7], or PEG.sub.30k-T-CB[7], All insulin
formulations reduced average blood glucose to a normoglycemic level
(<200 mg/dL for a mouse) following a single subcutaneous
administration. Animals that received either native insulin or
insulin formulated with unmodified CB[7] became hyperglycemic again
approximately 2 hours following administration. Animals that
received insulin formulated with PEG.sub.5k-T-CB[7] and
PEG.sub.10k-T-CB[7] remained normoglycemic for approximately 3
hours and 5 hours, respectively, following administration. Insulin
formulated with PEG.sub.30k-T-CB[7] preserved normoglycemia until
the 6-hour endpoint of the study, at which time the average blood
glucose level of mice in this group was 129 mg/dL, remaining well
within the normoglycemic range. Thus, the duration of activity for
native insulin is directly dependent on molecular weight of PEG in
the PEG-T-CB[7] conjugate. Pharmacological function of the
authentic native insulin protein can therefore be controlled by
non-covalent modification, enabling the duration of therapy to be
tuned through conjugate selection without a need for direct
modification to the therapeutic protein.
[0274] The mechanism by which PEG-T-CB[7] extends the activity of
insulin in vivo is likely multifactorial. First, insulin
administered subcutaneously must reach vascular circulation prior
to eliciting its function. Subcutaneous administration of compounds
<1 kDa in molecular weight are typically preferentially absorbed
directly via capillary circulation, while larger compounds
(including insulin) primarily leverage fluid flux of lymphatic
circulation to reach the vasculature, resulting in delayed systemic
bioavailability. See, e.g., McLennan et al. Drug discovery today
Technologies 2005, 2(1): 89-96; Swartz M A. Adv Drug Deliv Rev
2001, 50(1-2): 3-20; Chertok et al. Mol Pharm 2013, 10(10):
3531-3543. Moreover, it is known that molecular weight affects the
rate at which compounds can traffic within lymphatic circulation.
See, e.g., Supersaxo et al. Pharmaceutical research 1990, 7(2):
167-169. Therefore, supramolecular PEGylation of insulin with PEG
chains of various molecular weights could contribute to delayed
uptake and create a sustained depot of insulin in the subcutaneous
space by increasing the effective molecular weight of the
therapeutic protein. Once in circulation, native insulin is
primarily cleared through internalization following binding to the
insulin receptor. See, e.g., Di Guglielmo et al. Molecular and
cellular biochemistry 1998, 182(1-2): 59-63. Covalent PEGylation of
the B1 phenylalanine is known to induce a reduction in insulin
bioactivity and reduced receptor activation. Therefore,
supramolecular PEGylation could ensure insulin is cleared less
quickly via receptor internalization by sequestering the insulin in
a less active form for an extended time in circulation, whereby it
would remain in equilibrium with the active free insulin species on
account of the dynamic supramolecular interactions of CB[7] with
insulin. Native insulin (5.8 kDa) is also subject to glomerular
filtration, where it is subsequently degraded through enzymatic
action. See, e.g., Rabkin et al. Diabetologia 1984, 27(3): 351-357.
It is generally held that proteins or polymers (including PEG)
greater than approximately 30 kDa in molecular weight are too large
for significant uptake by the glomerulus. See, e.g., Caliceti et
al. Adv Drug Deliv Rev 2003, 55(10): 1261-1277. Therefore, the
supramolecular PEGylation of insulin with PEG-T-CB[7] could also
reduce renal clearance, again by increasing the effective molecular
weight of the protein. Additionally, PEGylation is well known to
provide a steric protective effect on proteins in limiting access
by proteolytic enzymes, a role that could also be filled by
supramolecular PEGylation. It has previously been speculated that
binding of CB[7] to the N-terminal phenylalanine is not likely to
occur to any significant extent in circulation in vivo because the
insulin concentration in normal serum is below the K.sub.D for
binding of CB[7] to insulin; additionally, a high concentration of
serum proteins that bind weakly to CB[7] could induce dissociation
by competition. In light of this, the underlying mechanism is
likely to favor a subcutaneous depot effect, whereby the
PEG-T-CB[7] conjugates slow the absorption of insulin into the
circulation and/or protect insulin from proteolysis when bound at
high concentration in the depot relative to systemic concentrations
upon uptake into the vasculature.
[0275] The PEG-T-CB[7] conjugates described here could be more
broadly used to promote stability and increase the duration of
action for a number of biopharmaceuticals. Insulin is an excellent
candidate to demonstrate this approach, as it is a widely used
biopharmaceutical, highly prone to amyloid formation, and possesses
an N-terminal aromatic residue necessary for strong binding to
CB[7]. See, e.g., Urbach et al. Israel Journal of Chemistry 2011,
51(5-6): 664-678. In fact, there is limited incidence of N-terminal
aromatic residues in the human proteome, meaning most
biopharmaceuticals would not have native sequences amenable to
binding with CB[7]. However, the installation of a phenylalanine or
tryptophan residue, or perhaps even a non-canonical amino acid
capable of binding to CB[7], onto the N-terminus of many
therapeutic proteins could improve compatibility with the
supramolecular PEGylation strategy outlined here. See, e.g.,
Logsdon el al. Journal of the American Chemical Society 2011,
133(42): 17087-17092. As described herein, this is an attractive
and useful strategy for modifying any protein.
PEGylation of Glucagon and Anti-CD20 Antibody
[0276] Glucagon and an antibody for human CD20--a variant of which
is used clinically as Rituximab--were also evaluated for stability
when formulated with CB[7]-PEG. Even in the absence of an
N-terminal aromatic, lower affinity interactions between CB[7] and
mid-chain residues were sufficient to confer improved stability.
Anti-CD20, when agitated in the presence of CB[7]-PEG, maintained
activity toward a B-cell lymphoma cell line (FIG. 9A). Glucagon,
which typically has aqueous solubility of less than one hour,
remained soluble and in its active .alpha.-helical conformation for
at least 24 hours when formulated with CB[7]-PEG (FIGS. 9B and
9C).
Methods
Synthesis of CB[7]-PEG Conjugates
[0277] Dibenzocyclooctyne-functional poly(ethylene glycol)
(PEG-DBCO) with approximate molecular weights of 5 kDa, 10 kDa, and
30 kDa were purchased from Click Chemistry Tools, LLC, whereas
monofunctional CB[7]--N.sub.3 was synthesized as described
previously. See, e.g., Vinciguerra et al. Journal of the American
Chemical Society 2012, 134(31): 13133-13140. Commercially sourced
PEG-DBCO derivatives (100 mg, 1.05 equivalent) were mixed with
CB[7]--N.sub.3 (1 equivalent) in H.sub.2O/DMSO (4 ml, 1:1 v/v) and
stirred at 60.degree. C. for 24 hours. The reaction mixture was
lyophilized and the product was characterized by MALDI-MS (FIGS.
2A-2D and FIG. 5) and .sup.1H-NMR (400 MHz; FIG. 6). Gel permeation
chromatography (GPC) was performed in water on a glucose-modified
divinylbenzene column utilizing a Malvern Viscotek.TM. system with
both refractive index and UV-visible detectors at a 1.0 mF/min flow
rate. Samples were filtered through 0.2 .mu.m PVDF filters prior to
injection onto the column.
Acridine Orange Competitive Binding Assay
[0278] Binding of PEG-T-CB[7] conjugates to insulin was assessed
using the acridine orange dye displacement assay, in accordance
with a previously described protocol. See, e.g., Chinai et al.
Journal of the American Chemical Society 2011, 133(23): 8810-8813
Briefly, 6 .mu.M of either unmodified CB[7] or the PEG-T-CB[7]
conjugates and 8 .mu.M acridine orange were combined with
recombinant insulin (0, 1, 2, 4, 6, 8, 10, 12.5, 15 .mu.M) in
H.sub.2O. Samples were incubated overnight in light-free conditions
and fluorescent spectra were collected on an Infinite.RTM. M1000
plate reader (Tecan Group, Ltd.), exciting at 485 nm and collecting
the resulting fluorescent spectra from 495-650 nm. The decay in the
peak of acridine orange fluorescent signal was fit to a one-site
competitive binding model (GraphPad Prism v6.0), using the CB[7] AO
equilibrium constant reported previously (K.sub.eq=2.times.10.sup.5
M.sup.-1), to determine binding constants of unmodified CB[7] and
PEG-T-CB[7] to native insulin. See, e.g., Shaikh et al.
Photochemical & photobiological sciences: Official journal of
the European Photochemistry Association and the European Society
for Photobiology 2008, 7(4): 408-414.
Circular Dichroism
[0279] Recombinant human insulin was dissolved at 0.25 mg/ml in
H.sub.2O in combination with 1 equivalent of PEG-T-CB[7] and
samples were left to equilibrate for 3 hours at room temperature.
Near-ultraviolet circular dichroism spectroscopy was performed with
a Jasco J-1500 high performance circular dichroism spectrometer
over a wavelength range of 190-250 nm using a 0.1 cm path-length
cell.
Insulin Aggregation Assay
[0280] Recombinant human insulin was dissolved at 4 mg/ml in 2 mM
HCl with 150 mM NaCl. Insulin was diluted to a final concentration
of 1 mg/ml in phosphate buffered saline in combination with 1
equivalent of unmodified CB[7] or PEG-T-CB[7] conjugates. Samples
were plated 150 .mu.l per well (n=4/group) in an clear 96-well
plate (Thermo Scientific Nunc) and sealed with optically clear and
thermally stable seal (VWR). The plate was immediately placed into
an Infinite.RTM. M1000 plate reader (Tecan Group, Ltd.) and shaken
continuously at 37.degree. C. Absorbance readings at 540 nm were
collected every 6 minutes for 100 hours, and absorbance values were
subsequently converted to transmittance. The aggregation of insulin
leads to light scattering, which results in reduction of sample
transmittance. The time for aggregation (t.sub.A) was defined as a
>10% reduction in transmittance from the initial transmittance.
Following the 100-hour kinetic study, the plate was maintained
under continuous agitation at 37.degree. C. and absorbance at 540
nm was monitored daily to approximate t.sub.A for the PEG-T-CB[7]
conjugates.
In Vitro Insulin Activity
[0281] C2C12 cells were purchased from the American Type Culture
Collection (ATCC) and confirmed free of mycoplasma contamination
prior to use. Cells were cultured in Dulbecco's modified Eagle
medium (DMEM) containing L-glutamine, 4.5 g/L D-glucose, and 110
mg/L sodium pyruvate, and supplemented with 10% fetal bovine serum
and 1% penicillin-streptomycin. Incubations occurred in a 5%
CO.sub.2/water-saturated incubator at 37.degree. C. Cells were
seeded in 96-well plates at a density of 5,000 cells per well.
Twenty-four hours after plating, the cells were washed twice with
200 .mu.L of DMEM containing L-glutamine, 4.5 g/L glucose, and 110
mg/L sodium pyruvate, and starved for 2 hours at 37.degree. C. in
the same serum-free conditions. After 2 hours the media was removed
and cells were stimulated with 100 .mu.L of insulin samples of
various concentrations for 30 minutes at 37.degree. C. After 30
minutes, cells were washed twice with 100 .mu.L of cold
Tris-buffered saline (IX), followed by lysing the cells for 10
minutes with 100 .mu.L of cold Lysis Buffer (Perkin Elmer). Levels
of phosphorylated AKT 1/2/3 (Ser473) and total AKT 1 were
determined from cell lysates using the AlphaLISA.RTM. SureFire.RTM.
ULTRA.TM. kits (Perkin Elmer) according to the manufacturer's
instructions. Intra-well normalized data of phosphorylated AKT
1/2/3 (Ser473) and total AKT 1 were analyzed using Graph Pad Prism
6.0, with dose-response curves fitted to a variable slope (three
parameter) stimulation model to determine the EC.sub.50 of each
insulin sample.
In Vivo Assessment of Performance
[0282] Male C57BL/6J, age 8 weeks, were purchased the Jackson
Laboratory. Upon acclimation for one week in the animal facility,
mice were fasted for 4 hours prior to intraperitoneal injection of
150 mg/kg streptozotocin (STZ, Sigma). STZ for injection was
dissolved at a concentration of 22.5 mg/ml in 2.94% (w/v) in pH 4.5
sodium citrate buffer immediately prior to injection. Mice were
allowed to eat ad libitum and glucose levels were monitored by
peripheral tail vein bleeds using a portable glucose meter
(Accu-Chek Aviva, Roche) daily until unfasted glucose levels
>400 mg/dL. STZ-induced diabetic mice were fasted overnight
prior to assessing insulin performance. Mice were bled at the
beginning of the study, and any mouse with a fasting blood glucose
level below 300 mg/dL was triaged from the study. Mice were then
randomized and injected subcutaneously with insulin dosed at 1
IU/kg (34.7 .mu.g/kg) either alone or formulated with 5 molar
equivalents of unmodified CB[7] or PEG-T-CB[7] conjugates. In all
case, insulin with and without CB[7] or PEG-T-CB[7] was injected in
a 200 .mu.L saline vehicle. Blood glucose readings were collected
every 30 minutes using a handheld glucose meter for 6 hours
following insulin injection.
Antibody Stability Assessment
[0283] Mouse anti-human CD20 IgG (Biolegend) was dissolved at 0.5
mg/ml in PBS alone, or with 2 equivalents of CB[7] or CB[7]-PEG
conjugates for a total volume of 200 .mu.l into 2 ml glass vials.
Into each vial, five 1 mm polystyrene beads (Sigma) were added. The
samples were subject to vortex agitation continuously for 24 hours
to induce aggregation. As a CD20-positive cell source, human Raji
B-cell lymphoma cells (ATCC) were grown in RPMI 1640 media
supplemented with 10% fetal bovine serum and passaged four times
prior to experiments. For each antibody sample, one million cells
were mixed in 100 .mu.l of various antibody formulations, diluted
to a concentration of 1 .mu.g/ml based on a dilution for this
antibody determined in separate controls to be an appropriate
dilution below saturation for these specific cells. Cells were
incubated with anti-CD20 antibody (100 .mu.l at 1 .mu.g/ml) for 25
minutes, following which they were washed, combined with a
secondary goat-anti-mouse IgG labeled with Alexa Fluor.RTM. 555
(1:500 dilution, Biolegend) and incubated for 25 minutes. Following
additional washes, cells were suspended in 500 .mu.l buffer and
analyzed on an LSRFortessa (BD Biosciences) flow cytometer. Data
was analyzed by FlowJo v10.1.
Glucagon Stability Assessment
[0284] Human recombinant glucagon (Sigma) was dissolved at 4 mg/ml
in PBS containing 1 mM HCl. This was mixed 1:1 with 1 equivalent of
CB[7]-PEG.sub.10k in PBS, or in PBS alone, for a final glucagon
concentration of 2 mg/ml. Vials were left to age at room
temperature for various times. Circular dichroism was performed on
samples without dilution, as well as on freshly dissolved glucagon,
by utilizing a 100 .mu.m-pathlength cuvette.
Equivalents and Scope
[0285] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process.
[0286] Furthermore, the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, each subgroup of the elements is
also disclosed, and any element(s) can be removed from the group.
It should it be understood that, in general, where the invention,
or aspects of the invention, is/are referred to as comprising
particular elements and/or features, certain embodiments of the
invention or aspects of the invention consist, or consist
essentially of, such elements and/or features. For purposes of
simplicity, those embodiments have not been specifically set forth
in haec verba herein. It is also noted that the terms "comprising"
and "containing" are intended to be open and permits the inclusion
of additional elements or steps. Where ranges are given, endpoints
are included. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or sub-range within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0287] This application refers to various issued patents, published
patent applications, journal articles, and other publications, all
of which are incorporated herein by reference. If there is a
conflict between any of the incorporated references and the instant
specification, the specification shall control. In addition, any
particular embodiment of the present invention that falls within
the prior art may be explicitly excluded from any one or more of
the claims. Because such embodiments are deemed to be known to one
of ordinary skill in the art, they may be excluded even if the
exclusion is not set forth explicitly herein. Any particular
embodiment of the invention can be excluded from any claim, for any
reason, whether or not related to the existence of prior art.
[0288] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation many
equivalents to the specific embodiments described herein. The scope
of the present embodiments described herein is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. Those of ordinary skill in the art will appreciate
that various changes and modifications to this description may be
made without departing from the spirit or scope of the present
invention, as defined in the following claims.
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