U.S. patent application number 15/066404 was filed with the patent office on 2016-09-08 for compositions and methods for diagnosing and treating diseases and disorders associated with d-dt.
The applicant listed for this patent is YALE UNIVERSITY. Invention is credited to Richard J. Bucala, Lawrence H. Young.
Application Number | 20160258966 15/066404 |
Document ID | / |
Family ID | 46146424 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160258966 |
Kind Code |
A1 |
Bucala; Richard J. ; et
al. |
September 8, 2016 |
Compositions and Methods for Diagnosing and Treating Diseases and
Disorders Associated With D-DT
Abstract
The present invention relates to the discovery that altered
levels of D-DT (also known as MIF-2) are associated with disorders
and diseases. Thus, the present invention relates to compositions
and methods useful of the assessment, diagnosis, characterization,
prevention and treatment of disorders and diseases associated with
an elevated level of D-DT. The present invention also relates to
compositions and methods useful of the assessment, diagnosis,
characterization, prevention and treatment of disorders and
diseases associated with a reduced level of D-DT.
Inventors: |
Bucala; Richard J.; (Cos
Cob, CT) ; Young; Lawrence H.; (Woodbridge,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YALE UNIVERSITY |
New Haven |
CT |
US |
|
|
Family ID: |
46146424 |
Appl. No.: |
15/066404 |
Filed: |
March 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13989182 |
Oct 7, 2013 |
9308255 |
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PCT/US11/62062 |
Nov 23, 2011 |
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15066404 |
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61416904 |
Nov 24, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/24 20130101;
C07K 16/40 20130101; C12N 15/1137 20130101; A61K 31/713 20130101;
C07K 2317/622 20130101; C12Q 2600/158 20130101; C12N 2310/14
20130101; G01N 33/6893 20130101; A61K 38/52 20130101; C07K 2317/76
20130101; A61K 38/02 20130101; C12Q 1/6883 20130101; C07K 2317/24
20130101; G01N 2333/99 20130101; C12Y 503/03012 20130101; C12N
2310/111 20130101; A61K 39/3955 20130101; C12N 2310/141 20130101;
A61K 2039/505 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C12Q 1/68 20060101 C12Q001/68; A61K 38/52 20060101
A61K038/52; C07K 16/40 20060101 C07K016/40; C12N 15/113 20060101
C12N015/113 |
Claims
1. A method of diagnosing a disease or disorder in a subject in
need thereof, the method comprising: a. determining the level of
D-DT in a biological sample from the subject, b. comparing the
level of D-DT in the biological sample with a comparator control,
and diagnosing the subject with a disease or disorder when the
level of D-DT in the biological sample is different than the level
of D-DT of the comparator control.
2. The method of claim 1, wherein the level of D-DT in the
biological sample is elevated when compared with the comparator
control.
3. The method of claim 1, wherein the level of D-DT in the
biological sample is reduced when compared with the comparator
control.
4. The method of claim 1, wherein the level of D-DT in the
biological sample is determined by measuring the level of D-DT mRNA
in the biological sample.
5. The method of claim 1, wherein the level of D-DT in the
biological sample is determined by measuring the level of D-DT
polypeptide in the biological sample.
6. The method of claim 1, wherein the level of D-DT in the
biological sample is determined by measuring an enzymatic activity
of D-DT polypeptide in the biological sample.
7. The method of claim 1, wherein the level of D-DT in the
biological sample is determined by measuring the binding of a
detectable molecule to the D-DT enzyme substrate binding site.
8. The method of claim 1, wherein the level of D-DT in the
biological sample is determined by measuring the displacement of a
detectable molecule from the D-DT enzyme substrate binding
site.
9. The method of claim 1, wherein the comparator control is at
least one selected from the group consisting of: a positive
control, a negative control, a historical control, a historical
norm, or the level of a reference molecule in the biological
sample.
10. (canceled)
11. The method of claim 2, wherein the disease or disorder is at
least one selected from the group consisting of: infection,
inflammatory disease, autoimmunity and cancer.
12. The method of claim 3, wherein the disease or disorder is an
ischemia-reperfusion injury.
13. The method of claim 1, further comprising the step of treating
the subject for the diagnosed disease or disorder.
14. A composition comprising a D-DT inhibitor.
15. The composition of claim 14, wherein the D-DT inhibitor is an
antibody that specifically binds to D-DT.
16. The composition of claim 15, wherein the antibody specifically
binds to D-DT and does not specifically bind to MIF.
17. The composition of claim 15, wherein the antibody specifically
binds to D-DT and also specifically binds to MIF.
18. The composition of claim 15, wherein the antibody is at least
one selected from the group consisting of: a polyclonal antibody, a
monoclonal antibody, an intracellular antibody, an antibody
fragment, a single chain antibody (scFv), a heavy chain antibody, a
synthetic antibody, a chimeric antibody, and humanized
antibody.
19. The composition of claim 14, wherein the D-DT inhibitor is an
antisense nucleic acid.
20. The composition of claim 19, wherein the antisense nucleic acid
is at least one selected from the group consisting of: siRNA and
miRNA.
21. The composition of claim 20, wherein the siRNA comprises the
nucleic acid sequence of SEQ ID NO: 2.
22. The composition of claim 14 wherein the D-DT inhibitor is at
least one selected from the group consisting of: a chemical
compound, a protein, a peptide, a peptidomemetic, a ribozyme, and a
small molecule chemical compound.
23. A method of treating or preventing a disease or disorder in a
subject in need thereof, the method comprising: administering to
the subject a therapeutically effective amount of a composition
comprising a D-DT inhibitor.
24. The method of claim 23, wherein the D-DT inhibitor is an
antibody that specifically binds to D-DT.
25. The method of claim 24, wherein the antibody specifically binds
to D-DT and does not specifically bind to MIF.
26. The method of claim 24, wherein the antibody specifically binds
to D-DT and also specifically binds to MIF.
27. The method of claim 24, wherein the antibody is at least one
selected from the group consisting of: a polyclonal antibody, a
monoclonal antibody, an intracellular antibodies, an antibody
fragment, a single chain antibody (scFv), a heavy chain antibody, a
synthetic antibody, a chimeric antibody, and humanized
antibody.
28. The method of claim 23, wherein the D-DT inhibitor is an
antisense nucleic acid.
29. The method of claim 28, wherein the antisense nucleic acid is
at least one selected from the group consisting of: siRNA and
miRNA.
30. The method of claim 29, wherein the siRNA comprises the nucleic
acid sequence of SEQ ID NO: 2.
31. The method of claim 23, wherein the D-DT inhibitor is at least
one selected from the group consisting of: a chemical compound, a
protein, a peptide, a peptidomemetic, a ribozyme, and a small
molecule chemical compound.
32. The method of claim 23, wherein the disease or disorder is at
least one selected from the group consisting of: infection,
inflammatory disease, autoimmunity and cancer.
33-44. (canceled)
45. A composition comprising a D-DT activator.
46. The composition of claim 45, wherein the D-DT activator is at
least one selected from the group consisting of: a chemical
compound, a protein, a peptide, a peptidomemetic, an antisense
nucleic acid, a ribozyme, or a small molecule chemical
compound.
47-63. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Macrophage migration inhibitory factor (MIF) is the first
cytokine activity described and a key regulatory mediator that is
released upon activation of different cell types (Bloom, et al.,
1966, Science 153:80-82; David, 1966, Proc Natl Acad Sci USA
56:72-77; Bernhagen, et al., 2003, Nature 365:756-759). MIF
increases macrophage antimicrobial responses and it is expressed
upstream of cytokines such as tumor necrosis factor (TNF)-.alpha.,
IFN-.gamma., and IL-1.beta. (Calandra, et al., 2003 Nat Rev Immunol
3:791-800). MIF activates immune cells by binding to CD74, leading
to the recruitment of CD44 into a signaling complex, the
stimulation of nonreceptor tyrosine kinases, and initiation of the
ERK1/2 MAP kinase pathway (Shi, et al., 2006 Immunity 25:595-606;
Leng, et al., 2003, J Exp Med 197:1467-1476). The chemokine
receptors CXCR2 and CXCR4 also become activated by MIF via
noncognate interactions that are reinforced in the presence of CD74
(Bernhagen, et al., 2007, Nat Med 13:587-596). Among mesenchymal
cell types, MIF binding to cardiomyocyte CD74 stimulates the
AMP-activated kinase (AMPK) cascade to mediate protection from
ischemic injury (Miller, et al., 2008, Nature 451:578-582; Qi, et
al., 2009, J Clin Invest 119: 3807-3816).
[0002] Although MIF receptor knockout mice (CD74-/-) phenocopy
features MIF deficiency (Meyer-Siegler, et al., 2006, J Immunol
177:8730-8739; Topilski, et al., 2002, J Immunol 168:1610-1617),
recent observations have led to the hypothesis that there may be a
second ligand for CD74. MIF-deficient B cells, for example, are
more sensitive to apoptosis than wild-type B cells, but the
magnitude of this defect is twofold more pronounced in
CD74-deficient cells (Gore, et al., 2008, J Biol Chem
283:2784-2792). Intravital microscopy studies also have shown a
more pronounced effect of antagonism of CD74 than MIF in monocyte
arrest (Bernhagen, et al., 2007, Nat Med 13:587-596). Anti-MIF
antibodies, although highly effective in experimental studies, do
not completely inhibit CD74-dependent cellular activation responses
(Chagnon, et al., 2005, Circ Res 96:1095-1102).
[0003] D-dopachrome tautomerase (D-DT) (also known as MIF-2) and
MIF show a conserved intron-exon structure and their coding regions
are highly homologous. The genes for MIF and D-DT are in close
apposition to each other and to two theta-class glutathione
S-transferases, suggesting that these gene clusters arose by an
ancestral duplication event. D-DT was named for its ability to
tautomerize the nonnaturally occurring, D-stereoisomer of
dopachrome, which is a catalytic property shared with MIF. This
activity has been hypothesized to be a vestigial function that
reflects MIF's ancestral origin in the invertebrate melanotic
encapsulation response (Fingerle-Rowson, et al., 2009, Mol Cell
Biol 29:1922-1932). A crystal structure of D-DT has verified its 3D
similarity with MIF (Sugimoto, et al., 1999, Biochemistry
38:3268-3279). With the exception of recent studies indicating an
interaction between the MIF and D-DT genes in the expression of
proangiogenic factors and COX-2 in adenocarcinoma cell lines (Xin,
et al., 2010, Mol Cancer Res 8:1601-1609; Coleman, et al., 2008, J
Immunol 181:2330-2337), there have been no studies of the biologic
functions of D-DT.
[0004] Despite the advances made in the art for detecting and
treating animation associated with MIF signaling through CD74,
there is a need in the art for the detection and treatment of
inflammation associated with other molecules that signal through
CD74. The present invention fulfills these needs.
SUMMARY OF THE INVENTION
[0005] The present invention relates to the discovery that altered
levels of D-DT (also known as MIF-2) are associated with disorders
and diseases. Thus, the present invention relates to compositions
and methods useful for the assessment, diagnosis, characterization,
prevention and treatment of disorders and diseases associated with
an elevated level of D-DT. The present invention also relates to
compositions and methods useful of the assessment, diagnosis,
characterization, prevention and treatment of disorders and
diseases associated with a reduced level of D-DT.
[0006] In one embodiment, the invention is a method of diagnosing a
disease or disorder in a subject including the steps of:
determining the level of D-DT in a biological sample from the
subject, comparing the level of D-DT in the biological sample with
a comparator control, and diagnosing the subject with a disease or
disorder when the level of D-DT in the biological sample is
different than the level of D-DT of the comparator control. In one
embodiment, the level of D-DT in the biological sample is elevated
when compared with the comparator control, while in another
embodiment, the level of D-DT in the biological sample is reduced
when compared with the comparator control. In one embodiment, the
level of D-DT in the biological sample is determined by measuring
the level of D-DT mRNA, while in another embodiment, the level of
the level of D-DT in the biological sample is determined by
measuring the level of D-DT polypeptide. In some embodiments, the
level of D-DT in the biological sample is determined by measuring
an enzymatic activity of D-DT polypeptide in the biological sample.
In another embodiment, the level of D-DT in the biological sample
is determined by measuring the binding of a detectable molecule to
the D-DT enzyme substrate binding site. In a further embodiment,
the level of D-DT in the biological sample is determined by
measuring the displacement of a detectable molecule from the D-DT
enzyme substrate binding site. In various embodiments, the
comparator control is at least one selected from the group
consisting of: a positive control, a negative control, a historical
control, a historical norm, or the level of a reference molecule in
the biological sample. In a particular embodiment, the reference
molecule is MIF. The method of diagnosing a disease or disorder of
the present invention useful in diagnosing a variety of diseases
and disorders associated with D-DT, including, for example,
infection, inflammatory disease, autoimmunity, cancer and
ischemia-reperfusion injury. In preferred embodiments, the subject
is human.
[0007] In another embodiment, the invention is a composition
comprising a D-DT inhibitor. In various embodiments, the D-DT
inhibitor is an antibody that specifically binds to D-DT, an
antibody that specifically binds to D-DT and does not specifically
bind to MIF, or an antibody that specifically binds to D-DT and
also specifically binds to MIF. In various embodiments, the D-DT
antibody is at least one of a polyclonal antibody, a monoclonal
antibody, an intracellular antibody, an antibody fragment, a single
chain antibody (scFv), a heavy chain antibody, a synthetic
antibody, a chimeric antibody, and humanized antibody. In another
embodiment, the D-DT inhibitor is an antisense nucleic acid. In
some embodiments, the antisense nucleic acid is an siRNA or an
miRNA. In a particular embodiment, the D-DT inhibitor is an siRNA
comprising the nucleic acid sequence of SEQ ID NO: 2. In other
various embodiments, the D-DT inhibitor is at least one of a
chemical compound, a protein, a peptide, a peptidomemetic, a
ribozyme, or a small molecule chemical compound.
[0008] In one embodiment, the invention is a method of treating a
disease or disorder in a subject by administering to the subject a
therapeutically effective amount of a composition comprising a D-DT
inhibitor. In various embodiments, the D-DT inhibitor is an
antibody that specifically binds to D-DT, an antibody that
specifically binds to D-DT and does not specifically bind to MIF,
or an antibody that specifically binds to D-DT and also
specifically binds to MIF. In various embodiments, the D-DT
antibody is at least one of a polyclonal antibody, a monoclonal
antibody, an intracellular antibody, an antibody fragment, a single
chain antibody (scFv), a heavy chain antibody, a synthetic
antibody, a chimeric antibody, and humanized antibody. In another
embodiment, the D-DT inhibitor is an antisense nucleic acid. In
some embodiments, the antisense nucleic acid is an siRNA or an
miRNA. In a particular embodiment, the D-DT inhibitor is an siRNA
comprising the nucleic acid sequence of SEQ ID NO: 2. In other
various embodiments, the D-DT inhibitor is at least one of a
chemical compound, a protein, a peptide, a peptidomemetic, a
ribozyme, or a small molecule chemical compound. In various
embodiments, the disease or disorder is at least one of infection,
inflammatory disease, autoimmunity and cancer. In preferred
embodiments, the subject is human.
[0009] In another embodiment, the invention is a method of
preventing a disease or disorder in a subject by administering to
the subject a therapeutically effective amount of composition
comprising a D-DT inhibitor. In various embodiments, the D-DT
inhibitor is an antibody that specifically binds to D-DT, an
antibody that specifically binds to D-DT and does not specifically
bind to MIF, or an antibody that specifically binds to D-DT and
also specifically binds to MIF. In various embodiments, the D-DT
antibody is at least one of a polyclonal antibody, a monoclonal
antibody, an intracellular antibody, an antibody fragment, a single
chain antibody (scFv), a heavy chain antibody, a synthetic
antibody, a chimeric antibody, and humanized antibody. In another
embodiment, the D-DT inhibitor is an antisense nucleic acid. In
some embodiments, the antisense nucleic acid is an siRNA or an
miRNA. In a particular embodiment, the D-DT inhibitor is an siRNA
comprising the nucleic acid sequence of SEQ ID NO: 2. In other
various embodiments, the D-DT inhibitor is at least one of a
chemical compound, a protein, a peptide, a peptidomemetic, a
ribozyme, or a small molecule chemical compound. In various
embodiments, the disease or disorder is at least one of infection,
inflammatory disease, autoimmunity and cancer. In preferred
embodiments, the subject is human.
[0010] In one embodiment, the invention is a composition comprising
a D-DT activator. In other various embodiments, the D-DT activator
is at least one of a chemical compound, a protein, a peptide, a
peptidomemetic, an antisense nucleic acid, a ribozyme, or a small
molecule chemical compound.
[0011] In another embodiment, the invention is a method of treating
ischemia-reperfusion injury in a subject by administering to the
subject a therapeutically effective amount of a composition
comprising at least one of a D-DT polypeptide, a recombinant D-DT
polypeptide, an active D-DT polypeptide fragment, or a D-DT
activator. In a preferred embodiment, the subject is a human.
[0012] In a further embodiment, the invention is a method of
preventing ischemia-reperfusion injury in a subject by
administering to the subject a therapeutically effective amount of
a composition comprising at least one of a D-DT polypeptide, a
recombinant D-DT polypeptide, an active D-DT polypeptide fragment,
or a D-DT activator. In a preferred embodiment, the subject is a
human.
[0013] In yet another embodiment, the invention is a method of
preventing ischemia-reperfusion injury in a tissue or organ by
administering to the tissue or organ a therapeutically effective
amount of a composition comprising at least one selected from the
group consisting of: a D-DT polypeptide, a recombinant D-DT
polypeptide, an active D-DT polypeptide fragment, or a D-DT
activator. In some embodiments, the tissue or organ is a
pre-transplant tissue or organ. In a preferred embodiment, the
tissue or organ is human.
[0014] In another embodiment, the invention is a method of
identifying a test compound as a modulator of D-DT including the
steps of: determining the level of D-DT in the presence of a test
compound, determining the level of D-DT in the absence of a test
compound, comparing the level of D-DT in the presence of the test
compound with the level of D-DT in the absence of the test
compound, and identifying the test compound as a modulator of D-DT
when the level of D-DT in the presence of the test compound is
different than the level of D-DT in the absence of the test
compound. In some embodiments, the test compound is identified as a
D-DT activator when the level of D-DT is higher in the presence of
the test compound. In other embodiments, the test compound is
identified as a D-DT inhibitor when the level of D-DT is lower in
the presence of the test compound. In some embodiments, the level
of D-DT is determined by measuring the level of D-DT mRNA. In other
embodiments, the level of D-DT is determined by measuring the level
of D-DT polypeptide. In some embodiments, the level of D-DT is
determined by measuring an enzymatic activity of D-DT polypeptide.
In certain embodiments, the enzymatic activity is tautomerase
activity. In some embodiments, the level of D-DT is determined by
measuring the binding of a detectable molecule to the D-DT enzyme
substrate binding site. In other embodiments, the level of D-DT is
determined by measuring the displacement of a detectable molecule
from the D-DT enzyme substrate binding site. In various
embodiments, the test compound is at least one of: a chemical
compound, a protein, a peptide, a peptidomemetic, an antibody, a
nucleic acid, an antisense nucleic acid, a ribozyme, and a small
molecule chemical compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0016] FIG. 1, comprising FIGS. 1A and 1B, is a schematic depicting
the genomic organization and protein homology of MIF and D-DT (also
known as MIF-2). (1A) Schematic diagrams showing the relationship
between human MIF and D-DT with matrix metalloprotease 11 and GST
genes (Upper) and the amino acid sequence and secondary structure
homologies of the two proteins (Lower). (1B) Mouse Mif, D-DT, and
adjacent genes (Upper) and the corresponding amino acid sequence
and secondary structure homologies of the two proteins (Lower).
Gene structure was compiled from www.ensembl.org and sequence
alignment performed using ClustalX and
espript.ibcp.fr/ESPript/ESPript.
[0017] FIG. 2 depicts the results of experiments characterizing the
D-DT protein. Characterization of the D-DT protein. (A) SDS/PAGE
and Coomassie analysis of sequential purification steps of
recombinant D-DT protein. The examples shown are for mouse D-DT but
qualitatively identical results were obtained for human D-DT. (B)
Electrospray ionization mass spectrometry of mouse D-DT showing a
molecular mass (m/z) that is within 0.02% accuracy of the predicted
m/z (12,946 Da). (C) Tautomerization activity of human MIF and D-DT
measured with the substrate, p-hydoxyphenylpyruvate. Results are
expressed as mean.+-.SD of duplicate measurements and are
representative of three experiments. (D) Anti-D-DT antibody
specifically recognizes D-DT. (Left) Anti-D-DT antibody recognizes
recombinant murine D-DT protein in Western blotting (1-100
ng/lane), but does not detect recombinant mouse MIF. (Right) D-DT
ELISA quantifies concentrations in the picogram range and shows no
crossreactivity to MIF. Results are expressed as mean.+-.SD of
duplicate measurements and are representative for two independent
experiments.
[0018] FIG. 3, comprising FIGS. 3A-3E, depicts the results of
experiments demonstrating the D-DT binds with high affinity to the
MIF receptor. D-DT binds with high affinity to the MIF receptor,
CD74. (3A) Concentration-dependent binding of D-DT and MIF to the
MIF receptor ectodomain, sCD74, using biotinylated human MIF as
competitor. Heat-denatured MIF served as a negative control. (3B)
Real-time surface plasmon resonance analysis (BIAcore) of the
interaction between D-DT and sCD74. (3C) Coimmunoprecipitation of
D-DT/JAB1 and MIF/JAB1. (Left) Cells were lysed and recombinant
D-DT was added. JAB1/D-DT-containing protein complexes were
coprecipitated by pull-down of JAB1, and D-DT was detected by
Western blot. (Right) Coimmunoprecipitation between JAB1 and MIF
following the same protocol. (3D) D-DT is differentially expressed
in mouse tissue. Protein lysates (75 .mu.g) were separated by
SDS/PAGE and analyzed by Western blot for D-DT, MIF, and CD74 (n=2
mice studied). (3E) D-DT protein expression analyzed by
immunostaining of five representative organs from a C57BL/6 mouse
(n=3 mice studied).
[0019] FIG. 4, comprising FIGS. 4A-4C, depicts the results of
experiments assessing the functional comparison of D-DT and MIF.
(4A) D-DT activates the sustained ERK1/2 MAP kinase pathway in a
MIF receptor complex (CD74/CD44)-dependent manner. (Top)
Macrophages (1.times.10.sup.6/mL) were treated with 0, 10, or 50
ng/mL of D-DT or MIF for 2 hours. Cell lysates were analyzed for
phosphorylation of ERK1/2. (Middle) Macrophages were treated with
the indicated concentrations of D-DT, MIF, or D-DT plus MIF.
Lysates were analyzed for the phosphorylation status of ERK1/2.
(Bottom) Wild-type and MIF receptor knockout (CD74-/- or CD44-/-)
macrophages were treated with 50 ng/mL of D-DT or MIF for 2 hours,
and cell lysates were analyzed by Western blot. Results are
representative of at least two independent experiments. (4B)
Increasing concentrations of D-DT or MIF inhibit the chemotaxis of
human peripheral blood monocytes to MCP-1. Data shown are
mean.+-.SD of quadruplicate assays and statistical significance for
the comparison of MIF vs. D-DT was analyzed by an unpaired
Student's t test; *P<0.01. (4C) D-DT or MIF inhibits
glucocorticoid-mediated suppression of TNF production. Macrophages
were preincubated for 1 hour with or without dexamethasone (Dex,
100 nM), MIF, or D-DT (100 ng/mL) and then stimulated with LPS (100
ng/mL). Supernatants were collected after 4 hours and TNF was
quantified by ELISA. Data shown are mean.+-.SD of triplicate
samples from one experiment and are representative of four
independent experiments. *P<0.005, **P<0.001 vs. LPS+Dex
condition by an unpaired Student's t test.
[0020] FIG. 5, comprising FIGS. 5A-5E, depicts the results of
experiments demonstrating that the neutralization of D-DT protects
from lethal endotoxic shock. (5A) D-DT is released from macrophages
after LPS stimulation. Peritoneal macrophages (1.times.106/mL) were
stimulated with LPS or PBS (control) and supernatants were analyzed
by ELISA for D-DT and MIF content. Results are expressed as
mean.+-.SD of duplicate assays and are representative of at least
three independent experiments. (5B) Reciprocal regulation of MIF
and D-DT. Macrophages were transfected with MIF, D-DT, or control
siRNA, respectively, and cultivated for 4 days. (Upper) Cells were
lysed and a Western blot was performed. (Lower) Macrophages were
stimulated with LPS (1 ng/mL) and after 6 hours the supernatants
were collected for ELISA. (5C) LPS challenge leads to increased
D-DT concentrations in serum. BALB/c mice (8 wk, female) were
challenged with 12.5 mg/kg of LPS, and blood was drawn 0, 6, 12,
and 24 hours after i.p. LPS administration. Serum was analyzed by
ELISA for D-DT and MIF content. The results are expressed as mean
values.+-.SD of two independent experiments (n=10), and statistical
significance was by Student's t test, **P<0.01, ***P<0.001.
(5D) Neutralization of D-DT protects from lethal endotoxemia.
BALB/c mice were injected i.p. with anti-D-DT antibody or nonimmune
antibody (control) 2 hours before LPS administration (20 mg/kg).
Data points are from three independent experiments. Survival was
75% (15 of 20) in mice treated with anti-D-DT antibody and 19% (4
of 21) in mice treated with control antibody. P<0.0001,
Kaplan-Meier test. (5E) Neutralization of D-DT influences serum
cytokine concentrations. Mice were treated with LPS as in C and
blood was drawn for cytokine analysis by Luminex. The results are
expressed as mean values.+-.SD of three independent experiments
(Student's t test, *P<0.05, **P<0.01).
[0021] FIG. 6, comprising FIGS. 6A-6D, depicts the results of
experiments demonstrating that human serum concentrations of D-DT
correlate with MIF, sepsis severity, and the presence of ovarian
cancer. (6A) D-DT and MIF are elevated in the serum of patients
with sepsis. Median concentrations of D-DT and MIF in healthy
controls were 6.9 ng/mL and 6.3 ng/mL, respectively. In patients
with sepsis, the median concentrations were 56 ng/mL for D-DT and
111 ng/mL for MIF (*P<0.0001 by nonparametric t test). ROC
analysis revealed an area under the curve of 0.99 for both
proteins. (6B) Positive correlation between the APACHE II (sepsis
severity) scores and the levels of either D-DT or MIF. (6C) D-DT
and MIF show a significant correlation both in the serum of healthy
individuals and in the serum of patients with severe sepsis. (6D)
D-DT and MIF are elevated in the serum of patients with ovarian
cancer and show a positive correlation. Statistical significance
between sera from healthy and diseased individuals was determined
by nonparametric t test, *P<0.001, and the significance of
correlation was by Pearson calculation.
[0022] FIG. 7 depicts the results of experiments demonstrating that
human serum concentrations of D-DT correlate with MIF and the
presence of vasculitis.
[0023] FIG. 8 depicts the results of an example western blot
experiment demonstrating that D-DT activates AMPK.
[0024] FIG. 9 depicts the results of experiments demonstrating that
endogenous DDT has a role in mediating AMPK activation during
hypoxia in isolated rat heart left ventricular muscles. The
addition of purified rabbit polyclonal neutralizing antibody for 30
minutes prior to and during 15 minutes of hypoxia significantly
reduced the critical phosphorylation of threonine 172 in the
activating domain of the alpha catalytic subunit of AMP-activated
protein kinase. DDT antibody also decreased the phosphorylation of
downstream acetyl-CoA carboxylase. Control was performed with
non-immune IgG incubation. FIG. 9B shows a comparison to incubation
with MIF neutralizing antibody. p=0.05.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention relates to the discovery that altered
levels of D-DT (also known as MIF-2) are associated with disorders
and diseases. Thus, the present invention relates to compositions
and methods useful for the assessment, diagnosis, characterization,
prevention and treatment of disorders and diseases associated with
an elevated level of D-DT. The present invention also relates to
compositions and methods useful of the assessment, diagnosis,
characterization, prevention and treatment of disorders and
diseases associated with a reduced level of D-DT.
[0026] In some embodiments, the compositions of the invention
relate to inhibitors of D-DT. The methods of the invention include
methods of diagnosing disorders and diseases associated with
elevated levels of D-DT, as well as methods of monitoring the
effectiveness of an applied treatment regimen of a disorder or
disease associated with an elevated level of D-DT. In various
embodiments, the disorders and diseases that can be diagnosed,
assessed, characterized, prevented or treated using the
compositions and methods of the invention include infection,
inflammatory disease, autoimmunity and cancer.
[0027] In other embodiments, the compositions of the invention
relate to activators of D-DT. The methods of the invention include
methods of diagnosing disorders and diseases associated with
reduced levels of D-DT, as well as methods of monitoring the
effectiveness of an applied treatment regimen of a disorder or
disease associated with a reduced level of D-DT. In various
embodiments, the disorders and diseases that can be diagnosed,
assessed, characterized, prevented or treated using the
compositions and methods of the invention include
ischemia-reperfusion injury.
DEFINITIONS
[0028] As used herein, each of the following terms has the meaning
associated with it in this section.
[0029] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0030] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0031] The term "abnormal" when used in the context of organisms,
tissues, cells or components thereof, refers to those organisms,
tissues, cells or components thereof that differ in at least one
observable or detectable characteristic (e.g., age, treatment, time
of day, etc.) from those organisms, tissues, cells or components
thereof that display the "normal" (expected) respective
characteristic. Characteristics which are normal or expected for
one cell or tissue type, might be abnormal for a different cell or
tissue type.
[0032] An "allele" refers to one specific form of a genetic
sequence (such as a gene) within a cell, an individual or within a
population, the specific form differing from other forms of the
same gene in the sequence of at least one, and frequently more than
one, variant sites within the sequence of the gene. The sequences
at these variant sites that differ between different alleles are
termed "variants." "polymorphisms," or "mutations."
[0033] As used herein, to "alleviate" a disease means reducing the
frequency or severity of at least one sign or symptom of a disease
or disorder.
[0034] As used herein the terms "alteration," "defect,"
"variation," or "mutation," refers to a mutation in a gene in a
cell that affects the function, activity, expression (transcription
or translation) or conformation of the polypeptide that it encodes.
Mutations encompassed by the present invention can be any mutation
of a gene in a cell that results in the enhancement or disruption
of the function, activity, expression or conformation of the
encoded polypeptide, including the complete absence of expression
of the encoded protein and can include, for example, missense and
nonsense mutations, insertions, deletions, frameshifts and
premature terminations. Without being so limited, mutations
encompassed by the present invention may alter splicing the mRNA
(splice site mutation) or cause a shift in the reading frame
(frameshift).
[0035] The term "amplification" refers to the operation by which
the number of copies of a target nucleotide sequence present in a
sample is multiplied.
[0036] The term "antibody," as used herein, refers to an
immunoglobulin molecule which is able to specifically bind to a
specific epitope on an antigen. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant
sources and can be immunoreactive portions of intact
immunoglobulins. The antibodies in the present invention may exist
in a variety of forms including, for example, polyclonal
antibodies, monoclonal antibodies, intracellular antibodies
("intrabodies"), Fv, Fab and F(ab)2, as well as single chain
antibodies (scFv), heavy chain antibodies, such as camelid
antibodies, synthetic antibodies, chimeric antibodies, and
humanized antibodies (Harlow et al., 1999, Using Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow
et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor,
N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; Bird et al., 1988, Science 242:423-426).
[0037] An "antibody heavy chain," as used herein, refers to the
larger of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
[0038] An "antibody light chain," as used herein, refers to the
smaller of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
.kappa. and .lamda. light chains refer to the two major antibody
light chain isotypes.
[0039] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0040] As used herein, an "immunoassay" refers to any binding assay
that uses an antibody capable of binding specifically to a target
molecule to detect and quantify the target molecule.
[0041] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes a
specific antigen, but does not substantially recognize or bind
other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more species. But, such cross-species
reactivity does not itself alter the classification of an antibody
as specific. In another example, an antibody that specifically
binds to an antigen may also bind to different allelic forms of the
antigen. However, such cross reactivity does not itself alter the
classification of an antibody as specific. In some instances, the
terms "specific binding" or "specifically binding," can be used in
reference to the interaction of an antibody, a protein, or a
peptide with a second chemical species, to mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0042] By the term "applicator," as the term is used herein, is
meant any device including, but not limited to, a hypodermic
syringe, a pipette, an iontophoresis device, a patch, and the like,
for administering the compositions of the invention to a
subject.
[0043] The term "auto-antigen" means, in accordance with the
present invention, any self-antigen which is mistakenly recognized
by the immune system as being foreign. Auto-antigens comprise, but
are not limited to, cellular proteins, phosphoproteins, cellular
surface proteins, cellular lipids, nucleic acids, glycoproteins,
including cell surface receptors.
[0044] The term "cancer," or "neoplasm" as used herein includes,
but is not limited to, benign and malignant cancers of the oral
cavity (e.g., mouth, tongue, pharynx, etc.), digestive system
(e.g., esophagus, stomach, small intestine, colon, rectum, liver,
bile duct, gall bladder, pancreas, etc.), respiratory system (e.g.,
larynx, lung, bronchus, etc.), bones, joints, skin (e.g., basal
cell, squamous cell, melanoma, etc.), breast, genital system,
(e.g., uterus, ovary, prostate, testis, etc.), urinary system (e.g,
bladder, kidney, ureter, etc.), eye, nervous system (e.g., brain,
etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic
system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic
leukemia, chronic lymphocytic leukemia, acute myeloid leukemia,
chronic myeloid leukemia, etc.).
[0045] The term "coding sequence," as used herein, means a sequence
of a nucleic acid or its complement, or a part thereof, that can be
transcribed and/or translated to produce the mRNA and/or the
polypeptide or a fragment thereof. Coding sequences include exons
in a genomic DNA or immature primary RNA transcripts, which are
joined together by the cell's biochemical machinery to provide a
mature mRNA. The anti-sense strand is the complement of such a
nucleic acid, and the coding sequence can be deduced therefrom. In
contrast, the term "non-coding sequence," as used herein, means a
sequence of a nucleic acid or its complement, or a part thereof,
that is not translated into amino acid in vivo, or where tRNA does
not interact to place or attempt to place an amino acid. Non-coding
sequences include both intron sequences in genomic DNA or immature
primary RNA transcripts, and gene-associated sequences such as
promoters, enhancers, silencers, and the like.
[0046] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "A-G-T," is complementary to the sequence
"T-C-A." Complementarity may be "partial," in which only some of
the nucleic acids' bases are matched according to the base pairing
rules. Or, there may be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, as well as
detection methods that depend upon binding between nucleic
acids.
[0047] As used herein, the term "diagnosis" refers to the
determination of the presence of a disease or disorder. In some
embodiments of the present invention, methods for making a
diagnosis are provided which permit determination of a the presence
of a particular disease or disorder.
[0048] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0049] An "effective amount" as used herein, means an amount which
provides a therapeutic or prophylactic benefit.
[0050] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0051] As used herein, the term "fragment," as applied to a nucleic
acid, refers to a subsequence of a larger nucleic acid. A
"fragment" of a nucleic acid can be at least about 15 nucleotides
in length; for example, at least about 50 nucleotides to about 100
nucleotides; at least about 100 to about 500 nucleotides, at least
about 500 to about 1000 nucleotides; at least about 1000
nucleotides to about 1500 nucleotides; about 1500 nucleotides to
about 2500 nucleotides; or about 2500 nucleotides (and any integer
value in between). As used herein, the term "fragment," as applied
to a protein or peptide, refers to a subsequence of a larger
protein or peptide. A "fragment" of a protein or peptide can be at
least about 20 amino acids in length; for example, at least about
50 amino acids in length; at least about 100 amino acids in length;
at least about 200 amino acids in length; at least about 300 amino
acids in length; or at least about 400 amino acids in length (and
any integer value in between).
[0052] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that includes coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., mRNA). The
polypeptide may be encoded by a full length coding sequence or by
any portion of the coding sequence so long as the desired activity
or functional property (e.g., enzymatic activity, ligand binding,
signal transduction, immunogenicity, etc.) of the full-length or
fragment is retained. The term also encompasses the coding region
of a structural gene and the sequences located adjacent to the
coding region on both the 5' and 3' ends for a distance of about 2
kb or more on either end such that the gene corresponds to the
length of the full-length mRNA and 5' regulatory sequences which
influence the transcriptional properties of the gene. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5'-untranslated sequences. The 5'-untranslated
sequences usually contain the regulatory sequences. Sequences
located 3' or downstream of the coding region and present on the
mRNA are referred to as 3'-untranslated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0053] A "genome" is all the genetic material of an organism. In
some instances, the term genome may refer to the chromosomal DNA.
Genome may be multichromosomal such that the DNA is cellularly
distributed among a plurality of individual chromosomes. For
example, in human there are 22 pairs of chromosomes plus a gender
associated XX or XY pair. DNA derived from the genetic material in
the chromosomes of a particular organism is genomic DNA. The term
genome may also refer to genetic materials from organisms that do
not have chromosomal structure. In addition, the term genome may
refer to mitochondria DNA. A genomic library is a collection of DNA
fragments representing the whole or a portion of a genome.
Frequently, a genomic library is a collection of clones made from a
set of randomly generated, sometimes overlapping DNA fragments
representing the entire genome or a portion of the genome of an
organism.
[0054] "Homologous" refers to the sequence similarity or sequence
identity between two polypeptides or between two nucleic acid
molecules. When a position in both of the two compared sequences is
occupied by the same base or amino acid monomer subunit, e.g., if a
position in each of two DNA molecules is occupied by adenine, then
the molecules are homologous at that position. The percent of
homology between two sequences is a function of the number of
matching or homologous positions shared by the two sequences
divided by the number of positions compared.times.100. For example,
if 6 of 10 of the positions in two sequences are matched or
homologous then the two sequences are 60% homologous. By way of
example, the DNA sequences ATTGCC and TATGGC share 50% homology.
Generally, a comparison is made when two sequences are aligned to
give maximum homology.
[0055] The term "housekeeping gene" as used herein refers to genes
that are generally always expressed and thought to be involved in
routine cellular metabolism. Housekeeping genes are well known and
include such genes as glyceraldehyde-3-phosphate dehydrogenase
(G3PDH or GAPDH), albumin, actins, tubulins, cyclophilin,
hypoxanthine phsophoribosyltransferase (HRPT), 28S, and 18S rRNAs
and the like.
[0056] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementarity between the nucleic acids,
stringency of the conditions involved, the T.sub.m of the formed
hybrid, and the G:C ratio within the nucleic acids. A single
molecule that contains pairing of complementary nucleic acids
within its structure is said to be "self-hybridized." A single DNA
molecule with internal complementarity could assume a variety of
secondary structures including loops, kinks or, for long stretches
of base pairs, coils.
[0057] The term "immunoglobulin" or "Ig," as used herein is defined
as a class of proteins, which function as antibodies. Antibodies
expressed by B cells are sometimes referred to as the BCR (B cell
receptor) or antigen receptor. The five members included in this
class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the
primary antibody that is present in body secretions, such as
saliva, tears, breast milk, gastrointestinal secretions and mucus
secretions of the respiratory and genitourinary tracts. IgG is the
most common circulating antibody. IgM is the main immunoglobulin
produced in the primary immune response in most subjects. It is the
most efficient immunoglobulin in agglutination, complement
fixation, and other antibody responses, and is important in defense
against bacteria and viruses. IgD is the immunoglobulin that has no
known antibody function, but may serve as an antigen receptor. IgE
is the immunoglobulin that mediates immediate hypersensitivity by
causing release of mediators from mast cells and basophils upon
exposure to allergen.
[0058] "Instructional material," as that term is used herein,
includes a publication, a recording, a diagram, or any other medium
of expression which can be used to communicate the usefulness of
the nucleic acid, peptide, and/or compound of the invention in the
kit for identifying, diagnosing or alleviating or treating the
various diseases or disorders recited herein. Optionally, or
alternately, the instructional material may describe one or more
methods of identifying, diagnosing or alleviating the diseases or
disorders in a cell or a tissue of a subject. The instructional
material of the kit may, for example, be affixed to a container
that contains the nucleic acid, peptide, and/or compound of the
invention or be shipped together with a container that contains the
nucleic acid, peptide, and/or compound. Alternatively, the
instructional material may be shipped separately from the container
with the intention that the recipient uses the instructional
material and the compound cooperatively.
[0059] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in a
living animal is not "isolated," but the same nucleic acid or
peptide partially or completely separated from the coexisting
materials of its natural state is "isolated." An isolated nucleic
acid or protein can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a host
cell.
[0060] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0061] The term "label" when used herein refers to a detectable
compound or composition that is conjugated directly or indirectly
to a probe to generate a "labeled" probe. The label may be
detectable by itself (e.g. radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition that is
detectable (e.g., avidin-biotin). In some instances, primers can be
labeled to detect a PCR product.
[0062] The terms "microarray" and "array" refers broadly to "DNA
microarrays," "DNA chip(s)," "protein microarrays" and "protein
chip(s)" and encompasses all art-recognized solid supports, and all
art-recognized methods for affixing nucleic acid, peptide, and
polypeptide molecules thereto. Preferred arrays typically comprise
a plurality of different nucleic acid or peptide probes that are
coupled to a surface of a substrate in different, known locations.
These arrays, also described as "microarrays" or colloquially
"chips" have been generally described in the art, for example, U.S.
Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 5,800,992,
6,040,193, 5,424,186 and Fodor et al., 1991, Science, 251:767-777,
each of which is incorporated by reference in its entirety for all
purposes. Arrays may generally be produced using a variety of
techniques, such as mechanical synthesis methods or light directed
synthesis methods that incorporate a combination of
photolithographic methods and solid phase synthesis methods.
Techniques for the synthesis of these arrays using mechanical
synthesis methods are described in, e.g., U.S. Pat. Nos. 5,384,261,
and 6,040,193, which are incorporated herein by reference in their
entirety for all purposes. Although a planar array surface is
preferred, the array may be fabricated on a surface of virtually
any shape or even a multiplicity of surfaces. Arrays may be nucleic
acids on beads, gels, polymeric surfaces, fibers such as fiber
optics, glass or any other appropriate substrate. (See U.S. Pat.
Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992,
which are hereby incorporated by reference in their entirety for
all purposes.) Arrays may be packaged in such a manner as to allow
for diagnostic use or can be an all-inclusive device; e.g., U.S.
Pat. Nos. 5,856,174 and 5,922,591 incorporated in their entirety by
reference for all purposes. Arrays are commercially available from,
for example, Affymetrix (Santa Clara, Calif.) and Applied
Biosystems (Foster City, Calif.), and are directed to a variety of
purposes, including genotyping, diagnostics, mutation analysis,
marker expression, and gene expression monitoring for a variety of
eukaryotic and prokaryotic organisms. The number of probes on a
solid support may be varied by changing the size of the individual
features. In one embodiment the feature size is 20 by 25 microns
square, in other embodiments features may be, for example, 8 by 8,
5 by 5 or 3 by 3 microns square, resulting in about 2,600,000,
6,600,000 or 18,000,000 individual probe features.
[0063] Assays for amplification of the known sequence are also
disclosed. For example primers for PCR may be designed to amplify
regions of the sequence. For RNA, a first reverse transcriptase
step may be used to generate double stranded DNA from the single
stranded RNA. The array may be designed to detect sequences from an
entire genome; or one or more regions of a genome, for example,
selected regions of a genome such as those coding for a protein or
RNA of interest; or a conserved region from multiple genomes; or
multiple genomes, arrays and methods of genetic analysis using
arrays is described in Cutler, et al., 2001, Genome Res. 11(11):
1913-1925 and Warrington, et al., 2002, Hum Mutat 19:402-409 and in
US Patent Pub No 20030124539, each of which is incorporated herein
by reference in its entirety.
[0064] By the term "modulating," as used herein, is meant mediating
a detectable increase or decrease in the level of a mRNA,
polypeptide, or a response in a subject compared with the level of
a mRNA, polypeptide or a response in the subject in the absence of
a treatment or compound, and/or compared with the level of a mRNA,
polypeptide, or a response in an otherwise identical but untreated
subject. The term encompasses perturbing and/or affecting a native
signal or response thereby mediating a beneficial therapeutic
response in a subject, preferably, a human.
[0065] A "mutation," as used herein, refers to a change in nucleic
acid or polypeptide sequence relative to a reference sequence
(which is preferably a naturally-occurring normal or "wild-type"
sequence), and includes translocations, deletions, insertions, and
substitutions/point mutations. A "mutant" as used herein, refers to
either a nucleic acid or protein comprising a mutation.
[0066] A "nucleic acid" refers to a polynucleotide and includes
poly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acids
according to the present invention may include any polymer or
oligomer of pyrimidine and purine bases, preferably cytosine,
thymine, and uracil, and adenine and guanine, respectively. (See
Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth
Pub. 1982) which is herein incorporated in its entirety for all
purposes). Indeed, the present invention contemplates any
deoxyribonucleotide, ribonucleotide or peptide nucleic acid
component, and any chemical variants thereof, such as methylated,
hydroxymethylated or glucosylated forms of these bases, and the
like. The polymers or oligomers may be heterogeneous or homogeneous
in composition, and may be isolated from naturally occurring
sources or may be artificially or synthetically produced. In
addition, the nucleic acids may be DNA or RNA, or a mixture
thereof, and may exist permanently or transitionally in
single-stranded or double-stranded form, including homoduplex,
heteroduplex, and hybrid states.
[0067] An "oligonucleotide" or "polynucleotide" is a nucleic acid
ranging from at least 2, preferably at least 8, 15 or 25
nucleotides in length, but may be up to 50, 100, 1000, or 5000
nucleotides long or a compound that specifically hybridizes to a
polynucleotide. Polynucleotides include sequences of
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or mimetics
thereof which may be isolated from natural sources, recombinantly
produced or artificially synthesized. A further example of a
polynucleotide of the present invention may be a peptide nucleic
acid (PNA). (See U.S. Pat. No. 6,156,501 which is hereby
incorporated by reference in its entirety.) The invention also
encompasses situations in which there is a nontraditional base
pairing such as Hoogsteen base pairing which has been identified in
certain tRNA molecules and postulated to exist in a triple helix.
"Polynucleotide" and "oligonucleotide" are used interchangeably in
this disclosure. It will be understood that when a nucleotide
sequence is represented herein by a DNA sequence (e.g., A, T, G,
and C), this also includes the corresponding RNA sequence (e.g., A,
U, G, C) in which "U" replaces "T".
[0068] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0069] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis (U.S. Pat. Nos. 4,683,195
4,683,202, and 4,965,188, hereby incorporated by reference), which
describe a method for increasing the concentration of a segment of
a target sequence in a mixture of genomic DNA without cloning or
purification. This process for amplifying the target sequence
consists of introducing a large excess of two oligonucleotide
primers to the DNA mixture containing the desired target sequence,
followed by a precise sequence of thermal cycling in the presence
of a DNA polymerase. The two primers are complementary to their
respective strands of the double stranded target sequence. To
effect amplification, the mixture is denatured and the primers then
annealed to their complementary sequences within the target
molecule. Following annealing, the primers are extended with a
polymerase so as to form a new pair of complementary strands. The
steps of denaturation, primer annealing and polymerase extension
can be repeated many times (i.e., denaturation, annealing and
extension constitute one "cycle"; there can be numerous "cycles")
to obtain a high concentration of an amplified segment of the
desired target sequence. The length of the amplified segment of the
desired target sequence is determined by the relative positions of
the primers with respect to each other, and therefore, this length
is a controllable parameter. By virtue of the repeating aspect of
the process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified". As used herein, the terms "PCR product," "PCR
fragment," "amplification product" or "amplicon" refer to the
resultant mixture of compounds after two or more cycles of the PCR
steps of denaturation, annealing and extension are complete. These
terms encompass the case where there has been amplification of one
or more segments of one or more target sequences.
[0070] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, that is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences.
[0071] The term "perfect match," "match," "perfect match probe" or
"perfect match control" refers to a nucleic acid that has a
sequence that is perfectly complementary to a particular target
sequence. The nucleic acid is typically perfectly complementary to
a portion (subsequence) of the target sequence. A perfect match
(PM) probe can be a "test probe," a "normalization control" probe,
an expression level control probe and the like. A perfect match
control or perfect match is, however, distinguished from a
"mismatch" or "mismatch probe." The term "mismatch," "mismatch
control" or "mismatch probe" refers to a nucleic acid whose
sequence is not perfectly complementary to a particular target
sequence. As a non-limiting example, for each mismatch (MM) control
in a high-density probe array there typically exists a
corresponding perfect match (PM) probe that is perfectly
complementary to the same particular target sequence. The mismatch
may comprise one or more bases. While the mismatch(es) may be
located anywhere in the mismatch probe, terminal mismatches are
less desirable because a terminal mismatch is less likely to
prevent hybridization of the target sequence. In a particularly
preferred embodiment, the mismatch is located at or near the center
of the probe such that the mismatch is most likely to destabilize
the duplex with the target sequence under the test hybridization
conditions.
[0072] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0073] As used herein, "polynucleotide" includes cDNA, RNA, DNA/RNA
hybrid, antisense RNA, ribozyme, genomic DNA, synthetic forms, and
mixed polymers, both sense and antisense strands, and may be
chemically or biochemically modified to contain non-natural or
derivatized, synthetic, or semi-synthetic nucleotide bases. Also,
contemplated are alterations of a wild type or synthetic gene,
including but not limited to deletion, insertion, substitution of
one or more nucleotides, or fusion to other polynucleotide
sequences.
[0074] The term "primer" refers to an oligonucleotide capable of
acting as a point of initiation of synthesis along a complementary
strand when conditions are suitable for synthesis of a primer
extension product. The synthesizing conditions include the presence
of four different deoxyribonucleotide triphosphates and at least
one polymerization-inducing agent such as reverse transcriptase or
DNA polymerase. These are present in a suitable buffer, which may
include constituents which are co-factors or which affect
conditions such as pH and the like at various suitable
temperatures. A primer is preferably a single strand sequence, such
that amplification efficiency is optimized, but double stranded
sequences can be utilized.
[0075] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
[0076] The term "reaction mixture" or "PCR reaction mixture" or
"master mix" or "master mixture" refers to an aqueous solution of
constituents in a PCR reaction that can be constant across
different reactions. An exemplary PCR reaction mixture includes
buffer, a mixture of deoxyribonucleoside triphosphates, primers,
probes, and DNA polymerase. Generally, template RNA or DNA is the
variable in a PCR.
[0077] "Sample" or "biological sample" as used herein means a
biological material isolated from a subject. The biological sample
may contain any biological material suitable for detecting a mRNA,
polypeptide or other marker of a physiologic or pathologic process
in a subject, and may comprise fluid, tissue, cellular and/or
non-cellular material obtained from the individual.
[0078] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. Under "low stringency conditions" a
nucleic acid sequence of interest will hybridize to its exact
complement, sequences with single base mismatches, closely related
sequences (e.g., sequences with 90% or greater homology), and
sequences having only partial homology (e.g., sequences with 50-90%
homology). Under "medium stringency conditions," a nucleic acid
sequence of interest will hybridize only to its exact complement,
sequences with single base mismatches, and closely related
sequences (e.g., 90% or greater homology). Under "high stringency
conditions," a nucleic acid sequence of interest will hybridize
only to its exact complement, and (depending on conditions such a
temperature) sequences with single base mismatches. In other words,
under conditions of high stringency the temperature can be raised
so as to exclude hybridization to sequences with single base
mismatches.
[0079] As used herein, "substantially purified" refers to being
essentially free of other components. For example, a substantially
purified cell is a cell which has been separated from other cell
types with which it is normally associated in its naturally
occurring state. In some instances, a population of substantially
purified cells refers to a homogenous population of cells. In other
instances, this term refers simply to a cell that have been
separated from the cells with which they are naturally associated
in their natural state.
[0080] The term "target" as used herein refers to a molecule that
has an affinity for a given probe. Targets may be
naturally-occurring or man-made molecules. Also, they can be
employed in their unaltered state or as aggregates with other
species. Targets may be attached, covalently or noncovalently, to a
binding member, either directly or via a specific binding
substance. Targets are sometimes referred to in the art as
anti-probes. As the term targets is used herein, no difference in
meaning is intended.
[0081] As used herein, the terms "therapy" or "therapeutic regimen"
refer to those activities taken to alleviate or alter a disorder or
disease state, e.g., a course of treatment intended to reduce or
eliminate at least one sign or symptom of a disease or disorder
using pharmacological, surgical, dietary and/or other techniques. A
therapeutic regimen may include a prescribed dosage of one or more
drugs or surgery. Therapies will most often be beneficial and
reduce or eliminate at least one sign or symptom of the disorder or
disease state, but in some instances the effect of a therapy will
have non-desirable or side-effects. The effect of therapy will also
be impacted by the physiological state of the subject, e.g., age,
gender, genetics, weight, other disease conditions, etc.
[0082] The term "therapeutically effective amount" refers to the
amount of the subject compound that will elicit the biological or
medical response of a tissue, system, or subject that is being
sought by the researcher, veterinarian, medical doctor or other
clinician. The term "therapeutically effective amount" includes
that amount of a compound that, when administered, is sufficient to
prevent development of, or alleviate to some extent, one or more of
the signs or symptoms of the disorder or disease being treated. The
therapeutically effective amount will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the subject to be treated.
[0083] To "treat" a disease as the term is used herein, means to
reduce the frequency or severity of at least one sign or symptom of
a disease or disorder experienced by a subject.
[0084] As used herein, the term "wild-type" refers to a gene or
gene product isolated from a naturally occurring source. A
wild-type gene is that which is most frequently observed in a
population and is thus arbitrarily designed the "normal" or
"wild-type" form of the gene. In contrast, the term "modified" or
"mutant" refers to a gene or gene product that displays
modifications in sequence and/or functional properties (i.e.,
altered characteristics) when compared to the wild-type gene or
gene product. It is noted that naturally occurring mutants can be
isolated; these are identified by the fact that they have altered
characteristics (including altered nucleic acid sequences) when
compared to the wild-type gene or gene product.
DESCRIPTION
[0085] The present invention relates to the discovery that altered
levels of D-DT (also known as MIF-2) are associated with disorders
and diseases. Thus, the present invention relates to compositions
and methods useful for the assessment, diagnosis, characterization,
prevention and treatment of disorders and diseases associated with
an elevated level of D-DT. The present invention also relates to
compositions and methods useful of the assessment, diagnosis,
characterization, prevention and treatment of disorders and
diseases associated with a reduced level of D-DT.
[0086] In some embodiments, the compositions of the invention
relate to inhibitors of D-DT. The methods of the invention include
methods of diagnosing disorders and diseases associated with
elevated levels of D-DT, as well as methods of monitoring the
effectiveness of an applied treatment regimen of a disorder or
disease associated with an elevated level of D-DT. In various
embodiments, the disorders and diseases that can be diagnosed,
assessed, characterized, prevented or treated using the
compositions and methods of the invention include infection,
inflammatory disease, autoimmunity and cancer. By way of
non-limiting examples, the disorders and diseases that can be
diagnosed, assessed, characterized, prevented or treated using the
compositions and methods of the invention include: asthma,
atheroma, atherosclerosis, autism, autoinflammatory disease,
autoimmune myocarditis, autoimmune hepatitis, bacterial infection,
cancer, celiac disease, cellular proliferative disorder, Crohn's
disease, colitis, diabetes, dermatitis, diverticulitis, dry
age-related macular degeneration (AMD), endotoxemia,
glomerulonephritis, graft versus host disease, Guillain-Barre
syndrome, heart disease, hepatitis, inflammation, inflammatory
breast cancer, inflammatory demyelinating polyneuropathy,
intestinal cystitis, irritable bowel disease (IBD), lupus
erythematous, microbial infection, multiple sclerosis, neoplasia,
ovarian cancer, pelvic inflammatory disease (PID), prostatitis,
psoriasis, reperfusion injury, rheumatoid arthritis, sarcoidosis,
sepsis, septic shock, transplant rejection, trauma-induced
inflammation, ulcerative colitis, vasculitis, Wegener's
granulomatous and wet age-related macular degeneration (AMD).
[0087] The present invention also relates to the discovery that
signaling by D-DT through CD74 activates AMPK in cardiomyocytes.
Thus, in other embodiments, the compositions of the invention
relate to activators of D-DT. The methods of the invention include
methods of diagnosing disorders and diseases associated with
reduced levels of D-DT, as well as methods of monitoring the
effectiveness of an applied treatment regimen of a disorder or
disease associated with a reduced level of D-DT. In various
embodiments, the disorders and diseases that can be diagnosed,
assessed, characterized, prevented or treated using the
compositions and methods of the invention include
ischemia-reperfusion injury in, for example, the heart and other
solid organs, including, but not limited to, the kidney, the liver
and the brain.
Assays
[0088] The present invention relates to the discovery that altered
levels of D-DT are associated with infection, inflammatory disease,
autoimmunity, cancer and ischemia-reperfusion injury. In some
embodiments, the invention relates to a screening assay of a
subject to determine whether the subject has an elevated level of
D-DT. In other embodiments, the invention relates to a screening
assay of a subject to determine whether the subject has a reduced
level of D-DT. The present invention provides methods of assessing
the level of D-DT in a subject. In various embodiments, the level
of D-DT in the biological sample can be determined by assessing the
amount of D-DT polypeptide present in the biological sample, the
amount of D-DT mRNA present in the biological sample, the amount of
D-DT enzymatic activity in the biological sample, the amount of
D-DT receptor binding activity in the biological sample, or a
combination thereof.
[0089] The present invention also provides methods of diagnosing a
subject having an elevated level of D-DT, with a disease or
disorder, such as infection, inflammatory disease, autoimmunity and
cancer. Further, the present invention provides methods of
diagnosing a subject having a reduced level of D-DT, with a disease
or disorder, such as ischemia-reperfusion injury.
[0090] In one embodiment, the method of the invention is a
diagnostic assay for diagnosing infection, inflammatory disease,
autoimmunity or cancer in a subject in need thereof, by determining
whether the level of D-DT is increased in a biological sample
obtained from the subject. In various embodiments, to determine
whether the level of D-DT is increased in a biological sample
obtained from the subject, the level of D-DT is compared with the
level of at least one comparator control, such as a positive
control, a negative control, a historical control, a historical
norm, or the level of another reference molecule in the biological
sample. In one embodiment, the reference molecule in the biological
sample is MIF. In certain embodiments, the ratio of D-DT and MIF is
determined to aid in the diagnosis. The results of the diagnostic
assay can be used alone, or in combination with other information
from the subject, or other information from the biological sample
obtained from the subject.
[0091] In another embodiment, the method of the invention is a
diagnostic assay for diagnosing ischemia-reperfusion injury in a
subject in need thereof, by determining whether the level of D-DT
is reduced in a biological sample obtained from the subject. In
various embodiments, to determine whether the level of D-DT is
reduced in a biological sample obtained from the subject, the level
of D-DT is compared with the level of at least one comparator
control, such as a positive control, a negative control, a
historical control, a historical norm, or the level of another
reference molecule in the biological sample. In one embodiment, the
reference molecule in the biological sample is MIF. In certain
embodiments, the ratio of D-DT and MIF is determined to aid in the
diagnosis. The results of the diagnostic assay can be used alone,
or in combination with other information from the subject, or other
information from the biological sample obtained from the
subject.
[0092] In a further embodiment, the method of the invention is an
assay for monitoring the effectiveness of a treatment administered
to a subject in need thereof, by determining whether the level of
D-DT in a biological sample obtained from the subject is modulated
upon administration of the treatment. The assay can be performed
before, during or after a treatment has been administered, or any
combination thereof. In various embodiments, to determine whether
the level of D-DT is modulated in a biological sample obtained from
the subject, the level of D-DT is compared with the level of at
least one comparator control, such as a positive control, a
negative control, a historical control, a historical norm, or the
level of another reference molecule in the biological sample. In
one embodiment, the reference molecule in the biological sample is
MIF. In certain embodiments, the ratio of D-DT and MIF is
determined to aid in the monitoring of the treatment. The results
of the assay can be used alone, or in combination with other
information from the subject, or other information from the
biological sample obtained from the subject.
[0093] In various embodiments of the assays of the invention, the
level of D-DT is determined to be elevated when the level of D-DT
is increased by at least 10%, by at least 20%, by at least 30%, by
at least 40%, by at least 50%, by at least 60%, by at least 70%, by
at least 80%, by at least 90%, by at least 100%, by at least 125%,
by at least 150%, by at least 175%, by at least 200%, by at least
250%, by at least 300%, by at least 400%, by at least 500%, by at
least 600%, by at least 700%, by at least 800%, by at least 900%,
by at least 1000%, by at least 1500%, by at least 2000%, by at
least 2500%, by at least 3000%, by at least 4000%, or by at least
5000%, when compared with a comparator control.
[0094] In other various embodiments of the assays of the invention,
the level of D-DT is determined to be reduced when the level of
D-DT is reduced by at least 10%, by at least 20%, by at least 30%,
by at least 40%, by at least 50%, by at least 60%, by at least 70%,
by at least 80%, by at least 90%, by at least 100%, by at least
125%, by at least 150%, by at least 175%, by at least 200%, by at
least 250%, by at least 300%, by at least 400%, by at least 500%,
by at least 600%, by at least 700%, by at least 800%, by at least
900%, by at least 1000%, by at least 1500%, by at least 2000%, by
at least 2500%, by at least 3000%, by at least 4000%, or by at
least 5000%, when compared with a comparator control.
[0095] In the assay methods of the invention, a test biological
sample from a subject is assessed for the level of D-DT in the
biological sample obtained from the patient. The level of D-DT in
the biological sample can be determined by assessing the amount of
D-DT polypeptide in the biological sample, the amount of D-DT mRNA
in the biological sample, the amount of D-DT enzymatic activity in
the biological sample, the amount of D-DT receptor binding activity
in the biological sample, or a combination thereof. In one
embodiment, the D-DT enzymatic activity assessed is D-DT
tautomerization activity. In another embodiment, the D-DT enzymatic
activity assessed is D-DT tautomerization of p-hydoxyphenylpyruvate
(HPP). In various embodiments, the subject is a human subject, and
may be of any race, sex and age. Representative subjects include
those who are suspected of having infection, inflammatory disease,
autoimmunity or cancer, those who have been diagnosed with
infection, inflammatory disease, autoimmunity or cancer, those who
have infection, inflammatory disease, autoimmunity or cancer, those
who have had infection, inflammatory disease, autoimmunity or
cancer, those who at risk of a recurrence of infection,
inflammatory disease, autoimmunity or cancer, and those who are at
risk of developing infection, inflammatory disease, autoimmunity or
cancer.
[0096] In various embodiments, the test sample is a sample
containing at least a fragment of a D-DT polypeptide or a D-DT
nucleic acid. The term, "fragment," as used herein, indicates that
the portion of the polypeptide, mRNA or cDNA is of a length that is
sufficient to identify the fragment as D-DT.
[0097] The test sample is prepared from a biological sample
obtained from the subject. The biological sample can be a sample
from any source which contains a polypeptide or a nucleic acid,
such as a body fluid or a tissue, or a combination thereof. A
biological sample can be obtained by appropriate methods, such as,
by way of examples, blood draw, fluid draw, or biopsy. A biological
sample can be used as the test sample; alternatively, a biological
sample can be processed to enhance access to the polypeptides or
nucleic acids, or copies of the nucleic acids, and the processed
biological sample can then be used as the test sample. For example,
in various embodiments, nucleic acid (e.g., mRNA, cDNA prepared
from mRNA, etc.) is prepared from a biological sample, for use in
the methods. Alternatively or in addition, if desired, an
amplification method can be used to amplify nucleic acids
comprising all or a fragment of a mRNA in a biological sample, for
use as the test sample in the assessment of the level of D-DT in
the biological sample.
[0098] In various embodiments of the invention, methods of
measuring D-DT polypeptide levels in a biological sample obtained
from a patient include, but are not limited to, an
immunochromatography assay, an immunodot assay, a Luminex assay, an
ELISA assay, an ELISPOT assay, a protein microarray assay, a
ligand-receptor binding assay, displacement of a ligand from a
receptor assay, displacement of a ligand from a shared receptor
assay, an immunostaining assay, a Western blot assay, a mass
spectrophotometry assay, a radioimmunoassay (RIA), a
radioimmunodiffusion assay, a liquid chromatography-tandem mass
spectrometry assay, an ouchterlony immunodiffusion assay, reverse
phase protein microarray, a rocket immunoelectrophoresis assay, an
immunohistostaining assay, an immunoprecipitation assay, a
complement fixation assay, FACS, an enzyme-substrate binding assay,
an enzymatic assay, an enzymatic assay employing a detectable
molecule, such as a chromophore, fluorophore, or radioactive
substrate, a substrate binding assay employing such a substrate, a
substrate displacement assay employing such a substrate, and a
protein chip assay (see also, 2007, Van Emon, Immunoassay and Other
Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay
Handbook, Gulf Professional Publishing; 1996, Diamandis and
Christopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays
in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti,
Proteomics Today, John Wiley and Sons; 2007).
[0099] In some embodiments, quantitative hybridization methods,
such as Southern analysis, Northern analysis, or in situ
hybridizations, can be used (see Current Protocols in Molecular
Biology, Ausubel, F. et al., eds., John Wiley & Sons, including
all supplements). A "nucleic acid probe," as used herein, can be a
DNA probe or an RNA probe. The probe can be, for example, a gene, a
gene fragment (e.g., one or more exons), a vector comprising the
gene, a probe or primer, etc. For representative examples of use of
nucleic acid probes, see, for example, U.S. Pat. Nos. 5,288,611 and
4,851,330. The nucleic acid probe can be, for example, a
full-length nucleic acid molecule, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and sufficient to specifically hybridize under stringent
conditions to appropriate target mRNA or cDNA. The hybridization
sample is maintained under conditions which are sufficient to allow
specific hybridization of the nucleic acid probe to mRNA or cDNA.
Specific hybridization can be performed under high stringency
conditions or moderate stringency conditions, as appropriate. In a
preferred embodiment, the hybridization conditions for specific
hybridization are high stringency. Specific hybridization, if
present, is then detected using standard methods. If specific
hybridization occurs between the nucleic acid probe having a mRNA
or cDNA in the test sample, the level of the mRNA or cDNA in the
sample can be assessed. More than one nucleic acid probe can also
be used concurrently in this method. Specific hybridization of any
one of the nucleic acid probes is indicative of the presence of the
mRNA or cDNA of interest, as described herein.
[0100] Alternatively, a peptide nucleic acid (PNA) probe can be
used instead of a nucleic acid probe in the quantitative
hybridization methods described herein. PNA is a DNA mimic having a
peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine
units, with an organic base (A, G, C, T or U) attached to the
glycine nitrogen via a methylene carbonyl linker (see, for example,
1994, Nielsen et al., Bioconjugate Chemistry 5:1). The PNA probe
can be designed to specifically hybridize to a target nucleic acid
sequence. Hybridization of the PNA probe to a nucleic acid sequence
is used to determine the level of the target nucleic acid in the
biological sample.
[0101] In another embodiment, arrays of oligonucleotide probes that
are complementary to target nucleic acid sequences in the
biological sample obtained from a subject can be used to determine
the level of D-DT in the biological sample obtained from a subject.
The array of oligonucleotide probes can be used to determine the
level of D-DT alone, or the level of D-DT in relation to the level
of one or more other nucleic acids in the biological sample.
Oligonucleotide arrays typically comprise a plurality of different
oligonucleotide probes that are coupled to a surface of a substrate
in different known locations. These oligonucleotide arrays, also
known as "Genechips," have been generally described in the art, for
example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO
90/15070 and 92/10092. These arrays can generally be produced using
mechanical synthesis methods or light directed synthesis methods
which incorporate a combination of photolithographic methods and
solid phase oligonucleotide synthesis methods. See Fodor et al.,
Science, 251:767-777 (1991), Pirrung et al., U.S. Pat. No.
5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et
al., PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186.
Techniques for the synthesis of these arrays using mechanical
synthesis methods are described in, e.g., U.S. Pat. No.
5,384,261.
[0102] After an oligonucleotide array is prepared, a nucleic acid
of interest is hybridized with the array and its level is
quantified. Hybridization and quantification are generally carried
out by methods described herein and also in, e.g., published PCT
Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No.
5,424,186. In brief, a target nucleic acid sequence is amplified by
well-known amplification techniques, e.g., PCR. Typically, this
involves the use of primer sequences that are complementary to the
target nucleic acid. Asymmetric PCR techniques may also be used.
Amplified target, generally incorporating a label, is then
hybridized with the array under appropriate conditions. Upon
completion of hybridization and washing of the array, the array is
scanned to determine the quantity of hybridized nucleic acid. The
hybridization data obtained from the scan is typically in the form
of fluorescence intensities as a function of quantity, or relative
quantity, of the target nucleic acid in the biological sample. The
target nucleic acid can be hybridized to the array in combination
with one or more comparator controls (e.g., positive control,
negative control, quantity control, etc.) to improve quantification
of the target nucleic acid in the sample.
[0103] The probes and primers according to the invention can be
labeled directly or indirectly with a radioactive or nonradioactive
compound, by methods well known to those skilled in the art, in
order to obtain a detectable and/or quantifiable signal; the
labeling of the primers or of the probes according to the invention
is carried out with radioactive elements or with nonradioactive
molecules. Among the radioactive isotopes used, mention may be made
of 32P, 33P, 35S or 3H. The nonradioactive entities are selected
from ligands such as biotin, avidin, streptavidin or digoxigenin,
haptenes, dyes, and luminescent agents such as radioluminescent,
chemoluminescent, bioluminescent, fluorescent or phosphorescent
agents.
[0104] Nucleic acids can be obtained from the cells using known
techniques. Nucleic acid herein refers to RNA, including mRNA, and
DNA, including cDNA. The nucleic acid can be double-stranded or
single-stranded (i.e., a sense or an antisense single strand) and
can be complementary to a nucleic acid encoding a polypeptide. The
nucleic acid content may also be an RNA or DNA extraction performed
on a biological sample, including a biological fluid and fresh or
fixed tissue sample.
[0105] There are many methods known in the art for the detection
and quantification of specific nucleic acid sequences and new
methods are continually reported. A great majority of the known
specific nucleic acid detection and quantification methods utilize
nucleic acid probes in specific hybridization reactions.
Preferably, the detection of hybridization to the duplex form is a
Southern blot technique. In the Southern blot technique, a nucleic
acid sample is separated in an agarose gel based on size (molecular
weight) and affixed to a membrane, denatured, and exposed to
(admixed with) the labeled nucleic acid probe under hybridizing
conditions. If the labeled nucleic acid probe forms a hybrid with
the nucleic acid on the blot, the label is bound to the
membrane.
[0106] In the Southern blot, the nucleic acid probe is preferably
labeled with a tag. That tag can be a radioactive isotope, a
fluorescent dye or the other well-known materials. Another type of
process for the specific detection of nucleic acids in a biological
sample known in the art are the hybridization methods as
exemplified by U.S. Pat. No. 6,159,693 and U.S. Pat. No. 6,270,974,
and related patents. To briefly summarize one of those methods, a
nucleic acid probe of at least 10 nucleotides, preferably at least
15 nucleotides, more preferably at least 25 nucleotides, having a
sequence complementary to a nucleic acid of interest is hybridized
in a sample, subjected to depolymerizing conditions, and the sample
is treated with an ATP/luciferase system, which will luminesce if
the nucleic sequence is present. In quantitative Southern blotting,
the level of the nucleic acid of interest can be compared with the
level of a second nucleic acid of interest, and/or to one or more
comparator control nucleic acids (e.g., positive control, negative
control, quantity control, etc.).
[0107] Many methods useful for the detection and quantification of
nucleic acid takes advantage of the polymerase chain reaction
(PCR). The PCR process is well known in the art (U.S. Pat. No.
4,683,195, U.S. Pat. No. 4,683,202, and U.S. Pat. No. 4,800,159).
To briefly summarize PCR, nucleic acid primers, complementary to
opposite strands of a nucleic acid amplification target sequence,
are permitted to anneal to the denatured sample. A DNA polymerase
(typically heat stable) extends the DNA duplex from the hybridized
primer. The process is repeated to amplify the nucleic acid target.
If the nucleic acid primers do not hybridize to the sample, then
there is no corresponding amplified PCR product. In this case, the
PCR primer acts as a hybridization probe.
[0108] In PCR, the nucleic acid probe can be labeled with a tag as
discussed elsewhere herein. Most preferably the detection of the
duplex is done using at least one primer directed to the nucleic
acid of interest. In yet another embodiment of PCR, the detection
of the hybridized duplex comprises electrophoretic gel separation
followed by dye-based visualization.
[0109] Typical hybridization and washing stringency conditions
depend in part on the size (i.e., number of nucleotides in length)
of the oligonucleotide probe, the base composition and monovalent
and divalent cation concentrations (Ausubel et al., 1994, eds
Current Protocols in Molecular Biology).
[0110] In a preferred embodiment, the process for determining the
quantitative and qualitative profile of the nucleic acid of
interest according to the present invention is characterized in
that the amplifications are real-time amplifications performed
using a labeled probe, preferably a labeled hydrolysis-probe,
capable of specifically hybridizing in stringent conditions with a
segment of the nucleic acid of interest. The labeled probe is
capable of emitting a detectable signal every time each
amplification cycle occurs, allowing the signal obtained for each
cycle to be measured.
[0111] The real-time amplification, such as real-time PCR, is well
known in the art, and the various known techniques will be employed
in the best way for the implementation of the present process.
These techniques are performed using various categories of probes,
such as hydrolysis probes, hybridization adjacent probes, or
molecular beacons. The techniques employing hydrolysis probes or
molecular beacons are based on the use of a fluorescence
quencher/reporter system, and the hybridization adjacent probes are
based on the use of fluorescence acceptor/donor molecules.
[0112] Hydrolysis probes with a fluorescence quencher/reporter
system are available in the market, and are for example
commercialized by the Applied Biosystems group (USA). Many
fluorescent dyes may be employed, such as FAM dyes
(6-carboxy-fluorescein), or any other dye phosphoramidite
reagents.
[0113] Among the stringent conditions applied for any one of the
hydrolysis-probes of the present invention is the Tm, which is in
the range of about 65.degree. C. to 75.degree. C. Preferably, the
Tm for any one of the hydrolysis-probes of the present invention is
in the range of about 67.degree. C. to about 70.degree. C. Most
preferably, the Tm applied for any one of the hydrolysis-probes of
the present invention is about 67.degree. C.
[0114] In one aspect, the invention includes a primer that is
complementary to a nucleic acid of interest, and more particularly
the primer includes 12 or more contiguous nucleotides substantially
complementary to the nucleic acid of interest. Preferably, a primer
featured in the invention includes a nucleotide sequence
sufficiently complementary to hybridize to a nucleic acid sequence
of about 12 to 25 nucleotides. More preferably, the primer differs
by no more than 1, 2, or 3 nucleotides from the target flanking
nucleotide sequence In another aspect, the length of the primer can
vary in length, preferably about 15 to 28 nucleotides in length
(e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27
nucleotides in length).
Kits
[0115] The present invention also pertains to kits useful in the
methods of the invention. Such kits comprise various combinations
of components useful in any of the methods described elsewhere
herein, including for example, hybridization probes or primers
(e.g., labeled probes or primers), antibodies, reagents for
detection of labeled molecules, materials for the amplification of
a subject's nucleic acids, a D-DT activator, a D-DT inhibitor,
materials for quantitatively analyzing D-DT polypeptide or D-DT
nucleic acid, materials for assessing the activity of a D-DT
polypeptide or a D-DT nucleic acid, and instructional material. For
example, in one embodiment, the kit comprises components useful for
the quantification of D-DT nucleic acid in a biological sample
obtained from a subject. In another embodiment, the kit comprises
components useful for the quantification of D-DT polypeptide in a
biological sample obtained from a subject. In a further embodiment,
the kit comprises components useful for the assessment of the
activity (e.g., enzymatic activity, receptor binding activity,
etc.) of a D-DT polypeptide in a biological sample obtained from a
subject.
[0116] In one embodiment, the kit comprises the components of a
diagnostic assay for diagnosing infection, inflammatory disease,
autoimmunity or cancer in a subject in need thereof, containing
instructional material and the components for determining whether
the level of D-DT is increased in a biological sample obtained from
the subject. In various embodiments, determining whether the level
of D-DT is increased in a biological sample obtained from the
subject, the level of D-DT is compared with the level of at least
one comparator control contained in the kit, such as a positive
control, a negative control, a historical control, a historical
norm, or the level of another reference molecule in the biological
sample. In one embodiment, the reference molecule in the biological
sample is MIF. In certain embodiments, the ratio of D-DT and MIF is
determined to aid in the diagnosis.
[0117] In another embodiment, the kit comprises the components of a
diagnostic assay for diagnosing ischemia-reperfusion injury in a
subject in need thereof, containing instructional material and the
components for determining whether the level of D-DT is reduced in
a biological sample obtained from the subject. In various
embodiments, determining whether the level of D-DT is reduced in a
biological sample obtained from the subject, the level of D-DT is
compared with the level of at least one comparator control
contained in the kit, such as a positive control, a negative
control, a historical control, a historical norm, or the level of
another reference molecule in the biological sample. In one
embodiment, the reference molecule in the biological sample is MIF.
In certain embodiments, the ratio of D-DT and MIF is determined to
aid in the diagnosis.
[0118] In a further embodiment, the kit comprises the components of
an assay for monitoring the effectiveness of a treatment
administered to a subject in need thereof, containing instructional
material and the components for determining whether the level of
D-DT in a biological sample obtained from the subject is modulated
during or after administration of the treatment. In various
embodiments, to determine whether the level of D-DT is modulated in
a biological sample obtained from the subject, the level of D-DT is
compared with the level of at least one comparator control
contained in the kit, such as a positive control, a negative
control, a historical control, a historical norm, or the level of
another reference molecule in the biological sample. In one
embodiment, the reference molecule in the biological sample is MIF.
In certain embodiments, the ratio of D-DT and MIF is determined to
aid in the monitoring of the treatment.
Therapeutic Inhibitor Compositions and Methods
[0119] In various embodiments, the present invention includes D-DT
inhibitor compositions and methods of treating infection,
inflammatory disease, autoimmunity and cancer. In various
embodiments, the D-DT inhibitor compositions and methods of
treatment of the invention diminish the amount of D-DT polypeptide,
the amount of D-DT mRNA, the amount of D-DT enzymatic activity, the
amount of D-DT receptor binding activity, or a combination
thereof.
[0120] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that a decrease in the level of
D-DT encompasses the decrease in D-DT expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that a decrease in the level of D-DT includes a decrease in D-DT
activity (e.g., enzymatic activity, receptor binding activity,
etc.). Thus, decreasing the level or activity of D-DT includes, but
is not limited to, decreasing transcription, translation, or both,
of a nucleic acid encoding D-DT; and it also includes decreasing
any activity of a D-DT polypeptide as well. The D-DT inhibitor
compositions and methods of the invention can selectively inhibit
D-DT, or can inhibit both D-DT and MIF.
[0121] Inhibition of D-DT can be assessed using a wide variety of
methods, including those disclosed herein, as well as methods known
in the art or to be developed in the future. That is, the routineer
would appreciate, based upon the disclosure provided herein, that
decreasing the level or activity of D-DT can be readily assessed
using methods that assess the level of a nucleic acid encoding D-DT
(e.g., mRNA), the level of a D-DT polypeptide present in a
biological sample, the level of D-DT activity (e.g., enzymatic
activity, receptor binding activity), or combinations thereof.
[0122] One skilled in the art, based upon the disclosure provided
herein, would understand that the invention is useful in treating
infection, inflammatory disease, autoimmunity or cancer in a
subject in need thereof, whether or not the subject also being
treated with other medication or therapy. Further, the skilled
artisan would further appreciate, based upon the teachings provided
herein, that the infection, inflammatory disease, autoimmunity or
cancer treatable by the compositions and methods described herein
encompass any pathology associated infection, inflammatory disease,
autoimmunity or cancer where D-DT plays a role.
[0123] The D-DT inhibitor compositions and methods of the invention
that decrease the level, enzymatic activity, or receptor binding
activity of D-DT include, but should not be construed as being
limited to, a chemical compound, a protein, a peptide, a
peptidomemetic, an antibody, a ribozyme, a small molecule chemical
compound, an antisense nucleic acid molecule (e.g., siRNA, miRNA,
etc.), or combinations thereof. One of skill in the art would
readily appreciate, based on the disclosure provided herein, that a
D-DT inhibitor composition encompasses a chemical compound that
decreases the level or activity of D-DT. Additionally, a D-DT
inhibitor composition encompasses a chemically modified compound,
and derivatives, as is well known to one of skill in the chemical
arts. In a particular embodiment, the D-DT inhibitor is an siRNA
comprising the nucleic acid sequence 5'-GCATGACCCTGTTGATGAA-3' (SEQ
ID NO: 2).
[0124] The D-DT inhibitor compositions and methods of the invention
that decrease the level or activity of D-DT include antibodies. The
antibodies of the invention include a variety of forms of
antibodies including, for example, polyclonal antibodies,
monoclonal antibodies, intracellular antibodies ("intrabodies"),
Fv, Fab and F(ab)2, single chain antibodies (scFv), heavy chain
antibodies (such as camelid antibodies), synthetic antibodies,
chimeric antibodies, and humanized antibodies. In one embodiment,
the antibody of the invention is an antibody that specifically
binds to D-DT, and does not substantially bind to MIF. In another
embodiment, the antibody of the invention is an antibody that
specifically binds to D-DT, and also specifically binds to MIF. In
a further embodiment, the antibody of the invention is a
dual-specific antibody that concurrently binds both D-DT and
MIF.
[0125] Further, one of skill in the art would, when equipped with
this disclosure and the methods exemplified herein, appreciate that
a D-DT inhibitor composition includes such inhibitors as discovered
in the future, as can be identified by well-known criteria in the
art of pharmacology, such as the physiological results of
inhibition of D-DT as described in detail herein and/or as known in
the art. Therefore, the present invention is not limited in any way
to any particular D-DT inhibitor composition as exemplified or
disclosed herein; rather, the invention encompasses those inhibitor
compositions that would be understood by the routineer to be useful
as are known in the art and as are discovered in the future.
[0126] Further methods of identifying and producing D-DT inhibitor
compositions are well known to those of ordinary skill in the art,
including, but not limited, obtaining an inhibitor from a naturally
occurring source (i.e., Streptomyces sp., Pseudomonas sp.,
Stylotella aurantium). Alternatively, a D-DT inhibitor can be
synthesized chemically. Further, the routineer would appreciate,
based upon the teachings provided herein, that a D-DT inhibitor
composition can be obtained from a recombinant organism.
Compositions and methods for chemically synthesizing D-DT
inhibitors and for obtaining them from natural sources are well
known in the art and are described in the art.
[0127] One of skill in the art will appreciate that an inhibitor
can be administered as a small molecule chemical, a protein, an
antibody, a nucleic acid construct encoding a protein, an antisense
nucleic acid, a nucleic acid construct encoding an antisense
nucleic acid, or combinations thereof. Numerous vectors and other
compositions and methods are well known for administering a protein
or a nucleic acid construct encoding a protein to cells or tissues.
Therefore, the invention includes a method of administering a
protein or a nucleic acid encoding a protein that is an inhibitor
of D-DT. (Sambrook et al., 2001, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al.,
1997, Current Protocols in Molecular Biology, John Wiley &
Sons, New York).
[0128] One of skill in the art will realize that diminishing the
amount or activity of a molecule that itself increases the amount
or activity of D-DT can serve in the compositions and methods of
the present invention to decrease the amount or activity of
D-DT.
[0129] Antisense oligonucleotides are DNA or RNA molecules that are
complementary to some portion of an RNA molecule. When present in a
cell, antisense oligonucleotides hybridize to an existing RNA
molecule and inhibit translation into a gene product. Inhibiting
the expression of a gene using an antisense oligonucleotide is well
known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as
are methods of expressing an antisense oligonucleotide in a cell
(Inoue, U.S. Pat. No. 5,190,931). The methods of the invention
include the use of an antisense oligonucleotide to diminish the
amount of D-DT, or to diminish the amount of a molecule that causes
an increase in the amount or activity of D-DT, thereby decreasing
the amount or activity of D-DT.
[0130] Contemplated in the present invention are antisense
oligonucleotides that are synthesized and provided to the cell by
way of methods well known to those of ordinary skill in the art. As
an example, an antisense oligonucleotide can be synthesized to be
between about 10 and about 100, more preferably between about 15
and about 50 nucleotides long. The synthesis of nucleic acid
molecules is well known in the art, as is the synthesis of modified
antisense oligonucleotides to improve biological activity in
comparison to unmodified antisense oligonucleotides (Tullis, 1991,
U.S. Pat. No. 5,023,243).
[0131] Similarly, the expression of a gene may be inhibited by the
hybridization of an antisense molecule to a promoter or other
regulatory element of a gene, thereby affecting the transcription
of the gene. Methods for the identification of a promoter or other
regulatory element that interacts with a gene of interest are well
known in the art, and include such methods as the yeast two hybrid
system (Bartel and Fields, eds., In: The Yeast Two Hybrid System,
Oxford University Press, Cary, N.C.).
[0132] Alternatively, inhibition of a gene expressing D-DT, or of a
gene expressing a protein that increases the level or activity of
D-DT, can be accomplished through the use of a ribozyme. Using
ribozymes for inhibiting gene expression is well known to those of
skill in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.
267:17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman et
al., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA
molecules with the ability to cleave other single-stranded RNA
molecules. Ribozymes are known to be sequence specific, and can
therefore be modified to recognize a specific nucleotide sequence
(Cech, 1988, J. Amer. Med. Assn. 260:3030), allowing the selective
cleavage of specific mRNA molecules. Given the nucleotide sequence
of the molecule, one of ordinary skill in the art could synthesize
an antisense oligonucleotide or ribozyme without undue
experimentation, provided with the disclosure and references
incorporated herein.
[0133] One of skill in the art will appreciate that inhibitors of
D-DT can be administered singly or in any combination. Further,
D-DT inhibitors can be administered singly or in any combination in
a temporal sense, in that they may be administered concurrently, or
before, and/or after each other. One of ordinary skill in the art
will appreciate, based on the disclosure provided herein, that D-DT
inhibitor compositions can be used to treat infection, inflammatory
disease, autoimmunity or cancer in a subject in need thereof, and
that an inhibitor composition can be used alone or in any
combination with another inhibitor to effect a therapeutic
result.
[0134] In various embodiments, any of the inhibitors of D-DT of the
invention described herein can be administered alone or in
combination with other inhibitors of other molecules associated
with infection, inflammatory disease, autoimmunity or cancer, such
as, for example, MIF. In some embodiments, the D-DT inhibitors of
the invention selectively inhibit D-DT and do not also inhibit MIF.
In other embodiments, the D-DT inhibitors of the invention inhibit
D-DT and also inhibit MIF.
[0135] It will be appreciated by one of skill in the art, when
armed with the present disclosure including the methods detailed
herein, that the invention is not limited to treatment of a disease
or disorder that is already established. Particularly, the disease
or disorder need not have manifested to the point of detriment to
the subject; indeed, the disease or disorder need not be detected
in a subject before treatment is administered. That is, significant
disease or disorder does not have to occur before the present
invention may provide benefit. Therefore, the present invention
includes a method for preventing infection, inflammatory disease,
autoimmunity or cancer in a subject, in that a D-DT inhibitor
composition, as discussed previously elsewhere herein, can be
administered to a subject prior to the onset of the infection,
inflammatory disease, autoimmunity or cancer, thereby preventing
the infection, inflammatory disease, autoimmunity or cancer. The
preventive methods described herein also include the treatment of a
subject that is in remission for the prevention of a recurrence of
an infection, inflammatory disease, autoimmunity or cancer.
[0136] One of skill in the art, when armed with the disclosure
herein, would appreciate that the prevention of infection,
inflammatory disease, autoimmunity or cancer encompasses
administering to a subject a D-DT inhibitor composition as a
preventative measure against infection, inflammatory disease,
autoimmunity or cancer. As more fully discussed elsewhere herein,
methods of decreasing the level or activity of D-DT encompass a
wide plethora of techniques for decreasing not only D-DT activity,
but also for decreasing expression of a nucleic acid encoding D-DT,
including either a decrease in transcription, a decrease in
translation, or both.
[0137] Additionally, as disclosed elsewhere herein, one skilled in
the art would understand, once armed with the teaching provided
herein, that the present invention encompasses a method of
preventing a wide variety of diseases, disorders and pathologies
where a decrease in expression and/or activity of D-DT mediates,
treats or prevents the disease, disorder or pathology. Methods for
assessing whether a disease relates to increased levels or activity
of D-DT are known in the art. Further, the invention encompasses
treatment or prevention of such diseases discovered in the
future.
[0138] The invention encompasses administration of an inhibitor of
D-DT to practice the methods of the invention; the skilled artisan
would understand, based on the disclosure provided herein, how to
formulate and administer the appropriate D-DT inhibitor to a
subject. Indeed, the successful administration of the D-DT
inhibitor has been reduced to practice as exemplified herein.
However, the present invention is not limited to any particular
method of administration or treatment regimen.
Therapeutic Activators and Methods
[0139] In various embodiments, the present invention includes D-DT
activator compositions and methods of treating ischemia-reperfusion
injury in a subject, a tissue, or an organ in need thereof. In
various embodiments, the D-DT activator compositions and methods of
treatment of the invention increase the amount of D-DT polypeptide,
the amount of D-DT mRNA, the amount of D-DT enzymatic activity, the
amount of D-DT receptor binding activity, or a combination
thereof.
[0140] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that an increase in the level of
D-DT encompasses the increase in D-DT expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that an increase in the level of D-DT includes an increase in D-DT
activity (e.g., enzymatic activity, receptor binding activity,
etc.). Thus, increasing the level or activity of D-DT includes, but
is not limited to, increasing the amount of D-DT polypeptide,
increasing transcription, translation, or both, of a nucleic acid
encoding D-DT; and it also includes increasing any activity of a
D-DT polypeptide as well. The D-DT activator compositions and
methods of the invention can selectively activate D-DT, or can
activate both D-DT and MIF.
[0141] Thus, the present invention relates to the prevention and
treatment of ischemia-reperfusion injury by administration of a
D-DT polypeptide, a recombinant D-DT polypeptide, an active D-DT
polypeptide fragment, or an activator of D-DT expression or
activity.
[0142] It is understood by one skilled in the art, that an increase
in the level of D-DT encompasses the increase of D-DT protein
expression. Additionally, the skilled artisan would appreciate,
that an increase in the level of D-DT includes an increase in D-DT
activity. Thus, increasing the level or activity of D-DT includes,
but is not limited to, increasing transcription, translation, or
both, of a nucleic acid encoding D-DT; and it also includes
increasing any activity of D-DT as well.
[0143] Activation of D-DT can be assessed using a wide variety of
methods, including those disclosed herein, as well as methods
well-known in the art or to be developed in the future. That is,
the routineer would appreciate, based upon the disclosure provided
herein, that increasing the level or activity of D-DT can be
readily assessed using methods that assess the level of a nucleic
acid encoding D-DT (e.g., mRNA) and/or the level of D-DT
polypeptide in a biological sample obtained from a subject.
[0144] One skilled in the art, based upon the disclosure provided
herein, would understand that the invention is useful in
ischemia-reperfusion injury in subjects who, in whole (e.g.,
systemically) or in part (e.g., locally, tissue, organ), are being
or will be, exposed to less than adequate oxygen levels.
Ischemia-reperfusion injury can occur through the loss of
ventilation, as well as through the loss of circulation, to all or
to part, of the subject's body. In one embodiment, the invention is
useful in treating or preventing ischemia-reperfusion injury in a
tissue or organ intended for transplantation. The skilled artisan
will appreciate, based upon the teachings provided herein, that the
ischemia-reperfusion injuries treatable by the compositions and
methods described herein encompass any ischemia-reperfusion
injury.
[0145] A D-DT activator can include, but should not be construed as
being limited to, a chemical compound, a protein, a peptidomemetic,
an antibody, a ribozyme, and an antisense nucleic acid molecule.
One of skill in the art would readily appreciate, based on the
disclosure provided herein, that a D-DT activator encompasses a
chemical compound that increases the level, enzymatic activity, or
receptor binding activity of D-DT. In some embodiments, the
enzymatic activity is tautomerase activity. Additionally, a D-DT
activator encompasses a chemically modified compound, and
derivatives, as is well known to one of skill in the chemical
arts.
[0146] In various embodiments, the present invention also includes
D-DT activator compositions and methods of immunostimulation in a
subject in need thereof. Immunostimulation is useful in a variety
of settings where an increase in the immunoactivity is desirable.
One such non-limiting example is the use of a D-DT activator
composition to increase immunoactivity before, during or after
vaccination. Another such non-limiting example is the use of a D-DT
activator composition to increase immunoactivity when the subject's
immune response is otherwise inadequate. In various embodiments,
the D-DT activator compositions and methods of immunostimulation of
the invention increase the amount of D-DT polypeptide, the amount
of D-DT mRNA, the amount of D-DT enzymatic activity, the amount of
D-DT receptor binding activity, or a combination thereof.
[0147] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that an increase in the level of
D-DT encompasses the increase in D-DT expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that an increase in the level of D-DT includes an increase in D-DT
activity (e.g., enzymatic activity, receptor binding activity,
etc.). Thus, increasing the level or activity of D-DT includes, but
is not limited to, increasing the amount of D-DT polypeptide,
increasing transcription, translation, or both, of a nucleic acid
encoding D-DT; and it also includes increasing any activity of a
D-DT polypeptide as well. The D-DT activator compositions and
methods of the invention can selectively activate D-DT, or can
activate both D-DT and MIF. Thus, the present invention relates to
immunostimulation by administration of a D-DT polypeptide, a
recombinant D-DT polypeptide, an active D-DT polypeptide fragment,
or an activator of D-DT expression or activity.
[0148] Further, one of skill in the art would, when equipped with
this disclosure and the methods exemplified herein, appreciate that
a D-DT activator includes such activators as discovered in the
future, as can be identified by well-known criteria in the art of
pharmacology, such as the physiological results of activation of
D-DT as described in detail herein and/or as known in the art.
Therefore, the present invention is not limited in any way to any
particular D-DT activator as exemplified or disclosed herein;
rather, the invention encompasses those activators that would be
understood by the routineer to be useful as are known in the art
and as are discovered in the future.
[0149] Further methods of identifying and producing a D-DT
activator are well known to those of ordinary skill in the art,
including, but not limited, obtaining an activator from a naturally
occurring source (i.e., Streptomyces sp., Pseudomonas sp.,
Stylotella aurantium). Alternatively, a D-DT activator can be
synthesized chemically. Further, the routineer would appreciate,
based upon the teachings provided herein, that a D-DT activator can
be obtained from a recombinant organism. Compositions and methods
for chemically synthesizing D-DT activators and for obtaining them
from natural sources are well known in the art and are described in
the art.
[0150] One of skill in the art will appreciate that an activator
can be administered as a small molecule chemical, a protein, a
nucleic acid construct encoding a protein, or combinations thereof.
Numerous vectors and other compositions and methods are well known
for administering a protein or a nucleic acid construct encoding a
protein to cells or tissues. Therefore, the invention includes a
method of administering a protein or a nucleic acid encoding an
protein that is an activator of D-DT. (Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York; Ausubel et al., 1997, Current Protocols in
Molecular Biology, John Wiley & Sons, New York).
[0151] One of skill in the art will realize that diminishing the
amount or activity of a molecule that itself diminishes the amount
or activity of D-DT can serve to increase the amount or activity of
D-DT. Antisense oligonucleotides are DNA or RNA molecules that are
complementary to some portion of a mRNA molecule. When present in a
cell, antisense oligonucleotides hybridize to an existing mRNA
molecule and inhibit translation into a gene product. Inhibiting
the expression of a gene using an antisense oligonucleotide is well
known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as
are methods of expressing an antisense oligonucleotide in a cell
(Inoue, U.S. Pat. No. 5,190,931). The methods of the invention
include the use of antisense oligonucleotide to diminish the amount
of a molecule that causes a decrease in the amount or activity
D-DT, thereby increasing the amount or activity of D-DT.
Contemplated in the present invention are antisense
oligonucleotides that are synthesized and provided to the cell by
way of methods well known to those of ordinary skill in the art. As
an example, an antisense oligonucleotide can be synthesized to be
between about 10 and about 100, more preferably between about 15
and about 50 nucleotides long. The synthesis of nucleic acid
molecules is well known in the art, as is the synthesis of modified
antisense oligonucleotides to improve biological activity in
comparison to unmodified antisense oligonucleotides (Tullis, 1991,
U.S. Pat. No. 5,023,243).
[0152] Similarly, the expression of a gene may be inhibited by the
hybridization of an antisense molecule to a promoter or other
regulatory element of a gene, thereby affecting the transcription
of the gene. Methods for the identification of a promoter or other
regulatory element that interacts with a gene of interest are well
known in the art, and include such methods as the yeast two hybrid
system (Bartel and Fields, eds., In: The Yeast Two Hybrid System,
Oxford University Press, Cary, N.C.).
[0153] Alternatively, inhibition of a gene expressing a protein
that diminishes the level or activity of D-DT can be accomplished
through the use of a ribozyme. Using ribozymes for inhibiting gene
expression is well known to those of skill in the art (see, e.g.,
Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989,
Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053).
Ribozymes are catalytic RNA molecules with the ability to cleave
other single-stranded RNA molecules. Ribozymes are known to be
sequence specific, and can therefore be modified to recognize a
specific nucleotide sequence (Cech, 1988, J. Amer. Med. Assn.
260:3030), allowing the selective cleavage of specific mRNA
molecules. Given the nucleotide sequence of the molecule, one of
ordinary skill in the art could synthesize an antisense
oligonucleotide or ribozyme without undue experimentation, provided
with the disclosure and references incorporated herein.
[0154] One of skill in the art will appreciate that a D-DT
polypeptide, a recombinant D-DT polypeptide, or an active D-DT
polypeptide fragment can be administered singly or in any
combination thereof. Further, a D-DT polypeptide, a recombinant
D-DT polypeptide, or an active D-DT polypeptide fragment can be
administered singly or in any combination thereof in a temporal
sense, in that they may be administered simultaneously, before,
and/or after each other. One of ordinary skill in the art will
appreciate, based on the disclosure provided herein, that a D-DT
polypeptide, a recombinant D-DT polypeptide, or an active D-DT
polypeptide fragment can be used to prevent or treat
ischemia-reperfusion injury, and that an activator can be used
alone or in any combination with another D-DT polypeptide,
recombinant D-DT polypeptide, active D-DT polypeptide fragment, or
D-DT activator to effect a therapeutic result.
[0155] One of skill in the art will also appreciate that activators
of D-DT gene expression can be administered singly or in any
combination thereof. Further, D-DT activators can be administered
singly or in any combination thereof in a temporal sense, in that
they may be administered simultaneously, before, and/or after each
other. One of ordinary skill in the art will appreciate, based on
the disclosure provided herein, that D-DT activators can be used to
prevent or treat ischemia-reperfusion injury, and that an activator
can be used alone or in any combination with another activator,
D-DT polypeptide, recombinant D-DT polypeptide, or active D-DT
polypeptide fragment to effect a therapeutic result.
[0156] It will be appreciated by one of skill in the art, when
armed with the present disclosure including the methods detailed
herein, that the invention is not limited to treatment of
ischemia-reperfusion injury once the ischemia-reperfusion injury is
established. Particularly, the symptoms of the ischemia-reperfusion
injury need not have manifested to the point of detriment to the
subject; indeed, the ischemia-reperfusion injury need not be
detected in a subject before treatment is administered. That is,
significant pathology from an ischemia-reperfusion injury does not
have to occur before the present invention may provide benefit.
Therefore, the present invention, as described more fully herein,
includes a method for preventing ischemia-reperfusion injury in a
subject, in that a D-DT molecule, or a D-DT activator, as discussed
elsewhere herein, can be administered to a subject prior to the
onset of an ischemia-reperfusion injury, thereby preventing the
ischemia-reperfusion injury.
[0157] One of skill in the art, when armed with the disclosure
herein, would appreciate that the prevention of
ischemia-reperfusion injury encompasses administering to a subject
a D-DT polypeptide, a recombinant D-DT polypeptide, an active D-DT
polypeptide fragment, or D-DT activator as a preventative measure
against ischemia-reperfusion injury. As more fully discussed
elsewhere herein, methods of increasing the level or activity of a
D-DT encompass a wide plethora of techniques for increasing not
only D-DT activity, but also for increasing expression of a nucleic
acid encoding D-DT. Additionally, as disclosed elsewhere herein,
one skilled in the art would understand, once armed with the
teaching provided herein, that the present invention encompasses a
method of preventing a wide variety of diseases where increased
expression and/or activity of D-DT mediates, treats or prevents the
disease. Further, the invention encompasses treatment or prevention
of such diseases discovered in the future.
[0158] The invention encompasses administration of a D-DT
polypeptide, a recombinant D-DT polypeptide, an active D-DT
polypeptide fragment, or a D-DT activator to practice the methods
of the invention; the skilled artisan would understand, based on
the disclosure provided herein, how to formulate and administer the
appropriate D-DT polypeptide, recombinant D-DT polypeptide, active
D-DT polypeptide fragment, or D-DT activator to a subject. However,
the present invention is not limited to any particular method of
administration or treatment regimen. This is especially true where
it would be appreciated by one skilled in the art, equipped with
the disclosure provided herein, including the reduction to practice
using an art-recognized model of ischemia-reperfusion injury, that
methods of administering a D-DT polypeptide, a recombinant D-DT
polypeptide, an active D-DT polypeptide fragment, or D-DT activator
can be determined by one of skill in the pharmacological arts.
[0159] As used herein, the term "pharmaceutically-acceptable
carrier" means a chemical composition with which an appropriate
D-DT polypeptide, recombinant D-DT polypeptide, active D-DT
polypeptide fragment, or D-DT activator, may be combined and which,
following the combination, can be used to administer the
appropriate D-DT polypeptide, recombinant D-DT polypeptide, active
D-DT polypeptide fragment, or D-DT activator to a subject.
Pharmaceutical Compositions
[0160] Compositions identified as modulators of D-DT can be
formulated and administered to a subject, as now described. For
example, compositions identified as useful D-DT inhibitors for the
treatment and/or prevention of infection, inflammatory disease,
autoimmunity or cancer, can be formulated and administered to a
subject, as now described. Further, compositions identified as
useful D-DT activators, as well as D-DT polypeptides, recombinant
D-DT polypeptides, and active D-DT polypeptide fragments, for the
treatment and/or prevention of ischemia-reperfusion injury can be
formulated and administered to a subject, as now described.
[0161] The invention encompasses the preparation and use of
pharmaceutical compositions comprising a composition useful for the
treatment of ischemia-reperfusion injury, or for the treatment of
infection, inflammatory disease, autoimmunity or cancer, disclosed
herein as an active ingredient. Such a pharmaceutical composition
may consist of the active ingredient alone, in a form suitable for
administration to a subject, or the pharmaceutical composition may
comprise the active ingredient and one or more pharmaceutically
acceptable carriers, one or more additional ingredients, or some
combination of these. The active ingredient may be present in the
pharmaceutical composition in the form of a physiologically
acceptable ester or salt, such as in combination with a
physiologically acceptable cation or anion, as is well known in the
art.
[0162] As used herein, the term "pharmaceutically-acceptable
carrier" means a chemical composition with which an appropriate
D-DT modulator thereof, may be combined and which, following the
combination, can be used to administer the appropriate D-DT
modulator thereof, to a subject.
[0163] The pharmaceutical compositions useful for practicing the
invention may be administered to deliver a dose of between about
0.1 ng/kg/day and 100 mg/kg/day.
[0164] In various embodiments, the pharmaceutical compositions
useful in the methods of the invention may be administered, by way
of example, systemically, parenterally, or topically, such as, in
oral formulations, inhaled formulations, including solid or
aerosol, and by topical or other similar formulations. In addition
to the appropriate therapeutic composition, such pharmaceutical
compositions may contain pharmaceutically acceptable carriers and
other ingredients known to enhance and facilitate drug
administration. Other possible formulations, such as nanoparticles,
liposomes, resealed erythrocytes, and immunologically based systems
may also be used to administer an appropriate modulator thereof,
according to the methods of the invention.
[0165] As used herein, the term "physiologically acceptable" ester
or salt means an ester or salt form of the active ingredient which
is compatible with any other ingredients of the pharmaceutical
composition, which is not deleterious to the subject to which the
composition is to be administered.
[0166] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of bringing the active ingredient into
association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0167] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for ethical administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation.
[0168] Pharmaceutical compositions that are useful in the methods
of the invention may be prepared, packaged, or sold in formulations
suitable for oral, rectal, vaginal, parenteral, topical, pulmonary,
intranasal, buccal, intravenous, ophthalmic, intrathecal and other
known routes of administration. Other contemplated formulations
include projected nanoparticles, liposomal preparations, resealed
erythrocytes containing the active ingredient, and
immunologically-based formulations.
[0169] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient. The amount of the
active ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0170] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient.
[0171] In addition to the active ingredient, a pharmaceutical
composition of the invention may further comprise one or more
additional pharmaceutically active agents.
[0172] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0173] A formulation of a pharmaceutical composition of the
invention suitable for oral administration may be prepared,
packaged, or sold in the form of a discrete solid dose unit
including, but not limited to, a tablet, a hard or soft capsule, a
cachet, a troche, or a lozenge, each containing a predetermined
amount of the active ingredient. Other formulations suitable for
oral administration include, but are not limited to, a powdered or
granular formulation, an aqueous or oily suspension, an aqueous or
oily solution, or an emulsion.
[0174] A tablet comprising the active ingredient may, for example,
be made by compressing or molding the active ingredient, optionally
with one or more additional ingredients. Compressed tablets may be
prepared by compressing, in a suitable device, the active
ingredient in a free-flowing form such as a powder or granular
preparation, optionally mixed with one or more of a binder, a
lubricant, an excipient, a surface active agent, and a dispersing
agent. Molded tablets may be made by molding, in a suitable device,
a mixture of the active ingredient, a pharmaceutically acceptable
carrier, and at least sufficient liquid to moisten the mixture.
Pharmaceutically acceptable excipients used in the manufacture of
tablets include, but are not limited to, inert diluents,
granulating and disintegrating agents, binding agents, and
lubricating agents. Known dispersing agents include, but are not
limited to, potato starch and sodium starch glycollate. Known
surface active agents include, but are not limited to, sodium
lauryl sulphate. Known diluents include, but are not limited to,
calcium carbonate, sodium carbonate, lactose, microcrystalline
cellulose, calcium phosphate, calcium hydrogen phosphate, and
sodium phosphate. Known granulating and disintegrating agents
include, but are not limited to, corn starch and alginic acid.
Known binding agents include, but are not limited to, gelatin,
acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and
hydroxypropyl methylcellulose. Known lubricating agents include,
but are not limited to, magnesium stearate, stearic acid, silica,
and talc.
[0175] Tablets may be non-coated or they may be coated using known
methods to achieve delayed disintegration in the gastrointestinal
tract of a subject, thereby providing sustained release and
absorption of the active ingredient. By way of example, a material
such as glyceryl monostearate or glyceryl distearate may be used to
coat tablets. Further by way of example, tablets may be coated
using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and
U.S. Pat. No. 4,265,874 to form osmotically-controlled release
tablets. Tablets may further comprise a sweetening agent, a
flavoring agent, a coloring agent, a preservative, or some
combination of these in order to provide pharmaceutically elegant
and palatable preparation.
[0176] Hard capsules comprising the active ingredient may be made
using a physiologically degradable composition, such as gelatin.
Such hard capsules comprise the active ingredient, and may further
comprise additional ingredients including, for example, an inert
solid diluent such as calcium carbonate, calcium phosphate, or
kaolin.
[0177] Soft gelatin capsules comprising the active ingredient may
be made using a physiologically degradable composition, such as
gelatin. Such soft capsules comprise the active ingredient, which
may be mixed with water or an oil medium such as peanut oil, liquid
paraffin, or olive oil.
[0178] Liquid formulations of a pharmaceutical composition of the
invention which are suitable for oral administration may be
prepared, packaged, and sold either in liquid form or in the form
of a dry product intended for reconstitution with water or another
suitable vehicle prior to use.
[0179] Liquid suspensions may be prepared using conventional
methods to achieve suspension of the active ingredient in an
aqueous or oily vehicle. Aqueous vehicles include, for example,
water and isotonic saline. Oily vehicles include, for example,
almond oil, oily esters, ethyl alcohol, vegetable oils such as
arachis, olive, sesame, or coconut oil, fractionated vegetable
oils, and mineral oils such as liquid paraffin. Liquid suspensions
may further comprise one or more additional ingredients including,
but not limited to, suspending agents, dispersing or wetting
agents, emulsifying agents, demulcents, preservatives, buffers,
salts, flavorings, coloring agents, and sweetening agents. Oily
suspensions may further comprise a thickening agent.
[0180] Known suspending agents include, but are not limited to,
sorbitol syrup, hydrogenated edible fats, sodium alginate,
polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose
derivatives such as sodium carboxymethylcellulose, methylcellulose,
and hydroxypropylmethylcellulose. Known dispersing or wetting
agents include, but are not limited to, naturally-occurring
phosphatides such as lecithin, condensation products of an alkylene
oxide with a fatty acid, with a long chain aliphatic alcohol, with
a partial ester derived from a fatty acid and a hexitol, or with a
partial ester derived from a fatty acid and a hexitol anhydride
(e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol,
polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan
monooleate, respectively). Known emulsifying agents include, but
are not limited to, lecithin and acacia. Known preservatives
include, but are not limited to, methyl, ethyl, or
n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid.
Known sweetening agents include, for example, glycerol, propylene
glycol, sorbitol, sucrose, and saccharin. Known thickening agents
for oily suspensions include, for example, beeswax, hard paraffin,
and cetyl alcohol.
[0181] Liquid solutions of the active ingredient in aqueous or oily
solvents may be prepared in substantially the same manner as liquid
suspensions, the primary difference being that the active
ingredient is dissolved, rather than suspended in the solvent.
Liquid solutions of the pharmaceutical composition of the invention
may comprise each of the components described with regard to liquid
suspensions, it being understood that suspending agents will not
necessarily aid dissolution of the active ingredient in the
solvent. Aqueous solvents include, for example, water and isotonic
saline. Oily solvents include, for example, almond oil, oily
esters, ethyl alcohol, vegetable oils such as arachis, olive,
sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin.
[0182] Powdered and granular formulations of a pharmaceutical
preparation of the invention may be prepared using known methods.
Such formulations may be administered directly to a subject, used,
for example, to form tablets, to fill capsules, or to prepare an
aqueous or oily suspension or solution by addition of an aqueous or
oily vehicle thereto. Each of these formulations may further
comprise one or more of dispersing or wetting agent, a suspending
agent, and a preservative. Additional excipients, such as fillers
and sweetening, flavoring, or coloring agents, may also be included
in these formulations.
[0183] A pharmaceutical composition of the invention may also be
prepared, packaged, or sold in the form of oil-in-water emulsion or
a water-in-oil emulsion. The oily phase may be a vegetable oil such
as olive or arachis oil, a mineral oil such as liquid paraffin, or
a combination of these. Such compositions may further comprise one
or more emulsifying agents such as naturally occurring gums such as
gum acacia or gum tragacanth, naturally-occurring phosphatides such
as soybean or lecithin phosphatide, esters or partial esters
derived from combinations of fatty acids and hexitol anhydrides
such as sorbitan monooleate, and condensation products of such
partial esters with ethylene oxide such as polyoxyethylene sorbitan
monooleate. These emulsions may also contain additional ingredients
including, for example, sweetening or flavoring agents.
[0184] Methods for impregnating or coating a material with a
chemical composition are known in the art, and include, but are not
limited to methods of depositing or binding a chemical composition
onto a surface, methods of incorporating a chemical composition
into the structure of a material during the synthesis of the
material (i.e. such as with a physiologically degradable material),
and methods of absorbing an aqueous or oily solution or suspension
into an absorbent material, with or without subsequent drying.
[0185] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, cutaneous, subcutaneous, intraperitoneal,
intravenous, intramuscular, intracisternal injection, and kidney
dialytic infusion techniques.
[0186] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in ampules
or in multi-dose containers containing a preservative. Formulations
for parenteral administration include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the active ingredient is
provided in dry (i.e. powder or granular) form for reconstitution
with a suitable vehicle (e.g., sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition.
[0187] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation may comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble
salt.
[0188] Formulations suitable for topical administration include,
but are not limited to, liquid or semi-liquid preparations such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, and solutions or suspensions.
Topically-administrable formulations may, for example, comprise
from about 1% to about 10% (w/w) active ingredient, although the
concentration of the active ingredient may be as high as the
solubility limit of the active ingredient in the solvent
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0189] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for pulmonary
administration via the buccal cavity. Such a formulation may
comprise dry particles which comprise the active ingredient and
which have a diameter in the range from about 0.5 to about 7
nanometers, and preferably from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant may be directed to disperse the powder
or using a self-propelling solvent/powder-dispensing container such
as a device comprising the active ingredient dissolved or suspended
in a low-boiling propellant in a sealed container. Preferably, such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by number have a diameter less than 7 nanometers.
More preferably, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions preferably include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
[0190] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic or solid anionic
surfactant or a solid diluent (preferably having a particle size of
the same order as particles comprising the active ingredient).
[0191] Pharmaceutical compositions of the invention formulated for
pulmonary delivery may also provide the active ingredient in the
form of droplets of a solution or suspension. Such formulations may
be prepared, packaged, or sold as aqueous or dilute alcoholic
solutions or suspensions, optionally sterile, comprising the active
ingredient, and may conveniently be administered using any
nebulization or atomization device. Such formulations may further
comprise one or more additional ingredients including, but not
limited to, a flavoring agent such as saccharin sodium, a volatile
oil, a buffering agent, a surface active agent, or a preservative
such as methylhydroxybenzoate. The droplets provided by this route
of administration preferably have an average diameter in the range
from about 0.1 to about 200 nanometers.
[0192] The formulations described herein as being useful for
pulmonary delivery are also useful for intranasal delivery of a
pharmaceutical composition of the invention.
[0193] Another formulation suitable for intranasal administration
is a coarse powder comprising the active ingredient and having an
average particle from about 0.2 to 500 micrometers.
[0194] Such a formulation is administered in the manner in which
snuff is taken i.e. by rapid inhalation through the nasal passage
from a container of the powder held close to the nares.
Formulations suitable for nasal administration may, for example,
comprise from about as little as 0.1% (w/w) and as much as 100%
(w/w) of the active ingredient, and may further comprise one or
more of the additional ingredients described herein.
[0195] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for buccal
administration. Such formulations may, for example, be in the form
of tablets or lozenges made using conventional methods, and may,
for example, contain 0.1 to 20% (w/w) active ingredient, the
balance comprising an orally dissolvable or degradable composition
and, optionally, one or more of the additional ingredients
described herein. Alternately, formulations suitable for buccal
administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient. Such
powdered, aerosolized, or aerosolized formulations, when dispersed,
preferably have an average particle or droplet size in the range
from about 0.1 to about 200 nanometers, and may further comprise
one or more of the additional ingredients described herein.
[0196] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for
ophthalmic administration. Such formulations may, for example, be
in the form of eye drops including, for example, a 0.1-1.0% (w/w)
solution or suspension of the active ingredient in an aqueous or
oily liquid carrier. Such drops may further comprise buffering
agents, salts, or one or more other of the additional ingredients
described herein. Other opthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form or in a liposomal preparation.
[0197] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Genaro, ed., 1985,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., which is incorporated herein by reference.
[0198] Typically dosages of the compound of the invention which may
be administered to an animal, preferably a human, range in amount
from about 0.01 mg to 20 about 100 g per kilogram of body weight of
the animal. While the precise dosage administered will vary
depending upon any number of factors, including, but not limited
to, the type of animal and type of disease state being treated, the
age of the animal and the route of administration. Preferably, the
dosage of the compound will vary from about 1 mg to about 100 mg
per kilogram of body weight of the animal. More preferably, the
dosage will vary from about 1 .mu.g to about 1 g per kilogram of
body weight of the animal. The compound can be administered to an
animal as frequently as several times daily, or it can be
administered less frequently, such as once a day, once a week, once
every two weeks, once a month, or even less frequently, such as
once every several months or even once a year or less. The
frequency of the dose will be readily apparent to the skilled
artisan and will depend upon any number of factors, such as, but
not limited to, the type and severity of the disease being treated,
the type and age of the animal, etc.
Methods of Identifying a D-DT Activators or D-DT Inhibitor
[0199] The current invention relates to a methods of identifying a
compound that modulates the level of D-DT, the enzymatic activity
of D-DT, the receptor binding activity of D-DT, or a combination
thereof. In some embodiments, the method of identifying of the
invention identifies a D-DT inhibitor compound that decreases the
level of D-DT, the enzymatic activity of D-DT, the receptor binding
activity of D-DT, or a combination thereof. In other embodiments,
the method of identifying of the invention identifies a D-DT
activator compound that increases the level of D-DT, the enzymatic
activity of D-DT, the receptor binding activity of D-DT, or a
combination thereof.
[0200] Other methods, as well as variation of the methods disclosed
herein will be apparent from the description of this invention. In
various embodiments, the test compound concentration in the
screening assay can be fixed or varied. A single test compound, or
a plurality of test compounds, can be tested at one time. Suitable
test compounds that may be used include, but are not limited to,
proteins, nucleic acids, antisense nucleic acids, small molecules,
antibodies and peptides.
[0201] The invention relates to a method for screening test
compounds to identify a modulator compound by its ability to
modulate (i.e., increase or decrease) the level of D-DT, the
enzymatic activity of D-DT, the receptor binding activity of D-DT,
or a combination thereof, by measuring the level of D-DT, the
enzymatic activity of D-DT, the receptor binding activity of D-DT,
or a combination thereof, in the presence and absence of the test
compound.
[0202] The test compounds can be obtained using any of the numerous
approaches in combinatorial library methods known in the art,
including: biological libraries; spatially addressable parallel
solid phase or solution phase libraries; synthetic library methods
requiring deconvolution; the "one-bead one-compound" library
method; and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam et al., 1997, Anticancer Drug Des. 12:45).
[0203] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example, in: DeWitt et al., 1993,
Proc. Natl. Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678;
Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem.
37:1233.
[0204] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith, 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J.
Mol. Biol. 222:301-310; and Ladner supra).
[0205] In situations where "high-throughput" modalities are
preferred, it is typical to that new chemical entities with useful
properties are generated by identifying a chemical compound (called
a "lead compound") with some desirable property or activity,
creating variants of the lead compound, and evaluating the property
and activity of those variant compounds.
[0206] In one embodiment, high throughput screening methods involve
providing a library containing a large number of test compounds
potentially having the desired activity. Such "combinatorial
chemical libraries" are then screened in one or more assays, as
described herein, to identify those library members (particular
chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
EXAMPLES
[0207] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0208] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
Example 1
The D-Dopachrome Tautomerase (D-DT) Gene Product is a Cytokine and
Functional Homolog of Macrophage Migration Inhibitory Factor
(MIF)
[0209] The functional characterization of human and mouse D-DT
(also known as MIF-2) is described herein. Recombinant D-DT was
produced and was demonstrated to bind with high affinity to CD74,
activate the ERK1/2 MAP kinase signaling cascade, and recapitulate
many of the inflammatory functions of MIF, including modulation of
macrophage migration and glucocorticoidinduced immunosuppression.
The administration of an anti-D-DT antibody, like anti-MIF,
protects mice from lethal endotoxic shock by reducing the
circulating levels of proinflammatory cytokines (TNF-.alpha.,
IFN-.gamma., IL-12p70, and IL-1.beta.) and increasing the
circulating concentration of the anti-inflammatory cytokine, IL-10.
An analysis of clinical samples from patients with sepsis or cancer
also revealed that D-DT is systemically expressed and that
circulating levels correlate with MIF and with the severity of
inflammatory disease and malignancy.
[0210] Both mouse and human D-DT proteins show conservation in
their catalytic, N-terminal proline but they lack MIF's -CXXC- and
pseudo(E)LRmotifs, the latter of which is known to mediate
noncognate interactions with CXCR2 and CXCR4 (Bernhagen, et al.,
2007, Nat Med 13:587-596; Weber, et al., 2008, Proc Natl Acad Sci
USA 105: 16278-16283). As described herein, a recombinant,
endotoxin-free D-DT protein was produced and it is shown that D-DT,
like MIF, tautomerizes the model substrate,
p-hydroxyphenylpyruvate, albeit the measured velocity is .about.10
times lower than that analyzed for MIF. D-DT is constitutively
expressed in different tissues and it is up-regulated by
proinflammatory activation. MIF is known to bind to the type II
transmembrane protein, CD74, leading to its intracellular
phosphorylation, the recruitment of the coreceptor CD44, and the
activation of SRC family kinases and the ERK1/2 MAPK pathway (Shi,
et al., 2006 Immunity 25:595-606; Leng, et al., 2003, J Exp Med
197:1467-1476). Although the interaction between D-DT and sCD74 was
reduced in comparison with MIF when measured by both competition
binding and BIAcore studies, the ERK1/2 activation potential of
D-DT and MIF appeared comparable by Western blotting. The KD of
sCD74 for D-DT was 60% of that measured for MIF; D-DT was found to
have an .about.3-fold higher association rate (ka) to sCD74 but to
dissociate (kd) .about.11-fold faster than MIF. Although not
wishing to be bound by any particular theory, these differences in
association and dissociation values may differentiate the signaling
action of the two proteins; e.g., D-DT binding to the receptor
might not always trigger a signaling cascade but might result in
its internalization. MIF showed a steeper dose response than D-DT
in measurements of macrophage migration inhibition and
glucocorticoid overriding, but the resolution of these cell-based
assays may be too limited to make precise, quantitative
conclusions. These results may support the notion that D-DT is a
less potent cytokine that might be associated with the
down-regulation of inflammation.
[0211] As described herein, anti-D-DT antibodies were produced that
do not cross-react with MIF; these antibodies allowed for
measurements of this protein in biologic specimens. D-DT was
expressed in the serum of endotoxemic mice at levels that were
.about.60% those of MIF. MIF is a high, upstream mediator of septic
shock, and immunoneutralization, genetic deletion, or pharmacologic
inhibition of MIF protects from lethal shock induced by different
microbial pathogens, even when administered many hours after
microbial invasion (Bernhagen, et al., 1993, Nature 365:756-759;
Bozza, et al., 1999, J Exp Med 189:341-346; Calandra, et al., 1998,
Proc Natl Acad Sci USA 95:11383-11388; Arjona, et al., 2007, J Clin
Invest 117: 3059-3066; McDevitt, et al., 2006, J Exp Med
203:1185-1196). Immunoneutralization of D-DT conferred protection
from lethal endotoxemia and this effect was accompanied by a broad
reduction in the systemic expression of TNF-.alpha., IL-1.beta.,
IL-12p70, and IFN-.gamma. and an increase in the circulating levels
of the anti-inflammatory mediator, IL-10. Given current development
of MIF inhibitors for the treatment of the inflammatory diseases
(Arjona, et al., 2007, J Clin Invest 117: 3059-3066; Lolis, et al.,
2003, Nat Rev Drug Discov 2:635-645; Cournia, et al. 2009, J Med
Chem 52:416-424), it was also of interest to determine whether the
combined neutralization of MIF and D-DT had a synergistic effect.
MIF levels are elevated in patients with systemic inflammatory
disease or with neoplasia, and in many instances a correlation
between disease severity and MIF levels has been observed (Grieb,
et al., 2010, Drug News Perspect 23:257-264). Like MIF, D-DT
circulates at baseline levels in healthy individuals and there is a
correlation in the plasma concentrations of the two mediators.
Circulating D-DT levels increase significantly in patients with
sepsis or ovarian cancer and show a strong correlation with MIF,
consistent with the explanation that the two mediators may be
coordinately expressed in response to similar activating stimuli.
The findings described herein include that D-DT is a close
structural and functional homolog of MIF, that it binds and
activates the MIF receptor, and that targeting D-DT, similar to
MIF, protects mice from lethal endotoxemia.
[0212] Like MIF (Bacher, et al., 1997, Am J Pathol 150:235-246),
D-DT was found to be widely expressed in tissues. As described
elsewhere herein, macrophages produce 20-fold more MIF than D-DT in
response to LPS, consistent with the explanation that there may be
cell-specific release of these mediators and that in contrast to
MIF, nonmacrophage sources of D-DT contribute more importantly to
the systemic expression of D-DT than MIF. Computational inspection
of the D-DT and MIF promoter regions shows similarities in the
presence of serum-responsive, SP-1 and AP-1 binding elements
(alggen.lsi.upc.es). Of note, the human D-DT gene lacks the
polymorphic, CATT5-8 microsatellite repeat that exists in the MIF
promoter (rs5844572) and that regulates MIF expression and is
associated with the severity of autoimmune inflammatory diseases
(Grieb, et al., 2010, Drug News Perspect 23:257-264). Although not
wishing to be bound by any particular theory, one difference
between MIF and D-DT may lie in the allele-specific regulation of
MIF expression but not D-DT. At the protein level, D-DT lacks MIF's
pseudo(E)LR domain, which is necessary for activation of CXCR2.
Bernhagen and colleagues have reported that MIF initiates
coordinated receptor interactions between CD74 and CXCR2/CXCR4
(Bernhagen, et al., 2007, Nat Med 13:587-596; Weber, et al., 2008,
Proc Natl Acad Sci USA 105: 16278-16283). D-DT's high binding
affinity to CD74 also may facilitate direct chemotactic effects,
perhaps in concert with MIF or IL-8, which is expressed upon CD74
signaling (Coleman, et al., 2008, J Immunol 181:2330-2337; Binsky,
et al., 2007, Proc Natl Acad Sci USA 104:13408-13413). D-DT binds
the intracellular protein, JAB1, indicating that it might affect
the regulation of cell cycle control and signalosome function,
similarly to MIF (Kleemann, et al., 2000, Nature 408:211-216). Like
MIF, D-DT lacks an N-terminal signal sequence and it may be
produced in sufficient intracytoplasmic concentrations to influence
these regulatory pathways. In summary, the data disclosed herein
identify D-DT to be a cytokine and a close functional homolog of
MIF.
[0213] The data described herein support the targeting of D-DT, as
well as the simultaneous targeting of D-DT and MIF, in various
diseases and disease models.
[0214] The materials and methods employed in this example are now
described.
Cloning and Purification of D-DT Protein
[0215] The cDNAs for the human and mouse D-DT proteins were
prepared by amplification of mRNA from human or murine monocytes
and subcloned into the pET22b expression vector. For native protein
expression, a stop codon was engineered. Bacterial extract was
purified using a Q Sepharose column (Amersham) and subsequent HPLC
using a C18 column (Amersham). D-DT was refolded using the protocol
established for MIF renaturation (Bernhagen, et al., 1994,
Biochemistry 33: 14144-14155). Protein purity was verified by
Coomassie and fidelity confirmed by mass spectroscopy. The
resulting proteins contained <1 pg LPS/.mu.g protein as
quantified by the PyroGene Recombinant Factor C assay (Cambrex).
MIF proteins were produced as described earlier (Bernhagen, et al.,
1994, Biochemistry 33: 14144-14155). D-dopachrome tautomerase
activity was assessed using the substrate HPP, measuring the change
in absorbance at 306 nm for 180 s (Stamps, et al., Biochemistry
39:9671-9678).
Anti-D-DT Antibody and ELISA
[0216] Polyclonal antibodies against recombinant mouse or human
D-DT were produced in rabbits. IgG antibody fractions were isolated
by Protein A agarose affinity chromatography (Pierce) and sterile
filtered. Microtiter plates (Nunc) were coated with 15 .mu.g/mL of
polyclonal anti-D-DT, washed, and blocked in 1% BSA and 1% sucrose.
Samples were added and incubated for 2 hours, followed by
biotinylated anti-D-DT antibody and a streptavidin-HRP conjugate.
The D-DT concentrations were calculated by extrapolation from a
sigmoidal quadratic standard curve using native D-DT protein
(dynamic range, 0-625 pg/mL). For mouse or human D-DT, the
detection limit was 15 pg/mL.
MIF Receptor Binding Studies
[0217] Binding of D-DT to the MIF receptor, CD74, was studied by
competition binding assay as described previously (Leng, et al.,
2003, J Exp Med 197:1467-1476). Real-time binding interaction of
MIF or D-DT with CD7473-232 was measured by surface plasmon
resonance using a BIAcore 2000 optical biosensor (Kamir, et al.,
2008, J Immunol 180:8250-8261). The MIF receptor sCD74 was
immobilized to the chip, and binding of the ligands D-DT and MIF
was measured in five serial dilutions, three times for each
dilution sample. Sensorgram response data were analyzed in the BIA
evaluation kinetics package and the equilibrium binding constants
calculated in the same experiment.
D-DT Protein Expression in Murine Tissues
[0218] Tissues were isolated from C57BL/6 mice, and proteins were
analyzed by Western blot (Bacher, et al., 1997, Am J Pathol
150:235-246). For immunohistochemistry, tissue sections from
C57BL/6 mice (Mizue, et al., 2005, Proc Natl Acad Sci USA
102:14410-14415) were deparaffinized and antigen retrieval was
performed using the Target Retrieval Solution (Dako). The
specificity of anti-D-DT antibody staining was established by
preabsorbing an aliquot of the antibody with a 1,000-fold molar
excess of either D-DT or MIF. Slides were incubated with rabbit
anti-D-DT or IgG control antibody (1:50) overnight and visualized
with the Liquid DAB+Substrate Chromogen System (Dako). To allow a
semiquantitative comparison of different tissues, all slides were
developed for 10 min.
siRNA-Mediated Knockdown of D-DT and MIF
[0219] Immortalized murine macrophages (Duewell, et al., 2010,
Nature 464:1357-1361) were transfected with 50 nM siRNA using
HiPerFect (Qiagen). Sequences used were 5'-TCAACTATTACGACATGAA-3'
(SEQ ID NO: 1) for MIF and 5'-GCATGACCCTGTTGATGAA-3' (SEQ ID NO: 2)
for D-DT.
Signal Transduction Studies
[0220] Mouse peritoneal macrophages (1.times.10.sup.6/well) were
rendered quiescent by incubation in 0.1% FBS before stimulation
with D-DT or MIF for 2 hours (Mitchell, et al., 1999, J Biol Chem
274:18100-18106). Cells were lysed in RIPA buffer and lysates were
run on a 4-12% Bis-Tris NuPage gel (Invitrogen). Immunoblotting was
conducted with Abs directed against total ERK1/2, and
phospho-ERK-1/2 (Cell Signaling).
Migration and Glucocorticoid Overriding Assays
[0221] Migration assays were performed as described previously
(Hermanowski-Vosatka, et al., 1999, Biochemistry 38:12841-12849).
Briefly, human monocytes were incubated for 20 min with MIF or
D-DT. Media with or without 25 ng/mL MCP-1 was added in the lower
compartment of migration chambers and monocytes was added to the
transwell (0.5-.mu.m pore size) for 90 min. Cells from the lower
migration chamber were lysed, and DNA was fluorescently labeled and
enumerated at 480/520 nm. Following the original glucocorticoid
overriding methodology of Calandra et al. (Calandra, et al., 1995,
Nature 377:68-71), macrophages were preincubated for 1 hour with
100 nM dexamethasone (Sigma) and MIF or D-DT before adding 100
ng/mL LPS (Sigma). TNF levels in supernatants were measured by
ELISA (eBioscience).
MIF/JAB1 Coimmunoprecipitation
[0222] Mif-/-MEFs were lysed in ice-cold buffer and incubated with
MIF or D-DT, respectively (Kleemann, et al., 2000, Nature
408:211-216). Two micrograms of anti-JAB1 (2A10.8; Gene Tex/Biozol)
or IgG1 control was added and the protein complexes were pulled
down with magnetic protein G beads (Invitrogen). Blotted proteins
were visualized using an anti-MIF or anti-D-DT antibodies and then
reprobed with anti-JAB1 antibody.
Endotoxemia Model.
[0223] Endotoxemia was induced in female BALB/c mice (8 wk old) by
i.p. administration of E. coli LPS 0111:B4 (Sigma) at a dose of
12.5 mg/kg for serum cytokine measurement and 20 mg/kg for
intervention experiments (LD80). For D-DT neutralization studies,
mice were injected i.p. with 200 .mu.L of rabbit anti-D-DT
antiserum or nonimmune serum 2 hours before administration of LPS.
Mice were monitored every 4 hours within the first 72 hours and
then twice daily until death or until 14 days. Cytokine levels were
obtained by bleeding mice 4, 24, and 36 hours after LPS challenge,
and serum cytokines were analyzed by Luminex (Bio-Rad).
Patient Samples
[0224] Serum concentrations of D-DT and MIF were measured in 85
healthy individuals and in 37 septic patients hospitalized in the
medical intensive care unit (Lesur, et al., 2010, Crit Care
14:R131). The median APACHE II score at the time of intensive care
unit admission was 22 points (range: 10-36 points). The mortality
rate was 27%. The etiologic agents of sepsis were Gram-negative
bacteria (43%) and Gram-positive bacteria (49%). Two patients had
an infection with Grampositive and Gram-negative bacteria and one
with fungi. Sera from women with biopsy-proven ovarian cancer
(n=21) were from Yale-New Haven Hospital.
[0225] The results of this example are now described.
Purification and Characterization of the D-DT Protein
[0226] The genes for MIF and D-DT lie within 0.1 kb of each other
in both the mouse and human genomes and have a similar
organizational relationship with nearby genes for matrix
metalloproteinase 11 and two theta class GSTs (FIG. 1). The amino
acid sequences show 34% identity between human MIF and human D-DT
and 27% identity between murine MIF and murine D-DT. The D-DT
proteins share with MIF a canonical N-terminal proline (formed
after posttranslational excision of the initiating methionine),
which catalyzes substrate tautomerization (Bendrat, et al., 1997,
Biochemistry 36:15356-15362), but they lack two of the three
conserved cysteines (Cys59 and Cys80) that appear in all known
mammalian MIF proteins. Murine and human D-DT also lack the
pseudo(E)LR (Arg11, Asp44) motif that mediates MIF's noncanonical
interactions with the CXCR2 chemokine receptor (Weber, et al.,
2008, Proc Natl Acad Sci USA 105: 16278-16283). The mRNA for D-DT
does not encode either an N-terminal or an internal secretory
signal sequence, suggesting that like MIF, D-DT is secreted by a
specialized, nonclassical export pathway (Merk, et al., 2009, J
Immunol 182:6896-6906).
[0227] The cDNAs for human and mouse D-DT were prepared from
monocytes and cloned into a bacterial expression vector for
recombinant protein production. Work was performed with native
sequence proteins because structure-function studies have shown
that modifications of the N or C termini interfere with trimer
formation, the functional unit of MIF (Bendrat, et al., 1997,
Biochemistry 36:15356-15362; Sun, et al., 1996, Proc Natl Acad Sci
USA 93: 5191-5196; El-Turk, et al., 2008, Biochemistry
47:10740-10756). Recombinant D-DT protein was purified by anion
exchange chromatography followed by high performance liquid
chromatography (HPLC) (FIG. 2A). Mass spectroscopy of purified
mouse D-DT protein gave an m/z of 12,947, which lies within 0.02%
of the calculated mass for D-DT (FIG. 2B). A minor peak of 13,079
Da also was detected; this peak corresponds to the molecular mass
of D-DT with an uncleaved N-terminal methionine (expected
m/z=13,077). MIF tautomerizes model substrates such as D-dopachrome
and p-hydoxyphenylpyruvate (HPP) (whether a physiological substrate
exists is unknown) (Fingerle-Rowson, et al., 2009, Mol Cell Biol
29:1922-1932), and D-DT purified from liver has tautomerization
activity (Rosengren, et al., 1996, Mol Med 2:143-149; Odh, et al.,
1993, Biochem Biophys Res Commun 197:619-624). It was verified that
recombinant D-DT tautomerizes HPP with a specific activity that is
.about.10 times lower than that measured for MIF (D-DT=0.5.+-.0.1
.DELTA.306min-1.mu.M-1, and MIF=4.3.+-.1.1 .DELTA.306min-1.mu.M-1,
P<0.001) (FIG. 2C). A possible explanation for the discrepancy
inactivity might be structural differences in the active site
regions of D-DT and MIF, resulting in a reduced affinity of D-DT to
its substrate (Sugimoto, et al., 1999, Biochemistry 38:3268-3279;
Sun, et al., 1996, Proc Natl Acad Sci USA 93: 5191-5196). A
polyclonal anti-D-DT antibody was prepared to establish an ELISA.
This antibody recognized both murine and human D-DT but did not
cross-react with MIF in its denatured form (assessed by Western
blot; FIG. 2D, Left) or in its native form (assessed by ELISA; FIG.
2D, Right).
D-DT Binds the MIF Receptor, CD74, and the Intracellular Protein,
JAB1
[0228] MIF activates ERK1/2 phosphorylation by engaging CD74, and a
high-affinity binding interaction between MIF and the CD74
ectodomain (CD7473-232 or sCD74) has been demonstrated by surface
plasmon resonance (Leng, et al., 2003, J Exp Med 197:1467-1476).
The interaction between D-DT and sCD74 in a competition binding
assay was studied. D-DT reduced MIF binding to the CD74 ectodomain
in a dose-dependent manner, with a maximal effect of .about.50%
compared with MIF (FIG. 3A). Measurement of the equilibrium
dissociation constants between human D-DT or MIF and sCD74 by
surface plasmon resonance (BIAcore analysis) revealed a
high-affinity binding interaction between D-DT and the MIF receptor
(KD of 5.42.times.10-9 M) (FIG. 3B), albeit 60% lower than for MIF
(KD=1.40.times.10-9 M). Detailed analysis revealed a ka of
1.2.times.105 M-1s-1 for D-DT and only 4.3.times.104 M-1s-1 for
MIF, whereas the dissociation rate (kd) was 11-fold lower for MIF
than for D-DT (6.times.10-5s-1 and 6.6.times.10-4s-1,
respectively). These measurements demonstrate that D-DT has an
.about.3-fold higher binding rate to the receptor CD74, but also
dissociates much faster than MIF. The intracellular transcriptional
regulator and COPS signalosome component JAB1 is a well
characterized binding partner of MIF that has been implicated in
MIF-dependent control of cell proliferation (Kleemann, et al.,
2000, Nature 408:211-216). D-DT binds to JAB1 as demonstrated by
coimmunoprecipation (FIG. 3C), and the interaction affinity between
JAB1 and D-DT appears comparable to that observed between JAB1 and
MIF.
Differential Expression of D-DT and MIF
[0229] The differential expression of D-DT in tissue compared with
MIF was assessed. Esumi et al. have published Northern blotting
data for D-DT expression that suggest enhanced expression in the
murine brain compared with Mif (Esumi, et al., 1998, Mamm Genome
9:753-757). Eight different mouse organs were analyzed by Western
blotting for D-DT, MIF, and CD74. D-DT and MIF were present in
constitutive and appreciable levels in all tissues examined, with
the greatest difference observed in the testis, where D-DT appeared
in severalfold higher concentrations compared with MIF (FIG. 3D).
Immunostaining of murine tissue confirmed these results and showed
that D-DT, like MIF (Bacher, et al., 1997, Am J Pathol
150:235-246), is detected in virtually all cells, with prominent
staining in the epithelia of the kidney, the lung, the bowel,
hepatocytes, and the follicular area of the spleen (FIG. 3E).
D-DT Initiates ERK-1/2 Phosphorylation in a MIF
Receptor-Complex-Dependent Manner, Mediates Macrophage Migration
Arrest, and Counterregulates Glucocorticoid-Induced
Immunosuppression
[0230] MIF binding to CD74 leads to the recruitment of CD44 and the
intracellular phosphorylation of these proteins, resulting in the
activation of SRC family nonreceptor tyrosine kinases and the
initiation of the ERK1/2 MAP kinase cascade (Shi, et al., 2006
Immunity 25:595-606; Leng, et al., 2003, J Exp Med 197:1467-1476).
Stimulation of cultured macrophages with increasing concentrations
of recombinant D-DT produced a dose-dependent phosphorylation of
ERK1/2, with an activating effect that was both sustained (2 h)
(Mitchell, et al., 1999, J Biol Chem 274:18100-18106) and
comparable to that observed for MIF (FIG. 4A, Top). Costimulation
with D-DT and MIF revealed an additive effect of the two proteins
in the ERK1/2 MAP kinase pathway (FIG. 4A, Middle). D-DT-induced
ERK1/2 phosphorylation was strictly dependent on the expression of
both CD74 and CD44, as previously reported for MIF (Shi, et al.,
2006 Immunity 25:595-606) (FIG. 4A, Bottom).
[0231] The biologic activity of recombinant D-DT was analyzed by
first assaying for MIF's effect on macrophage chemotaxis
(Hermanowski-Vosatka, et al., 1999, Biochemistry 38:12841-12849).
D-DT inhibited chemotaxis induced by CCL2/monocyte chemotactic
protein (MCP)-1, but with a less steep dose response and reduced
inhibitory action at 1 .mu.g/mL compared with MIF (FIG. 4B).
Although not wishing to be bound by any particular theory, this
observation may be explained by the reduced binding affinity of
D-DT for the MIF receptor or different rates of ligand
association/dissociation and a consequent reduction in the
downstream events necessary for CCL2 desensitization. MIF is unique
among immune mediators in its ability to counterregulate the
immunosuppressive action of glucocorticoids, which occurs via
intracellular pathways that involve cytoplasmic phospholipase A2,
I.kappa.B1, and MKP-1 (Calandra, et al., 1995, Nature 377:68-71;
Flaster, et al, 2007, Mol Endocrinol 21:1267-1280; Roger, et al.,
2005, Eur J Immunol 35:3405-3413). Using a standardized assay
(Calandra, et al., 1995, Nature 377:68-71), it was further observed
that D-DT, like MIF, counterregulated the inhibitory effect of
glucocorticoids on TNF production from lipopolysaccharide
(LPS)-stimulated macrophages (FIG. 4C). Similar to observations in
the migration assay, D-DT shows a decreased counterregulatory
potential at low concentrations compared with MIF, which may be
attributed to lower binding affinity to the MIF receptor.
D-DT is Produced in Response to LPS and Mediates Lethal
Endotoxemia
[0232] Whereas macrophages have been considered historically to be
a main target of MIF action, these cells also are a major source of
MIF production in response to microbial products and tissue
invasion in vivo (Calandra, et al, 1994, J Exp Med 179:1895-1902).
Cultured macrophages were stimulated with 1 .mu.g/mL of Escherichia
coli LPS and the secretion of D-DT and MIF was quantified by
specific ELISA. LPS-stimulated macrophages released D-DT into
conditioned medium with kinetics that were very similar to those of
MIF (FIG. 5A). Peak levels were detectable at 16 hours and
decreased thereafter. Unstimulated cells also slowly released these
proteins into supernatants, which in the case of MIF has been
attributed to a low level of constitutive secretion (Merk, et al.,
2009, J Immunol 182:6896-6906).
[0233] It was next assessed whether there is reciprocal regulation
of D-DT and MIF expression in macrophages. D-DT or MIF were
depleted in immortalized macrophages by siRNA-mediated knockdown
but detected no effect on the expression level of the reciprocal
protein in response to LPS stimulation (FIG. 5B). The
administration of LPS to mice resulted in a timedependent increase
in plasma D-DT concentrations (6.0.+-.4.3 ng/mL to 26.+-.12 ng/mL
at 24 h), and this increase mimicked the rise observed for MIF
(1.0.+-.0.9 ng/mL to 43.+-.28 ng/mL at 24 h) (FIG. 5C). D-DT also
is detectable in plasma under basal conditions and at comparable
levels to MIF (D-DT=6.0.+-.4.3 ng/mL and MIF=1.0.+-.0.9 ng/mL)
(Calandra, et al., 1995, Nature 377:68-71). It is noteworthy that
whereas plasma MIF and D-DT circulate in similar concentrations
under basal or stimulated conditions, LPS-stimulated macrophages
produce 20-fold more MIF than D-DT (FIG. 5A). Although not wishing
to be bound by any particular theory, this observation is
consistent with the explanation that nonmacrophage sources of D-DT
contribute significantly to plasma D-DT expression in vivo.
Immunoneutralization of D-DT Protects from Lethal Endotoxemia
[0234] Immunoneutralization or genetic deletion of MIF protects
mice from endotoxic shock (Bernhagen, et al., 1993, Nature
365:756-759; Bozza, et al., 1999, J Exp Med 189:341-346) and
subsequent studies demonstrated that this protective effect is due
to a reduction in the expression of tissue-damaging, effector
cytokines such as TNF and IL-1 (Mitchell, et al., 2002, Proc Natl
Acad Sci USA 99:345-350; Calandra, et al., 2000, Nat Med
6:164-170). The administration of a specific anti-D-DT antibody
before the injection of an LD80 dose of LPS increased survival from
20 to 79% (FIG. 5D), which is a level of protection comparable to
that of anti-MIF (Bernhagen, et al., 1993, Nature 365:756-759). An
analysis of plasma cytokine expression showed that D-DT
neutralization was associated with a significant reduction in the
circulating concentration of several proinflammatory cytokines
(TNF-.alpha., IL-1.beta., IFN-.gamma., and IL-12p70) implicated in
shock pathogenesis (FIG. 5E). In contrast, the concentration of the
anti-inflammatory cytokine IL-10 was increased in the
anti-D-DT-treated group compared with controls.
D-DT Expression is Up-Regulated in Patients with Sepsis, Invasive
Cancer, or Vasculitis
[0235] To determine whether D-DT is up-regulated in human disease
and to assess potential relationships between D-DT and MIF
production in vivo, the serum concentrations of these mediators
were analyzed in individuals with sepsis or with ovarian cancer,
which are two conditions characterized by high levels of MIF in
plasma (Calandra, et al., 2000, Nat Med 6:164-170; Visintin, et
al., 2008, Clin Cancer Res 14:1065-1072). There was a statistically
significant increase in circulating D-DT protein in patients with
sepsis compared with healthy controls (sepsis patients,
55.5.+-.61.3 ng/mL; control group, 5.9.+-.3.9 ng/mL; P<0.0001)
(FIG. 6A). MIF levels also were elevated, as expected from prior
work (Calandra, et al., 2000, Nat Med 6:164-170; Bozza, et al.,
2004, Shock 22:309-313; Lehmann, et al., 2001, Intensive Care Med
27:1412-1415) (sepsis patients, 111.0.+-.69.0 ng/mL; control group,
6.3.+-.6.2 ng/mL; P<0.0001). Receiver operator characteristic
(ROC) analysis revealed an area under the curve of 0.99 for MIF or
D-DT, indicating that both proteins show excellent sensitivity and
specificity for the diagnosis of sepsis. These measurements further
revealed that serum levels of D-DT, like MIF (Bozza, et al., 2004,
Shock 22:309-313; Emonts, et al., 2007, Clin Infect Dis
44:1321-1328), correlate with disease severity as determined by
APACHE II clinical severity scores (FIG. 6B). Both D-DT and MIF
show a significant association with outcome parameters; however, a
more precise assessment of the prognostic value of these proteins
may be obtained by serial measurements. Serum D-DT and MIF
concentrations also correlate in healthy individuals, and the
correlation coefficient increases from R=0.32 to R=0.5 for the
analysis of these mediators in patients with sepsis (FIG. 6C).
Circulating D-DT concentrations were also found to be elevated in
patients with ovarian cancer (cancer patients, 15.2.+-.13.8 ng/mL;
control group, 5.9.+-.3.9 ng/mL; P<0.0001) (FIG. 6D). ROC
analysis revealed an area under the curve of 0.8, which is
comparable to that observed for MIF (0.7). The correlation between
MIF and D-DT serum concentrations was stronger and showed greater
statistical significance than that observed for septic patients
(R=0.9, P=0.0001). D-DT expression is up-regulated in subjects with
vasculitis and D-DT concentrations correlate with MIF and the
presence of vasculitis (FIG. 7).
Example 2
Ischemia-Reperfusion Injury
[0236] As described herein, D-DT activates the cardioprotective
AMPK pathway in rat heart muscles. Mouse recombinant DDT activates
AMP-activated protein kinase in rat heart left ventricular
papillary muscles. DDT incubation leads to phosphorylation of
threonine 172, the major activating site in the catalytic alpha
subunit of AMPK, as well as downstream target acetyl-CoA
carboxylase (ACC). AMPK pathway activation by DDT is dose and time
dependent (FIGS. 8 and 9).
[0237] Moreover, endogenous DDT has a role in mediating AMPK
activation during hypoxia in isolated rat heart left ventricular
muscles (FIG. 9). The addition of purified rabbit polyclonal
neutralizing D-DT antibody for 30 minutes prior to and during 15
minutes of hypoxia significantly reduced the critical
phosphorylation of threonine 172 in the activating domain of the
alpha catalytic subunit of AMP-activated protein kinase. DDT
antibody also decreased the phosphorylation of downstream
acetyl-CoA carboxylase. Control was performed with non-immune IgG
incubation. FIG. 9B shows a comparison to incubation with MIF
neutralizing antibody. p=0.05.
[0238] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0239] While the invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
2119DNAHomo sapiens 1tcaactatta cgacatgaa 19219DNAHomo sapiens
2gcatgaccct gttgatgaa 19
* * * * *
References