Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of IκB kinase 2, TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation

Article abstract of DOI:10.1124/jpet.104.074484

Demonstration that IκB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-κB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-α and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC₅₀ = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-α, IL-6, and IL-8 with an IC₅₀ = 170 to 320 nM. Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1β, IL-6, TNF-α, and interferon-γ, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.

Full text of DOI:10.1124/jpet.104.074484

0
022-3565/05/3121-373–381$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics
JPET 312:373–381, 2005
Vol. 312, No. 1
74484/1181064
Printed in U.S.A.
Attenuation of Murine Collagen-Induced Arthritis by a Novel,
Potent, Selective Small Molecule Inhibitor of I␬B Kinase 2,
TPCA-1 (2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-
thiophenecarboxamide), Occurs via Reduction of Proinflammatory
Cytokines and Antigen-Induced T Cell Proliferation
Patricia L. Podolin, James F. Callahan, Brian J. Bolognese, Yue H. Li, Karey Carlson,
T. Gregg Davis, Geoff W. Mellor, Christopher Evans, and Amy K. Roshak
Respiratory and Inflammation Center of Excellence for Drug Discovery (P.L.P., J.F.C., B.J.B., Y.H.L., K.C., T.G.D.), Systems
Research (G.W.M), Drug Metabolism and Pharmacokinetics (C.E.), and Project and Portfolio Management (A.K.R.),
GlaxoSmithKline, King of Prussia, Pennsylvania
Received July 20, 2004; accepted August 9, 2004
ABSTRACT
Demonstration that I␬B kinase 2 (IKK-2) plays a pivotal role in the mg/kg, i.p., b.i.d. were comparable to the effects of the antirheu-
nuclear factor-␬B-regulated production of proinflammatory mole- matic drug, etanercept, when administered prophylactically at 4
cules by stimuli such as tumor necrosis factor (TNF)-␣ and inter- mg/kg, i.p., every other day. Nuclear localization of p65, as well as
leukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in levels of IL-1␤, IL-6, TNF-␣, and interferon-␥, were significantly
the treatment of rheumatoid arthritis. In the present study, we reduced in the paw tissue of TPCA-1- and etanercept-treated
demonstrate that a novel, potent (IC50 ϭ 17.9 nM), and selective mice. In addition, administration of TPCA-1 in vivo resulted in
inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluoro- significantly decreased collagen-induced T cell proliferation ex
phenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysac- vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at
charide-induced human monocyte production of TNF-␣, IL-6, and 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA,
IL-8 with an IC50 ϭ 170 to 320 nM. Prophylactic administration of as did etanercept administration at 12.5 mg/kg, i.p., every other
TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose- day. These results suggest that reduction of proinflammatory me-
dependent reduction in the severity of murine collagen-induced diators and inhibition of antigen-induced T cell proliferation are
arthritis (CIA). The significantly reduced disease severity and delay mechanisms underlying the attenuation of CIA by the IKK-2 inhib-
of disease onset resulting from administration of TPCA-1 at 10 itor, TPCA-1.
Rheumatoid arthritis (RA) is a disease characterized by characterizes the disease (Kingsley and Panayi, 1997; Ha-
chronic inflammation of the joint, leading to progressive de- sunuma et al., 1998). Many of the proinflammatory mole-
struction of cartilage and bone. Migration of leukocytes to the cules associated with RA, including TNF-␣, IL-1, IL-6, IL-8,
synovium results in synovial hypertrophy and the production
of proinflammatory mediators by both synoviocytes and leu-
kocytes. These mediators are believed to be responsible for
the subsequent cartilage destruction and bone erosion that
IFN-␥, intercellular adhesion molecule-1, vascular cell adhe-
sion molecule-1, cyclooxygenase (COX)-2, inducible nitric-
oxide synthase, matrix metalloproteinase (MMP)-1, and
MMP-9, are regulated by the Rel/NF-␬B family of transcrip-
tion factors (Pahl, 1999). Thus, members of this signaling
pathway are potential targets for the development of novel
RA therapeutics.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.104.074484.
ABBREVIATIONS: RA, rheumatoid arthritis; IKK, I␬B kinase; NF-␬B, nuclear factor-␬B; TNF, tumor necrosis factor; IL, interleukin; TPCA-1,
-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide; LPS, lipopolysaccharide; CIA, collagen-induced arthritis; IFN, interferon;
2
COX, cyclooxygenase; MMP, matrix metalloproteinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMSO, di-
methyl sulfoxide; DMA, dimethylacetoacetamide; LNC, lymph node cells; ELISA, enzyme-linked immunosorbent assay; JNK3, c-Jun N-terminal
kinase 3; AUC, area under the curve; NSAIDs, nonsteroidal anti-inflammatory drugs; DMARDs, disease-modifying antirheumatic drugs; GST,
glutathione S-transferase; BSA, bovine serum albumin; DTT, dithiothreitol; HBSS, Hanks’ balanced salt solution; APC, antigen-presenting cells;
Th1, subset 1 of T helper cells.
373

3
74
Podolin et al.
In mammals, the Rel/NF-␬B family consists of p50/p105 2001). These studies suggest that inhibition of IKK-2 is a
NF-␬B1), p52/p100 (NF-␬B2), p65 (RelA), c-Rel (Rel), and viable approach to the development of a novel therapeutic for
(
RelB, which exist in the cell cytoplasm as homodimeric or RA.
heterodimeric complexes. The NF-␬B dimer (classically p50/
In the current study, we characterize a novel, potent, se-
p65) is retained in the cytoplasm in an inactive form through lective small molecule inhibitor of IKK-2, TPCA-1 (2-[(ami-
its association with I␬B (inhibitor of NF-␬B) proteins. A nocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxam-
variety of stimuli, including TNF-␣ and IL-1, are capable of ide). Prophylactic or therapeutic administration of TPCA-1
inducing NF-␬B activation. These agents initiate a signaling significantly reduced the severity of murine CIA. This mod-
cascade leading to the phosphorylation of two N-terminal ulation of disease was accompanied by decreased tissue lev-
serine residues in I␬B, which facilitates the ubiquitination els of the proinflammatory cytokines, IL-1␤, IL-6, TNF-␣,
and subsequent degradation of I␬B by the 26S proteosome. and IFN-␥, as well as reduced T cell proliferation in response
Once released from I␬B, NF-␬B translocates to the nucleus, to antigen, suggesting that these mechanisms underlie the
where it binds to a ␬B consensus sequence encoded within its inhibition of CIA by TPCA-1.
target gene and initiates transcription (Makarov, 2001; Tak
and Firestein, 2001).
Because the enzymes responsible for the ubiquitination of
phosphorylated I␬B are constitutively active, the phosphor-
ylation of I␬B is a critical regulatory step in I␬B degradation
and subsequent NF-␬B activation. This phosphorylation
event is catalyzed by the I␬B kinase (IKK) complex, which
Materials and Methods
Synthesis of TPCA-1. TPCA-1 was synthesized at GlaxoSmith-
Kline by the Respiratory and Inflammation Center of Excellence for
Drug Discovery (King of Prussia, PA). The 2-amino-5-(4-fluorophe-
nyl)-3-thiophenecarboxamide precursor was prepared by the reac-
consists of two enzymatically active kinases, IKK-1 (IKK␣) tion of 4-fluorophenylacetaldehyde, 2-cyanoacetamide, sulfur, and
and IKK-2 (IKK␤), and a regulatory subunit, NEMO (IKK␥) triethylamine in dimethylformamide at 0°C, allowing the reaction to
(
Karin, 1999). Divergent physiological roles for the two ki- warm to room temperature overnight (Goudie, 1976). Treatment of
2-amino-5-(4-fluorophenyl)-3-thiophenecarboxamide with chlorosul-
nases are suggested by targeted gene deletion studies in
which IKK-2-deficient mice, but not IKK-1-deficient mice,
exhibited significantly impaired TNF-␣- and IL-1-induced
NF-␬B activation and IL-6 production (Li et al., 1999;
Tanaka et al., 1999). These results suggest that IKK-2,
rather than IKK-1, plays a critical role in the NF-␬B-regu-
lated production of proinflammatory molecules induced by
fonylisocyanate in methylene chloride at 0°C followed by aqueous
hydrolysis and subsequent recrystallization from ethanol provided
TPCA-1.
IKK-2 Assay. Recombinant human IKK-2 (residues 1–756) was
expressed in baculovirus as an N-terminal GST-tagged fusion pro-
tein, and its activity was assessed using a time-resolved fluorescence
resonance energy transfer assay. In brief, IKK-2 (5 nM final) diluted
stimuli such as TNF-␣ and IL-1 and thus is a relevant target in assay buffer (50 mM HEPES, 10 mM MgCl , 1 mM CHAPS, pH
2
for the development of an anti-inflammatory therapeutic.
7.4, with 1 mM DTT and 0.01% w/v BSA) was added to wells con-
Much evidence indicates a pivotal role for NF-␬B in the taining various concentrations of compound or dimethyl sulfoxide
(
DMSO; Sigma-Aldrich, St. Louis, MO) vehicle (3% final). The reac-
etiology of RA. Nuclear localization of p50 and p65 has been
shown to be significantly increased in synovial tissue from
RA patients, compared with synovium from normal controls
tion was initiated by the addition of GST-I␬B␣ substrate (25 nM
final)/ATP (1 ␮M final), in a total volume of 30 ␮l. The reaction was
incubated for 30 min at room temperature, then terminated by the
addition of 15 ␮l of 50 mM EDTA. Detection reagent (15 ␮l) in buffer
(
Handel et al., 1995; Han et al., 1998). Similarly, it was
demonstrated that fibroblast-like synoviocytes from RA sy-
novium contain constitutively active NF-␬B and spontane-
ously produce large quantities of IL-6, unlike fibroblast-like
(
100 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% w/v BSA) con-
taining antiphosphoserine-I␬B␣-32/36 monoclonal antibody 12C2
Cell Signaling Technology Inc., Beverly, MA), labeled with W-1024
(
synoviocytes from osteoarthritis synovium (Miyazawa et al., europium chelate (PerkinElmer Wallac, Turku, Finland), and an
1
998). In addition, a number of antirheumatic agents, includ- allophycocyanin-labeled anti-GST antibody (ProZyme, San Leandro,
ing glucocorticoids, sulfasalazine, gold salts, leflunomide, CA) was added, and the reaction was further incubated for 60 min at
room temperature. The degree of phosphorylation of GST-I␬B␣ was
measured as a ratio of specific 665-nm energy transfer signal to
and aspirin, are inhibitors of NF-␬B activation (Makarov,
2
001; Tak and Firestein, 2001), which may explain, at least
reference europium 620-nm signal, using a Packard Discovery plate
reader (PerkinElmer Life Sciences, Buckinghamshire, UK).
Lipopolysaccharide-Induced Cytokine/Chemokine Produc-
tion by Human Monocytes. Human monocytes were isolated from
heparinized whole blood by positive selection using CD14ϩ mi-
crobeads (Miltenyi Biotec Inc., Auburn, CA). In brief, human whole
in part, their anti-inflammatory effects.
Consistent with the data from human synovial tissue, in-
creased NF-␬B binding activity has been demonstrated in the
synovium of mice and rats following the development of CIA,
adjuvant arthritis, and streptococcal cell wall-induced arthri-
tis (Tak and Firestein, 2001). Additional evidence implicat- blood was collected from healthy volunteers and diluted with an
2ϩ
2ϩ
ing NF-␬B in animal models of RA comes from the demon- equal volume of HBSS (without Ca or Mg ) containing 1 mM
stration that in vivo administration of reagents exerting EGTA. Diluted blood was layered on a Ficoll-Hypaque gradient (Am-
ersham Biosciences AB, Uppsala, Sweden) and centrifuged at 900g
inhibitory effects at various points along the NF-␬B signaling
for 30 min. The resulting interface was removed and washed twice
pathway resulted in a reduction of disease (Tak and Firest-
ein, 2001). The results of studies specifically targeting IKK-2
suggest that this enzyme plays a pivotal role in the NF-␬B-
mediated inflammatory response underlying arthritis. Intra-
articular injection of a wild-type IKK-2 gene into the joints of
normal rats resulted in paw swelling and synovial inflamma-
with HBSS containing 1 mM EGTA. Cell pellets were resuspended at
8
1
.25 ϫ 10 cells/ml in PBS containing 0.5% BSA and 2 mM EDTA.
7
Cells were labeled with 20 ␮l CD14ϩ microbeads per 1 ϫ 10 cells
and incubated for 15 min at 6°C with frequent mixing. Labeled cells
8
were washed once and resuspended at 1 ϫ 10 cells/ml in chilled PBS
containing 0.5% BSA and 2 mM EDTA. Cells were applied to mag-
tion, whereas transfer of a dominant-negative IKK-2 gene netized columns using an autoMACS (Miltenyi Biotec Inc.), and
decreased the severity of rat adjuvant arthritis (Tak et al., separation was performed according to the manufacturer’s instruc-

Attenuation of Murine CIA by an IKK-2 Inhibitor
375
tions. The resulting monocyte population was Ͼ90% pure as as-
sessed by differential staining.
Preparation of Tissue for Evaluation of NF-␬B Activation
and Cytokine/Chemokine Measurement. Paw tissue was
Purified monocytes were washed twice with PBS containing 0.5% weighed and placed in a volume of PBS equal to 1 g/2 ml. The tissue
6
BSA and 2 mM EDTA and resuspended to 1 ϫ 10 cells/ml in warm was homogenized by Polytron (model PT 10/35; Brinkmann Instru-
RPMI 1640 medium containing 10% FBS and L-glutamine. Mono- ments, Westbury, NY) and kept on ice during the processing. Sam-
5
cytes were plated at 5 ϫ 10 cells/well in 48-well tissue culture plates ples were transferred to 1.5-ml conical tubes, and the tissue was
and incubated for 2 h at 37°C. Adhered monocytes were washed once centrifuged at 16,300g for 3 min at 4°C. The supernatant was then
with warm RPMI 1640 medium containing 10% fetal calf serum and collected for cytokine/chemokine analysis. The pellet was used for
L-glutamine, and TPCA-1 in 100% DMSO was added (0.1% DMSO preparation of nuclear extracts following published methods (Dig-
final concentration). The monocytes were incubated with compound nam et al., 1983; Osborn et al., 1989) with some modifications. In
for 30 min at 37°C and then stimulated with 200 ng/ml LPS for 24 h.
Plates were centrifuged at 500g for 10 min, and supernatants were
removed and stored at Ϫ20°C until cytokine/chemokine evaluation
was performed.
brief, the tissue was resuspended in 200 ␮l of buffer A [10 mM
HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl , 0.1% (w/v) Nonidet
2
P-40, 0.5 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride]. The
suspension was incubated on ice for 10 min and then centrifuged at
Mice. Male DBA/1 OlaHsd mice were obtained from Harlan UK
Ltd. (Bicester, Oxon, UK) at 6 to 8 weeks of age. Mice were housed
one per cage and fed standard rodent chow and water ad libitum.
Induction and Assessment of CIA. On day 0, 10- to 12-week-old
male DBA/1 mice were immunized intradermally at the base of the
tail with a total of 100 ␮l of complete Freund’s adjuvant (Sigma-
Aldrich) containing 200 ␮g of bovine type II collagen (Elastin Product
Co., Owensville, MO) and 250 ␮g of Mycobacterium tuberculosis
H37Ra (Difco, Detroit, MI). On day 21, mice were boosted intrader-
mally with 100 ␮l of PBS containing 200 ␮g of bovine type II colla-
gen. In all studies, TPCA-1 was administered in a vehicle consisting
of 0.9% DMSO, 7% dimethylacetoacetamide (DMA) (Aldrich Chem-
ical Co., Milwaukee, WI), and 10% Cremophor El (Sigma-Aldrich).
Etanercept (Enbrel) (purchased commercially) was administered in
PBS. Where the effects of TPCA-1 and etanercept were compared,
both treatment groups as well as their relevant vehicle-treated con-
trol groups were included in the same study. For prophylactic stud-
ies, TPCA-1 in vehicle or vehicle alone was administered i.p., b.i.d.,
beginning on day 1. Etanercept in PBS or PBS alone was adminis-
tered i.p. every other day beginning on day 1. The incidence of
disease exhibited by both vehicle-treated control groups (PBS- and
DMSO/DMA/Cremophor-treated mice) was 100%. For therapeutic stud-
ies, administration of TPCA-1, etanercept, or their respective vehicles,
as described above, was initiated (day 1) once an animal exhibited a
clinical score of “1” or greater for 2 consecutive days. Mice were scored
daily for clinical symptoms of disease using a micrometer caliper to
measure paw thickness. Each paw was assigned a score ranging from 0
to 4, based on the following criteria: 0, asymptomatic (paw thickness ϭ
1
020g for 10 min at 4°C. The supernatant was removed and labeled
as the cytoplasmic extract. The remaining pellet was resuspended in
25 ␮l of buffer C [20 mM HEPES, pH 7.9, 0.42 M NaCl, 1.5 mM
1
MgCl2, 25% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM
phenylmethylsulfonyl fluoride] and gently mixed for 20 min on ice.
The samples were then centrifuged at 16,300g for 10 min at 4°C, and
the supernatant was removed and labeled as the nuclear extract.
The samples were stored at Ϫ80°C until analysis.
Cytokine, Chemokine, and p65 Assays. Human TNF-␣, IL-6,
and IL-8 levels were measured using enzyme-linked immunosorbent
assay (ELISA) kits purchased from BD Biosciences PharMingen
(
San Diego, CA). Paw tissue mediators were measured using mouse
IL-1␤, IL-6, TNF-␣, and IFN-␥ ELISA kits purchased from BioSource
International (Camarillo, CA) and a mouse KC ELISA kit purchased
from R&D Systems (Minneapolis, MN). Nuclear p65 levels were
determined using the TransAM NF-␬B p65 kit (Active Motif, Carls-
bad, CA). Assays were performed according to the manufacturers’
instructions.
Lymph Node and Spleen Cell Cultures. Preparation and
culture of LNC and splenocytes were performed under sterile
conditions. Inguinal lymph nodes and spleens were rinsed in
HBSS containing penicillin (100 units/ml)-streptomycin (100 ␮g/ml)
(
Invitrogen, Carlsbad, CA) and gentamicin (50 ␮g/ml) (Sigma-
Aldrich), (HBSSϩ), teased apart in 5 ml of HBSSϩ, and filtered
through 50 ␮M nylon mesh. Samples were centrifuged at 500g for 10
min at 4°C, and the resulting LNC pellets were resuspended in 2 ml
of HBSSϩ. Splenocyte pellets were resuspended in 9 ml of H O for
3
were centrifuged at 500g for 10 min at 4°C, and the resulting pellets
were resuspended in 2 ml of HBSSϩ. LNC and splenocytes were
counted, and cells from three mice were combined at a ratio of 80%
LNC/20% splenocytes (each group of three mice considered an n ϭ 1).
In a volume of 200 ␮l of RPMI 1640 medium containing 10% FBS,
2
0 s, followed by the addition of 1 ml of 10ϫ PBS. Splenocyte samples
1.8 to 1.9 mm and no swollen digits); 1, paw thickness ϭ 1.8 to 1.9 mm
and one or more swollen digits; 2, paw thickness ϭ 2.0 to 2.5 mm and
one or more swollen digits; 3, paw thickness ϭ 2.6 to 3.0 mm and one or
more swollen digits; 4, paw thickness ϭ 3.0ϩ mm and one or more
swollen digits.
In addition to the mice that were scored throughout the experi-
ment, at the designated time points during the course of disease,
mice were removed from the experiment and used to measure cyto-
kine/chemokine levels and p65 levels in the paw and the ex vivo
antigen recall response by lymph node cells (LNC)/splenocytes.
These mice were scored on a daily basis until their removal from the
study, and the data were integrated into the analysis using the log
rank test.
penicillin (100 units/ml)-streptomycin (100 ␮g/ml), and gentamicin
6
(
50 ␮g/ml), 2 ϫ 10 cells/ml were cultured in the presence or absence
of bovine type II collagen (100 ␮g/ml). Following incubation for 72 h
at 37°C in 5% CO , 1 ␮Ci [ H]thymidine was added to each well and
the cells cultured for an additional 24 h. Cultures were harvested
using a Packard Filtermate 196 (PerkinElmer Life Sciences, Boston,
MA), and radioactivity was quantified using a Packard TopCount
liquid scintillation counter.
3
2
Quantitative Analysis of Blood TPCA-1 Levels. Quantita-
tive analysis of TPCA-1 in blood samples was performed utilizing
Statistical Analysis. Statistical differences in p65 levels, cyto-
a high-performance liquid chromatography/dual mass spectrome- kine/chemokine concentrations, and T cell proliferation were deter-
try method. TPCA-1 was isolated from 25 ␮l of mouse blood mined using the two-tailed Student’s t test. Analysis of CIA clinical
(
diluted with 25 ␮l of water) by protein precipitation and quanti- score data was performed by calculating the area under the curve
fied with a Sciex API 4000 instrument with a turbo ionspray (AUC) for each animal within a treatment group and then using the
interface. Samples were injected onto a Luna C18 column (2 ϫ 50 log rank test, a nonparametric test that allows for censored obser-
mm, 3 ␮m packing) under isocratic conditions [60:40, acetoni- vations (i.e., for any animal removed before the completion of the
trile/10 mM ammonium formate (pH 3.0)] at a flow rate of 350
l/min. Negative-ion multiple reaction monitoring was used for as the partial AUC exhibited). Analysis of CIA disease onset data
the dual mass spectrometry detection of TPCA-1, with a lower was performed using the log rank test. Values of p Ͻ 0.05 were
limit of quantitation of 10.0 ng/ml. considered significant.
study, the animal’s complete AUC is considered to be at least as large
␮

3
76
Podolin et al.
Results
Characterization of a Potent, Selective, and ATP-
Competitive Inhibitor of IKK-2. TPCA-1 (Fig. 1) was iden-
tified as a potent and selective inhibitor of IKK-2. In a time-
resolved fluorescence resonance energy transfer assay,
TPCA-1 inhibited human IKK-2 activity with an IC50
9.5 Ϯ 1.7 nM (representative experiment shown in Fig. 2).
The results from 57 assays gave a mean pIC50 ϭ 7.74 Ϯ 0.18
IC50 ϭ 17.9 nM). In addition, the compound was demon-
ϭ
1
(
strated to be ATP-competitive (data not shown).
Determination of the activity of TPCA-1 against ten se-
lected kinases, as well as COX-1 and COX-2, showed the
compound to be Ͼ550-fold selective for IKK-2 versus ten of
these enzymes (Table 1). TPCA-1 exhibited IC50 values ϭ 400
nM and 3600 nM against IKK-1 and JNK3, respectively,
demonstrating it to be 22- and 200-fold selective, respec-
tively, versus these kinases.
TPCA-1 Inhibits LPS-Induced TNF-␣, IL-6, and IL-8
Production by Human Monocytes. To confirm the cell-
based activity of TPCA-1, human peripheral blood monocytes taining europium-labeled antiphosphoserine-I␬B␣ antibody and an allo-
were stimulated with LPS in the absence or presence of phycocyanin-labeled anti-GST antibody was added. The degree of
Fig. 2. TPCA-1 is a potent inhibitor of IKK-2 activity. C-terminal GST-
tagged recombinant human IKK-2 (residues 1–756) was added to wells
containing various concentrations of TPCA-1 or vehicle (DMSO), and the
reaction was initiated by the addition of GST-I␬B␣ substrate/ATP. Fol-
lowing termination of the reaction using EDTA, detection reagent con-
phosphorylation of GST-I␬B␣ was measured as a ratio of specific 665 nm
varying concentrations of the inhibitor, and cell supernatants
were assayed for cytokine/chemokine content. As shown in
Fig. 3, TPCA-1 inhibited the production of TNF-␣, IL-6, and control group Ϯ S.D. (n ϭ 4 wells) and are representative of 57 indepen-
IL-8 in a concentration-dependent manner, exhibiting IC50
energy transfer signal to reference europium 620 nm signal. Data are
expressed as the mean percentage of inhibition of the vehicle-treated
dent experiments.
values of 170, 290, and 320 nM, respectively. These results
suggest that TPCA-1 effectively blocks the NF-␬B signaling
pathway in intact cells.
TABLE 1
Selectivity profile of 2-͓(aminocarbonyl)amino͔-5-(4-fluorophenyl)-3-
thiophenecarboxamide
Kinase assays were performed as described previously (Bain et al., 2003).
Increased Nuclear Localization of NF-␬B p65 in CIA.
Based on the finding that IKK-2 plays a critical role in the
NF-␬B-mediated transduction of signals generated by the
RA/CIA-associated cytokines, TNF-␣ and IL-1 (Li et al., 1999;
Tanaka et al., 1999), it was of interest to characterize the
kinetics of NF-␬B activation during the development of CIA.
DBA/1 mice were immunized (day 0) and boosted (day 21)
with type II collagen, and tissue from all front and hind paws
were collected on days 22, 30, and 40. As shown in Fig. 4A,
clinical symptoms of CIA appeared at day 26 and increased
thereafter, reaching peak severity between days 37 and 40.
At day 22, nuclear extracts of paw tissue from collagen-
immunized/boosted mice exhibited levels of p65 binding com-
parable to levels from naive mice (Fig. 4B). In contrast, p65
activity was significantly increased by day 30 of CIA, com-
Enzyme
IC50
␮
M
IKK-1
p38␣
0.40
Ͼ16
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ50
Ͼ50
Ͼ10
3.6
p38␤
p38␥
p38␦
MAPKAPK2
MKK1
MAPK2
COX-1
COX-2
JNK1
JNK3
Prophylactic Administration of TPCA-1 Reduces the
pared to naive controls, and elevated further by day 40 (Fig. Severity and Delays the Onset of CIA. Given the associ-
B). This indicates that the kinetics of NF-␬B activation ation between NF-␬B activation and the development of the
correlate closely with the appearance and progression of the clinical symptoms of CIA (Fig. 4), the effect of in vivo admin-
4
clinical symptoms of disease.
istration of TPCA-1 on murine CIA was explored. To deter-
mine the effect of prophylactic treatment of TPCA-1, the
inhibitor was administered to collagen-immunized/boosted
DBA/1 mice at 3, 10, or 20 mg/kg, i.p., b.i.d., from days 1 to
48. Blood concentrations of the inhibitor were measured in
samples from three mice per dose, at 2 to 2.5 h following the
first daily administration of TPCA-1, on days 4, 10, 15, 24, 31,
39, and 46. Administration of TPCA-1 at 3, 10, or 20 mg/kg
resulted in blood levels ranging from 0.07 Ϯ 0.01 to 0.17 Ϯ
0
0
.06, 0.25 Ϯ 0.04 to 0.44 Ϯ 0.09, and 0.79 Ϯ 0.26 to 1.14 Ϯ
.13 ␮M, respectively.
As shown in Fig. 5, the severity of arthritis, represented by
mean clinical score, was reduced in a dose-dependent man-
ner, with administration of TPCA-1 at 20 or 10 mg/kg (p Ͻ
0.01 and p Ͻ 0.05, respectively), but not at 3 mg/kg, resulting
Fig. 1. The chemical structure of the IKK-2 inhibitor, 2-[(aminocarbon-
yl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide.

Attenuation of Murine CIA by an IKK-2 Inhibitor
377
Fig. 3. TPCA-1 inhibits LPS-induced TNF-␣, IL-6,
and IL-8 production by human monocytes. Human
peripheral blood monocytes were incubated for 30
min with various concentrations of TPCA-1 or ve-
hicle (DMSO) and then stimulated for 24 h with
LPS (200 ng/ml). Cell supernatants were assayed
for TNF-␣, IL-6, and IL-8 content. Each data point
represents n ϭ 3 wells and is expressed as the
mean percentage of the vehicle-treated control
group Ϯ S.E.M. The results are representative of
three independent experiments.
in a significantly decreased mean clinical score, compared to CIA are regulated by NF-␬B, we hypothesized that inhibition
that of vehicle-treated mice.
of expression of these mediators may be one mechanism by
In a separate study, the effects of 10 mg/kg TPCA-1 ad- which TPCA-1 attenuates CIA. As illustrated in Fig. 7, fol-
ministered i.p., b.i.d. from days 1 to 47, were compared to lowing 38 days of prophylactic administration of TPCA-1 (10
those of etanercept (recombinant human TNF receptor p75 mg/kg, i.p., b.i.d.) to collagen-immunized/boosted DBA/1
Fc fusion protein; Enbrel), administered at 4 mg/kg, i.p., mice, paw tissue levels of IL-1␤, IL-6, TNF-␣, and IFN-␥ were
every other day from days 1 to 47. Previous studies estab- significantly inhibited compared to vehicle-treated control
lished that when administered prophylactically, etanercept mice. A trend toward reduction in the levels of KC, a murine
exhibits maximal efficacy in our CIA model under these chemokine with sequence and functional homology to the
conditions (data not shown). Similar to the results described human IL-8 family (Bozic et al., 1994), was observed in
above, administration of TPCA-1 at 10 mg/kg resulted in a TPCA-1-treated mice, although this decrease did not reach
significantly reduced mean clinical score compared to that of statistical significance. Similar to the IKK-2 inhibitor, ad-
vehicle-treated mice (p ϭ 0.001) (Fig. 6A). In addition, the ministration of etanercept (4 mg/kg, i.p., every other day)
time to onset of disease was significantly delayed as a result resulted in significantly decreased paw tissue levels of IL-1␤,
of treatment with TPCA-1 (p Ͻ 0.001) (Fig. 6B). Etanercept IL-6, TNF-␣, and IFN-␥, as well as significantly reduced KC
exhibited effects on disease comparable to those of TPCA-1 at levels (Fig. 7).
1
0
0
0 mg/kg, significantly reducing mean clinical score (p Ͻ
.001) (Fig. 6C) and delaying time to onset of disease (p Ͻ Ex Vivo Antigen-Induced T Cell Proliferation in CIA.
.001) (Fig. 6D), compared to control animals. Previous studies have demonstrated that inhibition of the
Prophylactic Administration of TPCA-1 Reduces Nu- NF-␬B signaling pathway in T cells via the T cell-specific
Prophylactic Administration of TPCA-1 Attenuates
clear Localization of p65 in CIA. To confirm the in vivo expression of an I␬B␣ transgene (Seetharaman et al., 1999)
inhibition of NF-␬B activation by TPCA-1, p65 levels were or the administration of a T cell-specific inhibitor of NF-␬B
measured in the nuclear extracts of paw tissue from collagen- (Gerlag et al., 2000) results in significant inhibition of mu-
immunized/boosted DBA/1 mice following 38 days of prophy- rine CIA. To determine whether the TPCA-1-induced reduc-
lactic administration of TPCA-1 (10 mg/kg, i.p., b.i.d.) or of tion in the severity of murine CIA and decrease in tissue
vehicle. As shown in Fig. 7, p65 nuclear localization was proinflammatory mediators are accompanied by an inhibi-
significantly inhibited in TPCA-1-treated mice compared to tion in antigen-induced T cell proliferation, LNC and spleno-
relevant vehicle-treated control mice. Mice receiving etaner- cytes were collected from collagen-immunized/boosted DBA/1
cept (4 mg/kg, i.p., every other day) also exhibited signifi- mice following 38 days of prophylactic administration of
cantly decreased levels of p65 binding. These results suggest TPCA-1 (10 mg/kg, i.p., b.i.d.) or of vehicle. Cells cultured in
that inhibition of NF-␬B activation is a likely mechanism the absence of the immunizing antigen, collagen, exhibited
through which TPCA-1, as well as etanercept, reduces the basal levels of proliferation that did not differ significantly
severity and delays the onset of CIA.
between the vehicle-treated and TPCA-1-treated groups (Fig.
Prophylactic Administration of TPCA-1 Reduces 8). In contrast, cells from vehicle-treated mice cultured in the
Proinflammatory Cytokine/Chemokine Levels in CIA. presence of collagen exhibited a robust antigen recall re-
Based on the fact that the genes encoding many of the proin- sponse, which was significantly reduced in cells derived from
flammatory cytokines/chemokines associated with RA and TPCA-1-treated mice (Fig. 8). These results indicate that in
Fig. 4. NF-␬B activation correlates with the de-
velopment of the clinical symptoms of CIA. A,
naive DBA/1 mice (f) (initial n ϭ 15; final n ϭ 6)
and DBA/1 mice immunized on day
0 and
boosted on day 21 with type II collagen (▫) (inital
n ϭ 15; final n ϭ 6) were scored for the clinical
symptoms of disease. B, on days 22, 30, and 40,
nuclear extracts of front and hind paw tissue
from naive mice (f) (4 paws/animal pooled, n ϭ 3
animals) and collagen-immunized/boosted mice
(
▫) (4 paws/animal pooled, n ϭ 3 animals) were
assayed for p65 binding. Data are expressed as
the mean Ϯ S.E.M. ء, p Ͻ 0.05; ءء, p Ͻ 0.01; and
ءءء, p Ͻ 0.001.

3
78
Podolin et al.
in blood levels ranging from 0.13 Ϯ 0.02 to 0.26 Ϯ 0.09,
.42 Ϯ 0.15 to 1.05 Ϯ 0.30, and 0.68 Ϯ 0.25 to 2.50 Ϯ 0.78 ␮M,
0
respectively.
As shown in Fig. 9, therapeutic administration of TCPA-1
at 20 mg/kg (p Ͻ 0.01) (Fig. 9A), but not 10 (Fig. 9B) or 3 (Fig.
9C) mg/kg, significantly reduced the mean clinical score com-
pared with that of vehicle-treated animals. Therapeutic ad-
ministration of etanercept (12.5 mg/kg, i.p., every other day)
also resulted in significant reduction of disease severity com-
pared to that exhibited by vehicle-treated control mice (p Ͻ
0.001) (Fig. 9D).
Discussion
In this report, we identify a novel inhibitor of IKK-2,
TPCA-1, and demonstrate its potency, selectivity, and cell-
based activity. TPCA-1 originated from the optimization of
an aminothiophene hit from high throughput screening of
our compound collection against IKK-2 homodimer. Two
Fig. 5. Prophylactic administration of TPCA-1 reduces the severity of
CIA in a dose-dependent manner. DBA/1 mice immunized on day 0 and other groups independently developed inhibitors in similar
boosted on day 21 with type II collagen were administered vehicle
aminothiophene series (Kishore et al., 2003; Baxter et al.,
(
DMSO/DMA/Cremophor) (F), or TPCA-1 at 20 (‚), 10 (f), or 3 (E) mg/kg,
2004). Subsequent structure-activity studies resulted in a
i.p., b.i.d., from days 1 to 48 and scored for the clinical symptoms of
series of 2-ureidothiophenes, an example of which is TPCA-1.
The substitution of the 2-amino group in the initial hit series
with a primary urea significantly increased the IKK-2 inhib-
disease. Data are expressed as the mean Ϯ S.E.M. (inital n ϭ 15; final n ϭ
6
).
vivo administration of TPCA-1 attenuates ex vivo antigen- itory activity. These studies also demonstrated that the
induced T cell proliferation in murine CIA.
Therapeutic Administration of TPCA-1 Reduces the
3-carboxamide significantly contributes to IKK-2 inhibition.
The identification of IKK-2 as the kinase primarily respon-
Severity of CIA. To determine whether TPCA-1 is capable sible for the NF-␬B-regulated production of proinflammatory
of modulating the severity of CIA when delivered therapeu- molecules induced by TNF-␣ and IL-1 suggested that TPCA-1
tically, administration of TPCA-1 (3, 10, or 20 mg/kg, i.p., may be beneficial in the treatment of inflammatory diseases
b.i.d.) or of vehicle was initiated following the onset of clinical such as RA, leading us to test its activity in CIA. TPCA-1
symptoms in collagen-immunized/boosted DBA/1 mice. Blood reduced the severity and delayed the onset of murine CIA. In
concentrations of TPCA-1 were measured in samples from our studies, the significantly reduced severity of murine CIA
three mice per dose at 2 to 2.5 h following the first daily resulting from prophylactic or therapeutic administration of
administration of inhibitor (day 1) on days 4, 8, 15, 21, and the IKK-2 inhibitor is consistent with the results of a recent
2
4. Administration of TPCA-1 at 3, 10, or 20 mg/kg resulted study demonstrating significant reduction of murine CIA
Fig. 6. Prophylactic administration of TPCA-1 reduces
the severity and delays the onset of CIA in a manner
comparable to a maximally effective dose of etanercept.
A and B, DBA/1 mice immunized on day 0 and boosted
on day 21 with type II collagen were administered
vehicle (DMSO/DMA/Cremophor) (F) or TPCA-1 at 10
(
f) mg/kg, i.p., b.i.d., from days 1 to 47 and scored for
the clinical symptoms of disease. C and D, DBA/1 mice,
immunized on day 0 and boosted on day 21 with type II
collagen, were administered vehicle (PBS) (ꢀ) or etan-
ercept at 4 (ꢁ) mg/kg, i.p., every other day from days 1
to 47 and scored for the clinical symptoms of disease.
For A and C, data are expressed as the mean Ϯ S.E.M.
(initial n ϭ 30, final n ϭ 15). For B and D, data are
expressed as the percentage of mice remaining free of
disease (i.e., not exhibiting clinical symptoms for 2
consecutive days).

Attenuation of Murine CIA by an IKK-2 Inhibitor
379
comparable to those of the antirheumatic drug, etanercept.
Collectively, these results suggest that potent, selective,
small molecule inhibitors of IKK-2 offer a promising ap-
proach to the development of novel therapeutics for RA.
The NF-␬B family of transcription factors regulates the
expression of a number of proinflammatory cytokines/chemo-
kines associated with RA and CIA, including TNF-␣, IL-1␤,
IL-6, IL-8/KC, and IFN-␥ (Pahl, 1999). The ability of TPCA-1
to inhibit the LPS-induced production of TNF-␣, IL-6, and
IL-8 by human monocytes in vitro is consistent with the
compound’s potent inhibitory activity against recombinant
human IKK-2 and demonstrates its cell-based activity. This
modulation of proinflammatory mediator expression by
TPCA-1 was observed in vivo as well, with TPCA-1-treated
animals exhibiting significantly reduced paw tissue levels of
IL-1␤, IL-6, TNF-␣, and IFN-␥, compared to vehicle-treated
control animals. This observation suggests that reduction of
Fig. 7. Prophylactic administration of TPCA-1 or etanercept reduces p65 proinflammatory mediators contributes to the TPCA-1-in-
nuclear translocation and proinflammatory cytokine/chemokine accumu-
duced attenuation of CIA. In addition to exerting a direct
lation in CIA. DBA/1 mice, immunized on day 0 and boosted on day 21
inhibitory effect on the transcriptional regulation of these
with type II collagen, were administered TPCA-1 in DMSO/DMA/Cremo-
phor vehicle at 10 mg/kg, i.p., b.i.d., from days 1 to 37 (f); alternatively, proinflammatory mediators, TPCA-1 may reduce cytokine
collagen-immunized/boosted mice received etanercept in PBS vehicle at 4 expression indirectly through attenuation of proinflamma-
mg/kg, i.p., every other day from days 1 to 37 (▫). On day 38, nuclear
tory cytokine cascades. In this regard, it has been demon-
extracts of front and hind paw tissue were assayed for p65 binding, and
strated that IL-1␤ stimulates the production of IL-1␤, TNF-␣,
supernatants from tissue homogenates were assayed for IL-1␤, IL-6,
TNF-␣, IFN-␥, and KC. For each treatment group, n ϭ 5, where n ϭ 1 is IL-6, and IFN-␥ (Dinarello, 1996), whereas TNF-␣ induces
defined as paws from three animals pooled. Data are expressed as the
IL-1␤, TNF-␣, and IL-6 expression (Aggarwal et al., 2001). In
addition, IFN-␥ has been shown to induce the production of
IL-1␤ and TNF-␣ (Collart et al., 1986).
mean percentage of inhibition of the relevant vehicle-treated control
group Ϯ S.E.M. ء, p Ͻ 0.05; ءء, p Ͻ 0.01; and ءءء, p Ͻ 0.001 compared to
the relevant vehicle-treated control group.
It is of interest that administration of etanercept resulted
in significantly reduced paw tissue levels of IL-1␤, IL-6,
TNF-␣, IFN-␥, and KC. The down-regulation of proinflam-
matory cytokine cascades is believed to be an important
mechanism underlying the clinical benefits of anti-TNF-␣
therapy in RA. It has been demonstrated that treatment of
RA patients with etanercept results in significantly reduced
plasma IL-6 levels (Feldmann et al., 1998); however, the
studies described herein are, to our knowledge, the first
comprehensive report of the effects of etanercept, as well as
those of an IKK-2 inhibitor, on RA/CIA-associated proinflam-
matory mediators in vivo.
following prophylactic or therapeutic administration of a se-
lective quinoxaline IKK-2 inhibitor, BMS-345541 (McIntyre
et al., 2003). We extend these observations, defining two
mechanisms by which this inhibition of disease occurs, dem-
onstrating that the TPCA-1-induced reduction of p65 nuclear
translocation in vivo was accompanied by decreased protein
levels of local NF-␬B-regulated proinflammatory mediators,
as well as by inhibition of collagen-induced T cell prolifera-
tion. In addition, the effects of TPCA-1 were shown to be
The fact that both TPCA-1 and etanercept significantly
reduced IL-1␤, IL-6, TNF-␣, and IFN-␥ levels in the paw
tissue suggests that these therapeutic agents may modulate
the same intracellular signaling pathway. This is supported
by the observed decreases in p65 nuclear localization follow-
ing in vivo administration of TPCA-1 and etanercept. While
TPCA-1 exerts its effect on the NF-␬B signaling pathway via
attenuation of IKK-2-mediated phosphorylation of I␬B, etan-
ercept is presumably acting at a site more distal to the
NF-␬B/I␬B complex, preventing TNF-␣-induced signaling at
the cell surface. Consistent with this hypothesis are reports
of decreased DNA binding activity of NF-␬B following in vitro
or in vivo treatment with anti-TNF-␣ antibodies (Pimentel-
Mui n˜ os et al., 1994; De Plaen et al., 2000).
Fig. 8. Prophylactic administration of TPCA-1 reduces ex vivo antigen-
induced T cell proliferation in CIA. On day 38, inguinal lymph nodes and
spleens were collected from collagen-immunized/boosted DBA/1 mice
treated from days 1 to 37 with vehicle (DMSO/DMA/Cremophor) (f) (n ϭ
It is widely recognized that CIA is a T cell-dependent,
antigen-specific disease (Myers et al., 1997). It has been
proposed that auto-antigen-specific T cells play a pivotal role
9
) or TPCA-1 at 10 mg/kg (▫) (n ϭ 9), i.p., b.i.d. Following preparation of
single cell suspensions, LNC and splenocytes from three mice were com-
bined at a ratio of 80% LNC/20% splenocytes, giving an n ϭ 3 pooled
groups. LNC/splenocytes were incubated in the presence or absence of in the etiology of RA as well (Panayi et al., 1992; Weyand and
type II collagen (100 ␮g/ml) for 96 h with the final 24 h of incubation Goronzy, 1997). In the studies described herein, the inhibi-
3
occurring in the presence of [ H]thymidine, and the incorporated radio-
tion of ex vivo collagen-induced T cell proliferation exhibited
activity was quantified. Data are expressed as the mean Ϯ S.E.M. ء, p Ͻ
by TPCA-1-treated mice, compared to vehicle-treated mice,
suggests that inhibition of antigen-induced T cell prolifera-
0
.05; ءء, p Ͻ 0.01; and ءءء, p Ͻ 0.001 compared with vehicle-treated
control mice.

3
80
Podolin et al.
Fig. 9. Therapeutic administration of TPCA-1 or
etanercept reduces the severity of CIA. Follow-
ing exhibition of clinical symptoms for 2 consec-
utive days (days Ϫ1 and 0), collagen-immunized/
boosted DBA/1 mice were administered vehicle
or drug from days 1 to 25 and were scored for the
clinical symptoms of disease. A–C, mice received
vehicle (DMSO/DMA/Cremophor) (F) or TPCA-1
at 20 (‚), 10 (f), or 3 (E) mg/kg, i.p., b.i.d. (n ϭ
3
0) D, mice received vehicle (PBS) (ꢀ) or etan-
ercept at 12.5 (ꢁ) mg/kg, i.p., every other day
n ϭ 18). Data are expressed as the mean Ϯ
S.E.M.
(
tion is a mechanism underlying the beneficial effects of the significant advances have been made in RA therapy. The
IKK-2 inhibitor in CIA. It has been demonstrated that the development of NSAIDs that selectively inhibit COX-2 and
decreased severity and incidence of murine CIA resulting spare COX-1 has reduced the gastrointestinal toxicity asso-
from inactivation of NF-␬B signaling, through the expression ciated with nonselective NSAIDs. In addition, the recognition
of an I␬B␣ transgene, were accompanied by significantly of the beneficial effects of aggressive DMARD therapy early
reduced ex vivo collagen-induced T cell proliferation and
IFN-␥ production. In this study, expression of the I␬B␣ trans-
gene was restricted to T cells, suggesting that NF-␬B signal-
ing in T cells is critical to antigen-induced T cell proliferation
in the course of the disease and the initiation of combination
DMARD therapy, such as using methotrexate with cyclospor-
ine or with sulfasalazine and hydroxychloroquine, has en-
abled DMARDs to be used to their maximum therapeutic
potential (Saravanan and Hamilton, 2002; Smolen and
Steiner, 2003). In the last 5 years, several new DMARDs
have been approved for clinical use, including the nonpeptide
immunomodulator leflunomide, the anti-TNF-␣ therapies et-
anercept, infliximab, and adalimumab, and the IL-1 receptor
antagonist anakinra. Although TNF-␣ blockade, the aggres-
sive use of DMARDs early in disease, and the initiation of
combination DMARD therapy have been major advance-
ments in the treatment of RA, it should be noted that all
available therapies to date are associated with issues of effi-
cacy and/or toxicity. Hence, a number of new targets, includ-
ing proinflammatory cytokines/chemokines, adhesion mole-
cules, and MMPs, are being pursued for the development of
RA therapeutics (Smolen and Steiner, 2003). The fact that
many of these molecules are regulated by the NF-␬B family
of transcription factors makes components of this signaling
pathway, such as IKK-2, intriguing potential targets.
(
Seetharaman et al., 1999). The results of other studies sup-
port this finding, demonstrating that T cell-intrinsic NF-␬B
activation is required for antigen-induced T cell proliferation
and the generation of a Th1 response (Artis et al., 2003; Corn
et al., 2003). It is possible that inhibition of NF-␬B signaling
in antigen-presenting cells (APC) instead of, or in addition to,
inhibition of NF-␬B induction in T cells is responsible for the
observed effects of TPCA-1 on collagen-induced T cell prolif-
eration in murine CIA. Activation of the NF-␬B signaling
pathway has been shown to play a pivotal role in the antigen-
presenting capacity of dendritic cells (Boffa et al., 2003; Ma et
al., 2003; Yoshimura et al., 2003). More specifically, IKK-2
has recently been shown to be essential for dendritic cell
antigen presentation to T cells (Andreakos et al., 2003). It is
of interest that reduced susceptibility of c-Rel-deficient mice
to the Th1-mediated disease, experimental autoimmune en-
cephalomyelitis, was found to be a result of both defective T
cell differentiation into Th1 cells and decreased IL-12 pro-
duction by APC (Hilliard et al., 2002). Whether IKK-2 inhib-
itors act in a similar fashion, abrogating both T cell and APC
function during antigen-induced T cell proliferation and dif-
ferentiation, remains to be determined.
In summary, the studies presented in this paper demon-
strate that a novel, potent, selective small molecule inhibitor
of IKK-2, TPCA-1, significantly reduces the severity of mu-
rine CIA following prophylactic or therapeutic administra-
tion, exhibiting effects comparable to those of the antirheu-
Currently, the two principal approaches to RA therapy
consist of nonsteroidal anti-inflammatory drugs (NSAIDs), matic drug etanercept. Inhibition of proinflammatory
which interfere with prostaglandin production through the mediator accumulation and inhibition of collagen-induced T
inhibition of COX enzymes, providing symptomatic relief, cell proliferation are likely mechanisms underlying modula-
and disease-modifying antirheumatic drugs (DMARDs), tion of CIA by the inhibitor. These results suggest that inhi-
which inhibit both the inflammatory and cartilage/bone de- bition of IKK-2 may be a beneficial approach in the treatment
structive processes of RA. Over the last 10 to 15 years, of human disease.

Attenuation of Murine CIA by an IKK-2 Inhibitor
381
Acknowledgments
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Kishore N, Sommers C, Mathialagan S, Guzova J, Yao M, Hauser S, Huynh K, Bonar
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J Biol Chem 278:32861–32871.
We thank John Peterson for guidance in statistical analysis of the
data and Bob Blade for large scale synthesis of TPCA-1.
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Address correspondence to: Dr. Patricia Podolin, GlaxoSmithKline, Mail
Code UW2532, 709 Swedeland Road, King of Prussia, PA 19406. E-mail:
patty_podolin@gsk.com
Hilliard BA, Mason N, Xu L, Sun J, Lamhamedi-Cherradi S-E, Liou H-C, Hunter C,