U.S. patent application number 15/605471 was filed with the patent office on 2017-09-21 for toll-like receptor 9 antagonist and methods of use thereof.
The applicant listed for this patent is IDERA PHARMACEUTICALS, INC.. Invention is credited to Sudhir Agrawal, Daqing Wang, Fu-Gang Zhu.
Application Number | 20170268006 15/605471 |
Document ID | / |
Family ID | 56615766 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170268006 |
Kind Code |
A1 |
Agrawal; Sudhir ; et
al. |
September 21, 2017 |
TOLL-LIKE RECEPTOR 9 ANTAGONIST AND METHODS OF USE THEREOF
Abstract
The invention provides antagonist of TLR9 and methods of use
thereof. These compounds inhibit or suppress TLR9-mediated
signaling. The methods may have use in the prevention and treatment
of diseases or disorders mediated by TLR9.
Inventors: |
Agrawal; Sudhir;
(Shrewsbury, MA) ; Wang; Daqing; (Bedford, MA)
; Zhu; Fu-Gang; (Bedford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IDERA PHARMACEUTICALS, INC. |
Cambridge |
MA |
US |
|
|
Family ID: |
56615766 |
Appl. No.: |
15/605471 |
Filed: |
May 25, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15041410 |
Feb 11, 2016 |
9688993 |
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15605471 |
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62115753 |
Feb 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/31 20130101;
C12N 2310/3521 20130101; C12N 2310/3125 20130101; C12N 2310/17
20130101; C12N 15/117 20130101; C12N 2310/321 20130101; A61K 45/06
20130101; C12N 2310/3341 20130101 |
International
Class: |
C12N 15/117 20060101
C12N015/117; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method for inhibiting a TLR-mediated immune response in a
mammal, the method comprising administering an effective amount of
a compound to inhibit the TLR-mediated immune response, wherein the
compound comprises the sequence
5'-G.sub.xTC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3',
wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein m is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto.
2. The method according to claim 1, wherein the compound is
selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
3. The method according to claim 1, wherein the route of
administration is parenteral, mucosal delivery, oral, sublingual,
transdermal, topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form.
4. The method according to claim 1, wherein the mammal is a
human.
5. The method according to claim 1, wherein the compound is
administered in combination with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonists, TLR antagonists, peptides,
proteins, gene therapy vectors, DNA vaccines, adjuvants, kinase
inhibitors or co-stimulatory molecules.
6. A method for inhibiting the activity of a TLR agonist comprising
administering an effective amount of a compound to inhibit the
activity of a TLR agonist, wherein the compound comprises the
sequence
5'-G.sub.xTC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3',
wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein in is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto.
7. The method according to claim 6, wherein the compound is
selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
8. The method according to claim 6, comprising administering the
compound prior to or at the same time as the TLR9 agonist.
9. The method according to claim 6, wherein the route of
administration is parenteral, mucosal delivery, oral, sublingual,
transdermal, topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form.
10. The method according to claim 6, wherein the compound is
administered in combination with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonists, TLR antagonists, peptides,
proteins, gene therapy vectors, DNA vaccines, adjuvants, kinase
inhibitors or co-stimulatory molecules.
11. A method for therapeutically treating a mammal, such method
comprising administering an effective amount of a compound
comprising the sequence
5'-G.sub.xTC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3',
wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein m is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto; and
wherein the compound inhibits a TLR-mediated immune response.
12. The method according to claim 11, wherein the compound is
selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
13. The method according to claim 11, wherein the autoimmune
disease is selected from psoriasis, rheumatoid arthritis, alopecia
universalis, acute disseminated encephalomyelitis, Addison's
disease, ankylosing spondylitis, antiphospholipid antibody
syndrome, autoimmune hemolytic anemia, autoimmune hepatitis,
Bullous pemphigoid, chagas disease, chronic obstructive pulmonary
disease, coeliac disease, dermatomyositis, endometriosis,
Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome,
Hashimoto's disease, hidradenitis suppurativa, idiopathic
thrombocytopenic purpura, interstitial cystitis, morphea,
myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious
anaemia, polymyositis, primary biliary cirrhosis, schizophrenia,
Sjogren's syndrome, temporal arteritis, vasculitis, vitiligo,
vulvodynia or Wegener's granulomatosis.
14. The method according to claim 11, wherein the compound is
administered in combination with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonists, TLR antagonists, peptides,
proteins, gene therapy vectors, DNA vaccines, adjuvants, kinase
inhibitors or co-stimulatory molecules.
15. The method according to claim 11, wherein the route of
administration is parenteral, mucosal delivery, oral, sublingual,
transdermal, topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form.
16. The method according to claim 11, wherein the mammal is a
human.
17. A method for preventing disease, such method comprising
administering to a mammal a compound comprising the sequence
5'-G.sub.xTC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3',
wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein m is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto, and
wherein the compound inhibits a TLR-mediated immune response.
18. The method according to claim 17, wherein the compound is
selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
19. The method according to claim 17, wherein the autoimmune
disease is selected from psoriasis, rheumatoid arthritis, alopecia
universalis, acute disseminated encephalomyelitis, Addison's
disease, ankylosing spondylitis, antiphospholipid antibody
syndrome, autoimmune hemolytic anemia, autoimmune hepatitis,
Bullous pemphigoid, chagas disease, chronic obstructive pulmonary
disease, coeliac disease, dermatomyositis, endometriosis,
Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome,
Hashimoto's disease, hidradenitis suppurativa, idiopathic
thrombocytopenic purpura, interstitial cystitis, morphea,
myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious
anaemia, polymyositis, primary biliary cirrhosis, schizophrenia,
Sjogren's syndrome, temporal arteritis, vasculitis, vitiligo,
vulvodynia or Wegener's granulomatosis.
20. The method according to claim 17, wherein the antagonist is
administered in combination with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonists, TLR antagonists, peptides,
proteins, gene therapy vectors, DNA vaccines, adjuvants or
co-stimulatory molecules.
21. The method according to claim 17, wherein the route of
administration is parenteral, mucosal delivery, oral, sublingual,
transdermal, topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form.
22. The method according to claim 17, wherein the mammal is a
human.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/115,753, filed on Feb. 13, 2015, the contents of
which are incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The invention generally relates to the field of immunology
and immunotherapy, and more specifically to the use of
oligonucleotides for inhibition and/or suppression of Toll-like
Receptor 9 (TLR9)-mediated immune responses.
SUMMARY OF THE RELATED ART
[0003] Toll-like receptors (TLRs) are present on many cells of the
immune system and have been shown to be involved in the innate
immune response (Hornung, V. et al., (2002) J. Immunol.
168:4531-4537). In vertebrates, or mammals, this family consists
often proteins called TLR1 to TLR10, which are known to recognize
pathogen associated molecular patterns from bacteria, fungi,
parasites, and viruses (Poltorak, A. et al. (1998) Science
282:2085-2088; Underhill, D. M., et al. (1999) Nature 401:811-815;
Hayashi, F. et al. (2001) Nature 410:1099-1103; Zhang, D. et al.
(2004) Science 303:1522-1526; Meier, A. et al. (2003) Cell.
Microbiol. 5:561-570; Campos, M. A. et al. (2001) J. Immunol. 167:
416-423; Hoebe, K. et al. (2003) Nature 424: 743-748; Lund, J.
(2003) J. Exp. Med. 198:513-520; Heil, F. et al. (2004) Science
303:1526-1529; Diebold, S. S., et al. (2004) Science 303:1529-1531;
Hornung, V. et al. (2004) J. Immunol. 173:5935-5943). TLRs are a
key means by which mammals recognize and mount an immune response
to foreign molecules and also provide a means by which the innate
and adaptive immune responses are linked (Akira, S. et al. (2001)
Nature Immunol. 2:675-680; Medzhitov, R. (2001) Nature Rev.
Immunol. 1:135-145). TLRs have also been shown to play a role in
the pathogenesis of many diseases, including autoimmunity,
infectious disease, and inflammation (Cook, D. N. et al. (2004)
Nature Immunol. 5:975-979) and the regulation of TLR-mediated
activation using appropriate agents may provide a means for disease
intervention.
[0004] Some TLRs are located on the cell surface to detect and
initiate a response to extracellular pathogens and other TLRs are
located inside the cell to detect and initiate a response to
intracellular pathogens. Table 1 provides a representation of TLRs,
their cellular location, and the known agonists therefore (Diebold,
S. S. et al. (2004) Science 303:1529-1531; Liew, F. et al. (2005)
Nature 5:446-458; Hemmi H. et al. (2002) Nat Immunol 3:196-200;
Jurk M. et al., (2002) Nat Immunol 3:499; Lee J. et al. (2003)
Proc. Natl. Acad. Sci. USA 100:6646-6651); (Alexopoulou, L. (2001)
Nature 413:732-738).
TABLE-US-00001 TABLE 1 TLR Molecule Agonist Cell Surface TLRs: TLR2
bacterial lipopeptides TLR4 gram negative bacteria TLR5 motile
bacteria TLR6 gram positive bacteria Endosomal TLRs: TLR3 double
stranded RNA viruses TLR7 single stranded RNA viruses TLR8 single
stranded RNA viruses TLR9 unmethylated DNA
[0005] Certain unmethylated CpG motifs present in bacterial and
synthetic DNA have been shown to activate the immune system and
induce antitumor activity. (Tokunaga T. et al., J. Natl. Cancer
Inst. (1984) 72:955-962; Shimada S., et al., Jpn. H Cancer Res,
1986, 77, 808-816; Yamamoto S., et al., Jpn. J. Cancer Res., 1986,
79, 866-73). Other studies using antisense oligonucleotides
containing CpG dinucleotides have been shown to stimulate immune
responses (Zhao Q., et al. (1996) Biochem. Pharmacol. 26:173-182).
Subsequent studies demonstrated that TLR9 recognizes unmethylated
CpG motifs present in bacterial and synthetic DNA (Hemmi, H. et al.
(2000) Nature 408:740-745). Other modifications of CpG-containing
phosphorothioate oligonucleotides can also affect their ability to
act as modulators of immune response through TLR9 (see, e.g., Zhao
et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao et al. (1996)
Biochem Pharmacol. 52:1537-1544; Zhao et al. (1997) Antisense
Nucleic Acid Drug Dev. 7:495-502; Zhao et al. (1999) Bioorg. Med.
Chem. Lett. 9:3453-3458; Zhao et al. (2000) Bioorg. Med. Chem.
Lett. 10:1051-1054; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett.
10:2585-2588; Yu, D. et al. (2001) Bioorg. Med. Chem. Lett.
11:2263-2267; and Kandimalla, E. et al. (2001) Bioorg. Med. Chem.
9:807-813). In addition, structure activity relationship studies
have allowed identification of synthetic motifs and novel DNA-based
compounds that induce specific immune response profiles that are
distinct from those resulting from unmethylated CpG dinucleotides.
(Kandimalla, E. et al. (2005) Proc. Natl. Acad. Sci. USA
102:6925-6930. Kandimalla, E. et al. (2003) Proc. Nat. Acad. Sci.
USA 100:14303-14308; Cong, Y. et al. (2003) Biochem Biophys Res.
Commun. 310:1133-1139; Kandimalla, E. et al. (2003) Biochem.
Biophys. Res. Commun. 306:948-953; Kandimalla, E. et al. (2003)
Nucleic Acids Res. 31:2393-2400; Yu, D. et al. (2003) Bioorg. Med.
Chem. 11:459-464; Bhagat, L. et al. (2003) Biochem. Biophys. Res.
Commun. 300:853-861; Yu, D. et al. (2002) Nucleic Acids Res.
30:4460-4469; Yu, D. et al. (2002) J. Med. Chem. 45:4540-4548. Yu,
D. et al. (2002) Biochem. Biophys. Res. Commun. 297:83-90;
Kandimalla. E. et al. (2002) Bioconjug. Chem. 13:966-974; Yu, D. et
al. (2002) Nucleic Acids Res. 30:1613-1619; Yu, D. et al. (2001)
Bioorg. Med. Chem. 9:2803-2808; Yu, D. et al. (2001) Bioorg. Med.
Chem. Lett. 11:2263-2267; Kandimalla, E. et al. (2001) Bioorg. Med.
Chem. 9:807-813; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett.
10:2585-2588; Putta, M. et al. (2006) Nucleic Acids Res.
34:3231-3238).
[0006] The selective localization of TLRs and the signaling
generated therefrom, provides some insight into their role in the
immune response. The immune response involves both an innate and an
adaptive response based upon the subset of cells involved in the
response. For example, the T helper (Th) cells involved in
classical cell-mediated functions such as delayed-type
hypersensitivity and activation of cytotoxic T lymphocytes (CTLs)
are Th1 cells. This response is the body's innate response to
antigen (e.g. viral infections, intracellular pathogens, and tumor
cells), and results in a secretion of IFN-gamma and a concomitant
activation of CTLs. Alternatively, the Th cells involved as helper
cells for B-cell activation are Th2 cells. Th2 cells have been
shown to be activated in response to bacteria and parasites and may
mediate the body's adaptive immune response (e.g. IgE production
and eosinophil activation) through the secretion of IL-4 and IL-5.
The type of immune response is influenced by the cytokines produced
in response to antigen exposure and the differences in the
cytokines secreted by Th1 and Th2 cells may be the result of the
different biological functions of these two subsets.
[0007] While activation of TLRs is involved in mounting an immune
response, an uncontrolled stimulation of the immune system through
TLRs may exacerbate certain diseases. Recent studies have shown
that toll-like receptor recognition of bacterial DNA from commensal
flora resulted in activation of an innate immune response leading
to the perpetuation of chronic inflammation in the gut (see e.g.,
Hiramatsu et al. (2014) International Immunopharmacology; Bouladoux
et al. (2012) Mucosal Immunol. 5(6): 623-634; Nagar et al. (2014)
International Immunopharmacology 21: 328-335; Sun et al., (2007)
Inflammation & Allergy--Drug Targets, Vol. 6, No. 4; and
Obermeier et al., (2005) GASTROENTEROLOGY, 129:913-927). Current
available therapies directed to treating this chronic inflammation
are limited due to their serious adverse effects. Thus there is a
need for compounds and compositions useful in many clinically
relevant applications for treating and preventing diseases and
disorders with an immune stimulatory component.
BRIEF SUMMARY OF THE INVENTION
[0008] The invention provides compounds that act as distinct
antagonists of TLR9 and methods of using such compounds to
antagonize, inhibit, suppress or prevent TLR9-mediated immune
stimulation. The compounds and compositions that preferentially
antagonize, inhibit, suppress or prevent the activity of TLR9 have
the sequence (SEQ ID NO: 1):
5'-(N.sub.1).sub.mCG(N.sub.2).sub.p-3', wherein N.sub.1 and N.sub.2
are independently any nucleotide, m is number from 0 to about 5, p
is a number from about 6 to about 35, C is a nucleotide comprising
a nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 and/or N.sub.2 comprise a CG
dinucleotide C is a nucleotide comprising a nitrogenous base
selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In some embodiments, the compounds and
compositions that preferentially antagonize, inhibit, suppress or
prevent the activity of TLR9 have the following sequence (SEQ ID
NO: 2): 5'-(N.sub.1).sub.nCG(N.sub.2).sub.rCG(N.sub.3).sub.z-3',
wherein N.sub.1, N.sub.2, and N.sub.3 are independently any
nucleotide, n is 2, r is 11, z is 3, C is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto, and provided that at each instance that N.sub.1,
N.sub.2 and/or N.sub.3 comprise a CG dinucleotide C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto.
[0009] In some embodiments, the compounds and compositions that
preferentially antagonize, inhibit, suppress or prevent the
activity of TLR9 comprise the sequence
5'-G.sub.xTC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3',
wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein m is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto.
[0010] Thus, the invention provides a method for inhibiting a
TLR9-mediated immune response in a vertebrate, or mammal, the
method comprising administering to the mammal a compound, or a
composition comprising the compound, according to the invention in
a pharmaceutically effective amount.
[0011] In some embodiments, the compounds may comprise at least two
oligonucleotides, wherein at least two oligonucleotides are
covalently linked via a direct nucleotide to nucleotide linkage at
their 3' ends through the 3' positions of the sugars or through a
modified sugar or modified nucleobase or via a non-nucleotide
linker at their 3' ends through the 3' positions of the sugars or
through a modified sugar or modified nucleobase.
[0012] The invention further provides a method for suppressing or
inhibiting the activity of a TLR9 agonist comprising administering
a compound according to the invention, wherein the compound is
administered at the same time, prior to or after the TLR9
agonist.
[0013] The invention further provides a method for therapeutically
treating a vertebrate, or mammal, having a disease mediated by
TLR9, such method comprising administering to the mammal a compound
according to the invention in a pharmaceutically effective amount.
In preferred embodiments, the disease is cancer, an autoimmune
disease or disorder, airway inflammation, an inflammatory disease
or disorder, infectious disease, malaria, Lyme disease, ocular
infections, conjunctivitis, skin disorders, psoriasis, scleroderma,
cardiovascular disease, atherosclerosis, chronic fatigue syndrome,
sarcoidosis, transplant rejection, allergy, asthma or a disease
caused by a pathogen. Preferred autoimmune diseases and disorders
include without limitation lupus erythematosus, multiple sclerosis,
type I diabetes mellitus, irritable bowel syndrome, Crohn's
disease, rheumatoid arthritis, septic shock, alopecia universalis,
acute disseminated encephalomyelitis, Addison's disease, ankylosing
spondylitis, antiphospholipid antibody syndrome, autoimmune
hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas
disease, chronic obstructive pulmonary disease, coeliac disease,
dermatomyositis, endometriosis, Goodpasture's syndrome, Graves'
disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis
suppurativa, idiopathic thrombocytopenic purpura, interstitial
cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, schizophrenia, Sjogren's syndrome, temporal arteritis
(also known as "giant cell arteritis"), vasculitis, vitiligo,
vulvodynia, and Wegener's granulomatosis. Preferred inflammatory
diseases and disorders include without limitation airway
inflammation, asthma, autoimmune diseases, chronic inflammation,
chronic prostatitis, glomerulonephritis, Behcet's disease,
hypersensitivities, inflammatory bowel disease, reperfusion injury,
rheumatoid arthritis, transplant rejection, ulcerative colitis,
uveitis, conjunctivitis, and vasculitis.
[0014] The invention further provides a method for preventing a
disease mediated by TLR9, such method comprising administering to
the mammal a compound according to the invention in a
pharmaceutically effective amount. In preferred embodiments, the
disease is cancer, autoimmune diseases or disorders, airway
inflammation, inflammatory diseases or disorders, infectious
disease, malaria, Lyme disease, ocular infections, conjunctivitis,
skin disorders, psoriasis, scleroderma, cardiovascular disease,
atherosclerosis, chronic fatigue syndrome, sarcoidosis, transplant
rejection, allergy, asthma or a disease caused by a pathogen in a
vertebrate, or mammal, such method comprising administering to the
mammal a compound according to the invention in a pharmaceutically
effective amount. Preferred autoimmune diseases and disorders
include without limitation lupus erythematosis, multiple sclerosis,
type I diabetes mellitus, irritable bowel syndrome, Crohn's
disease, rheumatoid arthritis, septic shock, alopecia universalis,
acute disseminated encephalomyelitis, Addison's disease, ankylosing
spondylitis, antiphospholipid antibody syndrome, autoimmune
hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, chagas
disease, chronic obstructive pulmonary disease, coeliac disease,
dermatomyositis, endometriosis, Goodpasture's syndrome, Graves'
disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis
suppurativa, idiopathic thrombocytopenic purpura, interstitial
cystitis, morphea, myasthenia gravis, narcolepsy, neuromyotonia,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, schizophrenia, Sjogren's syndrome, temporal arteritis
(also known as "giant cell arteritis"), vasculitis, vitiligo,
vulvodynia, and Wegener's granulomatosis. Preferred inflammatory
diseases and disorders include without limitation airway
inflammation, asthma, autoimmune diseases, chronic inflammation,
chronic prostatitis, glomerulonephritis, Behcet's disease,
hypersensitivities, inflammatory bowel disease, reperfusion injury,
rheumatoid arthritis, transplant rejection, ulcerative colitis,
uveitis, conjunctivitis, and vasculitis.
[0015] In some preferred embodiments, the compound according to the
invention is administered in combination with one or more vaccines,
antigens, antibodies, cytotoxic agents, allergens, antibiotics,
antisense oligonucleotides, TLR agonists, TLR antagonists,
peptides, proteins, gene therapy vectors, DNA vaccines, adjuvants,
kinase inhibitors or co-stimulatory molecules or combinations
thereof. In some preferred embodiments, the route of administration
is parenteral, mucosal delivery, oral, sublingual, transdermal,
topical, inhalation, intranasal, aerosol, intraocular,
intratracheal, intrarectal, intragastric, vaginal, by gene gun,
dermal patch or in eye drop or mouthwash form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
[0017] FIG. 1 shows that the compounds according to the invention
do not induce a TLR9-mediated immune response.
[0018] FIG. 2 depicts the ability of TLR9 antagonists according to
the invention to inhibit TLR9-induced IL-12 in vivo in mice.
[0019] FIG. 3 depicts the % body weight change in a TNBS-induced
colitis mouse model after administration of compounds according to
the invention.
[0020] FIG. 4 depicts the colon length in a TNBS-induced colitis
mouse model after administration of compounds according to the
invention.
[0021] FIG. 5 depicts colon myeloperoxidase levels in a
TNBS-induced colitis mouse model after administration of compounds
according to the invention.
[0022] FIG. 6 depicts the colon histological score in a
TNBS-induced colitis mouse model after administration of compounds
according to the invention.
[0023] FIG. 7 depicts the colonic gene expression in a TNBS-induced
colitis mouse model after administration of compounds according to
the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0024] The present invention relates to the therapeutic use of
oligonucleotide compounds as immune modulatory agents for
immunotherapy applications. The invention provides compounds that
provide immune inhibition through their interaction with TLR9.
Specifically, the invention provides compounds as antagonists of
toll-like receptor 9 (TLR9) to inhibit and/or suppress a
TLR9-mediated immune response. These compounds have chemical
modifications, and/or internucleotide linkages, and/or linkers
between oligonucleotides that provide their inhibition or
suppression of TLR9-mediated signaling in response to endogenous
and/or exogenous TLR9 ligands or agonists. The references cited
herein reflect the level of knowledge in the field and are hereby
incorporated by reference in their entirety. Any conflicts between
the teachings of the cited references and this specification shall
be resolved in favor of the latter.
[0025] The invention further provides methods for suppressing an
immune response induced by TLR9 and can be used for immunotherapy
applications such as, but not limited to, treatment of cancer,
autoimmune disorders, asthma, respiratory allergies, food
allergies, skin allergies, systemic lupus erythematosus (SLE),
arthritis, pleurisy, chronic infections, inflammatory diseases,
inflammatory bowel syndrome, sepsis, and bacteria, parasitic, and
viral infections in adult and pediatric human and veterinary
applications. Thus, the invention provides compounds having optimal
levels of immune modulatory effect for immunotherapy and methods
for making and using such compounds. In addition, compounds of the
invention are useful in combination with, for example, vaccines,
antigens, antibodies, allergens, chemotherapeutic agents (both
chemotherapy and targeted therapies), and/or antisense
oligonucleotides for prevention and treatment of diseases.
Definitions
[0026] The term "oligonucleotide" generally refers to a
polynucleoside comprising a plurality of linked nucleoside units.
Such oligonucleotides can be obtained from existing nucleic acid
sources, including genomic or cDNA, but are preferably produced by
synthetic methods. In preferred embodiments each nucleoside unit
can encompass various chemical modifications and substitutions as
compared to wild-type oligonucleotides, including but not limited
to modified nucleoside base and/or modified sugar unit. Examples of
chemical modifications are known to the person skilled in the art
and are described, for example, in Uhlmann E. et al. (1990) Chem.
Rev. 90:543; "Protocols for Oligonucleotides and Analogs" Synthesis
and Properties & Synthesis and Analytical Techniques, S.
Agrawal, Ed, Humana Press, Totowa, USA 1993; and Hunziker, J. et
al. (1995) Mod. Syn. Methods 7:331-417; and Crooke, S. et al.
(1996) Ann. Rev. Pharm. Tox. 36:107-129. The nucleoside residues
can be coupled to each other by any of the numerous known
internucleoside linkages. Such internucleoside linkages include,
without limitation, phosphodiester, phosphorothioate,
phosphorodithioate, alkylphosphonate, alkylphosphonothioate,
phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,
acetamidate, carbamate, morpholino, borano, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphorothioate, and sulfone internucleoside linkages. The term
"oligonucleotide" also encompasses polynucleosides having one or
more stereospecific internucleoside linkage (e.g., (R.sub.P)- or
(S.sub.P)-phosphorothioate, alkylphosphonate, or phosphotriester
linkages). As used herein, the terms "oligonucleotide" and
"dinucleotide" are expressly intended to include polynucleosides
and dinucleosides having any such internucleoside linkage, whether
or not the linkage comprises a phosphate group. In certain
preferred embodiments, these internucleoside linkages may be
phosphodiester, phosphorothioate or phosphorodithioate linkages or
combinations thereof.
[0027] The term "2'-substituted ribonucleoside" or "2'-substituted
arabinoside" generally includes ribonucleosides or
arabinonucleosides in which the hydroxyl group at the 2' position
of the pentose moiety is substituted to produce a 2'-substituted or
2'-O-substituted ribonucleoside. In certain embodiments, such
substitution is with a lower hydrocarbyl group containing 1-6
saturated or unsaturated carbon atoms, with a halogen atom, or with
an aryl group having 6-10 carbon atoms, wherein such hydrocarbyl,
or aryl group may be unsubstituted or may be substituted, for
example, with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl,
acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examples
of 2'-O-substituted ribonucleosides or
2'-O-substituted-arabinosides include, without limitation 2'-amino,
2'-fluoro, 2'-allyl, 2'-O-alkyl and 2'-propargyl ribonucleosides or
arabinosides, 2'-O-methylribonucleosides or 2'-O-methylarabinosides
and 2'-O-methoxyethoxyribonucleosides or
2'-O-methoxyethoxyarabinosides.
[0028] The term "3'", when used directionally, generally refers to
a region or position in a polynucleotide or oligonucleotide 3'
(downstream) from another region or position in the same
polynucleotide or oligonucleotide.
[0029] The term "5'", when used directionally, generally refers to
a region or position in a polynucleotide or oligonucleotide 5'
(upstream) from another region or position in the same
polynucleotide or oligonucleotide.
[0030] The term "about" generally means that the exact number is
not critical. Thus, the number of nucleoside residues in the
oligonucleotides is not critical, and oligonucleotides having one
or two fewer nucleoside residues, or from one to several additional
nucleoside residues are contemplated as equivalents of each of the
embodiments described above.
[0031] The term "agonist" generally refers to a substance that
binds to a receptor of a cell and induces a response. An agonist
can be a naturally occurring substance such as bacterial DNA or a
synthetic composition. A synthetic agonist often mimics the action
of a naturally occurring substance such as a ligand.
[0032] The term "antagonist" generally refers to a substance that
attenuates, inhibits or suppresses the effects of an agonist or
ligand.
[0033] The term "adjuvant" generally refers to a substance which,
when added to an immunogenic agent such as vaccine or antigen,
enhances or potentiates an immune response to the agent in the
recipient host upon exposure to the mixture.
[0034] The term "airway inflammation" generally includes, without
limitation, asthma.
[0035] The term "allergen" generally refers to an antigen or
antigenic portion of a molecule, usually a protein, which elicits
an allergic response upon exposure to a subject. Typically the
subject is allergic to the allergen as indicated, for instance, by
the wheal and flare test or any method known in the art. A molecule
is said to be an allergen even if only a small subset of subjects
exhibit an allergic immune response upon exposure to the
molecule.
[0036] The term "allergy" generally refers to an inappropriate
immune response characterized by inflammation and includes, without
limitation, food allergies and respiratory allergies.
[0037] The term "antigen" generally refers to a substance that is
recognized and selectively bound by an antibody or by a T cell
antigen receptor, resulting in induction of an immune response.
Antigens may include but are not limited to peptides, proteins,
nucleosides, nucleotides, and combinations thereof. Antigens may be
natural or synthetic and generally induce an immune response that
is specific for that antigen.
[0038] The terms "autoimmune disease" and autoimmune disorder"
generally refer to diseases or disorders in which "self' components
undergo attack by the immune system.
[0039] The term "TLR9-mediated disease" or TLR9-mediated disorder"
generally means any pathological condition for which activation of
one or more TLR9 is a contributing factor. Such conditions include
but are not limited, cancer, autoimmune diseases or disorders,
airway inflammation, inflammatory diseases or disorders, infectious
diseases, skin disorders, allergy, asthma or diseases caused by a
pathogen.
[0040] The term "physiologically acceptable" generally refers to a
material that does not interfere with the effectiveness of a
compound or composition according to the invention and that is
compatible with a biological system such as a cell, cell culture,
tissue or organism. Preferably, the biological system is a living
organism, such as a vertebrate, or mammal.
[0041] The term "carrier" generally encompasses any excipient,
diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid,
lipid containing vesicle, microspheres, liposomal encapsulation or
other material well known in the art for use in pharmaceutical
formulations. It will be understood that the characteristics of the
carrier, excipient or diluent will depend on the route of
administration for a particular application. The preparation of
pharmaceutically acceptable formulations containing these materials
is described in, for example, Remington's Pharmaceutical Sciences,
18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa.,
1990.
[0042] The term "co-administration" generally refers to the
administration of at least two different substances sufficiently
close in time. Co-administration refers to simultaneous
administration, as well as temporally spaced order of up to several
days apart, of at least two different substances in any order,
either in a single dose or separate doses.
[0043] The term "complementary" generally means having the ability
to hybridize to a nucleic acid. Such hybridization is ordinarily
the result of hydrogen bonding between complementary strands,
preferably to form Watson-Crick or Hoogsteen base pairs, although
other modes of hydrogen bonding, as well as base stacking can also
lead to hybridization.
[0044] The term an "effective amount" or a "sufficient amount"
generally refers to an amount sufficient to affect a desired
biological effect, such as beneficial results. Thus, an "effective
amount" or "sufficient amount" will depend upon the context in
which it is being administered. In the context of administering a
compound or composition that modulates an immune response to a
co-administered antigen, an effective amount of a compound or
composition according to the invention and antigen is an amount
sufficient to achieve the desired modulation, inhibition or
suppression as compared to the immune response obtained when the
antigen is administered alone. An effective amount may be
administered in one or more administrations.
[0045] The term "in combination with" generally means administering
two or more agents (e.g., a TLR9 antagonist compound according to
the invention and another agent) such that there is an overlap of
an effect of each agent on the patient. Such administration may be
done in any order, including simultaneous administration, as well
as temporally spaced order from a few seconds up to several days
apart. In some embodiments, the administration of the agents are
spaced sufficiently close together such that a combinatorial effect
is achieved. Such combination treatment may also include more than
a single administration of the compound according to the invention
and/or independently the other agent. The administration of the
compound according to the invention and the other agent may be by
the same or different routes. In some embodiments, administration
of at least one agent is made while the other agent is still
present at a therapeutic level in the subject.
[0046] The term "individual" or "patient" or "subject" or
"vertebrate" generally refers to a mammal. Mammals generally
include, but are not limited to, humans, non-human primates, rats,
mice, cats, dogs, horses, cattle, cows, pigs, sheep, and
rabbits.
[0047] The term "kinase inhibitor" generally refers to molecules
that antagonize or inhibit phosphorylation-dependent cell signaling
and/or growth pathways in a cell. Kinase inhibitors may be
naturally occurring or synthetic and include small molecules that
have the potential to be administered as oral therapeutics. Kinase
inhibitors have the ability to rapidly and specifically inhibit the
activation of the target kinase molecules. Protein kinases are
attractive drug targets, in part because they regulate a wide
variety of signaling and growth pathways and include many different
proteins. As such, they have great potential in the treatment of
diseases involving kinase signaling, including cancer,
cardiovascular disease, inflammatory disorders, diabetes, macular
degeneration and neurological disorders. Examples of kinase
inhibitors include sorafenib (NEXAVAR.RTM.), SUTENT.RTM.,
DASATINIB.TM., ZACTIMA.TM., TYKERB.TM. and STI571.
[0048] The term "nucleoside" generally refers to compounds
consisting of a sugar, usually ribose or deoxyribose, and a purine
or pyrimidine base.
[0049] The term "nucleotide" generally refers to a nucleoside
comprising a phosphate group attached to the sugar.
[0050] As used herein, the term "pyrimidine nucleoside" refers to a
nucleoside wherein the base component of the nucleoside is a
pyrimidine base (e.g., cytosine (C) or thymine (T) or Uracil (U)).
Similarly, the term "purine nucleoside" refers to a nucleoside
wherein the base component of the nucleoside is a purine base
(e.g., adenine (A) or guanine (G)).
[0051] The terms "analog" or "derivative" can be used
interchangeable to generally refer to any purine and/or pyrimidine
nucleotide or nucleoside that has a modified base and/or sugar. A
modified base is a base that is not guanine, cytosine, adenine,
thymine or uracil. A modified sugar is any sugar that is not ribose
or 2'deoxyribose and can be used in the backbone for an
oligonucleotide.
[0052] The term "inhibiting" or "suppressing" generally refers to a
decrease in or a prevention of a response or qualitative difference
in a response, which could otherwise arise from eliciting and/or
stimulation of a response.
[0053] The term "non-nucleotide linker" generally refers to any
linkage or moiety that can link or be linked to the
oligonucleotides other than through a phosphorous-containing
linkage. Preferably such linker is from about 2 angstroms to about
200 angstroms in length.
[0054] The term "nucleotide linkage" generally refers to a direct
3'-5' linkage that directly connects the 3' and 5' hydroxyl groups
of two nucleosides through a phosphorous-containing linkage.
[0055] The term "treatment" generally refers to an approach
intended to obtain a beneficial or desired results, which may
include alleviation of symptoms and/or delaying and/or ameliorating
the progression of a disease or disorder.
[0056] The invention provides methods for inhibiting or suppressing
TLR9-mediated induction of an immune response in a mammal, such
methods comprising administering to the mammal a compound according
to the invention comprising the sequence (SEQ ID NO: 1):
5'-(N.sub.1).sub.mCG(N.sub.2).sub.p-3', wherein N.sub.1 and N.sub.2
are independently any nucleotide, m is number from 0 to about 5, p
is a number from about 6 to about 35, C is a nucleotide comprising
a nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 and/or N.sub.2 comprise a CG
dinucleotide C is a nucleotide comprising a nitrogenous base
selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In some embodiments, the compounds and
compositions that preferentially antagonize, inhibit, suppress or
prevent the activity of TLR9 have the following sequence (SEQ ID
NO: 2): 5'-(N.sub.1).sub.nCG(N.sub.2).sub.1CG(N.sub.3).sub.z-3',
wherein N.sub.1, N.sub.2, and N.sub.3 are independently any
nucleotide, n is 2, r is 11, z is 3, C is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto, and provided that at each instance that N.sub.1,
N.sub.2 and/or N.sub.3 comprise a CG dinucleotide C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In some embodiments, the mammal is a human. In
preferred embodiments, the compound is administered to a mammal in
need of immune suppression. In preferred embodiments, the compound
is selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
[0057] In some embodiments, the invention provides methods for
inhibiting or suppressing TLR9-mediated induction of an immune
response in a mammal, such methods comprising administering to the
mammal a compound according to the invention comprising the
sequence
5'-G.times.TC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3'
(SEQ ID NO: 3), wherein N.sub.1 2'-deoxyguanosine or
2'-deoxyguanosine methylphosphonate and N.sub.2 is
2'-deoxycytosine, wherein m is 10 and p is 0 or 1, and wherein C*
is a nucleotide comprising a nitrogenous base selected from the
group consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G* is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; and provided that at each instance that N.sub.1
comprises a CG dinucleotide C is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In some embodiments, the mammal is a human. In
preferred embodiments, the compound is administered to a mammal in
need of immune suppression. In preferred embodiments, the compound
is selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
[0058] A method for inhibiting the activity of a TLR9 agonist
comprising administering a compound comprising the sequence (SEQ ID
NO: 1): 5'-(N.sub.1).sub.mCG(N.sub.2).sub.p-3', wherein N.sub.1 and
N.sub.2 are independently any nucleotide, m is number from 0 to
about 5, p is a number from about 6 to about 35, C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 and/or N.sub.2 comprise a CG
dinucleotide C is a nucleotide comprising a nitrogenous base
selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In some embodiments, the compounds and
compositions that preferentially antagonize, inhibit, suppress or
prevent the activity of TLR9 have the following sequence (SEQ ID
NO: 2): 5'-(N.sub.1).sub.nCG(N.sub.2).sub.rCG(N.sub.3).sub.z-3',
wherein N.sub.1, N.sub.2, and N.sub.3 are independently any
nucleotide, n is 2, r is 11, z is 3, C is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto, and provided that at each instance that N.sub.1,
N.sub.2 and/or N.sub.3 comprise a CG dinucleotide C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In preferred embodiments, the compound is
selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
[0059] In some embodiments, the invention provides a method for
inhibiting the activity of a TLR9 agonist comprising administering
a compound comprising the sequence
5'-G.sub.xTC*G*(N).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3' (SEQ ID
NO: 3), wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein m is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto. In
preferred embodiments, the compound is selected from the group
consisting of 5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate.
[0060] The invention further provides methods for therapeutically
treating a patient, such methods comprising administering to the
patient a compound according to the invention comprising the
sequence (SEQ ID NO: 1): 5'-(N.sub.1).sub.mCG(N.sub.2).sub.p-3',
wherein N.sub.1 and N.sub.2 are independently any nucleotide, m is
number from 0 to about 5, p is a number from about 6 to about 35, C
is a nucleotide comprising a nitrogenous base selected from the
group consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; and provided that at each instance that N.sub.1
and/or N.sub.2 comprise a CG dinucleotide C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In some embodiments, the compounds and
compositions that preferentially antagonize, inhibit, suppress or
prevent the activity of TLR9 have the following sequence (SEQ ID
NO: 2): 5'-(N.sub.1).sub.nCG(N.sub.2).sub.rCG(N.sub.3).sub.r-3',
wherein N.sub.1, N.sub.2, and N.sub.3 are independently any
nucleotide, n is 2, r is 11, z is 3, C is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto, and provided that at each instance that N.sub.1,
N.sub.2 and/or N.sub.3 comprise a CG dinucleotide C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In preferred embodiments, the compound is
selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate. In some embodiments the patient has a disease or
disorder. In some embodiments, the disease or disorder is mediated
by a toll-like receptor (TLR). In some embodiments, the disease or
disorder is mediated by TLR9.
[0061] In some embodiments, the invention provides methods for
therapeutically treating a patient, such methods comprising
administering to the patient a compound according to the invention
comprising the sequence
5'-G.sub.xTC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3' (SEQ
ID NO: 3), wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein m is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto. In
preferred embodiments, the compound is selected from the group
consisting of 5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate. In some embodiments the patient has a disease or
disorder. In some embodiments, the disease or disorder is mediated
by a toll-like receptor (TLR). In some embodiments, the disease or
disorder is mediated by TLR9.
[0062] In various embodiments, the disease or disorder to be
treated is cancer, an autoimmune disorder, airway inflammation,
inflammatory disorders, infectious disease, malaria, Lyme disease,
ocular infections, conjunctivitis, skin disorders, psoriasis,
scleroderma, cardiovascular disease, atherosclerosis, chronic
fatigue syndrome, sarcoidosis, transplant rejection, allergy,
asthma or a disease caused by a pathogen. Preferred autoimmune
disorders include without limitation lupus erythematosus, multiple
sclerosis, type I diabetes mellitus, irritable bowel syndrome,
Crohn's disease, rheumatoid arthritis, septic shock, alopecia
universalis, acute disseminated encephalomyelitis, Addison's
disease, ankylosing spondylitis, antiphospholipid antibody
syndrome, autoimmune hemolytic anemia, autoimmune hepatitis,
Bullous pemphigoid, chagas disease, chronic obstructive pulmonary
disease, coeliac disease, dermatomyositis, endometriosis,
Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome,
Hashimoto's disease, hidradenitis suppurativa, idiopathic
thrombocytopenic purpura, interstitial cystitis, morphea,
myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious
anaemia, polymyositis, primary biliary cirrhosis, schizophrenia,
Sjogren's syndrome, temporal arteritis ("giant cell arteritis"),
vasculitis, vitiligo, vulvodynia and Wegener's granulomatosis.
Preferred inflammatory disorders include without limitation airway
inflammation, asthma, autoimmune diseases, chronic inflammation,
chronic prostatitis, glomerulonephritis, Behcet's disease,
hypersensitivities, inflammatory bowel disease, reperfusion injury,
rheumatoid arthritis, transplant rejection, ulcerative colitis,
uveitis, conjunctivitis and vasculitis. Pathogens include bacteria,
parasites, fungi, viruses, viroids, and prions. In some
embodiments, the disease or disorder is mediated by a TLR9-induced
immune response. In some embodiments the disease is selected from
ulcerative colitis, Crohn's disease, and inflammatory bowel
disease. In some embodiments the disease is ulcerative colitis. In
some embodiments the disease is Crohn's disease. In some
embodiments the disease is inflammatory bowel disease.
[0063] The invention further provides methods for preventing
disease or disorder, such methods comprising administering to the
patient a compound according to the invention comprising the
sequence (SEQ ID NO: 1): 5'-(N.sub.1).sub.mCG(N.sub.2).sub.p-3',
wherein N.sub.1 and N.sub.2 are independently any nucleotide, m is
number from 0 to about 5, p is a number from about 6 to about 35, C
is a nucleotide comprising a nitrogenous base selected from the
group consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; and provided that at each instance that N.sub.1
and/or N.sub.2 comprise a CG dinucleotide C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In some embodiments, the compounds and
compositions that preferentially antagonize, inhibit, suppress or
prevent the activity of TLR9 have the following sequence (SEQ ID
NO: 2): 5'-(N.sub.1).sub.nCG(N.sub.2).sub.rCG(N.sub.3).sub.z-3',
wherein N.sub.1, N.sub.2, and N.sub.3 are independently any
nucleotide, n is 2, r is 11, z is 3, C is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto, and provided that at each instance that N.sub.1,
N.sub.2 and/or N.sub.3 comprise a CG dinucleotide C is a nucleotide
comprising a nitrogenous base selected from the group consisting of
cytosine, 5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G
is a nucleotide comprising a nitrogenous base selected from the
group consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; or the complementary
sequence thereto. In preferred embodiments, the compound is
selected from the group consisting of
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate. In some embodiments, the disease or disorder is
mediated by a toll-like receptor (TLR). In some embodiments, the
disease or disorder is mediated by TLR9.
[0064] The invention further provides methods for preventing
disease or disorder, such methods comprising administering to the
patient a compound according to the invention comprising the
sequence
5'-G.sub.xTC*G*(N.sub.1).sub.mCC*G*CAG.sub.x(N.sub.2).sub.p-3' (SEQ
ID NO: 3), wherein N.sub.1 2'-deoxyguanosine or 2'-deoxyguanosine
methylphosphonate and N.sub.2 is 2'-deoxycytosine, wherein m is 10
and p is 0 or 1, and wherein C* is a nucleotide comprising a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and 2'-O-methylcytosine and G* is a
nucleotide comprising a nitrogenous base selected from the group
consisting of guanine, 5-methyl-2'-deoxyguanosine and
2'-O-methylguanine, provided that at least one of the nucleotides C
or G comprises a methylated nitrogenous base; and provided that at
each instance that N.sub.1 comprises a CG dinucleotide C is a
nucleotide comprising a nitrogenous base selected from the group
consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosine and G is a nucleotide comprising a nitrogenous
base selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and 2'-O-methylguanine, provided that at
least one of the nucleotides C or G comprises a methylated
nitrogenous base; or the complementary sequence thereto. In
preferred embodiments, the compound is selected from the group
consisting of 5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3,
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3',
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3',
5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3', and
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3', wherein
C.sub.1 is 5-methyl-dC and G.sub.1 is 2'-deoxyguanosine
methylphosphonate. In some embodiments, the disease or disorder is
mediated by a toll-like receptor (TLR). In some embodiments, the
disease or disorder is mediated by TLR9.
[0065] In various embodiments, the disease or disorder to be
prevented is cancer, an autoimmune disorder, airway inflammation,
inflammatory disorders, infectious disease, malaria, Lyme disease,
ocular infections, conjunctivitis, skin disorders, psoriasis,
scleroderma, cardiovascular disease, atherosclerosis, chronic
fatigue syndrome, sarcoidosis, transplant rejection, allergy,
asthma or a disease caused by a pathogen. Preferred autoimmune
disorders include without limitation lupus erythematosus, multiple
sclerosis, type I diabetes mellitus, irritable bowel syndrome,
Crohn's disease, rheumatoid arthritis, septic shock, alopecia
universalis, acute disseminated encephalomyelitis, Addison's
disease, ankylosing spondylitis, antiphospholipid antibody
syndrome, autoimmune hemolytic anemia, autoimmune hepatitis,
Bullous pemphigoid, chagas disease, chronic obstructive pulmonary
disease, coeliac disease, dermatomyositis, endometriosis,
Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome,
Hashimoto's disease, hidradenitis suppurativa, idiopathic
thrombocytopenic purpura, interstitial cystitis, morphea,
myasthenia gravis, narcolepsy, neuromyotonia, pemphigus, pernicious
anaemia, polymyositis, primary biliary cirrhosis, schizophrenia,
Sjogren's syndrome, temporal arteritis ("giant cell arteritis"),
vasculitis, vitiligo, vulvodynia and Wegener's granulomatosis.
Preferred inflammatory disorders include without limitation airway
inflammation, asthma, autoimmune diseases, chronic inflammation,
chronic prostatitis, glomerulonephritis, Behcet's disease,
hypersensitivities, inflammatory bowel disease, reperfusion injury,
rheumatoid arthritis, transplant rejection, ulcerative colitis,
uveitis, conjunctivitis and vasculitis. Pathogens include bacteria,
parasites, fungi, viruses, viroids, and prions. In some
embodiments, the disease or disorder is mediated by a TLR9-induced
immune response. In some embodiments the disease is selected from
ulcerative colitis, Crohn's disease, and inflammatory bowel
disease. In some embodiments the disease is ulcerative colitis. In
some embodiments the disease is Crohn's disease. In some
embodiments the disease is inflammatory bowel disease.
[0066] Certain compounds according to the invention are shown in
Table 2. In this table, the compounds have all phosphorothioate
(PS) linkages, except where indicated. Except where indicated, all
nucleotides are deoxyribonucleotides.
TABLE-US-00002 TABLE 2 Compound # Sequence / SEQ ID NO 1
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAG-3 / SEQ ID NO: 4 2
5'-GTC.sub.1GCCCCTTCTCCCC.sub.1GCAGC-3' / SEQ ID NO: 5 3
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1-3' / SEQ ID NO: 6 4
5'-G.sub.1TC.sub.1GCCCCTTCTCCCC.sub.1GCAG.sub.1C-3' / SEQ ID NO: 7
5 5'-GTC.sub.1GTTTACCTCTTCC.sub.1GCAGC-3' / SEQ ID NO: 8 C.sub.1 =
5-methyl 2'-deoxycytidine 5'-monophosphate; G.sub.1 =
2'-deoxyguanosine methylphosphonate
[0067] Certain control compounds are shown in Table 3. In this
table, the compounds have all phosphorothioate (PS) linkages,
except where indicated. Except where indicated, all nucleotides are
deoxyribonucleotides.
TABLE-US-00003 TABLE 3 Compound # Sequence / SEQ ID NO 6
5'-GTCGCCCCTTCTCCCCGCAG-3 / SEQ ID NO: 9 7
5-GTCGCCCCTTCTCCCCGCAGC-3 / SEQ ID NO: 10 8
5'-5'-C.sub.1TATC.sub.1TGUC.sub.1G.sub.2TTC.sub.1TC.sub.1TGU-3' /
SEQ ID NO: 11 (TLR antagonist) C.sub.1 = 5-methyl 2'-deoxycytidine
5'-monophosphate; G.sub.2 = 7-deaza-dG; G = 2'-O-Me-G; U =
2'-O-Me-U.
[0068] In some embodiments of any of the methods according to the
invention, two oligonucleotides are covalently linked via a direct
nucleotide to nucleotide linkage at their 3' ends through the 3'
positions of the sugars or through a modified sugar or modified
nucleobase or via a non-nucleotide linker at their 3' ends through
the 3' positions of the sugars or through a modified sugar or
modified nucleobase. In preferred aspects of this embodiment, at
least one of the oligonucleotide is a compound according to the
invention. In preferred embodiment, both oligonucleotides are
compounds according to the invention. In preferred embodiments, the
two oligonucleotides are covalently linked directly via a
nucleotide linkage. In more preferred embodiments, the two
oligonucleotides are covalently linked via a non-nucleotide
linker.
[0069] As a non-limiting example, the non-nucleotide linker
covalently linking the two oligonucleotides may be attached to the
3'-hydroxyl of the sugar. In such embodiments, the linker comprises
a functional group, which is attached to the 3'-hydroxyl by means
of a phosphate-based linkage like, for example, phosphodiester,
phosphorothioate, phosphorodithioate, methylphosphonate, or by a
non-phosphate-based linkage. Possible sites of conjugation for the
linker to the 3' end of the oligonucleotide are indicated in
Formula I, below, wherein B represents a heterocyclic base and
wherein the arrow pointing to P indicates any attachment to
phosphorous.
##STR00001##
[0070] In certain embodiments, the non-nucleotide linker is a small
molecule, macromolecule or biomolecule, including, without
limitation, polypeptides, antibodies, lipids, antigens, allergens,
and oligosaccharides. In certain other embodiments, the
non-nucleotide linker is a small molecule. For purposes of the
invention, a small molecule is an organic moiety having a molecular
weight of less than 1,000 Da. In some embodiments, the small
molecule has a molecular weight of less than 750 Da.
[0071] In some embodiments, the small molecule is an aliphatic or
aromatic hydrocarbon, either of which optionally can include,
either in the linear chain connecting the oligonucleotides or
appended to it, one or more functional groups including, but not
limited to, hydroxy, amino, thiol, thioether, ether, amide,
thioamide, ester, urea, or thiourea. The small molecule can be
cyclic or acyclic. Examples of small molecule linkers include, but
are not limited to, amino acids, carbohydrates, cyclodextrins,
adamantane, cholesterol, haptens, and antibiotics. However, for
purposes of describing the non-nucleotide linker, the term "small
molecule" is not intended to include a nucleoside.
[0072] In some embodiments, the non-nucleotide linker is an alkyl
linker or amino linker. The alkyl linker may be branched or
unbranched, cyclic or acyclic, substituted or unsubstituted,
saturated or unsaturated, chiral, achiral or racemic mixture. The
alkyl linkers can have from about 2 to about 18 carbon atoms. In
some embodiments such alkyl linkers have from about 2 to about 9
carbon atoms. In other embodiments, the alkyl linker has less than
3 carbon atoms. In further embodiments, the alkyl linker has at
least 3 carbon atoms and preferentially more than three carbon
atoms. Some alkyl linkers include one or more functional groups
including, but not limited to, hydroxy, amino, thiol, thioether,
ether, amide, thioamide, ester, urea, and thioether. Such alkyl
linkers can include, but are not limited to, 1,2 propanediol, 1,2,3
propanetriol, 1,3 propanediol, 1,2,4-Butanetriol,
1,3,5-Pentanetriol, 3-trimethylamino-1,2-propanediol,
Bis-1,5-O-(3'thymidyl(-1,3,5-pentanetriol,
Bis-1,5-O-[3'-(1,2-dideoxy-D-robosyl)]-1,3,5-pentanetriol,
3-(2-Hydroxyethyl)-1,5-pentanediol, triethylene glycol hexaethylene
glycol, polyethylene glycollinkers (e.g. [--O--CH2-CH2-].sub.n
(n=1-9)), methyl linkers, ethyl linkers, propyl linkers, butyl
linkers or hexyl linkers. In some embodiments, such alkyl linkers
may include peptides or amino acids.
[0073] In some embodiments, the non-nucleotide linker may include,
but are not limited to, those listed in Table 3.
TABLE-US-00004 TABLE 3 Representative Non-Nucleotidic Linkers
##STR00002## 1,2,3-Propanediol linker (glycerol) ##STR00003##
1,2,4-Butanetriol Linker ##STR00004## 1,3,5-Pentanetriol Linker
##STR00005## 3-Trimethylamino-1,2-propanediol Linker ##STR00006##
Bis-1,5-O-(3'-thymidyl)-1,3,5-pentanetriol Linker ##STR00007##
Bis-1,5-O-[3'-(1,2-dideoxy-D-ribosyl)]-1,3,5- pentanetriol Linker
##STR00008## 3-(2-Hydroxyethyl)-1,5-pentanediol Linker
[0074] In some embodiments, the small molecule linker is glycerol
or a glycerol homolog of the formula
HO--(CH.sub.2).sub.o--CH(OH)--(CH.sub.2).sub.p--OH, wherein o and p
independently are integers from 1 to about 6, from 1 to about 4, or
from 1 to about 3. In some other embodiments, the small molecule
linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such
derivatives have the formula
HO--(CH.sub.2).sub.m--C(O)NH--CH.sub.2--CH(OH)--CH.sub.2--NHC(O)--(CH.sub-
.2).sub.m--OH, wherein m is an integer from 0 to about 10, from 0
to about 6, from 2 to about 6, or from 2 to about 4.
[0075] Some non-nucleotide linkers permit attachment of more than
two oligonucleotides. For example, the small molecule linker
glycerol has three hydroxyl groups to which oligonucleotides may be
covalently attached. Some compounds according to the invention,
therefore, comprise two or more oligonucleotides linked to a
nucleotide or a non-nucleotide linker. Such compounds are referred
to as being "branched".
[0076] In any of the methods according to the invention, the
compounds can be combined with one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonist, TLR antagonist, peptides, proteins,
gene therapy vectors, DNA vaccines, adjuvants or kinase inhibitors
to enhance the specificity or magnitude of the immune response, or
co-stimulatory molecules such as cytokines, chemokines, protein
ligands, trans-activating factors, peptides and peptides comprising
modified amino acids.
[0077] In any of the methods according to the invention,
administration of the compound according to the invention can be by
any suitable route, including, without limitation, parenteral,
mucosal delivery, oral, sublingual, transdermal, topical,
inhalation, intragastric, intranasal, aerosol, intraocular,
intratracheal, intrarectal, vaginal, by gene gun, dermal patch or
in eye drop or mouthwash form. Administration of the therapeutic
compositions of the compound can be carried out using known
procedures at dosages and for periods of time effective to reduce
symptoms or surrogate markers of the disease. When administered
systemically, the therapeutic composition is preferably
administered at a sufficient dosage to attain a blood concentration
of compound from about 0.0001 micromolar to about 100 micromolar.
More preferably, systemic administration would be at a sufficient
dosage to attain a blood concentration of the compound from about
0.001 micromolar to about 10 micromolar. For localized
administration, much lower concentrations than this may be
effective, and much higher concentrations may be tolerated.
Preferably, a total dosage of the compound ranges from about 0.001
mg per patient per day to about 200 mg per kg body weight per day.
It may be desirable to administer the compound according to the
invention daily, every second day, every third day, every fourth
day, every fifth day, every sixth day or weekly. It may be
desirable to administer simultaneously, or sequentially, a
therapeutically effective amount of one or more of the containing
therapeutic compositions of the invention to an individual as a
single treatment episode.
[0078] The methods according to the invention are useful for model
studies of the immune system. The methods are also useful for the
prophylactic or therapeutic treatment of human or animal disease.
For example, the methods are useful for pediatric, adult, and
veterinary vaccine applications.
[0079] In any of the methods according to the invention, the
compound can be administered in combination with any other agent
useful for treating or preventing the disease or condition that
does not abolish the immune antagonist, inhibitory, suppression or
prevention effect or activity of the compound. In any of the
methods according to the invention, the agent useful for treating
or preventing the disease or condition includes, but is not limited
to, one or more vaccines, antigens, antibodies, cytotoxic agents,
allergens, antibiotics, antisense oligonucleotides, TLR agonist,
TLR antagonist, peptides, proteins, gene therapy vectors, DNA
vaccines, adjuvants or kinase inhibitors to enhance the specificity
or magnitude of the immune response, or co-stimulatory molecules
such as cytokines, chemokines, protein ligands, trans-activating
factors, peptides and peptides comprising modified amino acids. For
example, in the treatment of cancer, it is contemplated that the
compound may be administered in combination with one or more
chemotherapeutic compound, targeted therapeutic agent and/or
monoclonal antibody; and in preventing a disease, it is
contemplated that the compound may be administered in combination
with one or more vaccine.
[0080] In some embodiments, the invention provides a pharmaceutical
composition comprising a compound according to the invention and a
physiologically acceptable carrier. In some embodiments, the
composition can further comprise one or more vaccines, antigens,
antibodies, cytotoxic agents, allergens, antibiotics, antisense
oligonucleotides, TLR agonist, TLR antagonist, peptides, proteins,
gene therapy vectors, DNA vaccines, adjuvants or kinase inhibitors
to enhance the specificity or magnitude of the immune response, or
co-stimulatory molecules such as cytokines, chemokines, protein
ligands, trans-activating factors, peptides and peptides comprising
modified amino acids.
[0081] The following examples are intended to further illustrate
certain exemplary embodiments of the invention and are not intended
to limit the scope of the invention. For example, representative
TLR-ligands are shown in the following examples, but do not limit
the scope of ligands to which the compounds of the invention act as
antagonists.
Example 1
Compounds of the Invention do not Induce a TLR9-Mediated Immune
Response
[0082] C57 BL/6 mice (female, n=3) were injected subcutaneously
with 40 mg/kg of compound 8 (control TLR antagonist), compound 2,
compound 7, compound 5 (10 nt mismatch with compound 2) or PBS.
Serum samples were collected 2 hours after injection. Serum IL-12
level was determined by a mouse specific IL-12 ELISA. Results are
shown in FIG. 1.
Example 2
Compounds of the Invention are Antagonist of TLR9
[0083] C57 BL/6 mice (female, n=3) were injected subcutaneously
with 10 mg/kg of compound 8 (control TLR antagonist), compound 2,
compound 5 (10 nt mismatch with compound 2) or PBS. All mice were
injected subcutaneously with 0.25 mg/kg of TLR9 agonist. Serum
samples were collected 2 hours after TLR9 agonist administration.
Serum IL-12 level was determined by a mouse specific IL-12 ELISA.
Results are shown in FIG. 2.
[0084] SJL/J mice (female, n=10) were intrarectally administered
with 2.5 mg 2,4,6-trinitrobenzene sulfonic acid (TNBS,
Sigma-Aldrich, St. Louis, Mo.) in 100 .mu.l of 50% ethanol on day
0. On day 1, mice were intragastrically administered with 200 ml
(15 mg/kg) of compound 8 (TLR antagonist), compound 2, compound 5
(10 nt mismatch with compound 2) or PBS. All mice were monitored
for body weight change daily. Results are shown in FIG. 3.
[0085] On day 7, all mice were sacrificed, colon length were
measured by a caliper and colon samples were collected. Some of the
colon samples were homogenated and colon myeloperoxidase (MPO)
levels were evaluated using a MPO colorimetric assay kit
(Sigma-Aldrich). Asterisk indicates p<0.05 when compared to PBS
group. Statistical analysis was done by unpaired two-tailed
Student's t test. Results are shown in FIGS. 4 and 5.
[0086] Additionally, some of the colon samples were fixed with 10%
formalin and sectioned. Colon histology was evaluated (0: no
evidence of inflammation; 1: leucocyte infiltration in a <10%
high-power field (hpf), no structural changes observed; 2:
leucocyte infiltration in 10-25% hpf, crypt elongation, intestinal
wall thickening which does not extend beyond mucosal layer; 3:
leucocyte infiltration in 25-50% hpf, thickening of intestinal wall
which extends beyond mucosal layer; 4: leucocyte infiltration in
>50% hpf, mucosal structure distortion, transmural intestinal
wall thickening with ulceration.) Asterisk indicates p<0.05 when
compared to PBS group. Statistical analysis was done by unpaired
two-tailed Student's t test. Results are shown in FIG. 6.
[0087] Some of the colon samples were stored in RNAlater (Life
Technologies, Grand Island, N.Y.). Total RNA was isolated using
MagMax (Life Technologies) according to manufacturer's suggestion.
1 mg RNA was reverse transcribed to cDNA using High-Capacity
RNA-to-cDNA kit (Life Technologies). Gene expression was determined
using a mouse Crohn's disease PCR array (QIAGEN, Valencia, Calif.).
Gene expression levels were normalized to naive SJL/J mice (n=3).
Mean of Log 2 fold change were shown in FIG. 7.
[0088] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
1114DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(1)N = A,T,C or
Gmisc_feature(2)..(2)C is a nitrogenous base selected from the
group consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosinemisc_feature(3)..(3)G is a nitrogenous base
selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and
2'-O-methylguaninemisc_feature(4)..(4)N = A,T,C or G 1ncgn 4
27DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(1)N = A, T, C or
Gmisc_feature(2)..(2)C is a nitrogenous base selected from the
group consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosinemisc_feature(3)..(3)G is a nitrogenous base
selected from the group consisting of guanine,
5-methyl-2'-deomisc_feature(4)..(4)N = A, T, C or
Gmisc_feature(5)..(5)C is a nitrogenous base selected from the
group consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosinemisc_feature(6)..(6)G is a nitrogenous base
selected from the group consisting of guanine,
5-methyl-2'-deomisc_feature(7)..(7)N = A, T, C or G 2ncgncgn 7
312DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(1)2'-deoxyguanosine or
2'-deoxyguanosine methylphosphonatemisc_feature(3)..(3)C is a
nitrogenous base selected from the group consisting of cytosine,
5-methyl-2'-deoxycytosine and
2'-O-methylcytosinemisc_feature(4)..(4)G is a nitrogenous base
selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and
2'-O-methylguaninemisc_feature(5)..(5)n is a, c, g, or
tmisc_feature(7)..(7)C is a nitrogenous base selected from the
group consisting of cytosine, 5-methyl-2'-deoxycytosine and
2'-O-methylcytosinemisc_feature(8)..(8)G is a nitrogenous base
selected from the group consisting of guanine,
5-methyl-2'-deoxyguanosine and
2'-O-methylguaninemisc_feature(11)..(11)2'-deoxyguanosine or
2'-deoxyguanosine
methylphosphonatemisc_feature(12)..(12)2'-deoxycytosine 3gtcgnccgca
gn 12420DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(3)..(3)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(16)..(16)C = 5-methyl 2'-deoxycytidine
5'-monophosphate 4gtcgcccctt ctccccgcag 20521DNAArtificial
SequenceSynthetic oligonucleotidemisc_feature(3)..(3)C = 5-methyl
2'-deoxycytidine 5'-monophosphatemisc_feature(16)..(16)C = 5-methyl
2'-deoxycytidine 5'-monophosphate 5gtcgcccctt ctccccgcag c
21620DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(1)2'-deoxyguanosine
methylphosphonatemisc_feature(3)..(3)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(16)..(16)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(20)..(20)2'-deoxyguanosine
methylphosphonate 6gtcgcccctt ctccccgcag 20721DNAArtificial
SequenceSynthetic oligonucleotidemisc_feature(1)..(1)G =
2'-deoxyguanosine methylphosphonatemisc_feature(3)..(3)C = 5-methyl
2'-deoxycytidine 5'-monophosphatemisc_feature(16)..(16)C = 5-methyl
2'-deoxycytidine 5'-monophosphatemisc_feature(20)..(20)G =
2'-deoxyguanosine methylphosphonate 7gtcgcccctt ctccccgcag c
21821DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(3)..(3)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(16)..(16)C = 5-methyl 2'-deoxycytidine
5'-monophosphate 8gtcgtttacc tcttccgcag c 21920DNAArtificial
SequenceSynthetic oligonucleotide 9gtcgcccctt ctccccgcag
201021DNAArtificial SequenceSynthetic oligonucleotide 10gtcgcccctt
ctccccgcag c 211118DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(1)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(5)..(5)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(7)..(7)G =
2'-O-Me-Gmisc_feature(8)..(8)U = 2'-O-Me-Umisc_feature(9)..(9)C =
5-methyl 2'-deoxycytidine 5'-monophosphatemisc_feature(10)..(10)G
=7-deaza-dGmisc_feature(13)..(13)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(15)..(15)C = 5-methyl 2'-deoxycytidine
5'-monophosphatemisc_feature(17)..(17)G =
2'-O-Me-Gmisc_feature(18)..(18)U = 2'-O-Me-U 11ctatctgucg ttctctgu
18
* * * * *