U.S. patent application number 10/821319 was filed with the patent office on 2004-10-14 for delivery of immune response modifier compounds using metal-containing particulate support materials.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to Jing, Naiyong, Liu, Jie J., Wightman, Paul D..
Application Number | 20040202720 10/821319 |
Document ID | / |
Family ID | 33136302 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040202720 |
Kind Code |
A1 |
Wightman, Paul D. ; et
al. |
October 14, 2004 |
Delivery of immune response modifier compounds using
metal-containing particulate support materials
Abstract
The present invention provides immune response modifiers (IRMs)
on particulate support materials that includes one or more metals,
including alloys or complexes thereof.
Inventors: |
Wightman, Paul D.;
(Woodbury, MN) ; Liu, Jie J.; (Woodbury, MN)
; Jing, Naiyong; (Woodbury, MN) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
33136302 |
Appl. No.: |
10/821319 |
Filed: |
April 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10821319 |
Apr 9, 2004 |
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10640904 |
Aug 14, 2003 |
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60462140 |
Apr 10, 2003 |
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60545542 |
Feb 18, 2004 |
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60515256 |
Oct 29, 2003 |
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60545424 |
Feb 18, 2004 |
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Current U.S.
Class: |
424/489 ;
424/130.1; 514/44R |
Current CPC
Class: |
A61K 47/6957 20170801;
A61K 47/50 20170801; A61P 35/00 20180101; A61P 37/08 20180101; A61K
47/6903 20170801; A61P 9/00 20180101; A61P 15/00 20180101 |
Class at
Publication: |
424/489 ;
424/130.1; 514/044 |
International
Class: |
A61K 048/00; A61K
039/395; A61K 009/14 |
Claims
What is claimed is:
1. An IRM-support complex comprising at least one IRM compound on
particulate support material comprising at least one metal.
2. The IRM-support complex of claim 1 wherein the IRM compound is
attached to the support material.
3. The IRM-support complex of claim 2 wherein the IRM compound is
covalently attached to the support material.
4. The IRM-support complex of claim 3 wherein the IRM compound is
covalently attached to least one of the metals of the support
material.
5. The IRM-support complex of claim 1 wherein the support material
is in the form of porous particles.
6. The IRM-support complex of claim 1 wherein the metal is coated
on the support material.
7. The IRM-support complex of claim 6 wherein the support material
comprises an organic polymer or an inorganic polymer.
8. The IRM-support complex of claim 7 wherein the particulate
support material comprises a metal oxide.
9. The IRM-support complex of claim 8 wherein the particulate
support material comprises a glass or a ceramic.
10. The IRM-support complex of claim 1 wherein the support material
is in the form of solid metal particles.
11. The IRM-support complex of claim 1 wherein the metal forms the
core of the particulate support material.
12. The IRM-support complex of claim 1 wherein the particulate
support material has an average density of 0.1 g/cm.sup.3 to 25
g/cm.sup.3.
13. The IRM-support complex of claim 12 wherein the particulate
support material has an average density of 5 g/cm.sup.3 to 20
g/cm.sup.3.
14. The IRM-support complex of claim 1 wherein the particulate
support material has an average particle size of 1 nanometer to 250
microns.
15. The IRM-support complex of claim 14 wherein the particulate
support material has an average particle size of 0.1 micron to 20
microns.
16. The IRM-support complex of claim 14 wherein the particulate
support material has an average particle size of 0.2 micron to 5
microns.
17. The IRM-support complex of claim 1 wherein the particulate
support material is magnetic.
18. The IRM-support complex of claim 17 wherein the particulate
support material is superparamagnetic.
19. The IRM-support complex of claim 1 wherein the IRM compound is
an agonist of at least one TLR.
20. The IRM-support complex of claim 19 wherein the TLR is selected
from the group consisting of TLR6, TLR7, TLR8, and combinations
thereof.
21. The IRM-support complex of claim 1 wherein the IRM compound is
a small molecule immune response modifier.
22. The IRM-support complex of claim 1 wherein at least one IRM
compound is selected from the group consisting of imidazoquinoline
amines, amide substituted imidazoquinoline amines, sulfonamide
substituted imidazoquinoline amines, urea substituted
imidazoquinoline amines, aryl ether substituted imidazoquinoline
amines, heterocyclic ether substituted imidazoquinoline amines,
amido ether substituted imidazoquinoline amines, sulfonamido ether
substituted imidazoquinoline amines, urea substituted
imidazoquinoline ethers, thioether substituted imidazoquinoline
amines, 6-, 7-, 8-, or 9-aryl or heteroaryl substituted
imidazoquinoline amines, tetrahydroimidazoquinoline amines, amide
substituted tetrahydroimidazoquinoline amines, sulfonamide
substituted tetrahydroimidazoquinoline amines, urea substituted
tetrahydroimidazoquinoline amines, aryl ether substituted
tetrahydroimidazoquinoline amines, heterocyclic ether substituted
tetrahydroimidazoquinoline amines, amido ether substituted
tetrahydroimidazoquinoline amines, sulfonamido ether substituted
tetrahydroimidazoquinoline amines, urea substituted
tetrahydroimidazoquinoline ethers, thioether substituted
tetrahydroimidazoquinoline amines, imidazopyridine amines, amide
substituted imidazopyridine amines, sulfonamide substituted
imidazopyridine amines, urea substituted imidazopyridine amines,
aryl ether substituted imidazopyridine amines, heterocyclic ether
substituted imidazopyridine amines, amido ether substituted
imidazopyridine amines, sulfonamido ether substituted
imidazopyridine amines, urea substituted imidazopyridine ethers,
thioether substituted imidazopyridine amines, 1,2-bridged
imidazoquinoline amines, 6,7-fused cycloalkylimidazopyridine
amines, imidazonaphthyridine amines, tetrahydroimidazonaphthyridine
amines, oxazoloquinoline amines, thiazoloquinoline amines,
oxazolopyridine amines, thiazolopyridine amines,
oxazolonaphthyridine amines, thiazolonaphthyridine amines,
1H-imidazo dimers fused to pyridine amines, quinoline amines,
tetrahydroquinoline amines, naphthyridine amines, or
tetrahydronaphthyridine amines; pharmaceutically acceptable salts
thereof; and combinations thereof.
23. The IRM-support complex of claim 1 wherein the IRM is selected
from the group consisting of amide substituted imidazoquinoline
amines, sulfonamide substituted imidazoquinoline amines, urea
substituted imidazoquinoline amines, aryl ether substituted
imidazoquinoline amines, heterocyclic ether substituted
imidazoquinoline amines, amido ether substituted imidazoquinoline
amines, sulfonamido ether substituted imidazoquinoline amines, urea
substituted imidazoquinoline ethers, thioether substituted
imidazoquinoline amines, 6-, 7-, 8-, or 9-aryl or heteroaryl
substituted imidazoquinoline amines, tetrahydroimidazoquinolin- e
amines, amide substituted tetrahydroimidazoquinoline amines,
sulfonamide substituted tetrahydroimidazoquinoline amines, urea
substituted tetrahydroimidazoquinoline amines, aryl ether
substituted tetrahydroimidazoquinoline amines, heterocyclic ether
substituted tetrahydroimidazoquinoline amines, amido ether
substituted tetrahydroimidazoquinoline amines, sulfonamido ether
substituted tetrahydroimidazoquinoline amines, urea substituted
tetrahydroimidazoquinoline ethers, thioether substituted
tetrahydroimidazoquinoline amines, imidazopyridine amines, amide
substituted imidazopyridines, sulfonamide substituted
imidazopyridines, urea substituted imidazopyridines, 1,2-bridged
imidazoquinoline amines, 6,7-fused cycloalkylimidazopyridine
amines, imidazonaphthyridine amines, tetrahydroimidazonaphthyridine
amines, oxazoloquinoline amines, thiazoloquinoline amines,
oxazolopyridine amines, thiazolopyridine amines,
oxazolonaphthyridine amines, thiazolonaphthyridine amines,
1H-imidazo dimers fused to pyridine amines, quinoline amines,
tetrahydroquinoline amines, naphthyridine amines, or
tetrahydronaphthyridine amines; pharmaceutically acceptable salts
thereof; and combinations thereof.
24. The IRM-support complex of claim 1 wherein at least one IRM
compound is selected from the group consisting of purines,
imidazoquinoline amides, benzimidazoles, 1H-imidazopyridines,
adenines, and derivatives thereof.
25. The IRM-support complex of claim 1 wherein at least one IRM
compound comprises a 2-aminopyridine fused to a five-membered
nitrogen-containing heterocyclic ring.
26. The IRM-support complex of claim 1 wherein the metal is a
transition metal, a metalloid, or a rare earth metal.
27. The IRM-support complex of claim 26 wherein the metal is
selected from the group consisting of Groups 6-11 of the Periodic
Table.
28. The IRM-support complex of claim 27 wherein the metal is
selected from the group consisting of tungsten, iron, gold, silver,
platinum, zirconium, nickel, cobalt, rhodium, titanium, and
combinations thereof.
29. The IRM-support complex of claim 1 wherein the metal is a
zero-valent metal.
30. The IRM-support complex of claim 29 wherein the zero
valent-metal is in the form of an alloy.
31. The IRM-support complex of claim 1 further comprising at least
one additional drug.
32. The IRM-support complex of claim 31 wherein the additional drug
is a vaccine.
33. The IRM-support complex of claim 32 wherein the vaccine is a
DNA vaccine.
34. An IRM-support complex comprising at least one IRM compound
covalently attached to particulate support material comprising at
least one zero-valent transition metal, wherein the particulate
support material has an average density of 5 g/cm.sup.3 to 20
g/cm.sup.3.
35. The IRM-support complex of claim 34 contained in a delivery
gun.
36. The IRM-support complex of claim 34 wherein the metal is
selected from the group consisting of tungsten, iron, gold, silver,
platinum, zirconium, nickel, cobalt, rhodium, titanium, and
combinations thereof.
37. An IRM-support complex comprising at least one IRM compound
covalently attached to particulate support material comprising at
least one zero-valent transition metal, wherein the particulate
support material has an average particle size of 0.2 micron to 5
microns.
38. The IRM-support complex of claim 37 wherein the metal is
selected from the group consisting of tungsten, iron, gold, silver,
platinum, zirconium, nickel, cobalt, rhodium, titanium, and
combinations thereof.
39. An IRM-support complex comprising at least one IRM compound
covalently attached to particulate support material comprising at
least one zero-valent transition metal selected from the group
consisting of Groups 6-11 of the Periodic Table.
40. The IRM-support complex of claim 39 wherein the metal is
selected from the group consisting of tungsten, iron, gold, silver,
platinum, zirconium, nickel, cobalt, rhodium, titanium, and
combinations thereof.
41. The IRM-support complex of claim 40 wherein the wherein the
particulate support material has an average particle size of 5 nm
to 100 nm.
42. An IRM-support complex comprising at least one IRM compound
covalently attached to an oligonucleotide, which is attached to
particulate support material comprising at least one metal.
43. The IRM-support complex of claim 42 wherein the particulate
support material has an average particle size of 2 microns to 5
microns.
44. A method of delivering an IRM to a subject, the method
comprising delivering the IRM-support complex of claim 1.
45. A method of delivering an IRM to a subject, the method
comprising delivering the IRM-support complex of claim 34.
46. A method of delivering an IRM to a subject, the method
comprising delivering the IRM-support complex of claim 37.
47. A method of delivering an IRM to a subject, the method
comprising delivering the IRM-support complex of claim 39.
48. A method of delivering an IRM to a subject, the method
comprising delivering the IRM-support complex of claim 42.
49. A delivery device comprising a reservoir containing an
IRM-support complex comprising at least one IRM compound on
particulate support material comprising at least one metal.
50. The delivery device of claim 49 wherein the IRM compound is
covalently attached to the particulate support material.
51. The delivery device of claim 50 wherein the particulate support
material comprises at least one zero-valent transition metal.
52. The delivery device of claim 50 wherein the particulate support
material has an average density of 10 g/cm.sup.3 to 20
g/cm.sup.3.
53. The delivery device of claim 50 wherein the particulate support
material has an average particle size of 0.2 micron to 5
microns.
54. The delivery device of claim 50 wherein the zero-valent
transition metal is selected from the group consisting of Groups
6-11 of the Periodic Table.
55. The delivery device of claim 50 which is a ballistic
device.
56. A method of making an IRM-support complex comprising attaching
an immune/response modifier to a particulate support material
comprising at least one metal.
57. The method of claim 56 wherein the immune response modifier is
covalently attached to the particulate support material.
58. The method of claim 57 wherein the method comprises modifying
the IRM to comprise an alkoxysilane moiety.
59. The method of claim 58 wherein the IRM-modified alkoxysilane is
attached to a silicon-containing particulate support material.
60. The method of claim 59 wherein the silicon-containing
particulate support material comprises silica particles.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation-In-Part of U.S.
patent application Ser. No. 10/640,904, filed on Aug. 14, 2003, and
claims priority to U.S. Provisional Patent Application Serial Nos.
60/462,140, filed on Apr. 10, 2003, 60/545,424, filed on Feb. 18,
2004, 60/515,256, filed on Oct. 29, 2003, and 60/545,542, filed on
Feb. 18, 2004, each of which is incorporated herein by reference in
their entirety.
BACKGROUND
[0002] There has been a major effort in recent years, with
significant successes, to discover new drug compounds that act by
stimulating certain key aspects of the immune system, as well as by
suppressing certain other aspects (see, e.g., U.S. Pat. Nos.
6,039,969 and 6,200,592). These compounds, referred to as immune
response modifiers (IRMs), appear to act through basic immune
system mechanisms known as toll-like receptors to induce selected
cytokine biosynthesis and may be used to treat a wide variety of
diseases and conditions. For example, certain IRMs may be useful
for treating viral diseases (e.g., human papilloma virus,
hepatitis, herpes), neoplasias (e.g., basal cell carcinoma,
squamous cell carcinoma, actinic keratosis), and T.sub.H2-mediated
diseases (e.g., asthma, allergic rhinitis, atopic dermatitis), and
are also useful as vaccine adjuvants. Many of the IRM compounds are
small organic molecule imidazoquinoline amine derivatives (see,
e.g., U.S. Pat. No. 4,689,338), but a number of other compound
classes are known as well (see, e.g., U.S. Pat. No. 5,446,153) and
more are still being discovered. Other IRMs have higher molecular
weights, such as oligonucleotides, including CpGs (see, e.g., U.S.
Pat. No. 6,194,388. In view of the great therapeutic potential for
IRMs, and despite the important work that has already been done,
there is a substantial ongoing need for new means of controlling
the delivery and activity of IRMs in order to expand their uses and
therapeutic benefits.
SUMMARY
[0003] It has now surprisingly been found that immune response
modifiers (IRMs) of the invention can be attached to support
materials that include a metal and, importantly, that they retain
biological activity even while they remain attached to such
material. This ability to attach IRMs to metal-containing supports,
such as gold particles, and to form biologically active IRM-support
complexes allows for a tremendous range of useful applications. For
example, where one may wish to use metal-containing complexes to
deliver the IRMs, such as gold particles used in certain needless
injection devices, and/or where one may not wish to release all the
IRM compound to be effective.
[0004] That is, in contrast to eluting drug from a coated surface
or delivering drug from a formulation, the IRMs here can be active
while attached to particulate support materials that include a
metal. This approach can be used, e.g., to help reduce systemic
absorption through dermal, mucosal and other tissues, as well as to
maintain extended deposition of the IRM at an intended site of
action, such as implanted in a solid tumor mass.
[0005] Moreover, not only has it been found that the IRMs are still
biologically active when attached to a support complex, but
surprisingly, the cytokine induction profile of the IRM can be
altered in potentially desirable ways by virtue of such
attachment.
[0006] The IRM may be covalently or non-covalently bound,
preferably covalently bound, to the particulate support material.
Attachment of an IRM to a particulate support material provides for
the localized biological activity of the IRM and typically
prevents, or at least reduces the occurrence of, the systemic
distribution of the IRM.
[0007] Accordingly, the present invention provides an IRM-support
complex that includes at least one IRM compound attached to
particulate support material including at least one metal. In some
embodiments, the IRM compound is covalently attached to the support
material that includes the metal. Typically, the IRM compound is
covalently attached to at least one of the metals.
[0008] In some embodiments, the support material is in the form of
porous particles or solid particles. The solid particles are
typically in the form of solid metal particles.
[0009] In certain embodiments of the present invention, the support
material is coated with one or more metals or alloys thereof. In
other embodiments, the metal forms the core of the support material
and is coated with another material, which may be an organic
polymer, for example.
[0010] In certain embodiments, the particles may be solid metal
particles. In other embodiments, the support material includes an
organic polymer or an inorganic polymer, the latter of which is
typically in the form of a metal oxide, such as a glass or a
ceramic.
[0011] In certain embodiments of the present invention, the
particulate support material (including one or more metals) has an
average density of 0.1 grams per cubic centimeter (g/cm.sup.3) to
25 g/cm.sup.3. For certain applications, the particulate support
material has an average density of 5 g/cm.sup.3 to 20 g/cm.sup.3
(preferably, 10 g/cm.sup.3 to 20 g/cm.sup.3).
[0012] In certain embodiments, the particulate support material
(including one or more metals) has an average particle size of 1
nanometer (nm) to 250 microns (micrometers, .mu.m). For certain
applications, the particulate support material has an average
particle size of 5 nm to 100 nm. For certain applications, the
particulate support material has an average particle size of 10 nm
to 50 microns. For certain applications, the particulate support
material has an average particle size of 0.1 micron to 20 microns.
For certain applications, the particulate support material has an
average particle size of 0.2 micron to 5 microns.
[0013] The metal is typically a transition metal, preferably
selected from the group consisting of Groups 6-11 of the Periodic
Table, and more preferably selected from the group consisting of
tungsten, iron, gold, silver, platinum, nickel, cobalt, rhodium,
zirconium, titanium, and combinations thereof. For certain
embodiments, silicon-based materials (e.g., silica-based materials
can be used). Thus, herein, the term "metal" includes metalloids
such as silicon. Alternatively, rare earth elements (i.e., the
lanthamides and actinides) can be used. The metal can be in the
form of an alloy or a complex (e.g., a metal-organic complex or a
metal oxide), for example.
[0014] In certain embodiments, the particulate support material has
magnetic properties, either permanent magnetic, paramagnetic, or
superparagmetic, preferably, superparamagnetic. The particulate
support material for such embodiments preferably include iron,
nickel, cobalt, tungsten, titanium, rare earth elements, or
combinations thereof. For such embodiments, the IRM-support complex
can be guided into the host, relocated, redistributed inside the
host, and/or removed from the host by an external magnetic field.
The particulate support material for such embodiments can also be
used to enhance the capacity of radiological diagnostics such as in
magnetic resonance imaging.
[0015] In some embodiments, the IRM-support complex may further
include an additional drug. The IRM compound and the additional
drug may be coated onto at least a portion of the particulate
support material. The additional drug may be a vaccine, including,
for example, a DNA vaccine. The IRM compound may be physically or
chemically linked to the vaccine so as to form a unit. The
additional drug may be linked directly to the particulate support
material separately from the directly linked IRM.
[0016] In some embodiments of the present invention, the IRM
compound may be an agonist of at least one TLR, preferably an
agonist of TLR6, TLR7, or TLR8. The IRM may also in some cases be
an agonist of TLR 9. In some embodiments of the present invention,
the IRM compound may be a small molecule immune response modifier
(e.g., molecular weight of less than about 1000 daltons).
[0017] In some embodiments of the present invention, the IRM
compound may comprise a 2-aminopyridine fused to a five membered
nitrogen-containing heterocyclic ring, or a 4-aminopyrimidine fused
to a five membered nitrogen-containing heterocyclic ring.
[0018] In some embodiments of the present invention, at least one
IRM compound may be an imidazoquinoline amine such as, for example,
an amide substituted imidazoquinoline amine, a sulfonamide
substituted imidazoquinoline amine, a urea substituted
imidazoquinoline amine, an aryl ether substituted imidazoquinoline
amine, a heterocyclic ether substituted imidazoquinoline amine, an
amido ether substituted imidazoquinoline amine, a sulfonamido ether
substituted imidazoquinoline amine, a urea substituted
imidazoquinoline ether, a thioether substituted imidazoquinoline
amine, or a 6-, 7-, 8-, or 9-aryl or heteroaryl substituted
imidazoquinoline amine; a tetrahydroimidazoquinoline amine such as,
for example, an amide substituted tetrahydroimidazoquinoline amine,
a sulfonamide substituted tetrahydroimidazoquinoline amine, a urea
substituted tetrahydroimidazoquinoline amine, a aryl ether
substituted tetrahydroimidazoquinoline amine, a heterocyclic ether
substituted tetrahydroimidazoquinoline amine, an amido ether
substituted tetrahydroimidazoquinoline amine, a sulfonamido ether
substituted tetrahydroimidazoquinoline amine, a urea substituted
tetrahydroimidazoquinoline ether, or a thioether substituted
tetrahydroimidazoquinoline amine; an imidazopyridine amine such as,
for example, an amide substituted imidazopyridine amine, a
sulfonamide substituted imidazopyridine amine, a urea substituted
imidazopyridine amine, an aryl ether substituted imidazopyridine
amine, a heterocyclic ether substituted imidazopyridine amine, an
amido ether substituted imidazopyridine amine, a sulfonamido ether
substituted imidazopyridine amine, a urea substituted
imidazopyridine ether, or a thioether substituted imidazopyridine
amine; a 1,2-bridged imidazoquinoline amine; a 6,7-fused
cycloalkylimidazopyridine amine; an imidazonaphthyridine amine; a
tetrahydroimidazonaphthyridine amine; an oxazoloquinoline amine; a
thiazoloquinoline amine; an oxazolopyridine amine; a
thiazolopyridine amine; an oxazolonaphthyridine amine; a
thiazolonaphthyridine amine; a 1H-imidazo dimer fused to a pyridine
amine, a quinoline amine, a tetrahydroquinoline amine, a
naphthyridine amine, or a tetrahydronaphthyridine amine;
pharmaceutically acceptable salts thereof; and combinations
thereof.
[0019] In some embodiments, at least one IRM compound may be a
purine, imidazoquinoline amide, benzimidazole, 1H-imidazopyridine,
adenine, or a derivative thereof.
[0020] In certain embodiments, the present invention provides an
IRM-support complex that includes at least one IRM compound
covalently attached to particulate support material including at
least one zero-valent transition metal, wherein the particulate
support material has an average density of 10 g/cm.sup.3 to 20
g/cm.sup.3.
[0021] The IRM-support complex may be contained in a delivery
device, such as a so-called gene gun or needle-free injection
system. The IRM-support complex can be delivered by ballistic force
or magnetic acceleration, for example. Thus, in one aspect of the
invention there is provided a delivery device that includes a
reservoir containing an IRM-support complex comprising at least one
IRM compound on particulate support material comprising at least
one metal. After delivery, e.g., from a DNA vaccine gene gun or
other needle-free injection system, the IRM may be active while
remaining attached and/or may be active after detachment from the
support complex. Also, particles used in such devices may have both
an IRM and vaccine, e.g., DNA or other vaccine, attached to the
same particles, or the IRM and vaccine may be separated, for
example each on separate particles.
[0022] In certain embodiments, the present invention provides an
IRM-support complex that includes at least one IRM compound
covalently attached to particulate support material including at
least one zero-valent transition metal, wherein the particulate
support material has an average particle size of 0.2 micron to 5
microns. Such IRM-support complexes are particularly desirable for
deposition of an IRM into the lungs of a subject. Such IRM-support
complexes are also desirable for deposition in solid tumors
following intravenous administration due to the increased tumor
capillary permeability. Particles useful for targeting delivery to
tumors can have an average particle size of 5 nm to 100 nm.
[0023] In certain embodiments, the present invention provides an
IRM-support complex that includes at least one IRM compound
covalently attached to particulate support material including at
least one zero-valent transition metal selected from the group
consisting of Groups 6-11 of the Periodic Table. Such IRM-support
complexes are particularly desirable for visualization of the
location of an IRM. In certain embodiments, the signal (e.g., a
magnetic resonance signal) from the IRM-support complex can be
recorded to generate 2- or 3-dimensional images and used as
diagnostics for the host.
[0024] In certain embodiments, the present invention provides an
IRM-support complex that includes at least one IRM compound
covalently attached to a tether, such as an oligonucleotide, which
attaches by physical attraction (e.g., static forces, hydrogen
bonding, hydrophobic-hydrophilic interactions) to the particulate
support material. Preferably, for this embodiment, the particulate
support material has an average particle size of 2 microns to 5
microns.
[0025] The present invention also provides methods of delivering an
IRM to a subject that includes delivering an IRM-support complex of
the present invention. Delivery devices having a reservoir that
includes one or more of the IRM-support complexes of the present
invention is also provided.
[0026] A method of making an IRM-support complex is also provided,
wherein the method includes attaching an immune response modifier
to a particulate support material that includes at least one metal.
Preferably, the method of attaching includes covalently attaching
the IRM. This can occur by modifying the IRM to include an
alkoxysilane moiety. The IRM-modified alkoxysilane is attached to a
silicon-containing particulate support material, which can include
silica particles.
[0027] The term "comprises" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims,
[0028] As used herein, "a," "an," "the," "at least one," and "one
or more" are used interchangeably. Thus, for example, an
IRM-support complex comprising "an" IRM compound can be interpreted
to mean that the complex includes at least one IRM compound.
Similarly, for example, particulate support material comprising "a"
metal can be interpreted to mean that the particulate support
material includes at least one metal.
[0029] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0030] The above summary of the present invention is not intended
to describe each disclosed embodiment or every implementation of
the present invention. The description that follows more
particularly exemplifies illustrative embodiments. In several
places throughout the application, guidance is provided through
lists of examples, which examples can be used individually and in
various combinations. In each instance, the recited list serves
only as a representative group and should not be interpreted as an
exclusive list.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE
INVENTION
[0031] The present invention is directed to the attachment of
cytokine inducing and/or suppressing immune response modifiers
(IRMs) to particulate support materials that include a metal to
form IRM-support complexes. The IRMs retain biological activity
following such attachment to a particulate support material.
IRM-support complexes allow for the localized delivery of an IRM to
a desired location in the body of a subject and typically prevent,
or at least reduce the occurrence of, the systemic distribution of
the IRM.
[0032] Significant advantages can be realized from the present
invention. For example, the metal-containing IRM-support complex
can be used with a delivery device, such as a gene gun, for
delivery of the IRM. The metal of the metal-containing IRM-support
material can be used for visualization of the location of
deposition of the IRM-support complex. The metal of the
metal-containing IRM-support complex can be used for absorption of
energy from an external energy source (e.g., microwave, x-ray, UV
light) to break the linkage with the IRM.
[0033] As used herein, "particulate support material" is a
particulate material (i.e., material in the form of particles) that
is itself generally biologically inactive. As used herein,
"generally biologically inactive" means that cellular interaction
with the material does not appreciably alter the phenotype of the
cell. The particulate support material may be of a size and
chemical nature to prevent the engulfment or penetration of the
particulate material into cells, in which case the IRM-support
complex retains an extracellular location. Alternatively, the
macromolecular support material may be of a size and chemical
nature to allow engulfment by cells. For example, the
macromolecular support material may be of a size and chemical
nature to allow selective deposition in solid tumors on the basis
of the tumor's increased vascular permeability. The terms
"substrate," "support material," or "support," may also be used
herein to refer to a particulate support material that includes a
metal, an alloy, or a metal complex.
[0034] Typically, the metal-containing particulate support material
is in the form of porous or solid particles. The solid particles
are typically in the form of solid metal-containing particles,
which may be zero-valent metal particles (e.g., gold
particles).
[0035] The support material can be coated or impregnated with one
or more metals. Alternatively, the support material can include one
or more metals as the core. Alternatively, the support material can
be in the form of metallic particles (e.g., gold particles), which
may be porous or solid.
[0036] The support material can include an organic polymer or an
inorganic polymer, the latter of which is typically in the form of
a metal oxide, which can be in the form of a glass or a ceramic. If
the support includes an organic polymer it also includes a metal,
which can be a zero-valent metal. If the support includes an
inorganic polymer, there may be no need for an additional metal.
Alternatively, a different material containing a metal, such as a
zero-valent metal (although other oxidations states of the metal
are also possible), may be included in the support material. Other
inorganic and/or organic materials can be used as the support
material as long as it includes a metal, in any of a variety of
oxidation states.
[0037] The particulate support material can possess a wide range of
densities. For certain embodiments, the particles have an average
density of at least 0.1 gram per cubic centimeter (g/cm.sup.3), and
for certain embodiments at least 5 g/cm.sup.3, and for certain
embodiments at least 10 g/cm.sup.3. For certain applications, the
particles have an average density of no greater than 25 g/cm.sup.3,
and for certain embodiments at no greater than 20 g/cm.sup.3. These
values of densities are for the particulate support material that
includes one or more metals.
[0038] The particulate support material can possess a wide range of
particle shapes and sizes. Herein, the average particle size is the
average of the longest dimension of the particles. The particles
are preferably spherical and the average particle size is the
average diameter. The particles preferably have an average particle
size of at least 1 nanometer (nm), although in certain situations
it may even be as low as 0.1 nm. For certain embodiments the
average particle size is at least 2 nanometers; for certain
embodiments it is at least 5 nm; for certain embodiments it is at
least 10 nm; for certain embodiments it is at least 0.1 micron; for
certain embodiments it is at least 0.2 micron; and for certain
embodiments it is at least 2 microns. For certain embodiments the
average particle size is no greater than 250 microns; for certain
embodiments it is no greater than 50 microns; for other embodiments
it is no greater than 20 microns; for other embodiments it is no
greater than 10 microns; for certain other embodiments the average
particle size is no greater than 5 microns; for certain embodiments
it is no greater than 100 nm; for other embodiments it is no
greater than 10 nm; and for other embodiments it is no greater than
5 mm. These values of particle sizes are for the particulate
support material that includes one or more metals, which can be in
the form of zero-valent metal or in the form of a metal-containing
compound having a non-zero valency, for example.
[0039] The metal can possess a wide range of electron densities,
depending on the desired application. The metal is typically a
transition metal, preferably selected from the group consisting of
Groups 6-11 of the Periodic Table, and more preferably selected
from the group consisting of tungsten, iron, gold, silver,
platinum, nickel, cobalt, rhodium, zirconium, titanium, and
combinations thereof. For certain embodiments, silicon-based
materials (e.g., silica-based materials can be used). Thus, herein,
the term "metal" includes metalloids such as silicon.
Alternatively, rare earth elements (i.e., the lanthamides and
actinides) can be used as the metal. The metal can be in the form
of an alloy or a complex (e.g., a metal-organic complex or a metal
oxide), for example. Thus, herein, the term "metal" includes
metalloids such as silicon in addition to transition metals, main
group metals, rare earth metals, which my or may not be in their
zero-valent state.
[0040] In certain embodiments, the particulate support material has
magnetic properties, either permanent magnetic, paramagnetic, or
supermagnetic, preferably, superparamagnetic. The particulate
support material for such embodiments preferably include iron,
nickel, cobalt, tungsten, titanium, rare earth elements, or
combinations thereof. The particulate support material for such
embodiments can also be used to enhance the capacity of
radiological diagnostics such as in magnetic resonance imaging.
[0041] In an IRM-support complex, preferably an IRM is attached to
a particulate support material. This attachment may be directly to
the metal incorporated in the particulate support material. As used
herein, the term "attached" includes both covalent bonding and
non-covalent chemical association (e.g., ionic bonding, hydrophobic
bonding, and hydrogen bonding) of an immune response modifier with
a particulate support material. Preferably, the immune response
modifiers are attached to a particulate support material by means
of covalent bonding and hydrogen bonding. Preferably, this
attachment is to the metal present in the particulate support
material. The terms "coupled," "conjugated," "bonded," or
"immobilized" may also be used herein to represent "attached."
[0042] The IRM is coated on, impregnated within, or attached to the
support material by a sufficiently strong bond (which sometimes may
require a covalent bond) so that under the circumstances of
intended use the IRM is biologically active during use while it is
attached to the support. It should also be understood that for each
of the uses described herein an IRM may be provided in an
unattached, releasable form, or become unattached over time, so
that the IRM can be released and function in that manner. Mixtures
of the two types can also be used where desirable.
[0043] The IRM-support complex of the present invention provides
for the localized biological activity of the IRM. Preferably, the
IRM is attached to the particulate support material. For example,
the IRM can be attached as a side group to a polymer, and the
polymer coated onto a metal core. In certain embodiments, the
present invention provides an IRM-support complex that includes at
least one IRM compound covalently attached to a tether, such as an
oligonucleotide, or an antibody, or an antigen, which couples by
physical attraction (e.g., static forces, hydrogen bonding,
hydrophobic-hydrophilic interactions) to the particulate support
material.
[0044] Although the IRM may eventually detach from the particulate
support material (e.g., through biodegradation of a polymer to
which the IRM is attached, for example), the IRM preferably does
not detach during a suitable period of use while it is active
(although it may of course also be active after detachment). Such
attachment of an IRM to a particulate support material can be used
to reduce the occurrence of, or prevent, the systemic absorption of
the IRM, and can minimize the systemic side effects sometimes
observed with the systemic administration of an IRM. Also, such
attachment of an IRM to a substrate can serve to limit or focus the
effect of the IRM to a localized region for a desired duration, and
if the support material can be removed, the IRM can then be easily
removed at will along with it. This provides very important control
over where and how long the IRM is applied.
[0045] The substrate having the IRM attached thereto can be used in
a variety of medical applications, which can be therapeutic,
prophylactic (e.g., as a vaccine adjuvant), or diagnostic. As used
herein, "treating" a condition or a subject includes therapeutic,
prophylactic, and diagnostic treatments.
[0046] In some embodiments, an IRM-support complex of the present
invention can be used in, e.g., ex-vivo treatment of immune cells,
experimental testing, or a diagnostic assay in which an IRM is a
component. For example, use of an IRM-support complex can enhance
cellular contact with an IRM, can facilitate the removal of an IRM
from a diagnostic assay, can allow for the concentrated delivery of
an IRM, and can assist in the conservation of IRM reagents.
[0047] In addition to one or more IRM compounds, the IRM-support
complexes and methods of the present invention can include
additional agents administered in a composition with the
IRM-support complexes. Alternatively, the additional agent(s) can
be administered separately from the IRM-support complexes. Such
additional agents may be additional drugs, including, for example,
a vaccine, a tumor necrosis factor (TNF) agonist, or a tumor
necrosis factor receptor (TNFR) agonist. Vaccines include any
material that raises either humoral and/or cell mediated immune
response, such as live or attenuated viral and bacterial immunogens
and inactivated viral, tumor-derived, protozoal, organism-derived,
fungal, and bacterial immunogens, toxoids, toxins, polysaccharides,
proteins, glycoproteins, peptides, cellular vaccines, such as using
dendritic cells, DNA vaccines, recombinant proteins, glycoproteins,
and peptides, and the like, for use in connection with, e.g., BCG,
cholera, plague, typhoid, hepatitis A, B, and C, influenza A and B,
parainfluenza, polio, rabies, measles, mumps, rubella, yellow
fever, tetanus, diphtheria, hemophilus influenza b, tuberculosis,
meningococcal and pneumococcal vaccines, adenovirus, HIV, chicken
pox, cytomegalovirus, dengue, feline leukemia, fowl plague, HSV-1
and HSV-2, hog cholera, Japanese encephalitis, respiratory
syncytial virus, rotavirus, papilloma virus, severe acute
respiratory syndrome (SARS) virus (coronavirus), anthrax, and
yellow fever. Such additional agents can include, but are no
limited to, drugs, such as antiviral agents or cytokines. The
vaccine may be separate or may be physically or chemically linked
to the IRM, such as by chemical conjugation or other means, so that
they are delivered as a unit. TNFR agonists that may be delivered
in conjunction with include, but are not limited to, CD40 receptor
agonists.
[0048] The methods, materials, and articles of the present
invention may be applicable for any suitable subject. Suitable
subjects include, but are not limited to, animals such as, but not
limited to, humans, non-human primates, rodents, dogs, cats,
horses, pigs, sheep, goats, cows, or birds.
[0049] Suitable Immune Response Modifiers:
[0050] Immune response modifiers ("IRM") useful in the present
invention include compounds that act on the immune system by
inducing and/or suppressing cytokine biosynthesis. IRM compounds
possess potent immunostimulating activity including, but not
limited to, antiviral and antitumor activity, and can also
down-regulate other aspects of the immune response, for example
shifting the immune response away from a TH-2 immune response,
which is useful for treating a wide range of TH-2 mediated
diseases. IRM compounds can also be used to modulate humoral
immunity by stimulating antibody production by B cells. Further,
various IRM compounds have been shown to be useful as vaccine
adjuvants (see, e.g., U.S. Pat. Nos. 6,083,505 and 6,406,705, and
International Publication No. WO 02/24225).
[0051] In particular, certain IRM compounds effect their
immunostimulatory activity by inducing the production and secretion
of cytokines such as, e.g., Type I interferons, TNF-.alpha., IL-1,
IL-6, IL-8, IL-10, IL-12, MIP-1, and/or MCP-1, and can also inhibit
production and secretion of certain TH-2 cytokines, such as IL-4
and IL-5. Some IRM compounds are said to suppress IL-1 and TNF
(U.S. Pat. No. 6,518,265).
[0052] For some embodiments, the preferred IRM compounds are
so-called small molecule IRMs, which are relatively small organic
compounds (e.g., molecular weight under about 1000 daltons,
preferably under about 500 daltons, as opposed to large biologic
protein, peptides, and the like).
[0053] Although not bound by any single theory of activity, some
IRMs are known to be agonists of at least one Toll-like receptor
(TLR). IRM compounds that are agonists for TLRs selected from 6, 7,
8, and/or 9 may be particularly useful for certain applications. In
some applications, for example, the preferred IRM compound is not a
TLR7 agonist and is a TLR 8 or TLR 9 agonist. Some small molecule
IRM compounds are agonists of TLRs such as 6, 7, and/or 8, while
oligonucleotide IRM compounds are agonists of TLR9, and perhaps
others. Thus, in some embodiments, the IRM that is attached to a
particulate support material may be a compound identified as an
agonist of one or more TLRs.
[0054] For example, without being bound to any particular theory or
mechanism of action, IRM compounds that activate a strong cytotoxic
lymphocyte (CTL) response may be particularly desirable as vaccine
adjuvants, especially for therapeutic viral and/or cancer vaccines
because a therapeutic effect in these settings is dependent on the
activation of cellular immunity. For example, studies have shown
that activation of T cell immunity in a given patient has a
significant positive effect on the prognosis of the patient.
Therefore the ability to enhance T cell immunity is believed to be
critical to producing a therapeutic effect in these disease
settings.
[0055] IRM compounds that are TLR 8 agonists may be particularly
desirable for use with therapeutic cancer vaccines because antigen
presenting cells that express TLR8 have been shown to produce IL-12
upon stimulation through TLR8. IL-12 is believed to play a
significant role in activation of CTLs, which are important for
mediating therapeutic efficacy as described above.
[0056] IRM compounds that are TLR 7 agonists and/or TLR 9 agonists
may be particularly desirable for use with prophylactic vaccines
because the type I interferon induced by stimulation through these
TLRs is believed to contribute to the formation of neutralizing
T.sub.H1-like humoral and cellular responses.
[0057] IRM compounds that are both TLR 7 and TLR 8 agonists may be
particularly desirable for use with therapeutic viral vaccines
and/or cancer vaccines because TLR7 stimulation is believed to
induce the production of type I IFN and activation of innate cells
such as macrophages and NK cells, and TLR8 stimulation is believed
to activate antigen presenting cells to initiate cellular adaptive
immunity as described above. These cell types are able to mediate
viral clearance and/or therapeutic growth inhibitory effects
against neoplasms.
[0058] IRM compounds that are non-TLR 7 agonists, and do not induce
substantial amounts of interferon alpha, may be desirable for use
with certain vaccines such as bacterial vaccines because TLR7
induces type I IFN production, which down-regulates the production
of IL-12 from macrophages and DCs. IL-12 contributes to the
subsequent activation of macrophages, NK cells and CTLs, all of
which contribute to anti-bacterial immunity. Therefore the
induction of anti-bacterial immunity against some kinds of bacteria
may be enhanced in the absence of IFNa.
[0059] For purposes of the present application, one way to
determine if an IRM compound is considered to be an agonist for a
particular TLR is if it activates an NFkB/luciferase reporter
construct through that TLR from the target species more than about
1.5 fold, and usually at least about 2 fold, in TLR transfected
host cells such as, e.g., HEK293 or Namalwa cells relative to
control transfectants. For information regarding TLR activation,
see, e.g., International Publication Nos. WO 03/043573 and WO
03/043588, U.S. patent application Ser. Nos. 10/777,310,
10/732,563, 10/732,796, and 10/788,731, U.S. Patent Publication No.
US2004/0014779, and the other IRM patents and applications
disclosed herein.
[0060] Preferred IRM compounds include a 2-aminopyridine fused to a
five-membered nitrogen-containing heterocyclic ring.
[0061] Certain IRMs are small organic molecules (e.g., molecular
weight under about 1000 Daltons, preferably under about 500
Daltons, as opposed to large biologic protein, peptides, and the
like) such as those disclosed in, for example, U.S. Pat. Nos.
4,689,338; 4,929,624; 4,988,815; 5,037,986; 5,175,296; 5,238,944;
5,266,575; 5,268,376; 5,346,905; 5,352,784; 5,367,076; 5,389,640;
5,395,937; 5,446,153; 5,482,936; 5,693,811; 5,741,908; 5,756,747;
5,939,090; 6,039,969; 6,083,505; 6,110,929; 6,194,425; 6,245,776;
6,331,539; 6,376,669; 6,451,810; 6,525,064; 6,545,016; 6,545,017;
6,558,951; 6,573,273; 6,656,938; 6,660,735; 6,660,747; 6,664,260;
6,664,264; 6,664,265; 6,667,312; 6,670,372; 6,677,347; 6,677,348;
6,677,349; 6,683,088; European Patent 0 394 026; U.S. Patent
Publication Nos. 2002/0016332; 2002/0055517; 2002/0110840;
2003/0133913; 2003/0199538; and 2004/0014779; and International
Patent Publication Nos. WO 02/102377 and WO 03/103584.
[0062] Examples of classes of small molecule IRM compounds include,
but are not limited to, derivatives of imidazoquinoline amines such
as, for example, amide substituted imidazoquinoline amines,
sulfonamide substituted imidazoquinoline amines, urea substituted
imidazoquinoline amines, aryl ether substituted imidazoquinoline
amines, heterocyclic ether substituted imidazoquinoline amines,
amido ether substituted imidazoquinoline amines, sulfonamido ether
substituted imidazoquinoline amines, urea substituted
imidazoquinoline ethers, thioether substituted imidazoquinoline
amines, and 6-7-, 8-, or 9-aryl or heteroaryl substituted
imidazoquinoline amines; tetrahydroimidazoquinoline amines
including but not limited to amide substituted
tetrahydroimidazoquinoline amines, sulfonamide substituted
tetrahydroimidazoquinoline amines, urea substituted
tetrahydroimidazoquinoline amines, aryl ether substituted
tetrahydroimidazoquinoline amines, heterocyclic ether substituted
tetrahydroimidazoquinoline amines, amido ether substituted
tetrahydroimidazoquinoline amines, sulfonamido ether substituted
tetrahydroimidazoquinoline amines, urea substituted
tetrahydroimidazoquinoline ethers, and thioether substituted
tetrahydroimidazoquinoline amines; imidazopyridine amines including
but not limited to amide substituted imidazopyridine amines,
sulfonamido substituted imidazopyridine amines, urea substituted
imidazopyridine amines, aryl ether substituted imidazopyridine
amines, heterocyclic ether substituted imidazopyridine amines,
amido ether substituted imidazopyridine amines, sulfonamido ether
substituted imidazopyridine amines, urea substituted
imidazopyridine ethers, and thioether substituted imidazopyridine
amines; 1,2-bridged imidazoquinoline amines; 6,7-fused
cycloalkylimidazopyridine amines; imidazonaphthyridine amines;
tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines;
thiazoloquinoline amines; oxazolopyridine amines; thiazolopyridine
amines; oxazolonaphthyridine amines; thiazolonaphthyridine amines;
and 1H-imidazo dimers fused to pyridine amines, quinoline amines,
tetrahydroquinoline amines, naphthyridine amines, or
tetrahydronaphthyridine amines.
[0063] Additional examples of small molecule IRMs include certain
purine derivatives (such as those described in U.S. Pat. Nos.
6,376,501, and 6,028,076), certain imidazoquinoline amide
derivatives (such as those described in U.S. Pat. No. 6,069,149),
certain imidazopyridine derivatives (such as those described in
U.S. Pat. No. 6,518,265), 1H-imidazopyridine derivatives (such as
those described in Japanese Patent Application No. 9-255926),
certain benzimidazole derivatives (such as those described in U.S.
Pat. No. 6,387,938), certain derivatives of a 4-aminopyrimidine
fused to a five membered nitrogen containing heterocyclic ring
(such as adenine derivatives described in U.S. Pat. Nos. 6,376,501;
6,028,076 and 6,329,381; and in International Publication No. WO
02/08595), and certain 3-.beta.-D-ribofuranosylthiazolo[4,5-d]pyri-
midine derivatives (such as those described in U.S. Patent
Publication No. 2003/0199461). 1H-imidazopyridine derivatives (such
as those described in U.S. Pat. No. 6,518,265 and European Patent
Application EP No. 1 256 582)) are said to inhibit TNF and IL-1
cytokines.
[0064] Examples of small molecule IRMs that comprise a
4-aminopyrimidine fused to a five-membered nitrogen-containing
heterocyclic ring include adenine derivatives (such as those
described in U.S. Pat. Nos. 6,376,501; 6,028,076 and 6,329,381; and
in International Publication No. WO 02/08595).
[0065] In some applications, for example, the preferred IRM
compound is other than imiquimod or S-28463 (i.e., resiquimod:
4-Amino-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol).
[0066] Examples of particular IRM compounds include
2-propyl[1,3]thiazolo[4,5-c]quinolin-4-amine, which is considered
predominantly a TLR 8 agonist (and not a substantial TLR 7
agonist),
4-amino-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol,
which is considered predominantly a TLR 7 agonist (and not a
substantial TLR 8 agonist), and
4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-6,7-
,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-1-ethanol, which is a
TLR 7 and TLR 8 agonist. In addition to its TLR 7 activity (and TLR
6 activity, but low TLR 8 activity),
4-amino-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]qu-
inoline-1-ethanol has beneficial characteristics, including that it
has a much lower CNS effect when delivered systemically compared to
imiquimod. Other examples of specific IRM compounds include, e.g.,
N-[4-(4-amino-2-butyl-1H-imidazo[4,5-c][1,5]naphthyridin-1-yl)butyl]-N'-c-
yclohexylurea,
2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyri-
din-4-amine,
1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridin-4-amine- ,
N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimet-
hylethyl} methanesulfonamide,
N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quino-
lin-1-yl)butyl]methanesulfonamide,
2-methyl-1-[5-(methylsulfonyl)pentyl]-1-
H-imidazo[4,5-c]quinolin-4-amine,
N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]-
quinolin-1-yl)butyl]methanesulfonamide,
2-butyl-1-[3-(methylsulfonyl)propy-
l]-1H-imidazo[4,5-c]quinoline-4-amine,
2-butyl-1-{2-[(1-methylethyl)sulfon-
yl]ethyl}-1H-imidazo[4,5-c]quinolin-4-amine,
N-{2-[4-amino-2-(ethoxymethyl-
)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}-N'-cyclohexylurea,
N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimeth-
ylethyl} cyclohexanecarboxamide,
N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo-
[4,5-c]quinolin-1-yl]ethyl}-N'-isopropylurea. Resiquimod,
4-amino-2-ethoxymethyl-.alpha.,.alpha.-dimethyl-1H-imidazo[4,5-c]quinolin-
e-1-ethanol, may also be used in certain situations where a
combination TLR 7 and TLR 8 agonist is desired.
[0067] Other IRM compounds include large biological molecules such
as oligonucleotide sequences. Some IRM oligonucleotide sequences
contain cytosine-guanine dinucleotides (CpG) and are described, for
example, in U.S. Pat. Nos. 6,194,388; 6,207,646; 6,239,116;
6,339,068; and 6,406,705. Some CpG-containing oligonucleotides can
include synthetic immunomodulatory structural motifs such as those
described, for example, in U.S. Pat. Nos. 6,426,334 and 6,476,000.
Other IRM nucleotide sequences lack CpG and are described, for
example, in International Patent Publication No. WO 00/75304.
[0068] Various combinations of IRMs can be used if desired.
[0069] Exemplary Applications:
[0070] The metal-containing IRM-support complexes of the present
invention can be used with a delivery device, particularly a high
pressure (e.g., ballistic, or magnetic accelleration) delivery
device, such as a gene gun, for delivery of the IRM. Typically, an
IRM-support complex for use with delivery devices includes at least
one IRM compound covalently attached to particulate support
material including at least one metal (e.g., zero-valent transition
metal), wherein the particulate support material has an average
density of 10 g/cm.sup.3 to 20 g/cm.sup.3.
[0071] Delivery devices such as gene guns can be used for delivery
of the IRM by propelling the IRM-support complex, which is
contained in a reservoir in the delivery device, at cells or
tissues at a speed sufficient for the particles to penetrate the
surface barrier and become incorporated into the interior of the
host. Such devices are disclosed, for example, in U.S. Pat. No.
5,371,015, for example. Other delivery devices are disclosed in
U.S. Pat. No. 5,630,796 that can be used for delivery of powdered
material using gas pressure to generate a supersonic gas flow. Such
delivery devices are well-known to one of skill in the art.
[0072] The IRM-support complexes of the present invention are
particularly useful for local delivery of an IRM. Local delivery of
an IRM-support complex would allow for concentration of its
biological activity to the site of application. Immobilization of
the IRM, as well as an antigen, would allow for maintaining these
components at high concentration relative to one another without
dilution into the periphery. In such applications, e.g., with an
associated antigen, an IRM can be attached to a particle
accompanied by a specific immunizing antigen on the same particle.
Alternatively, an IRM can be attached to a particle while the
immunizing antigen is attached to a second particle. The latter
case would allow for admixture of the IRM-support complex with any
one of many possible immunizing antigens. These could be
administered simultaneously or sequentially with a delivery device,
such as a gene gun. The initial targeted layers can be fine-tuned
by the size and the density of the support materials and the
applied force.
[0073] IRM-support complexes can also be used in deposition
applications, particularly for inhalation into the lungs of a
subject. For such applications, the particulate support material
typically has an average particle size of 0.2 micron to 5 microns
(preferably 2-5 microns), although larger particle sizes can be use
as well. Targeted areas can include proximal, medial, or distal
regions of the lungs. Selection of particle size would allow for
zonal selectivity in deposition. For example, the 2-5 micron
particles would allow for deposition into the distal airways of the
lung. Larger particles would be limited to the proximal airways of
the lung.
[0074] In certain embodiments, IRM-support complexes can also be
used in targeting solid tumors. Typically, such particles have an
average particle size of 5 nm (the permeable upper limit of a
healthy blood vessel) to 100 nm (the permeable upper limit of a of
tumor blood vessel). The particulate-IRM complex can be selectively
delivered to the tumor site through the hypropermeated endothelium
liner of the blood vessel.
[0075] The metal of the metal-containing IRM-support complex can be
used for visualization of the location of deposition of the
IRM-support material. Visualization, for example, can be
accomplished by techniques such as x-ray or magnetic resonance
imaging. The metal should be of sufficient electron density for the
desired visualization technique. Typically, an IRM-support complex
that can be visualized includes at least one metal-containing
material (e.g., a zero-valent transition metal or metal oxide).
Preferably, the metal is selected from the group consisting of
Groups 6-11 and rare earth elements of the Periodic Table.
Preferably, such complex has magnetic properties (preferably,
superparamagnetic), fluorescent properties, or relatively high
electron density. An ability to visualize the administration of an
IRM can be of advantage in monitoring the targeting of an IRM to a
desired site.
[0076] If the support material is magnetic (e.g., either
permanently magnetic, paramagnetic, or supermagnetic, preferably,
superparamagnetic), an additional signal can result in magnetic
resonance imaging. In certain embodiments, the resonance magnetic
signal from the IRM-support complex can be recorded to generate 2-
or 3-dimensional images and used as diagnostics for the host.
[0077] A magnetic metal-containing IRM-support complex (or just the
metal after the IRM has detached, for example) can be further
manipulated if desired. For example, the complex (or just the
metal) can be relocated or redistributed inside the host by an
external magnetic field to maximize the effects of the IRM. In some
cases, the magnetic metal-containing IRM-support complex (or just
the metal) can be removed from the host to minimize the long effect
of the material by the external magnetic field, such as can be
applied with a wearable magnetic collar.
[0078] The metal of the metal-containing IRM-support material can
be used for absorption of energy from an external energy source
(e.g., microwave) to break the linkage with the IRM and release the
IRM. For example, an IRM can be covalently bonded to a
single-stranded oligonucleotide, which can hybridize with the
complementary oligonucleotide immobilized on the support. Upon the
absorption of microwave energy from an external energy source, the
temperature would increase to denature the oligonucleotide (see,
for example, J. Nam et al., Science, 301, 1884-1886 (2003)) and
release the IRMs as desired.
[0079] The metal-containing IRM-support complex can be used in a
wide variety of applications, such as in the treatment of a wide
variety of conditions. For example, IRMs such as imiquimod--a small
molecule, imidazoquinoline IRM, marketed as ALDARA (3M
Pharmaceuticals, St. Paul, Minn.)--have been shown to be useful for
the therapeutic treatment of warts, as well as certain cancerous or
pre-cancerous lesions (See, e.g., Geisse et al., J. Am. Acad.
Dermatol., 47(3): 390-398 (2002); Shumack et al., Arch. Dermatol.,
138: 1163-1171 (2002); U.S. Pat. No. 5,238,944 and International
Publication No. WO 03/045391.
[0080] Conditions that may be treated by administering an
IRM-support complex of the present invention include, but are not
limited to:
[0081] (a) viral diseases such as, for example, diseases resulting
from infection by an adenovirus, a herpesvirus (e.g., HSV-I,
HSV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus such as
variola or vaccinia, or molluscum contagiosum), a picomavirus
(e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g.,
influenzavirus), a paramyxovirus (e.g., parainfluenzavirus, mumps
virus, measles virus, and respiratory syncytial virus (RSV)), a
coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses,
such as those that cause genital warts, common warts, or plantar
warts), a hepadnavirus (e.g., hepatitis B virus), a flavivirus
(e.g., hepatitis C virus or Dengue virus), or a retrovirus (e.g., a
lentivirus such as HIV);
[0082] (b) bacterial diseases such as, for example, diseases
resulting from infection by bacteria of, for example, the genus
Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella,
Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus,
Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus,
Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium,
Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium,
Brucella, Yersinia, Haemophilus, or Bordetella;
[0083] (c) other infectious diseases, such chlamydia, fungal
diseases including but not limited to candidiasis, aspergillosis,
histoplasmosis, cryptococcal meningitis, or parasitic diseases
including but not limited to malaria, pneumocystis carnii
pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and
trypanosome infection; and
[0084] (d) neoplastic diseases, such as intraepithelial neoplasias,
cervical dysplasia, actinic keratosis, basal cell carcinoma,
squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma,
melanoma, renal cell carcinoma, leukemias including but not limited
to myelogeous leukemia, chronic lymphocytic leukemia, multiple
myeloma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, B-cell
lymphoma, and hairy cell leukemia, and other cancers;
[0085] (e) T.sub.H2-mediated, atopic diseases, such as atopic
dermatitis or eczema, eosinophilia, asthma, allergy, allergic
rhinitis, and Ommen's syndrome;
[0086] (f) certain autoimmune diseases such as systemic lupus
erythematosus, essential thrombocythaemia, multiple sclerosis,
discoid lupus, alopecia greata; and
[0087] (g) diseases associated with wound repair such as, for
example, inhibition of keloid formation and other types of scarring
(e.g., enhancing would healing, including chronic wounds).
[0088] Additionally, an IRM-support complex of the present
invention may be useful as a vaccine adjuvant for use in
conjunction with any material that raises either humoral and/or
cell mediated immune response, such as, for example, live viral,
bacterial, or parasitic immunogens; inactivated viral,
tumor-derived, protozoal, organism-derived, fungal, or bacterial
immunogens, toxoids, toxins; self-antigens; polysaccharides;
proteins; glycoproteins; peptides; cellular vaccines; DNA vaccines;
autologous vaccines; recombinant proteins; glycoproteins; peptides;
and the like, for use in connection with, for example, BCG,
cholera, plague, typhoid, hepatitis A, hepatitis B, hepatitis C,
influenza A, influenza B, parainfluenza, polio, rabies, measles,
mumps, rubella, yellow fever, tetanus, diphtheria, hemophilus
influenza b, tuberculosis, meningococcal and pneumococcal vaccines,
adenovirus, HIV, chicken pox, cytomegalovirus, dengue, feline
leukemia, fowl plague, HSV-1 and HSV-2, hog cholera, Japanese
encephalitis, respiratory syncytial virus, rotavirus, papilloma
virus, yellow fever, and Alzheimer's Disease.
[0089] Certain IRM-support complexes of the present invention may
be particularly helpful in individuals having compromised immune
function. For example, certain complexes may be used for treating
the opportunistic infections and tumors that occur after
suppression of cell mediated immunity in, for example, transplant
patients, cancer patients and HIV patients.
[0090] Particulate Support Material:
[0091] Selection of a particulate support material to serve as a
substrate for attachment of an IRM can vary widely within the scope
of the invention. A particulate support material can be porous or
nonporous, depending on preferred final use. A particulate support
material can be made of a variety of materials as long as a portion
of it includes a metal. The metal can be coated on or impregnated
in particles of another material. The metal can form the core of
the particulate support material. For example, such particulate
support material includes substrates made of inorganic or organic
materials, typically polymeric materials, or combinations of
materials, as long as they include (e.g., are coated with or
impregnated with) a metal (e.g., a transition metal, metalloid, or
a rare earth metal), which can be a zero-valent metal (although
this is not a requirement). The inorganic particles can be made of
metal oxides (e.g., TiO.sub.2 or SiO.sub.2) and can be in the form
of ceramics (e.g., alumina or zirconia) or glasses, for example.
Other compounds or complexes containing a metal, whether it be in a
zero-valent oxidation state or not, can be used as particles in the
present invention.
[0092] In certain embodiments, the selected particulate support
materials, such as iron oxide or ferritin, can eventually be
degraded, broken down, or secreted by the host after a desired
duration.
[0093] In certain embodiments, the selected particulate support
materials, such superparamagnetic beads, can be energized by an
external magnetic source, which makes remote manipulation
possible.
[0094] In certain embodiments, the selected particulate support
materials, such metal oxide, can be heated by a remote energy
source such as microwaves.
[0095] Ceramic, glass, and metallic particulate materials are all
known in the art and are commercially available or can be prepared
by a variety of known techniques. For example, a variety of
colloidal gold particles are available commercially from ICN
Biomedicals, Inc., Aurora, Ohio. Magnetic beads (such as those
available under the trade designation DYNABEADS), metal particles,
and metal oxides, are available from Dynal Biotech (Lake Success,
N.Y.), Argonide (Sanford, N.Y.), and NanoSource Technologies
(Oklahoma City, Okla.). A variety of silica particles are available
from Naclo, Naperville, Ill. Silica coated superparamagnetic
particles are available from Chemicell Gmbh, Berlin, Germany). Also
suitable are quantum dots, such as CdSe particles, which typically
have a particle size of 10 nm or less, and often have a particle
size of 2 nm to 5 nm.
[0096] Suitable polymers for use in the particulate support
material may be natural or synthetic polymers. The polymers can
form the core of the particles with a metal coated thereon or the
polymers can form a coating on a metal core material. Methods for
making metal-coated particles include, for example, metal plasma
vacuum deposition, and electric plating. Methods for making
polymer-coated metal particles include, for example, solvent
coating, and techniques for preparing self-assembled monolayers.
Such methods are well-known to one of skill in the art.
[0097] Synthetic polymers are preferred. Herein, a polymer includes
homopolymers and copolymers. A copolymer is used to refer to a
polymer prepared from two or more monomers, and includes
terpolymers, tetrapolymers, etc.
[0098] Exemplary synthetic polymers include, but are not limited
to: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols
(i.e., polyalkylene oxides), polyalkylene terepthalates, polyvinyl
alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides,
polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes
and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses,
cellulose ethers, cellulose esters, nitro celluloses, polymers of
acrylic and methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly(methyl methacrylate), poly(ethyl
methacrylate), poly(butyl methacrylate), poly(isobutyl
methacrylate), poly(hexyl methacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl
acetate), poly(vinyl chloride), polystyrene, polyamides,
polyvinylpyrrolidone, and polymers of lactic acid and glycolic
acid, polyanhydrides, poly(ortho)esters, poly(butic acid),
poly(valeric acid), poly(lactide-cocaprolactone), and fluorinated
polymers.
[0099] Exemplary natural polymers include, but are not limited to:
alginate and other polysaccharides including dextran and cellulose,
collagen, chemical derivatives thereof (substitutions, additions of
chemical groups, for example, alkyl, alkylene, hydroxylations,
oxidations, and other modifications routinely made by those skilled
in the art), zein, and other prolamines and hydrophobic proteins,
copolymers and mixtures thereof. Copolymers and mixtures of any of
these polymers could be used if desired. Polysciences, Inc.
(Warrington, Pa.) supplies many types of polymeric beads.
[0100] Other examples of particulate support materials include, but
are not limited to, carbohydrate beads and latex beads, such as
those commercially available from many suppliers, including, for
example Biorad and Pierce. The particles can also be in the form of
microparticles, such as microspheres, microcapsules, etc. The
particles can be quantum dots.
[0101] The particles having a metal and an IRM associated therewith
can include a combination of materials. For example, they can
include a combination of inorganic and organic materials. This can
occur by layering the materials, for example. One or more of the
materials can be associated (e.g., attached) to the particulate
support material on the outermost surface such that an IRM is
masked or hidden from a body's immune system until it reaches its
targeted site of action. For example, gold particles having one or
more IRMs attached thereto can have a coating of a polyalkylene
oxide polymer (e.g., polyethylene glycol) thereon (see, e.g., Gref
et al., Colloids and Surfaces B: Biointerfaces 18, 301-313, 2000).
The polyalkylene oxide can function to mask the IRM from the body's
immune system until it reaches its targeted site of action.
[0102] Attachment to Substrates:
[0103] IRMs can be attached to a particulate support material
through either covalent attachment or non-covalent attachment.
Non-covalent attachment of an IRM to a particulate support material
includes attachment by ionic interaction or hydrogen bonding, for
example.
[0104] One example of a non-covalent attachment included in the
present invention is the well-know biotin-avidin system.
Avidin-biotin affinity-based technology has found wide
applicability in numerous fields of biology and biotechnology since
the pioneering work by Dr. Edward Bayer and Dr. Meier Wilchek in
the 1970's. The affinity constant between avidin and biotin is
remarkably high (the dissociation constant, Kd, is approximately
10.sup.-15 M, see, Green, Biochem. J., 89, 599, 1963) and is not
significantly lessened when biotin is coupled to a wide variety of
biomolecules. Numerous chemistries have been identified for
coupling biomolecules to biotin with minimal or negligible loss in
the activity or other desired characteristics of the biomolecule. A
review of the biotin-avidin technology can be found in Applications
of Avidin-Biotin Technology to Affinity-Based Separation, Bayer, et
al., J. of Chromatography, 1990, pgs. 3-11.
[0105] Streptavidin, and its functional homolog avidin, are
tetrameric proteins, having four identical subunits. Streptavidin
is secreted by the actinobacterium Streptomyces avidinii. A monomer
of streptavidin or avidin contains one high-affinity binding site
for the water-soluble vitamin biotin and a streptavidin or avidin
tetramer binds four biotin molecules.
[0106] Biotin, also known as vitamin H or
cis-hexahydro-2-oxo-1H-thieno-[3- ,4]-imidazole-4-pentanoic acid,
is a basic vitamin which is essential for most organisms including
bacteria and yeast. Biotin has a molecular weight of about 244
daltons, much lower than its binding partners avidin and
streptavidin. Biotin is also an enzyme cofactor of pyruvate
carboxylase, trans-carboxylase, acetyl-CoA-carboxylase and
beta-methylcrotonyl-CoA carboxylase which together carboxylate a
wide variety of substrates.
[0107] Both streptavidin and avidin exhibit extremely tight and
highly specific binding to biotin which is one of the strongest
known non-covalent interactions between proteins and ligands, with
a molar dissociation constant of 10.sup.-15 molar (M) (Green,
Advances in Protein Chemistry, Vol. 29, pp. 85-133, 1975), and a
t1/2 of ligand dissociation of 89 days (Green, N.M., Advances in
Protein Chemistry, Vol. 29, pp. 85-133, 1975). The avidin-biotin
bond is stable in serum and in the circulation (Wei et al.,
Experientia, Vol. 27, pp. 366-368, 1970). Once formed, the
avidin-biotin complex is unaffected by most extremes of pH, organic
solvents and denaturing conditions. Separation of streptavidin from
biotin requires conditions, such as 8M guanidine, pH 1.5, or
autoclaving at 121.degree. C. for 10 minutes (min).
[0108] IRMs may be biotinylated using any known methodologies. For
example, IRMs may be biotinylated chemically, using activated
biotin analogues, such as N-hydroxysuccinimidobiotin (NHS-biotin),
which is commercially available from Pierce Chemical Company,
Rockford, Ill., and requires the presence of a free primary amino
group on the IRM.
[0109] Representative methods for covalent attaching an IRM to a
particulate support material include chemical crosslinkers, such as
heterobifunctional crosslinking compounds (i.e., "linkers") that
react to form a bond between reactive groups (such as hydroxyl,
amino, amido, or sulfhydryl groups) in a the immune response
modifier and other reactive groups (of a similar nature) in the
support material. This bond may be, for example, a peptide bond,
disulfide bond, thioester bond, amide bond, thioether bond, and the
like.
[0110] Immune response modifiers may be covalently bonded to a
particulate support material by any of the methods known in the
art. For example, U.S. Pat. Nos. 4,722,906, 4,979,959, 4,973,493,
and 5,263,992 relate to devices having biocompatible agents
covalently bound via a photoreactive group and a chemical linking
moiety to the biomaterial surface. U.S. Pat. Nos. 5,258,041 and
5,217,492 relate to the attachment of biomolecules to a surface
through the use of long chain chemical spacers. U.S. Pat. Nos.
5,002,582 and 5,263,992 relate to the preparation and use of
polymeric surfaces, wherein polymeric agents providing desirable
properties are covalently bound via a photoreactive moiety to the
surface. Others have used photochemistry to modify the surfaces of
biomedical devices, e.g., to coat vascular grafts. (See, e.g., Kito
et al., ASAIO Journal 39, M506-M511, 1993; and Clapper et al.,
Trans. Soc. Biomat. 16, 42, 1993). Cholakis and Sefton synthesized
a polymer having a polyvinyl alcohol (PVA) backbone and heparin
bioactive groups. The polymer was coupled to polyethylene tubing
via nonlatent reactive chemistry, and the resultant surface was
evaluated for thromboresistance in a series of in vitro and in vivo
assays (Cholakis et al., J. Biomed. Mater. Res., 23, 399-415, 1989
and Cholakis et al., J. Biomed. Mater. Res., 23, 417-441, 1989).
Finally, Kinoshita et al. disclose the use of reactive chemistry to
generate polyacrylic acid backbones on porous polyethylene, with
collagen molecules being subsequently coupled to carboxyl moieties
on the polyacrylic acid backbones. (See Kinoshita et al.,
Biomaterials 14, 209-215, 1993). U.S. Pat. No. 6,127,448 discusses
the preparation of biocompatible polymeric coatings.
[0111] In a preferred embodiment, the IRM can be attached to a
particulate support material using a linking group. The linking
group can be any suitable organic linking group that allows the
substrate to be covalently coupled to the immune response modifier
moiety while preserving an effective amount of IRM activity. In
some embodiments, the linking group may be selected to create
sufficient space between the active core of the immune response
modifier moiety and the substrate that the substrate does not
interfere with a biologically effective interaction between the
active core and the T cells that results in IRM activity such as
cytokine production.
[0112] The linking group includes a reactive group capable of
reacting with a reactive group on the substrate to form a covalent
bond. Suitable reactive groups include those discussed in
Hermanson, Bioconjugate Techniques, Academic Press, Chapter 2 "The
Chemistry of Reactive Functional Groups", 137-166, 1996. For
example, the linking group may react with a primary amine (e.g., an
N-hydroxysuccinimidyl ester or an N-hydroxysulfosuccinimidyl
ester); it may react with a sulfhydryl group (e.g., a maleimide or
an iodoacetyl), or it may be a photoreactive group (e.g. a phenyl
azide including 4-azidophenyl, 2-hydroxy-4-azidophenyl,
2-nitro-4-azidophenyl, and 2-nitro-3-azidophenyl). The linking
group may also be an alkoxysilyl group (e.g., a triethyoxysilyl
group) that can be covalently coupled to an IRM. The alkoxysilyl
group can then be covalently coupled to a silicon-containing
particulate support material such as silica particles.
[0113] The substrate includes a chemically active group accessible
for covalent coupling to the linking group. A chemically active
group accessible for covalent coupling to the linking group
includes groups that may be used directly for covalent coupling to
the linking group or groups that may be modified to be available
for covalent coupling to the linking group. For example, suitable
chemically active groups include, but are not limited to, primary
amines and sulfhydryl groups.
[0114] Typically, attachment may occur by reacting an immune
response modifier with a crosslinker and then reacting the
resulting intermediate with a substrate. Many crosslinkers suitable
for preparing bioconjugates are known and many are commercially
available. See for example, Hermanson, Bioconjugate Techniques,
Academic Press, 1996.
[0115] Attachment also may occur, for example, according to the
method shown in Reaction Scheme I in which the substrate is linked
to the IRM moiety through R.sub.1. In step (1) of Reaction Scheme I
a compound of Formula III is reacted with a heterobifunctional
crosslinker of Formula IV to provide a compound of II. R.sub.A and
R.sub.B each contain a functional group that is selected to react
with the other. For example, if R.sub.A contains a primary amine,
then a heterobifunctional crosslinker may be selected in which
R.sub.B contains an amine-reactive functional group such as an
N-hydroxysulfosuccinimidyl ester. R.sub.A and R.sub.B may be
selected so that they react to provide the desired linker group in
the conjugate.
[0116] Methods for preparing compounds of Formula III where R.sub.A
contains a functional group are known. See, for example, U.S. Pat.
Nos. 4,689,338; 4,929,624; 5,268,376; 5,389,640; 5,352,784;
5,494,916; 4,988,815; 5,367,076; 5,175,296; 5,395,937; 5,741,908;
5,693,811; 6,069,149; 6,194,425; 6,331,539; 6,451,810; 6,525,064;
6,541,485; 6,545,016; 6,545,017; 6,573,273; 6,656,938; 6,660,735;
6,660,747; 6,664,260; 6,664,264; 6,664,265; 6,667,312; 6,670,372;
6,677,347; 6,677,348; 6,677,349; 6,683,088; and International
Publication No. WO 03/103584.
[0117] Many heterobifunctional crosslinkers are known and many are
commercially available. See for example, Hermanson, Bioconjugate
Techniques, Academic Press, Chapter 5 "Heterobifunctional
Cross-Linkers", 229-285, 1996. The reaction generally can be
carried out by combining a solution of the compound of Formula III
in a suitable solvent such as N,N-dimethylformamide with a solution
of the heterobifunctional cross-linker of Formula IV in a suitable
solvent such as N,N-dimethylformamide. The reaction may be run at
ambient temperature. The product of Formula II may then be isolated
using conventional techniques.
[0118] In step (2) of Reaction Scheme I a compound of Formula II
that contains reactive group Z.sub.A is reacted with the substrate
to provide the IRM-couples substrate of Formula I. In one
embodiment the reaction can be carried out by combining a solution
of the compound of Formula II in a suitable solvent such as
dimethyl sulfoxide with the substrate. The reaction may be run at
ambient temperature or at a reduced temperature (approximately
4.degree. C.). If Z.sub.A is a photoreactive group such as a phenyl
azide then the reaction mixture will be exposed to long wave UV
light for a length of time adequate to effect cross-linking (e.g.,
10-20 minutes). The average number of immune response modifier
moieties per substrate surface area may be controlled by adjusting
the amount of compound of Formula II used in the reaction. 1
[0119] Alternatively, a compound of Formula II may be synthesized
without using a heterobifunctional crosslinker. So long as the
compound of Formula II contains the reactive group Z.sub.A, it may
be reacted with the substrate using the method of step (2) above to
provide an IRM-coupled substrate.
[0120] The R groups can be hydrogen or organic groups that can
optionally include various substitutions. They can include alkyl
groups, alkenyl groups, including haloalkyl groups, aryl groups,
heteroaryl groups, heterocyclyl groups, and the like.
[0121] For example, preferred R.sub.2 groups include hydrogen,
alkyl groups having 1 to 4 carbon atoms (i.e., methyl, ethyl,
propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and
cyclopropylmethyl), and alkoxyalkyl groups (e.g., methoxyethyl and
ethoxymethyl). Preferably R.sub.3 and R.sub.4 are independently
hydrogen or methyl or R.sub.3 and R.sub.4 join together to form a
benzene ring, a pyridine ring, a 6-membered saturated ring or a
6-membered saturated ring containing a nitrogen atom. One or more
of these preferred substituents, if present, can be present in the
compounds of the invention in any combination.
[0122] As used herein, the terms "alkyl," "alkenyl," and the prefix
"alk-" include straight chain, branched chain, and cyclic groups,
i.e. cycloalkyl and cycloalkenyl. Unless otherwise specified, these
groups contain from 1 to 20 carbon atoms, with alkenyl groups
containing from 2 to 20 carbon atoms. Preferred groups have a total
of up to 10 carbon atoms. Cyclic groups can be monocyclic or
polycyclic and preferably have from 3 to 10 ring carbon atoms.
Exemplary cyclic groups include cyclopropyl, cyclopentyl,
cyclohexyl, cyclopropylmethyl, and adamantyl.
[0123] The term "haloalkyl" is inclusive of groups that are
substituted by one or more halogen atoms, including perfluorinated
groups. This is also true of groups that include the prefix
"halo-". Examples of suitable haloalkyl groups are chloromethyl,
trifluoromethyl, and the like.
[0124] The term "aryl" as used herein includes carbocyclic aromatic
rings or ring systems. Examples of aryl groups include phenyl,
naphthyl, biphenyl, fluorenyl and indenyl. The term "heteroaryl"
includes aromatic rings or ring systems that contain at least one
ring hetero atom (e.g., O, S, N). Suitable heteroaryl groups
include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl,
indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl,
pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl,
carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl,
quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl,
isothiazolyl, purinyl, quinazolinyl, and so on.
[0125] "Heterocyclyl" includes non-aromatic rings or ring systems
that contain at least one ring hetero atom (e.g., O, S, N) and
includes all of the fully saturated and partially unsaturated
derivatives of the above mentioned heteroaryl groups. Exemplary
heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl,
morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl,
thiazolidinyl, isothiazolidinyl, and imidazolidinyl.
[0126] The aryl, heteroaryl, and heterocyclyl groups can be
unsubstituted or substituted by one or more substituents
independently selected from the group consisting of alkyl, alkoxy,
methylenedioxy, ethylenedioxy, alkylthio, haloalkyl, haloalkoxy,
haloalkylthio, halogen, nitro, hydroxy, mercapto, cyano, carboxy,
formyl, aryl, aryloxy, arylthio, arylalkoxy, arylalkylthio,
heteroaryl, heteroaryloxy, heteroarylthio, heteroarylalkoxy,
heteroarylalkylthio, amino, alkylamino, dialkylamino, heterocyclyl,
heterocycloalkyl, alkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl,
haloalkylcarbonyl, haloalkoxycarbonyl, alkylthiocarbonyl,
arylcarbonyl, heteroarylcarbonyl, aryloxycarbonyl,
heteroaryloxycarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl,
alkanoyloxy, alkanoylthio, alkanoylamino, arylcarbonyloxy,
arylcarbonythio, alkylaminosulfonyl, alkylsulfonyl, arylsulfonyl,
heteroarylsulfonyl, aryldiazinyl, alkylsulfonylamino,
arylsulfonylamino, arylalkylsulfonylamino, alkylcarbonylamino,
alkenylcarbonylamino, arylcarbonylamino, arylalkylcarbonylamino,
heteroarylcarbonylamino, heteroarylalkycarbonylam- ino,
alkylsulfonylamino, alkenylsulfonylamino, arylsulfonylamino,
arylalkylsulfonylamino, heteroarylsulfonylamino,
heteroarylalkylsulfonyla- mino, alkylaminocarbonylamino,
alkenylaminocarbonylamino, arylaminocarbonylamino,
arylalkylaminocarbonylamino, heteroarylaminocarbonylamino,
heteroarylalkylaminocarbonylamino and, in the case of heterocyclyl,
oxo. If other groups are described as being "substituted" or
"optionally substituted," then those groups can also be substituted
by one or more of the above-enumerated substituents.
[0127] In Reaction Scheme I the IRM is attached to the substrate
through a linking group at the N.sup.1 nitrogen of the imidazole
ring. Alternatively the linking can occur at different positions on
the ring system. Examples of which are shown below for
imidazoquinoline amines, imidazonaphthyridine amines and
imidazopyridine amines respectively. 2
[0128] The attachment is effected using the method of Reaction
Scheme I starting with an IRM containing reactive group R.sub.A at
the desired attachment point.
[0129] An amount of an IRM-support complex effective for a given
therapeutic or prophylactic application is an amount sufficient to
achieve the intended therapeutic or prophylactic application. The
precise amount of IRM-support complex used will vary according to
factors known in the art including but not limited to the physical
and chemical nature of the IRM compound, the nature of the
particulate support material, the intended dosing regimen, the
state of the subject's immune system (e.g., suppressed,
compromised, stimulated), the method of administering the IRM
compound, and the species to which the formulation is being
administered. Accordingly it is not practical to set forth
generally the amount that constitutes an amount of IRM-support
complex effective for all possible applications. Those of ordinary
skill in the art, however, can readily determine the appropriate
amount with due consideration of such factors.
[0130] The dosing regimen may depend at least in part on many
factors known in the art including but not limited to the physical
and chemical nature of the IRM compound, the nature of the
particulate support material, the amount of IRM being administered,
the state of the subject's immune system (e.g., suppressed,
compromised, stimulated), the method of administering the
IRM-support complex, and the species to which the formulation is
being administered. Accordingly it is not practical to set forth
generally the dosing regimen effective for all possible
applications. Those of ordinary skill in the art, however, can
readily determine the appropriate amount with due consideration of
such factors.
EXAMPLES
[0131] The following examples have been selected merely to further
illustrate features, advantages, and other details of the
invention. It is to be expressly understood, however, that while
the examples serve this purpose, the particular materials and
amounts used as well as other conditions and details are not to be
construed in a matter that would unduly limit the scope of this
invention.
[0132] Preparation of
N-[3-(4-Amino-2-ethoxymethyl-6,7-dimethyl-1H-imidazo-
[4,5-c]pyridin-1-yl)propyl]-N'-[3-(triethoxysilyl)propyl]urea 3
[0133] Into a flask was placed
1-(3-aminopropyl)-2-ethoxymethyl-6,7-dimeth-
yl-1H-imidazo[4,5-c]pyridin-4-amine (100 milligrams (mg), 0.36
millimole (mmol); Example 21 in U.S. Pat. No. 6,545,016) and 5
milliliters (mL) anhydrous dimethyl sulfoxide (DMSO). The mixture
was stirred until the solid was completely dissolved. To the
solution was slowly added 3-(triethoxysilyl) propyl isocyanate
(89.1 mg, 0.36 mmol) in DMSO (1.5 mL at room temperature. After the
addition, the reaction solution was stirred overnight. The reaction
solution was sampled and analyzed by NMR. The spectra showed the
desired addition product, N-[3-(4-amino-2-ethoxyme-
thyl-6,7-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)propyl]-N'-[3-(triethoxysi-
lyl)propyl]urea, at 100% conversion. The sample was also analyzed
by liquid chromatography, the spectrum showed a single product peak
with the disappearance of the starting materials.
[0134] Preparation of
N-[4-(4-Amino-2-propyl-H-imidazo[4,5-c]quinolin-1-yl-
)butyl]-N'-[3-(triethoxysilyl)propyl]urea 4
[0135] Into a flask was placed
1-(4-aminobutyl)-2-propyl-1H-imidazo[4,5-c]- quinolin-4-amine (100
mg, 0.336 mmol; which can be prepared using the methods disclosed
in U.S. Pat. No. 6,069,149) and 5 mL anhydrous dimethyl sulfoxide
(DMSO). The mixture was stirred until the solid was completely
dissolved. To the solution was slowly added 3-(triethoxysilyl)
propyl isocyanate (83.2 mg, 0.336 mmol) in DMSO (1.5 mL) at room
temperature. After the addition, the reaction solution was stirred
overnight. The reaction solution was sampled and analyzed by NMR.
The spectra showed the desired addition product,
N-[4-(4-amino-2-propyl-H-imidazo[4,5-c]quinolin-
-1-yl)butyl]-N'-[3-(triethoxysilyl)propyl]urea, at 100% conversion.
The sample was also analyzed by liquid chromatography, the spectrum
showed a single product peak with the disappearance of the starting
materials.
[0136] The reaction was repeated using 15 mL of anhydrous
tetrahydrofuran (THF) in place of the DMSO. Analysis of the
resulting product by NMR showed 97% conversion of the starting
material to the desired addition product.
[0137] Preparation of
2-ethoxymethyl-1-((3-{2-hydroxy-3-[3-(trimethoxysily-
l)propoxy]propyl}
amino))propyl-6,7-dimethyl-1H-imidazo[4,5-c]pyridine-4-a- mine
5
[0138] Into a flask was placed
1-(3-aminopropyl)-2-ethoxymethyl-6,7-dimeth-
yl-1H-imidazo[4,5-c]pyridin-4-amine (10 mg, 0.036 mmol; Example 21
in U.S. Pat. No. 6,545,016) and 2.5 mL anhydrous tetrahydrofuran.
The mixture was stirred until the solid was completely dissolved.
To the solution was slowly added 3-glycidoxypropyltrimethoxysilane
(8.51 mg, 0.036 mmol) at room temperature. After the addition, the
reaction solution was stirred overnight. The reaction solution was
sampled and analyzed by NMR. The spectra showed the desired
addition product, 2-ethoxymethyl-1-((3-{2-hydr-
oxy-3-[3-(trimethoxysilyl)propoxy]propyl}
amino))propyl-6,7-dimethyl-1H-im- idazo[4,5-c]pyridine-4-amine, at
100% conversion.
Example 1
[0139] IRMs were covalently coupled to gold particles to form
nanometer-sized IRM-gold conjugates through a two-step reaction:
the gold surface was functionalized with carbonate by reacting with
thiol carbonate; the carbonate functional group was then coupled to
the primary amine group of an IRM catalyzed by a carbodiimide.
[0140] Briefly, ten micro-liters of 100 mM solution of
mercaptoacetic acid (catalog no. 10,900-2, Aldrich, Milwaukee,
Wis.) were added to one mL of colloidal gold particles solution
(approximately 10 nanomolar (nM), catalog no. 154015, average
size=40 nm, from ICN Biomedicals Inc., Aurora, Ohio). Under a
nitrogen atmosphere, the mixture was shaken at 3 Hz for 3 hours
(hr) at room temperature.
[0141] Twenty micro-liters of 10 mg/L PBS buffer (pH 7.2) solution
of an imidazoquinoline IRM compound
(4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]qu- inoline-1-ethanamine,
disclosed in U.S. Pat. No. 6,069,149), 20 microliters of 50
milligrams per liter (mg/L) PBS buffer solution of
1-ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDC, HCl salt,
Pierce, Rockford, Ill.), and one drop of approximately 1N HCl, were
then added to the mixture. The final mixture was shaken at 3 Hz at
room temperature for another 12 hours (hr) followed by
centrifugation at 14,000 revolutions per minute (rpm) for 30
minutes (min). After removing the supernatant, the precipitant was
washed with 0.5 mL of PBS buffer twice before being redispersed in
1 millilter (mL) of PBS. A field emission SEM micrograph showed
that, the modified particles were well separated and distributed.
The infrared spectrum showed that there was a significant increase
at the --NH-signal, indicating the successful coupling of IRM to
the colloidal gold.
Example 2
[0142] Similarly, a gold conjugate was also made with 10 nm
colloidal gold (catalog number 154011, ICN Biomedicals).
Example 3
[0143] IRM-gold particles were also made from avidin-biotin or anti
biotin-biotin coupling: reacting the commercially available
gold-strepavidin (Amersham Biosciences, Nanoprobes, Inc. Stoney
Brook, N.Y.) or anti-biotin Nanogold Fab' conjugate (Nanoprobes,
Inc. Stoney Brook, N.Y.) with the biotin complex of Example 29 of
U.S. Pat. No. 6,451,810, which is comparable to the uncomplexed IRM
in stimulating TNF release, but superior in IL-6 stimulation.
Example 4
[0144] An IRM conjugate of ferritin, a metaloprotein containing
4000 to 5000 Fe.sup.3+ ions, was synthesized through direct
coupling between the carboxyl group of
[(4-amino-1-isobutyl-1H-imidazo[4,5-c]quinolin-2-yl)met-
hoxy]acetic acid and the primary amine of ferritin catalyzed by
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
[0145] Five milliliters of a solution of
[(4-amino-1-isobutyl-1H-imidazo[4-
,5-c]quinolin-2-yl)methoxy]acetic acid in pH 7.4 PBS buffer (0.4
g/L) was added to a mixture of 3 ml of 50 g/L of ferritin in pH 7.4
PBS buffer solution from ICN Biochemedicals Inc., Aurora, Ohio, 2
mL of freshly made 20 mM EDC in PBS, and 10 drops of 1N HCl. After
a 5-second vortex mixing, the mixture was allowed to react
overnight. The mixture was then eluted through a size-exclusion
liquid chromatography (PD-10) column. The brown-colored fraction
was collected. The average ratio of
[(4-amino-1-isobutyl-1H-imidazo[4,5-c]quinolin-2-yl)methoxy]acetic
acid to ferritin in the conjugate was determined to be 0.6 (M/M),
based on the UV spectrum measurement of
[(4-amino-1-isobutyl-1H-imidaz[4,5-c]quinolin-- 2-yl)methoxy]acetic
acid in the initial solution and the eluted solution. The recovery
rate of ferritin was 95% after passing through the column. The
eluted fraction was verified by HPLC, which showed a single peak.
No significant lost of iron was observed during the modification.
The conjugate showed biological activities in a test with RAW
cells.
Example 5
[0146] An IRM was covalently immobilized onto functionalized
superparamagnetic particles using a modified protocol based on the
manufacturer's suggested protocol. Briefly, one hundred milligrams
of freeze-dried DYNABEADS M-270 Epoxy (from Dynal Biotect, Lake
Success, N.Y., containing approximately 6.7.times.10.sup.9 beads)
was suspended in 7 mL of de-ionized water. After being vortexed for
30 seconds and incubated for 10 minutes, the mixture was
centrifuged at 3000 Gravity (G) for 10 min and the supernatant was
discarded.
[0147] Three milliliters of a freshly prepared solution (0.4 grams
per liter (g/L)) of
1-(4-aminobutyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amin- e (which
can be prepared using the methods disclosed in U.S. Pat. No.
6,069,149) in carbonate-bicarbonate buffer (0.1 M, pH 9.4) and 5 mL
of 4 M ammonium sulfate in de-ionized water were added to the
beads. The mixture was vortexed for 30 seconds and then placed on a
shaker operating at 10 Hz at room temperature for 24 hours.
[0148] The mixture was centrifuged at 3000 gauss (G) for 10 min.
The supernatant was removed and the IRM concentration was
determined by UV absorption at 247 nm. The beads were washed with 7
mL of methanol 3 times and 7 ml of Dulbecco's PBS 3 times. The IRM
content in the modified beads was calculated by subtracting the
amount of IRM found in the supernatant and washes from the amount
of IRM that was initially combined with the beads.
Example 6
An IRM was covalently immobilized onto nanosized superparamagnetic
particles using the following procedure.
[0149] A portion (0.1 mL) of water-based ferrofluid (EMG 304,
Nashua, N.H.), a water based dispersion of iron oxide particles
with dimensions in the range of 5-15 nm, was diluted with 4 mL
de-ionized water and 20 mL 2-propanol. Under continuous mechanical
stirring, 0.3 mL ammonia (30 wt-%, Aldrich) and 8.5 mg of
N-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-1H-
-imidazo[4,5-c]pyridin-1-yl)propyl]-N'-[3-(triethoxysilyl)propyl]urea
was slowly added to the dispersion. The reaction was allowed to
proceed at room temperature for 4 hours under continuous stirring.
After the reaction was complete, the IRM-attached magnetic
particles were concentrated using a magnet.
Example 7
[0150] An IRM was covalently attached to core shell
superparamagnetic particles using the following procedure. A
portion (1 mL) of water-based silica coated superparamagnetic
particles (50 mg, SiMAG-1, Chemicell Gmbh, Berlin, Germany) a water
based dispersion of core shell magnetic particles with dimensions
in the range of 100 nm, was diluted with 5 mL de-ionized water and
15 mL 2-propanol. Under continuous mechanical stirring, 0.3 mL
ammonia (30 wt-%, Aldrich) and 8.5 mg of
N-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)p-
ropyl]-N'-[3-(triethoxysilyl)propyl]urea was slowly added to the
dispersion. The reaction was allowed to proceed at room temperature
for 4 hours under continuous stirring. After the reaction was
complete, the IRM-attached magnetic particles were concentrated
using a magnet.
Example 8
Preparation of IRM Grafted Nanoparticles
[0151] A dispersion of SiO.sub.2 particles (0.49 grams (g) of 2327,
20 nm ammonium stabilized colloidal silica sol, 41% solids; Nalco,
Naperville, Ill.) was placed in a 5 mL vial. The dispersion was
diluted with 0.2 g of deionized water and 0.5 g of DMSO. To the
stirred dispersion was added 33 mg of
N-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-1H-imidazo[4,5-c]pyridin--
1-yl)propyl]-N'-[3-(triethoxysilyl)propyl]urea in 2 g of DMSO.
After the addition, the dispersion was placed in an ultrasonic bath
at 40.degree. C. for 2 hours. The vial was then capped and placed
in an oven at 80.degree. C. for 24 hours. The resulting dispersion
was analyzed by liquid chromatography. The spectrum showed a broad
peak with different retention time compared to that of the starting
IRM silane. The dispersion was centrifuged to remove the
solvents.
Example 9
Preparation of IRM Grafted Nanoparticles
[0152] A dispersion of SiO.sub.2 particles (0.49 g of 2327, 20 nm
ammonium stabilized colloidal silica sol, 41% solids; Nalco,
Naperville, Ill.) was placed in a 5 mL vial. The dispersion was
diluted with 0.2 g of deionized water and 0.5 g of DMSO. To the
stirred dispersion was added 33 mg of
N-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)p-
ropyl]-N'-[3-(triethoxysilyl)propyl]urea in 2 g of DMSO. After the
addition, the dispersion was placed in an ultrasonic bath at
40.degree. C. for 2 hours. The vial was then capped and placed in
an oven at 80.degree. C. for 24 hours. The vial was cooled to room
temperature and to the vial was added PEG triethoxysilane (12.4 mg,
0.0248 mmol available from GELEST, INC., Morrisville, Pa.). After
the addition, the vial was capped and placed in an ultrasonic bath
for 2 hours. The vial was then placed in an oven at 80.degree. C.
for 24 hours. The dispersion was then centrifuged to remove the
solvents.
Example 10
Preparation of IRM Grafted Nanoparticles
[0153] The procedure of Example 9 was repeated except that the
amount of
N-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)p-
ropyl]-N'-[3-(triethoxysilyl)propyl]urea was reduced from 33 mg to
17 mg.
Example 11
Preparation of IRM Grafted Nanoparticles
[0154] The procedure of Example 9 was repeated except that the
amount of
N-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)p-
ropyl]-N'-[3-(triethoxysilyl)propyl]urea was reduced from 33 mg to
8.5 mg.
Example 12
Preparation of IRM Grafted Nanoparticles
[0155] The procedure of Example 9 was repeated except that the
amount of PEG triethoxysilane was increased from 12.4 mg to 31.0
mg.
Example 13
Preparation of IRM Grafted Nanoparticles
[0156] The procedure of Example 8 was repeated except that 34 mg of
N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]-N'-[3-(triet-
hoxysilyl)propyl]urea was used in lieu of
N-[3-(4-amino-2-ethoxymethyl-6,7-
-dimethyl-H-imidazo[4,5-c]pyridin-1-yl)propyl]-N'-[3-(triethoxysilyl)propy-
l]urea.
Example 14
Preparation of IRM Grafted Nanoparticles
[0157] The procedure of Example 9 was repeated except that 34 mg of
N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]-N'-[3-(triet-
hoxysilyl)propyl]urea was used in lieu of
N-[3-(4-amino-2-ethoxymethyl-6,7-
-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)propyl]-N'-[3-(triethoxysilyl)prop-
yl]urea.
Example 15
Preparation of IRM Grafted Nanoparticles
[0158] The procedure of Example 14 was repeated except that the
amount of
N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]-N'-[3-(triet-
hoxysilyl)propyl]urea was reduced from 34 mg to 17 mg.
Example 16
Preparation of IRM Grafted Nanoparticles
[0159] The procedure of Example 14 was repeated except that the
amount of
N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]-N'-[3-(triet-
hoxysilyl)propyl]urea was reduced from 34 mg to 8.5 mg.
Example 17
Preparation of IRM Grafted Nanoparticles
[0160] The procedure of Example 14 was repeated except that the
amount of PEG triethoxysilane was increased from 12.4 mg to 31.0
mg.
[0161] Test Data
[0162] The beads prepared in Example 5 were tested for their
ability to induce cytokines in the following manner. Twenty
microliters (20 .mu.L) of a slurry of the beads (80 mg beads/mL
PBS) was added to 250 .mu.L of human peripheral blood mononuclear
cells (5.times.10.sup.5 cells) in RPMI complete media and incubated
overnight. 1:1 dilution duplicates were assayed for IFNa and
TNF.alpha. concentrations by ELISA. The results are shown in the
table below where IFN and TNF are reported in picograms/mL and sd
is the standard deviation. Control DYNABEADS are beads that were
treated with buffer alone.
1 IFN.alpha. IFN.alpha. Ave TNF.alpha. TNF.alpha. Ave (1) (2)
IFN.alpha. sd (1) (2) TNF.alpha. sd IRM on 1148.7 888.6 1018.7
130.0 33.2 45.8 39.5 6.3 DYNABEADS Control 5.7 1.4 3.5 2.1 26.1
17.2 21.7 4.5 DYNABEADS
[0163] The particles of Examples 1, 2, and 8-17 were tested in a
single experiment using the method described above and did not
induce significant amounts of either interferon alpha or tumor
necrosis factor alpha at the concentrations tested.
[0164] The complete disclosures of the patents, patent documents
and publications cited herein are incorporated by reference in
their entirety as if each were individually incorporated. In case
of conflict, the present specification, including definitions,
shall control. Various modifications and alterations to this
invention will become apparent to those skilled in the art without
departing from the scope and spirit of this invention. Illustrative
embodiments and examples are provided as examples only and are not
intended to limit the scope of the present invention. The scope of
the invention is limited only by the claims set forth as
follows.
* * * * *