DBM1285 Suppresses Tumor Necrosis Factor Production by 3

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DBM1285 Suppresses Tumor Necrosis Factor Production byBlocking p38 Mitogen-Activated Protein Kinase/Mitogen-ActivatedProtein Kinase-Activated Protein Kinase 2 Signaling Pathway□S

Jong Soon Kang, Hwan Mook Kim, In Young Choi, Sang-Bae Han, Yeo Dae Yoon,Hyunju Lee, Ki Hwan Park, Ig Jun Cho, Chang Woo Lee, Kiho Lee, Ki Hoon Lee,and Song-Kyu Park

Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Chungbuk, Republic of Korea (J.S.K., H.M.K., Y.D.Y., H.L., K.H.P., I.J.C., C.W.L., K.L., K.H.L., S.-K.P.); Dongbu HiTek Co., Ltd., Daeduck ScienceTown, Daejeon, Republic of Korea (I.Y.C.); and College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk,Republic of Korea (S.-B.H.)

Received September 15, 2009; accepted April 28, 2010

ABSTRACT

Tumornecrosis factor (TNF- ) is a major inflammatory cytokine thatplays an important role in the development of various inflammatorydiseases. TNF- has been considered as a potential therapeutictarget for the treatment of chronic inflammatory diseases, includingrheumatoid arthritis and inflammatory bowel disease. In this study,we report that cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}amine (DBM1285) is a novel inhibitor ofTNF- production. DBM1285 concentration-dependently inhibitedlipopolysaccharide (LPS)-induced TNF- secretion in various cells ofmacrophage/monocyte lineage, including mousebone marrowmac-rophages, THP-1 cells, andRAW 264.7 cells. However, LPS-induced

mRNA expression of TNF-

was not affected by DBM1285 in thesecells. Further studies demonstrated that the inhibitory effect ofDBM1285 on TNF- production might be mediated by post-tran-

scriptional regulation through the modulation of the p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2(MK2) signaling pathway. We also confirmed that DBM1285 directlyinhibits p38 MAPK enzymatic activity. In vivo administration ofDBM1285 inhibited LPS-induced increase in the plasma level ofTNF- in mice. Whole-blood in vivo target inhibition assay also re-vealed that DBM1285 attenuates p38 MAPK activity after oral ad-ministration in mice. Moreover, DBM1285 suppressed zymosan-in-duced inflammation and adjuvant-induced arthritis in murine models.Collectively, these results suggest that DBM1285 inhibits TNF- pro-duction, at least in part, by blocking the p38 MAPK/MK2 pathway.

Furthermore, in vivo results suggest that DBM1285 might be a pos-sible therapeutic candidate for the treatment of TNF--relatedchronic inflammatory diseases.

Tumor necrosis factor (TNF-) is a multifunctional proin-flammatory cytokine produced mainly by cells of the mono-cyte/macrophage lineage, which plays an important role inchronic inflammatory diseases, such as rheumatoid arthritis(RA) and inflammatory bowel disease, by amplifying an in-

flammatory signal in multiple pathways (Montecucco andMach, 2009). Human TNF- is expressed as a 26-kDa proteinon the plasma membrane, cleaved by TNF--converting en-zyme (TACE), and released as a mature 17-kDa protein (Pal-ladino et al., 2003). The expression of the TNF- gene iscontrolled at both the transcriptional and post-transcrip-

tional levels, and a variety of signaling pathways are involved in these processes. Mitogen-activated protein kinases(MAPKs) are a family of serine/threonine kinases involved ina variety of cellular processes, such as inflammation, cellgrowth/differentiation, and cell survival/death (Chang andKarin, 2001). Three kinds of MAPKs, including extracellularsignal-regulated kinase, c-Jun N-terminal kinase, and p38MAPK, have been identified so far. Among these, p38 MAPK

This work was supported in part by grants from the Korea Research Insti-tute of Bioscience and Biotechnology Research Initiative Program and KoreaHealth 21 Research and Development Project A062254 of the Ministry of Health and Welfare, Republic of Korea.

J.S.K., H.M.K. and I.Y.C. contributed equally to this work. Article, publication date, and citation information can be found at

http://jpet.aspetjournals.org.doi:10.1124/jpet.109.161687.□S The online version of this article (available at http://jpet.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: TNF-, tumor necrosis factor ; DBM1285, cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}

amine; BIRB796, 1–5-tert -butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalene-1-yl]urea; LPS, lipopolysaccharide; MAPK, mito-

gen-activated protein kinase; MK2, mitogen-activated protein kinase-activated protein kinase 2; RA, rheumatoid arthritis; NSAID, nonsteroidal anti-

inflammatory drug; DMARD, disease-modifying antirheumatic drug; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-

polymerase chain reaction; DMSO, dimethyl sulfoxide; TACE, TNF--converting enzyme; BM, bone marrow; ARE, AU-rich element.

0022-3565/10/3342-657–664$20.00THE JOURNAL OF PHARMACOLOGY AND E XPERIMENTAL THERAPEUTICS Vol. 334, No. 2Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 161687/3603439JPET 334:657–664, 2010 Printed in U.S.A.

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DC1.htmlhttp://jpet.aspetjournals.org/content/suppl/2010/04/28/jpet.109.161687.Supplemental Material can be found at:



is a key molecule involved in the induction of inflammatorygenes such as TNF-, inducible nitric oxide synthase, cyclo-oxygenase-2, and various adhesion molecules (Saklatvala,2004; Schindler et al., 2007). The p38 MAPK is comprised of four different isoforms (p38, p38, p38, and p38); p38 isthe main isoform involved in the regulation of TNF- produc-tion (Schindler et al., 2007).

Among TNF--related diseases, RA is one of the most prev-

alent systemic autoimmune disorders (Smolen and Steiner,2003). It affects approximately 1% of the population and ismore frequent in women than men (Smolen and Steiner,2003; Brennan and McInnes, 2008). Therapeutic approachesfor RA are divided into two main categories: treatment withnonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs). NSAIDs affectonly a small portion of the inflammatory cascade, such asprostaglandin generation by inhibition of cyclooxygenases,and have not been a major treatment option for RA patients.Recently, it has been suggested that aggressive treatmentwith DMARDs, rather than NSAIDs, is required for betterlong-term life quality for RA patients by attenuating disease

progression and retaining joint function. Small-moleculeDMARDs, such as methotrexate and sulfasalazine, havebeen used for a long time clinically. Although DMARDs re-main the most common therapy for RA, they provide onlypartial clinical benefits and cause significant toxicity (Bren-nan and McInnes, 2008). However, the recent development of biological DMARDs targeting TNF- and interleukin-1 hasrevolutionized RA treatment (Segal et al., 2008).

Although biological DMARDs have proven to be clinicallysuccessful, they have innate limitations that include the re-quirement for injection and reduced efficacy after repeatedtreatment caused by antibody formation against the applieddrug (Radstake et al., 2009). Therefore, the development of new orally available small-molecule DMARDs with enhancedefficacy and reduced toxicity is continuously required, andnumerous small-molecule DMARDs targeted for RA treat-ment are currently under development (Pettus and Wurz,2008; Stanczyk et al., 2008). To develop a small-moleculeinhibitor of TNF- production we screened chemical librariesdeveloped by Dongbu HiTek Co., Ltd. Among the chemicalsexamined, DBM1285 [cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}amine; Fig. 1] dis-played the most potent inhibition of TNF- production in vitro and in vivo. In this study, we examined the effect of DBM1285 on lipopolysaccharide (LPS)-induced TNF- pro-duction in vitro and in vivo and arthritis progression in ananimal model of RA. We also elucidated the mechanism re-

sponsible for DBM1285-mediated inhibition of TNF- pro-

duction. Our results indicate DBM1285 is a potential thera-peutic candidate for the treatment of RA.

Materials and Methods

Chemicals, Cells, and Animals. All reagents were purchasedfrom Sigma-Aldrich (St. Louis, MO) unless otherwise noted.DBM1285 and 1-5-tert-butyl-2-p-tolyl-2 H -pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalene-1-yl]urea (BIRB796) were

synthesized and supplied by Dongbu HiTek Co., Ltd. DBM1285 ischaracterized as a small lipophilic compound with molecularweight of 395.5 and cLogP of 3.68 (calculated by ChemBioDrawUltra 12.0; CambridgeSoft Corporation, Cambridge, MA).DBM1285 and BIRB796 were dissolved in dimethyl sulfoxide(DMSO) and freshly diluted in culture medium for in vitro exper-iments (final DMSO concentration of 0.1%). For in vivo experi-ments, DBM1285 was dissolved in phosphate-buffered saline sup-plemented with 5% Tween 80. Six- to 8-week-old female C57BL/6mice and Sprague-Dawley rats were purchased from Koatech(Pyungtaek, Gyeonggi, Korea) and cared for as described previ-ously (Kang et al., 2009). All animals were allowed to acclimate tothe local environment for 1 week. All animal experiments wereapproved by the Institutional Animal Care and Use Committee at

the Korea Research Institute of Bioscience and Biotechnology.THP-1 cells (TIB-202; American Type Culture Collection, Manas-sas, VA) and RAW 264.7 cells (TIB-71; American Type CultureCollection) were grown in RPMI medium (Invitrogen, Carlsbad,CA) and Dulbecco’s modified Eagle’s medium (Invitrogen) supple-mented with 10% fetal bovine serum, 100 U/ml penicillin, and 100g/ml streptomycin. Bone marrow (BM) macrophages were gen-erated from BM cells obtained from female C57BL/6 mice. In brief,BM cells were flushed out from femurs and tibias. After lysing redblood cells, whole BM cells (2 105 cells/ml) were cultured in100-mm2 culture dishes in 10 ml/dish of RPMI complete mediumcontaining 10 ng/ml macrophage colony-stimulating factor. Onculture day 3, another 10 ml of fresh RPMI complete mediumcontaining 10 ng/ml macrophage colony-stimulating factor wasadded, and half of the medium was changed on day 6. On day 8,

cells were harvested and used as BM macrophages. In general,90% of cells recovered from these cultures were CD11b, and95% of cells were F4/80. Cell viability was determined withCell Proliferation Kit II (Roche Applied Science, Mannheim, Ger-many) according to the manufacturer’s instructions. In brief, thelabeling mixture was prepared by mixing 50 volumes of 1 mg/mlsodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate with 1 volume of 0.383 mg/ml N-methyldibenzopyrazine methyl sulfate. This mix-ture was added to the cultures and incubated for 2 h at 37°C.

Absorbance was measur ed at 490 nm with a refer ence wavel engthat 650 nm.

Enzyme-Linked Immunosorbent Assay. BM macrophages,THP-1 cells, and RAW 264.7 cells were stimulated with LPS (1g/ml) for 6 h in the presence or absence of 0.001, 0.01, 0.1 or 1 MDBM1285 or BIRB796. Culture supernatants were recovered, andthe concentration of TNF- was determined by using the mouseTNF- DuoSet (for BM macrophages and RAW 264.7 cells; R&DSystems, Minneapolis, MN) or the human TNF- DuoSet (for THP-1cells; R&D Systems) according to the manufacturer’s instructions.

Real-Time Reverse Transcription-Polymerase Chain Reac-

tion. The expression of mRNA transcripts was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) asdescribed previously (Han et al., 2007). Total RNAs were isolated byusing TRIzol reagent (Invitrogen) as described previously (Chom-czynski and Mackey, 1995). Equal amounts of RNA were reverse-transcribed into cDNA by using oligo(dT)15 primers. The primersequences used were: mouse TNF-, sense 5-CCT GTA GCC CACGTC GTA GC-3, antisense 5-TTG ACC TCA GCG CTG AGT TG-3;

human TNF-, sense 5-GAG TGA CAA GCC TGT AGC CCA TGT

N

S

NN

NH

NH

F

Fig. 1. Chemical structure of DBM1285.

658 Kang et al.



TGT AGC A-3, antisense 5-GGC AAT GAT GAT CCC AAA GTA GAC CTG CCC AGA CT-3; mouse -actin, sense 5-TGG AAT CCTGTG GCA TCC ATG AAA C-3, antisense 5-TAA AAC GCA GCTCAG TAA CAG TCC G-3; and human -actin, sense 5-GGG TCA GAA GGA TTC CTA TG-3, antisense 5-GGT CTC AAA CAT GATCTG GG-3. iQ SYBR Green Supermix (Bio-Rad Laboratories, Her-cules, CA) and the iCycler iQ Real-Time PCR Detection System(Bio-Rad Laboratories) were used for real-time PCR analysis. Foramplification, samples were heated to 94°C for 8 min and cycled 45

times at 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s. Using standards the amount of each gene was determined and normalizedby the amount of -actin.

TACE Assay. Enzyme assay was performed by using recombi-nant human TACE and substrate (MCA-Pro-Leu-Ala-Gln-Ala-

Val-DPA- Arg-Ser- Ser- Ser-Arg- NH2) purchased from R&D Sys-tems. Recombinant human TACE (0.01 g) was loaded in a F16Black Maxisorp Plate (Nunc, Roskilde, Denmark), and DBM1285(0.001, 0.01, 0.1, or 1 M) was added to each well. Enzyme reac-tion was started by adding substrate (10 M), and fluorescencewas measured at excitation and emission wavelengths of 320 and405 nm, respectively, in the kinetic mode. Specific enzyme activitywas measured according to the manufacturer’s instructions andexpressed as percentage of the vehicle-treated group.

Western Immunoblot Analysis. Twenty micrograms of whole-cell lysates was separated by 10% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and electrotransferred to a nitrocellulosemembrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK).Each membrane was preincubated for 1 h at room temperature inTris-buffered saline, pH 7.6, containing 0.05% Tween 20 and 5%nonfat milk. Each nitrocellulose membrane was incubated with spe-cific antibodies against phospho-p38 MAPK, nonphospho-p38MAPK, phospho-MK2, and nonphospho-MK2 (Cell Signaling Tech-nology, Danvers, MA). Immunoreactive bands were then detected byincubating with secondary anti-rabbit IgG antibody conjugated withhorseradish peroxidase and visualizing with enhanced chemilumi-nescence reagents (GE Healthcare).

Kinase Assay. Recombinant human p38 MAPK (active), recombi-nant human MK2 (active), recombinant human MK2 (inactive), and

MK2 substrate peptide were purchased from Millipore Corporation(Billerica, MA). Kinase assay was performed according to the manufac-turer’s instructions by serially adding substrate, recombinant enzymes,DBM 1285 (0.001, 0.01, 0.1, or 1 M), and [-32P]ATP and incubating itfor 10 min at 30°C. Twenty microliters of reaction product was trans-ferred to 2 2-cm P81 paper (Millipore Corporation) and washed threetimes with 0.75% phosphoric acid. The paper was washed once withacetone and subjected to scintillation counting.

In Vivo TNF- Assay. Mice were fasted for 24 h before the startof the experiment. DBM1285 (0.1, 1, or 10 mg/kg) was given by oraladministration. After 15 min, LPS (2 mg/kg) was intraperitoneallyinjected into each mouse. After 90 min, blood samples were collectedby retro-orbital puncture, and plasmas were prepared. The amountof TNF- in plasma was measured by enzyme-linked immunosorbentassay (ELISA) as described earlier.

In Vivo Target Inhibition Assay. The in vivo validation of inhibitory effect of DBM1285 on p38 MAPK activity was performedas described previously (Zhao et al., 2008). In brief, mice were pre-treated with vehicle or 10 mg/kg DBM1285. After 15 min, wholeblood was collected in heparin tubes (BD Biosciences, San Jose, CA)by retro-orbital puncture. Fluorescein isothiocyanate-conjugated ratanti-mouse Ly-6G antibody (BD Biosciences), allophycocyanin-con-

jugated rat anti-mouse CD11b antibody (BD Biosciences), and LPS(1 g/ml) were added to the blood and incubated at 37°C for 15 min.Twenty volumes of BD Phosflow Lyse/Fix buffer (BD Biosciences)was added to lyse red blood cells and fix white blood cells. Cells werecollected by centrifugation and resuspended in BD Phosflow Permbuffer III (BD Biosciences) with anti-phospho-MK2 antibody(Thr334; Cell Signaling Technology) for intracellular staining of

phospho-MK2. Cells were collected by centrifugation and finally

stained with anti-rabbit immunoglobulin-phycoerythrin (BD Bio-sciences) and incubated at room temperature for 30 min. Stainedcells were collected and analyzed by flow cytometry (FACSCalibur;BD Biosciences).

Zymosan-Induced Inflammation. Inflammation was inducedby subcutaneous injection of 50 l of 3 mg/ml zymosan A isolatedfrom Saccharomyces cerevisiae (Sigma-Aldrich) into the hind footpads of C57BL/6 mice. DBM1285 was orally administered from days0 to 6. On day 7, popliteal lymph nodes were isolated and weighed as

described previously (Frasnelli et al., 2005). Adjuvant-Induced Arthritis. The adjuvant-induced arthritis

model used has been described previously (Ishikawa et al., 2005).Paw volume was measured 1 day before the start of the experiment.

Arthritis was induced by subcutaneous injection of 0.5 mg of Myco-

bacterium tuberculosis (Difco, Detroit, MI) in 50 l of mineral oil intothe right hind footpads of Sprague-Dawley rats on day 0. DBM1285(1, 3, or 10 mg/kg) was administrated orally daily from days 0 to 25.Left hind paw volumes were measured by the water displacementmethod using a model MK-550 plethysmometer (Muramachi Kikai,Tokyo, Japan). Changes in paw volume were expressed as percent-age change of initial paw volume of each rat.

Statistical Analysis. Results are expressed as mean S.D.One-way analysis of variance and Dunnett’s t test were used formultiple comparisons by using Prism (GraphPad Software Inc., LaJolla, CA). The criterion for statistical significance was set at p

0.05.

Results

Effect of DBM1285 on LPS-Induced TNF- Secretion

in Cells of Macrophage/Monocyte Lineage. We first ex-amined the effect of DBM1285 on LPS-induced TNF- secre-tion in various cells, including mouse BM macrophages,THP-1 human monocyte cells, and RAW 264.7 mouse mac-rophage-like cells. As shown in Fig. 2A, DBM1285 concentra-tion-dependently inhibited LPS-induced TNF- secretion inBM macrophages. Treatment of BM macrophages with 0.01,0.1, and 1 M of DBM1285 caused 28, 84, and 100% inhibi-tion of TNF- secretion, respectively. In THP-1 cells andRAW 264.7 cells, DBM1285 also suppressed LPS-inducedTNF- secretion in a dose-dependent manner (Fig. 2, B andC). Treatment of cells with 0.01, 0.1, and 1 M DBM1285inhibited TNF- secretion in THP-1 cells by 50, 62, and 84%,respectively, and inhibited TNF- secretion in RAW 264.7cells by 18, 55, and 91%, respectively. IC50 for TNF- secre-tion was 0.023, 0.01, and 0.073 M in BM macrophages,THP-1 cells, and RAW 264.7 cells, respectively. The concen-tration and duration of DBM1285 treatment used in thisstudy had no significant effect on the viability of BM macro-phages, THP-1 cells, and RAW 264.7 cells (data not shown).

In addition, concanavalin A-induced T cell proliferation andLPS-induced B cell proliferation were not affected even at 10M DBM1285 (Supplemental Fig. 1).

Effect of DBM1285 on LPS-Induced mRNA Expres-

sion of TNF- in BM Macrophages and THP-1 Cells. Toinvestigate whether the DBM1285-mediated inhibition of TNF- secretion might be mediated by transcriptional regu-lation of cognate genes, we examined the effect of DBM1285on the mRNA expression of TNF- by real-time RT-PCR.LPS-induced TNF- mRNA expression was not affected byDBM1285 treatment in BM macrophages (Fig. 3A), THP-1cells (Fig. 3B), and RAW 264.7 cells (Supplemental Fig. 2).

Effect of DBM1285 on TACE Activity. To investigate

whether TACE might be involved in the inhibitory effect of

DBM-1285 Inhibits TNF- Production 659



TNF- secretion by DBM1285, we examined the effect of DBM1285 on TACE activity by performing an enzyme assay.DBM1285 had no effect on the enzymatic activity of TACE(Fig. 4).

Effect of DBM1285 on p38 MAPK Activity. It is wellknown that the p38 MAPK/MK2 signaling pathway is in-

volved in post-transcriptional regulation of TNF- biosynthe-sis by modulation of the activity of AU-binding proteins in AU-rich regions of the 3 untranslated region of TNF-mRNA (Kotlyarov and Gaestel, 2002). Therefore, we exam-ined the effect of DBM1285 on LPS-induced phosphorylationof p38 MAPK in BM macrophages and THP-1 cells to char-acterize whether the p38 MAPK pathway could be involvedin the DBM1285-mediated inhibition of TNF- production.DBM1285 blocked the LPS-induced phosphorylation of p38MAPK in BM macrophages (Fig. 5A) and THP-1 cells (Fig.5B), although the degree of inhibition did not correspond tothat of TNF- secretion. In addition, LPS-induced phosphor-ylation of p38 MAPK was not affected by DBM1285 in RAW

264.7 cells (Supplemental Fig. 2). However, phosphorylation

UN VH 0.001 0.01 0.1 10

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+ DBM1285 ( M)

T N F - ( n g / m l )

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+ DBM1285 ( M)

T N F - ( p g / m l )

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T N F - ( n g / m l )

UN VH 0.01 0.1 1

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+ DBM1285 ( M)

*

*

*

*

*

*

*

*

*

A B

C

Fig. 2. Effect of DBM1285 on LPS-induced TNF- secre-tion. Mouse BM macrophages (A), THP-1 cells (B), andRAW264.7 cells (C) were pretreated with vehicle (VH;DMSO) or the indicated concentrations of DBM1285(DBM) for 1 h before being incubated with LPS (1 g/ml)for 6 h. The culture supernatants were subsequently iso-lated and analyzed for TNF- secretion by ELISA. UN,untreated. Each column shows the mean S.D. of tripli-cate determinations. Statistical significance was deter-mined by using Dunnett’s t test versus the vehicle-treatedgroup (, p 0.05).

A B

UN VH 0.1 10

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o l d i n d u c t i o n

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+ DBM1285 ( M)

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+ DBM1285 ( M)

0

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25 Fig. 3. Effect of DBM1285 on LPS-induced TNF- mRNA expression. Mouse BM macrophages (A) and THP-1 cells(B) were pretreated with vehicle (VH: DMSO) or the indi-cated concentrations of DBM1285 (DBM) for 1 h beforebeing incubated with LPS (1 g/ml) for 6 h. Total RNAswere isolated, and the mRNA expression of TNF- and-actin was determined by real-time RT-PCR. The amount

of TNF- mRNA was normalized by that of -actin andexpressed as fold induction versus untreated group. UN,untreated. Each column shows the mean S.D. of tripli-cate determinations. Statistical significance was deter-mined by using Dunnett’s t test versus the vehicle-treatedgroup (, p 0.05).

VH 0.001 0.01 0.1 10

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T A C E a c t i v i t y

( % o

f v e h i

c l e )

DBM1285 ( M)

Fig. 4. Effect of DBM1285 on TACE activity. Recombinant human TACEwas treated with vehicle (VH: DMSO) or the indicated concentrations of DBM1285 (DBM). TACE activity was measured as described under Ma-terials and Methods by measuring fluorescence signal. Each column

shows the mean S.D. of triplicate determinations.

660 Kang et al.



of MK2, a downstream target of p38 MAPK and a regulatorof TNF- biosynthesis, was dramatically suppressed byDBM1285 treatment in LPS-stimulated BM macrophages(Fig. 5C). To further characterize the molecular target of DBM1285, we examined the effect of DBM1285 on kinaseactivity of p38 MAPK and MK2 by in vitro enzyme assay.

DBM1285 potently suppressed kinase activity of p38 MAPK (Fig. 6, A and B). However, MK2 activity was not affected byDBM1285 treatment (Fig. 6C).

To compare the potency and mode of action of DBM1285with a previously reported p38 MAPK inhibitor, we exam-ined the effect of BIRB796 (Regan et al., 2003) on LPS-induced TNF- secretion, TNF- mRNA expression, and p38MAPK phosphorylation. As shown in Fig. 7A, BIRB796 alsosuppressed TNF- secretion in a concentration-dependentmanner in our system. Consistent with the results obtainedwith DBM1285, BIRB796 treatment also had no effect onLPS-induced mRNA expression of TNF- (Fig. 7B). However,BIRB796 potently suppressed LPS-induced phosphorylation

of p38 MAPK (Fig. 7C) and MK2 (Supplemental Fig. 3).

Inhibition of LPS-Induced TNF- Production and Val-

idation of p38 MAPK Inhibition by Oral Administration

of DBM1285 in Mice. To investigate whether DBM1285 couldalso exert its effect in vivo, we first examined the effect of DBM1285 on LPS-induced TNF- production in mice. As shownin Fig. 8A, oral administration of DBM1285 suppressed LPS-

induced increases in the serum level of TNF- in a dose-relatedmanner. To further confirm the inhibition of p38 MAPK in vivoby DBM1285 treatment, we treated mice with DBM1285 for 15min and analyzed p38 MAPK activity by measuring intracellu-lar MK2 phosphorylation in a monocyte population of wholeblood cells. As shown in Fig. 8B, MK2 phosphorylation wasdetected in LPS-treated samples. However, DBM1285 treat-ment markedly suppressed phosphorylation of MK2 by LPStreatment (Fig. 8B).

Effect of DBM1285 on Zymosan-Induced Inflamma-

tion and Adjuvant-Induced Arthritis in Murine Mod-

els. We investigated the effect of DBM1285 in a zymosan-induced inflammation model. Popliteal lymph node weight

was substantially increased in vehicle-treated mice and sup-

UN VH 0.001 0.01 0.1 1

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+ DBM1285 ( M)

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p-p38 MAPK

p38 MAPK

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p38 MAPK

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+ DBM1285 ( M)

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p-MK-2

MK-2

UN VH 0.001 0.01 0.1 1

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+ DBM1285 ( M)

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Fig. 5. Effect of DBM1285 on LPS-induced phosphoryla-tion of p38 MAPK and MK-2. Mouse BM macrophages (A and C) and THP-1 cells (B) were pretreated with vehicle(VH: DMSO) or the indicated concentrations of

DBM1285 (DBM) for 1 h before being incubated withLPS (1 g/ml) for 30 min. Cells were lysed and phosphor-ylated, and nonphosphorylated forms of p38 MAPK (A and B) and MK2 (C) were detected by Western immuno-blot analysis. UN, untreated.

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MK-2 substrate

DBM1285 ( M)

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-

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*

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+

Fig. 6. Effect of DBM1285 on p38 MAPK and MK2 activity. A, active human p38 MAPK was treated with vehicle (VH:DMSO) or the indicated concentrations of DBM1285(DBM). Activating transcription factor 2 (ATF-2) and[-32p]ATP were added and incubated for 10 min at 30°C.B, active human p38 MAPK was treated with vehicle (VH:DMSO) or the indicated concentrations of DBM1285. Inac-tive MK2 and ATP were added and incubated for 15 min at30°C. Subsequent reaction was started by adding MK2substrate peptide and [-32p]ATP, and the reaction mixturewas incubated for 10 min at 30°C. C, active human MK2was treated with vehicle (VH: DMSO) or the indicatedconcentrations of DBM1285. MK2 substrate peptide and[-32p]ATP were added and incubated for 10 min at 30°C.Final reaction mixtures were transferred to P81 paper, andradiolabeled substrates were detected by scintillationcounting. Each column shows the mean S.D. of triplicate

determinations. Statistical significance was determined byusing Dunnett’s t test versus the vehicle-treated group (,

p 0.05).

DBM-1285 Inhibits TNF- Production 661



pressed by oral administration of DBM1285 in a concentra-tion-dependent manner (Fig. 9A). In addition, we demon-strated that DBM1285 potently suppressed the plasma levelof TNF- in zymosan-treated mice (Fig. 9B). Moreover, we

examined the effect of DBM1285 on RA development in anadjuvant-induced arthritis model. Rats were injected subcu-taneously with adjuvant in their right rear feet on day 0, anddisease progression was monitored in their left rear feet for26 days. Vehicle-treated rats showed severe inflammation intheir left rear feet; paw volume was increased to 280.4% of initial volume at day 18 and gradually decreased thereafter(Fig. 9C). However, daily oral administration of 10 mg/kg DBM1285 caused 49% inhibition at day 18 and 41% inhibi-tion at day 26 (Fig. 8C). Body weights of DBM1285-treatedrats were not significantly decreased compared with those of vehicle-treated rats during experiment, and LD50 was esti-mated to be over 300 mg/kg according to single and 14-day

repeated-dose oral toxicity experiments (data not shown).

Discussion

The results of the present study support the potential of DBM1285 as an inhibitor of TNF- production and a thera-

peutic candidate for TNF--related diseases. Here, we haveshown that DBM1285 significantly inhibits LPS-induced se-cretion of TNF- in mouse and human cells of macrophage/ monocyte lineage, including mouse BM macrophages, THP-1cells, and RAW 264.7 cells. Our results also demonstratethat, although submicromolar concentrations of DBM1285can markedly suppress TNF- secretion in LPS-stimulatedcells of macrophage/monocyte lineage, treatment with 10 MDBM1285 has no effect on concanavalin A-induced T cellproliferation and LPS-induced B cell proliferation, suggest-ing that the effect of DBM1285 might be cell type-specific. Ingeneral, high specificity is associated with reduced adverseeffect. Therefore, it is assumed that DBM1285 might be a

good therapeutic candidate for TNF--related diseases. It is

0

50

100

150

T N F - α ( p g / m l )

UNVH 0

. 0 0 1

0 . 0

1 0.1 1

BIRB796 ( M)

0.01 1

DBM1285 ( M)

A

*

*

**

*

*

LPS (1 g/ml)

B

p-p38 MAPK

p38 MAPK

UN VH 0.001 0.01 0.1 1

LPS (1 g/ml)+ BIRB796 ( M)

C

0

5

10

15

20

25

F o l d i n d u c t i o n

UN VH 0.1 1

LPS (1 g/ml)

+ BIRB796 ( M)

Fig. 7. Effect of BIRB796 on LPS-induced TNF- secre-tion, TNF- mRNA expression, and p38 MAPK phos-phorylation. THP-1 cells were pretreated with vehicle(VH: DMSO) or the indicated concentrations of BIRB796for 1 h before being incubated with LPS (1 g/ml) for 6 hfor measurement of TNF- secretion and TNF- mRNA

expression and 30 min for measurement of p38 MAPK phosphorylation. A, the culture supernatants were sub-sequently isolated and analyzed for TNF- secretion byELISA. UN, untreated. Each column shows the mean S.D. of triplicate determinations. B, total RNAs wereisolated, and the mRNA expression of TNF- and -actinwas determined by real-time RT-PCR. The amount of TNF- mRNA was normalized by that of -actin andexpressed as fold induction versus the untreated (UN)group. C, cells were lysed, and phosphorylated and non-phosphorylated forms of p38 MAPK were detected byWestern immunoblot analysis. Statistical significancewas determined by using Dunnett’s t test versus the

vehicle-treated group (, p 0.05).

0

0.5

1.0

1.5

2.0

2.5A

T N F - α

( n g / m l )

UN VH 0. 1 1 10

LPS (2 mg/kg)

+ DBM1285 (mg/kg)

*

*

*

Phospho-MK2

UN

VH

+ LPS (1 g/ml)

DBM1285 (10 mg/kg)

+ LPS (1 g/ml)

B

Fig. 8. Effect of DBM1285 on TNF- production and p38MAPK activity in vivo. A, mice were pretreated orally with

vehicle (VH: 5% Tween 80 in PBS) or the indicated concen-trations of DBM1285 (DBM) for 15 min, and LPS (2 mg/kg)was injected intraperitoneally into each mouse. After 90min, blood samples were collected, plasmas were prepared,and TNF- level was determined by ELISA. UN, un-treated. Each column shows the mean S.D. of triplicatedeterminations. Statistical significance was determined by

using Dunnett’s t test versus the vehicle-treated group (

, p 0.05). B, mice were pretreated with vehicle (VH: 5%Tween 80 in PBS) or 10 mg/kg DBM1285 for 15 min. Wholeblood was collected and stimulated with 1 g/ml LPS for 15min. Monocytes (CD11bMedLy-6G) in whole blood cellswere analyzed for MK2 phosphorylation by flow cytometry.

662 Kang et al.



BIRB796 is a well known inhibitor of p38 MAPK (Kumar etal., 2003). To compare the potency and mode of action of

DBM1285 with those of BIRB796, we examined the effect of BIRB796 on LPS-induced TNF- expression and p38 MAPK phosphorylation. Our results showed that BIRB796 dose-dependently suppressed LPS-induced secretion of TNF-. Itwas also demonstrated that the potency of DBM1285 in theinhibition of TNF- secretion was comparable with or betterthan that of BIRB796. As in the case of DBM1285, BIRB796did not affect LPS-induced mRNA expression of TNF-, sug-gesting that BIRB796 might also regulate TNF- secretion atthe post-transcriptional level. However, analysis of the effectof BIRB796 on p38 MAPK phosphorylation revealed a dra-matic inhibition of LPS-induced phosphorylation of p38MAPK. This result is different from that obtained withDBM1285, which showed slight inhibition of p38 MAPK

phosphorylation and suggests that these two compounds donot share the same mechanism to regulate the activity of p38MAPK. A previous report described that BIRB796 inhibitedp38 MAPK activity through direct binding (Regan et al.,2003). From these results, we can speculate that bothDBM1285 and BIRB796 bind directly to p38 MAPK to exerttheir inhibitory effect, but their binding sites and exact in-hibitory mechanisms are different (Fig. 10). Further studiesare required to fully address this question.

In summary, the results presented in this article demonstratethat DBM1285 inhibits TNF- production in vitro and in vivo andexerts an antiarthritic effect in an animal model. We also showthat the inhibitory effect of DBM1285 on TNF- production is

mediated by the inhibition of p38 MAPK activity and the down-stream signaling cascade. Our results indicate DBM1285 is apromising drug candidate for RA treatment.

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Address correspondence to: Dr. Song-Kyu Park, Bioevaluation Center,Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheong-won, Chungbuk, Republic of Korea. E-mail: [email protected]

p38

MK2

Receptor

MAPKKK

MAPKK

Environmental stress/

Inflammatory cytokines

26-kDa TNF-α

17-kDa TNF-α

cap

AAAA

cap

AAAA

cap AAAAcap AAAA

p

AU-binding protein

translation

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MK2 MK2

MAPKK

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DBM1285 BIRB796

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Fig. 10. Schematic diagram showing the mode of action of DBM1285 on TNF- production. A, the p38 MAPK/MK2pathway is implicated in the post-transcriptional regula-

tion of the TNF- gene. DBM1285 and BIRB796 inhibitMK2 phosphorylation and activation by p38 MAPK through the modulation of p38 MAPK activity. B, differen-tial mode of action of DBM1285 and BIRB796 on the inhi-bition of the p38 MAPK/MK2 pathway.

664 Kang et al.

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