Synthesized pyridine compound derivatives decreased TNF alpha and adhesion molecules and ameliorated HSV-induced inflammation in a mouse model

Article abstract of DOI:10.1016/j.ejphar.2011.01.062

Synthesized pyridine compound derivatives (SK94, SK126) from a natural lead source were administered to mice to test for possible anti-TNF alpha and anti-inflammatory activities. Lipopolysaccharide (LPS)-induced TNF alpha production was analyzed in the endothelial cells, Raw 264.7 cells, and serum of normal mice after treatment with SK compounds. These compounds were also orally administered to a herpes simplex virus (HSV)-induced Behcet's disease mouse model to investigate their anti-inflammatory therapeutic effect. TNF alpha production was inhibited in a dose-dependent manner in the SK94 treated cells. E-selectin, VCAM-1, and ICAM-1 mRNA levels were also down-regulated. Treatment with 30 mg/kg SK94 inhibited 55% of the TNF alpha production in LPS challenged Balb/c mice (n = 8). SK94 and SK126 were administered to the Behcet's disease-like mice for five consecutive days and SK94 improved in five out of six mice (83%), while it only improved in one out of nine mice (11%) in the pH 1.2 saline (artificial gastric juice) group (P < 0.005), four out of ten mice (40%) in the thalidomide group (P < 0.05), and six out of seven (86%) in the SK126 group (P < 0.005). Soluble ICAM-1 was inhibited by 23.8% in the sera of SK94 treated mice and by 34.6% in SK126 treated mice when compared to artificial gastric juice. Based on these findings, SK compounds could be candidates for clinical trials.

Full text of DOI:10.1016/j.ejphar.2011.01.062

European Journal of Pharmacology 657 (2011) 167–172
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and Inflammation
Synthesized pyridine compound derivatives decreased TNF alpha and adhesion
molecules and ameliorated HSV-induced inflammation in a mouse model
Bunsoon Choi a, Joohyon Kim b, Eun-So Lee c, Dongsik Bang d, Seonghyang Sohn a,e,
⁎
a
Laboratory of Cell Biology, Ajou University Institute for Medical Sciences, Republic of Korea
SK Chemical Life Science R&D Center, Republic of Korea
Department of Dermatology, Ajou University School of Medicine, Republic of Korea
Department of Dermatology, Yonsei University, Republic of Korea
b
c
d
e
Brain Korea Project for Medical Science, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2010
Received in revised form 5 January 2011
Accepted 25 January 2011
Available online 17 February 2011
Synthesized pyridine compound derivatives (SK94, SK126) from a natural lead source were administered to
mice to test for possible anti-TNF alpha and anti-inflammatory activities. Lipopolysaccharide (LPS)-induced
TNF alpha production was analyzed in the endothelial cells, Raw 264.7 cells, and serum of normal mice after
treatment with SK compounds. These compounds were also orally administered to a herpes simplex virus
(HSV)-induced Behcet's disease mouse model to investigate their anti-inflammatory therapeutic effect. TNF
alpha production was inhibited in a dose-dependent manner in the SK94 treated cells. E-selectin, VCAM-1,
and ICAM-1 mRNA levels were also down-regulated. Treatment with 30 mg/kg SK94 inhibited 55% of the TNF
alpha production in LPS challenged Balb/c mice (n=8). SK94 and SK126 were administered to the Behcet's
disease-like mice for five consecutive days and SK94 improved in five out of six mice (83%), while it only
improved in one out of nine mice (11%) in the pH 1.2 saline (artificial gastric juice) group (Pb0.005), four out
of ten mice (40%) in the thalidomide group (Pb0.05), and six out of seven (86%) in the SK126 group
(Pb0.005). Soluble ICAM-1 was inhibited by 23.8% in the sera of SK94 treated mice and by 34.6% in SK126
treated mice when compared to artificial gastric juice. Based on these findings, SK compounds could be
candidates for clinical trials.
Keywords:
Pyridine compound derivative
Behcet's disease
Herpes simplex virus
TNF alpha
Oral administration
Anti-inflammation
Mouse model
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Meide and Schellekens, 1996); therefore, developers of treatments for
inflammatory disease have focused on inhibitors of TNFα production.
Tumor necrosis factor-α (TNFα) is a potent paracrine and endocrine
mediator of inflammatory and immune functions. TNFα over expression
has been implicated in acute and chronic inflammatory diseases, such as
septic shock, bowel disease, Crohn's disease, rheumatoid arthritis, atopic
dermatitis, psoriasis, and Behcet's disease (Edwards, 2004). TNFα is
primarily produced in T cells, polymorphonuclear cells, dendritic cells,
and macrophages (Wallach and Kovalenko, 2009). In macrophages, TNFα
gene expression is induced by physical, chemical, and biological stimuli
that include ischemia, trauma, irradiation, viruses, bacteria, tumor cells,
complement, and cytokines such as intreleukin-1β (IL-1β), intreleukin-2
(IL-2), interferon-γ (IFNγ), granulocyte-macrophage colony stimulating
factor (GM-CSF), macrophage CSF (M-CSF), and TNFα itself (Brouckaert
et al., 1993). TNFα is also known to induce other inflammatory cytokines
including IL-6 (Brouckaert et al., 1993) and IL-8 (Williams et al., 2008).
TNFα plays a central role in a variety of inflammatory responses (Van der
Behcet's disease is a chronic, multi-systemic disorder with arthritic,
gastrointestinal, mucocutaneous, ocular, vascular, and central nervous
system involvement. This disease has a chronic course with periodic
exacerbations and progressive deterioration (Shimizu, 1979). Although
the etiology of Behcet's disease is unclear, viral infection has long been
postulated as one of the main factors. Since Hulusi Behcet first proposed
a viral etiology (Behcet, 1937), the viral hypothesis has been verified by
the detection of viruses in the saliva (Lee et al., 1996b), intestinal ulcers
(Lee et al., 1993), genital ulcers (Bang et al., 1997; Lee et al., 1996a), and
serum (Hamzaoui et al., 1990; Sánchez Román et al., 1992) of patients
with Behcet's disease. Subsequent to this finding, herpes simplex virus
(HSV) inoculation of the earlobes of ICR mice was found to induce the
development of Behcet's disease-like symptoms (Sohn et al., 1998).
Manifestations in mice following HSV inoculation included multiple
symptoms such as oral ulcers, genital ulcers, skin ulcers, eye symptoms,
gastrointestinal ulcers, arthritis, and neural involvement, as well as skin
crusting. The frequency of these symptoms was similar to that of
patients with Behcet's disease (Kim et al., 1988). Anti-viral drugs such as
Famcyclovir have been used to treat Behcet's disease, but with limited
success. Specifically, these drugs have had only 40% efficacy in a Behcet's
⁎
Corresponding author at: Laboratory of Cell Biology, Ajou University Institute for
Medical Sciences, Suwon, 443–721, Republic of Korea. Tel.: +82 31 219 4510; fax: +82
31 219 4514.
E-mail address: sohnsh@ajou.ac.kr (S. Sohn).
0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.01.062

168
B. Choi et al. / European Journal of Pharmacology 657 (2011) 167–172
disease mouse model, compared to a 60% success rate when used to
treat HSV (Sohn et al., 2001). Anti-TNFα antibody, Infliximab, and
soluble TNF receptor (Etanercept) have also been used to treat Behcet's
disease patients.
The pyridine compound derivatives of the SK chemicals SK126 and
SK94 (Fig. 1) are synthetic compounds based on gentianine, which is one
of the major components of Gentiana Macrophylla Radix. SK126 was
shown to effectively inhibit the production of inflammatory cytokines
including TNFα in antigen presenting cells (Kang et al., 2008). In this
study, low-molecular weight, orally administered TNF alpha inhibitors
were tested for their ability to ameliorate HSV-induced inflammation in a
Behcet's disease mouse model.
used to determine the severity score of Behcet's disease. Symptomatic
mice were photographed on the starting day of drug administration and
once every week for five weeks on the same day of the week using a
digital camera. SK compounds (1 mg) or thalidomide (50 μg) were orally
administered to five to ten Behcet's disease-like mice for five consecutive
days. Placebo was administered to ten Behcet's disease mice in an
identical manner.
2.3. Preparation of SK126 and SK94
SK126 and SK94, which are pyridine derivatives based on lead
compounds derived from a natural product, were provided by SK
Chemicals (Suwon, Republic of Korea). Briefly, SK126, 2-ethyl-8-(4-
fluorophenyl)-6-methyl-3,4-dihydro-2H[2,7] naphthyridin-1-one was
synthesized using a five-step procedure. 2-Chloro-4,6-dimethylnicoti-
nonitrile readily underwent Suzuki-coupling with 4-fluorophenylboro-
nic acid to give 2-(4-fluorophenyl)-4,6-dimethyl-nicotinonitrile.
Treatment of this coupled product with N,N-dimethylformamide
dimethyl acetal followed by sulfuric acid yielded the cyclized compound,
8-(4-fluorophenyl)-6-methyl-2H-[2,7]naphthyridin-1-one. Finally,
ethylation and hydrogenation of the lactam gave SK126, which was
purified by silica gel column chromatography and subsequent recrystal-
lization. NMR data describing the final compound revealed that the
purity was greater than 95%.
2. Materials and methods
2.1. In vitro analysis of SK-compounds for cell adhesion molecules
Human Umbilical Vein Endothelial Cells (HUVEC) were treated
with SK compounds for 30 min and then stimulated with TNFα for
4 h. The cells were then harvested, after which the RNA was
extracted and subjected to RT-PCR.
2.2. Animals, introduction of Behcet's disease symptoms and treatment of
Behcet's disease mice with placebo or SK-compounds
2.4. ELISA
Four to five-week-old male ICR mice were used for this study. The
earlobes of the mice were scratched with a needle and then inoculated
with 1.0×106 plaque forming units/ml of HSV type 1 (F strain). Virus
inoculation was conducted twice with a 10-day interval between
treatments, after which the mice were observed for 16 weeks. Mice
were bred in temperature- and light-controlled conventional rooms
(20–22 °C, 12 h light cycle starting at 8:00 A.M.) with free access to food
and water. During the experimental period, the animals were closely
observed and photographed. Animals were handled in accordance to a
protocol approved by our institutional animal care committee.
A revised Japanese classification with minor modifications was used
to classify symptomatic mice with Behcet's disease. Briefly, oral, genital
and other skin ulcers (including bulla and crust) and eye symptoms were
classified as major symptoms, while arthritis, gastrointestinal ulcers and
neurological disorders were identified as minor symptoms. Mice with at
least one major and one minor symptom were classified as having
Behcet's disease. Of the total number of HSV-injected mice, 15%
developed Behcet's disease-like symptoms. Treatments that led to the
disappearance of symptoms or a decrease in the lesion size of greater
than 20% were classified as effective. Scoring of the severity of Behcet's
disease was followed by determination of the Behcet's disease activity
index, as outlined in the Behcet's disease Activity Form (www.behcet.ws/
pdf/BehcetsDiseaseActivityForm.pdf). Among the symptoms in patients,
mouth ulceration, genital ulceration, erythema, skin pustules, skin
ulceration, joints-arthritis, diarrhea, red eye (right, left), reduced vision
(right, left), loss of balance, discoloration, and swelling of the face were
selected and analyzed in the Behcet's disease mouse model. The score of
each symptom was one, and after the score was computed the total was
For detection of soluble ICAM-1, soluble VCAM-1, soluble E-selectin,
soluble TNF receptor 1, and MCP-1, the drug treated mice were
sacrificed on the last day of the drug treatment and the serum and
spleens were collected for analysis. ELISA was applied using commercial
kits for the detection of mouse soluble type 1 TNFα receptor (R&D
system, Minneapolis, MN), TNFα (R&D system), soluble ICAM-1, soluble
VCAM-1, soluble E-selectin (R&D system), and MCP-1 (R&D system).
ELISA analysis was conducted on spleen tissue from each mouse. Briefly,
the spleen was excised, finely minced with scissors, and then suspended
in 3 mL of 10 mM potassium phosphate buffer (pH 6.0). The tissue
samples were then homogenized and centrifuged at 9000×g. A micro
BCA protein assay kit (Pierce, Rockford, Illinois, USA) was used to
quantify each protein in the supernatants, after which ELISA was
conducted according to the manufacturer's instructions. The ELISA
reader was a Bio-Rad model 170–6850 microplate reader and the
samples were read at a wavelength of 450 nm.
2.5. Administration of SK compounds to BALB/c mice
SK94 (1–70 mg/kg) or SK126 (0.1–100 mg/kg) were orally adminis-
tered to BALB/c mice. Two hours after administration, mice were
challenged intraperitoneally with LPS (1 mg/kg). Two hours after LPS
challenge, blood was collected from the mice, and the sera were subjected
to ELISA (n=8 for LPS-treated groups, n=6 for saline-treated group).
2.6. Statistical analysis
All data shown are the means S.D. Statistical differences
between the experimental groups were determined according to a
Student's t test and the Bonferroni correction. Statistical analyses
were performed using MedCalc® version 9.3.0.0.
3. Results
3.1. mRNA expression of adhesion molecules were inhibited by SK
compounds
Fig. 1. Synthesized pyridine compound derivative structures (SK126, SK94). These are
synthetic compounds based on gentianine, one of the major components of Gentiana
Macrophylla Radix.
Cell adhesion molecules are located on the cell surface and involved in
the binding of other cells or extracellular matrix. Cell–cell and cell–matrix

B. Choi et al. / European Journal of Pharmacology 657 (2011) 167–172
169
interactions play vital roles in inflammation. In leukocytes and platelets,
cell adhesion molecules interact with each other or with the extracellular
matrix to bring circulating cells to areas of inflammation (Arnaout, 1993).
Tissue- and inflammation-specific leukocyte/endothelial cell adhesion
molecules constitute attractive targets for suppression or manipulation of
the early stages of tissue inflammation (Jutila et al., 1989). To evaluate the
inhibitory effects of SK compounds on cell adhesion molecules such as
vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion
molecule-1 (ICAM-1), and E-selectin, HUVEC cultures were treated with
SK compounds. The VCAM-1 and E-selectin mRNA expression was down-
regulated in the 0.1–10 μM treated groups by reverse transcriptase PCR
and real time PCR, while ICAM-1 expression was not affected by treatment
with SK compounds (Fig. 2A).
untreated control group (100.0%) (control vs. TNFα, TNFα vs.
TNFα+SK94, TNFα vs. TNFα+SK126, all P values are b0.01). The
VCAM-1 expression was 771.3 14.3% in the TNFα treated group,
211.3 11.1% in the combined TNFα and SK94 treated group, and
223.7 6.1% in the combined TNFα and SK126 treated group when
compared to the untreated control group (100.0%) (control vs. TNFα,
TNFα vs. TNFα+SK94, TNFα vs. TNFα+SK126, all P values b0.001).
E-selectin expression was 169.3 8.2% in the TNFα treated group,
100.3 7.8% in the combined TNFα and SK94 treated group, and
101.7 11.1% in the combined TNFα and SK126 treated group when
compared to the untreated control group (100.0%) (control vs. TNFα,
TNFα vs. TNFα+SK94, TNFα vs. TNFα+SK126, all P values are
b0.002) (Fig. 2B). Additionally, SK compounds were found to suppress
the protein expression of ICAM-1, VCAM-1, and E-selectin in in vitro
TNFα stimulated HUVEC cultures.
3.2. Protein levels of adhesion molecules were inhibited by SK compounds
To confirm the inhibitory effect of SK compounds at the protein
level, HUVEC were treated with SK compounds and stimulated with
TNFα for 12 h. The levels of adhesion molecules were measured in
conditioned media by ELISA. The comparative expression of ICAM-1
was 882.0 16.1% in the TNFα treated group, 798.7 11.7% in the
combined TNFα and SK94 treated group, and 675.0 16.7% in the
combined TNFα and SK126 treated group when compared to the
3.3. SK-compounds inhibit TNFα production in RAW 264.7 cells, Balb/c
mice, and Behcet's disease mice
RAW 264.7 cells were treated with SK94 (1–70 μM) or SK126 (1–
8 μM) for 30 min, after which they were stimulated with LPS for 18 h. The
expression of TNFα was then quantified in conditioned media by ELISA.
SK94 inhibited TNFα expression in cultures of RAW 264.7 cells in a dose
Fig. 2. A. mRNA expression of adhesion molecules inhibited by SK compounds. HUVEC were treated with SK compounds for 30 min and then stimulated with TNFα for 4 h. Cells were
harvested, after which the RNA was extracted and applied to RT-PCR and real time PCR. N: negative control, P: positive control, FBP (far-upstream element binding protein):
experimental control. B. Adhesion protein expression was inhibited by the effects of SK compounds in endothelial cells. Endothelial cells (HUVEC) were treated with SK compounds
and stimulated with TNFα for 12 h. The quantities of adhesion molecules were determined by ELISA.

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B. Choi et al. / European Journal of Pharmacology 657 (2011) 167–172
70
P=0.01
A120
60
50
40
30
20
10
0
P=0.02
100
80
60
40
20
0
saline
thalidomide
SK-94
SK-126
0
1.87
3.75
7.5
15
30
60
SK-94 (µM)
Fig. 4. SK compounds inhibit TNFα production in Behcet's disease mice. SK94 (1 mg/
mouse) or SK126 (1 mg/mouse) was orally administered to Behcet's disease mice for
five consecutive days. At 4 h after the last administration, the sera were collected and
analyzed by ELISA.
80
B
60
40
20
0
(Bang, 1997) and Behcet's disease mice (Lee et al., 2004); however, SK126
more effectively down-regulated the serum level of TNFα than
thalidomide in Behcet's disease mice (Fig. 4).
3.4. SK compounds down-regulate adhesion molecules in Behcet's disease
mice
0
0.125 0.25 0.5
1
2
4
8
SK-126 (µM)
SK94 (1 mg/mouse) or SK126 (1 mg/mouse) was orally adminis-
tered to Behcet's disease mice for five consecutive days to determine if
it down-regulated soluble adhesion molecules (n=7 per group).
Soluble forms of ICAM-1, VCAM-1 and E-selectin are elevated during
inflammatory conditions (Miles et al., 2001); however, SK126
significantly down-regulated soluble ICAM-1 in the sera of Behcet's
disease mice (36.4 5.6 ng/ml) when compared to the control
(55.7 17.6 ng/ml) (Pb0.05). SK94 also down-regulated the soluble
E-selectin (1048 56 pg/ml) when compared to the control
(1981 221 pg/ml) (Pb0.05) (Table 2). In spleen tissues, SK126
significantly down-regulated the soluble E-selectin (372 397 pg/
ml) when compared to the control (1100 727 pg/ml) (Pb0.05).
Monocyte chemoattractant protein-1 (MCP-1) is an essential
chemokine involved in monocyte traffic across endo- and epithelial
barriers both in vitro and in vivo (Maus et al., 2002). MCP-1 also
triggers firm adhesion of monocytes to vascular endothelium
(Gerszten et al., 1999). SK126 inhibited MCP-1 (52.0 12.3 pg/ml)
when compared to the control (91.9 73.0 pg/ml), but this inhibition
was not statistically significant (Table 3).
Fig. 3. SK compounds inhibit TNFα production in the RAW 264.7 cell line. SK94 (A) or
SK126 (B) was applied to the cultures of RAW 264.7 cells for 30 min, after which they
were stimulated for 18 h with LPS to express TNFα. The expression of TNFα was
quantified by ELISA.
dependent manner. SK126 also inhibited TNFα expression more
efficiently than SK94 at lower concentrations (Fig. 3). To determine if
the SK compounds down-regulated the TNFα levels in serum, normal
BALB/c mice were orally administered SK94 (1–70 mg/kg) or SK126 (0.1–
100 mg/kg) (Table 1) (n=8 per dose). SK94 and SK126 down-regulated
the level of TNFα in the serum of Balb/c mice. SK94 (1 mg/mouse) or
SK126 (1 mg/mouse) was also orally administered to Behcet's disease
mice for five consecutive days to determine if it down-regulated the
serum level of TNFα. At4 hafterthelasttreatment, theserawerecollected
and analyzed by ELISA. SK126 down-regulated TNFα (11.1 13.4 pg/ml)
when compared to the saline administered control (49.2 9.2 pg/ml)
(P=0.01) (n=5 per group). In addition, SK126 more efficiently down-
regulated TNFα when compared to the thalidomide administered group
(24.6 13.9 pg/ml) (P=0.02). Thalidomide is highly effective at
suppressing oral and genital manifestations of Behcet's disease patients
3.5. SK compounds improved Behcet's disease mice
SK94 (1 mg/mouse) or SK126 (1 mg/mouse) was orally adminis-
tered to Behcet's disease mice for five consecutive days to determine if
they improved Behcet's disease symptoms. Behcet's disease symptoms
were ameliorated after SK94 or SK126 administration. Specifically,
SK94 improved the symptoms of Behcet's disease in five out of six mice
(83%) and six out of seven mice (86%) in the SK126 group (Pb0.005),
while improvements only occurred in one of nine (11%) mice in the pH
Table 1
The inhibition of TNFα expression by oral administration of SK compounds in LPS
challenged Balb/c mice. Balb/c mice were administered with SK94 (1–70 mg/kg) (A) or
SK126 (0.1–100 mg/kg) (B). Two hours after the administration, mice were challenged
intraperitoneally with LPS (1 mg/kg). Two hours after the LPS challenge, blood was
collected and the sera were subjected to ELISA (n=8 per dose).
Compound
Dose
(mg/kg)
Inhibition
(%)
ED50
Table 2
A.
SK compounds down-regulated adhesion molecules in the sera of BD mice. SK
compounds were orally administered to BD mice for 5 consecutive days, and then sera
were obtained from each mouse and subjected to ELISA (n=7).
SK-94
70
50
30
10
3
84
64
55
40
28
16
14.2 mg/kg
Groups
Saline
SK-94
SK-126
Thalidomide
100 μL/mouse 1 mg/mouse
1 mg/mouse
50 μg/mouse
1
(inhibition %) (inhibition %) (inhibition %)
B.
Soluble ICAM-1
(ng/ml)
Soluble VCAM-1
(ng/ml)
Soluble E-selectin 1981 221
(pg/ml)
55.7 17.6
46.7 8.4
42.5 7.3
(23.8%)
36.4 5.6a
(34.6%)
40.2 4.3
(14.1%)
1182 669
(40.3%)
47.8 9.5
(14.2%)
43.2 3.5
(7.6%)
1843 708
(6.9%)
SK-126
100
10
3
1
0.3
0.1
82
78
71
66
55
22
0.48 mg/kg
43.1 3.7
(7.8%)
1048 56a
(47.1%)
a
Pb0.05.

B. Choi et al. / European Journal of Pharmacology 657 (2011) 167–172
171
Table 3
disease mice and ameliorated the symptoms. TNFα and adhesion
molecules have been shown to be up-regulated in Behcet's disease
patients (Sayinalp et al., 1996; Kose et al., 2008). In addition, down-
regulation of TNFα using anti-TNFα antibody (Infliximab) was found to
suppress various Behcet's disease symptoms including sight-threaten-
ing panuveitis (Sfikakis et al., 2001) and intestinal ulcers (Ju et al., 2007)
in patients and mice (Choi et al., 2008). Another TNFα blocker,
Etanercept, is also known to be an effective therapeutic agent for
Behcet's disease patients and mice (Cantarini et al., 2009; Choi et al.,
2008). All TNFα blockers are administered by injection; however, if
orally administered TNFα blockers were available it would increase
comfort for the patients. Orally administered SK compounds were found
to effectively down-regulate the serum level of TNFα in LPS treated
Balb/c mice and Behcet's disease mice. Additionally, SK126 more
efficiently down-regulated the serum level of TNFα, soluble ICAM-1
(sICAM-1), and soluble E-selectin (sE-selectin) when compared to
thalidomide in Behcet's disease mice. Thalidomide is known to be a
TNFα inhibitor (Schmidt et al., 1996) that is effective in Behcet's disease
patients (Hamuryudan et al., 1998) and Behcet's disease mice (Lee et al.,
2004).
Infliximab treatment reduced the serum concentrations of soluble
ICAM-1 and E-selectin in patients with juvenile idiopathic arthritis
(Levälampi et al., 2007). In addition, subcutaneous injection of
Etanercept down regulated the level of sICAM-1, sVCAM-1, and sE-
selectin in the serum of rheumatoid arthritis patients (Klimiuk et al.,
2009). SK126 and SK94 more efficiently down-regulated sICAM-1,
sVCAM-1, and sE-selectin when compared to thalidomide in the sera
of Behcet's disease mice, and it down-regulated sE-selectin when
compared to the thalidomide treated group in the spleen tissues of
Behcet's disease mice. SK126 and SK94 decreased monocyte
chemotactic protein-1 (MCP-1) in the spleen tissues of Behcet's
disease mice. MCP-1 has been found in the joints of people with
rheumatoid arthritis, where it may serve to recruit macrophages and
perpetuate inflammation (Harigai et al., 1993). Plasma MCP-1 levels
have also been shown to be elevated in Behcet's disease patients
(Kaburaki et al., 2003). In addition, MCP-1 inhibition ameliorated rat
SK compounds down-regulated adhesion molecules, cytokine receptor, and chemokine
in the spleen tissue of BD mice. SK compounds were orally administered to BD mice for
5 consecutive days, and then spleen tissues were obtained from each mouse and
homogenized, and supernatant was subjected to ELISA (n=7).
Groups
Saline
SK-94
SK-126
1 mg/mouse
Thalidomide
50 μg/mouse
100 μL/mouse 1 mg/mouse
(inhibition %) (inhibition %) (inhibition %)
Soluble ICAM-1
(ng/ml)
Soluble VCAM-1
(ng/ml)
Soluble E-selectin 1100 727
(pg/ml)
Soluble TNFR1
(pg/ml)
608 140
423 115
622 221
469 200
1226 1295
2500 721
637 118
590 192
575 142
477 192
973 764
2012 475
372 397a
(66.2%)
2217 1260
2277 121
MCP-1
(pg/ml)
91.9 73.0
69.6 28.7
(24.3%)
52.0 12.3
(43.4%)
81.5 11.6
(11.3%)
a
Pb0.05.
1.2 saline (artificial gastric juice) group (Pb0.005) and four of ten
(40%) mice in the thalidomide group (Pb0.05). The disappearance of
symptoms or a decrease in the lesion size of more than 20% was
classified as improvement. Photographs of the mice taken before and
three weeks after administration were also compared (Fig. 5A), as was
the severity score (Fig. 5B). In the SK126 treated group, the severity
score was decreased from 2.56 0.5 to 1.63 1.02 (P=0.03). In SK94
and thalidomide treated Behcet's disease mice the severity decreased
from 2.17 0.35 to 1.5 0.76 and 2.61 0.86 to 2.05 1.47, respec-
tively, but this change was not statistically significant. Conversely, the
severity score in the saline injected group increased from 2.14 to 2.98.
4. Discussion
The results of the present study showed that synthesized pyridine
compound derivatives can down-regulate the expression of TNFα and
adhesion molecules in HUVEC and normal mice. Furthermore, these
compounds down-regulated TNFα and adhesion molecules in Behcet's
Fig. 5. Administration of SK compounds to Behcet's disease mice. A. Change in symptoms, B. change in severity score.

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B. Choi et al. / European Journal of Pharmacology 657 (2011) 167–172
Hamzaoui, K., Ayed, K., Slim, A., Hamza, A., Touraine, J., 1990. Natural killercell activity,
interferon-gamma and antibodies to herpes virus in patients with Behcet's disease.
Clin. Exp. Immunol. 79, 28–34.
Harigai, M., Hara, M., Yoshimura, T., Leonard, E.J., Inoue, K., Kashiwazaki, S., 1993.
Monocyte chemoattractant protein-1 (MCP-1) in inflammatory joint diseases and
its involvement in the cytokine network of rheumatoid synovium. Clin. Immunol.
Immunopathol. 69, 83–91.
Ju, J.H., Kwok, S.K., Seo, S.H., Yoon, C.H., Kim, H.Y., Park, S.H., 2007. Successful treatment
of life-threatening intestinal ulcer in Behçet's disease with infliximab: rapid healing
of Behçet's ulcer with infliximab. Clin. Rheumatol. 26, 1383–1385.
Jutila, M.A., Berg, E.L., Kishimoto, T.K., Picker, L.J., Bargatze, R.F., Bishop, D.K., Orosz, C.G.,
Wu, N.W., Butcher, E.C., 1989. Inflammation-induced endothelial cell adhesion to
lymphocytes, neutrophils, and monocytes. Role of homing receptors and other
adhesion molecules. Transplantation 48, 727–731.
rheumatoid arthritis (Guglielmotti et al., 2002). Taken together,
these findings indicate that the decrease in MCP-1 in response to
treatment with SK compounds may contribute to the amelioration of
Behcet's disease symptoms.
Thalidomide has been shown to effectively improve symptoms in
Behcet's disease patients (Direskeneli et al., 2008) and mice (Lee et al.,
2004). In the present study, the change in the severity score of SK94
treated Behcet's disease mice was similar to that of the thalidomide
treated group. The decreasing inclination of severity score was steeper
in the SK126 treated group than the thalidomide treated group, which
demonstrates that SK 126 more effectively improves Behcet's disease
symptoms than thalidomide. Thalidomide efficiently decreases TNFα
and improves Behcet's disease symptoms, but it should not be
prescribed during pregnancy. Overall, SK126 is more effective and
safer than thalidomide and more convenient than Infliximab or
Etanercept as a treatment for Behcet's disease.
In conclusion, SK compounds effectively down-regulated cell
adhesion molecules at the mRNA and protein levels in HUVEC. SK
compounds also inhibited TNFα in an in vitro culture of RAW 264.7
cells and in vivo in Balb/c mice. The down-regulated cell adhesion
molecules and TNFα were correlated with the amelioration of
Behcet's disease symptoms in mice. These findings suggest that SK
compounds can be used as therapeutic agents to reduce the levels of
TNFα and adhesion molecules during the treatment of inflammatory
disease, especially through oral administration.
Kaburaki, T., Fujino, Y., Kawashima, H., Merino, G., Numaga, J., Chen, J., Matsushima, K.,
2003. Plasma and whole-blood chemokine levels in patients with Behcet's disease.
Graefes Arch. Clin. Exp. Ophthalmol. 241, 353–358.
Kang, K., Kim, H., Kim, K.I., Yang, Y., Yoon, D.Y., Kim, J.H., Ryu, J.H., Noh, E.J., Jeon, S.D.,
Lim, J.S., 2008. SK-126,
a synthetic compound, regulates the production of
inflammatory cytokines induced by LPS in antigen-presenting cells. Biochem.
Pharmacol. 75, 1054–1064.
Kim, H.J., Bang, D., Lee, S.H., Yang, D.S., Kim, D.H., Lee, K.H., Lee, S., Kim, H.B., Hong, W.P.,
1988. Behcet's syndrome in Korea: a look at the clinical picture. Yonsei Med. J. 29,
72–78.
Klimiuk, P.A., Sierakowski, S., Domyslawska, I., Chwiecko, J., 2009. Effect of etanercept
on serum levels of soluble cell adhesion molecules (sICAM-1, sVCAM-1, and sE-
selectin) and vascular endothelial growth factor in patients with rheumatoid
arthritis. Scand. J. Rheumatol. 38, 439–444.
Kose, O., Stewart, J., Waseem, A., Lalli, A., Fortune, F., 2008. Expression of cytokeratins,
adhesion and activation molecules in oral ulcers of Behçet's disease. Clin. Exp.
Dermatol. 33, 62–69.
Lee, E.S., Lee, S., Bang, D., Sohn, S., Park, C., Lee, K., 1993. Herpes simplex virus detection
by polymerase chain reaction in intestinal ulcer of patients with Behcet's disease. J.
Invest. Dermatol. 101, 474.
Lee, E.S., Lee, S., Bang, D., Sohn, S., 1996a. Detection of herpes simplex virus DNA by
polymerase chain reaction in genital ulcer of patients with Behcet's disease. Rev.
Rhum. 63, 532.
Acknowledgements
Lee, S., Bang, D., Cho, Y.H., Lee, E.S., Sohn, S., 1996b. Polymerase chain reaction reveals
herpes simplex virus DNA in saliva of patients with Behcet's disease. Arch.
Dermatol. Res. 288, 179–183.
This work was supported by grant no. R01-2008-000-20474-0 from
the Basic Research Program of the Korea Science & Engineering
Foundation, KRF-2008-531-E00024 from the Korea Research Foundation,
KRF-2007-313-E00349 from MOEHRD, 2010–0011130 from the Ministry
of Education, Science and Technology, and the SK Chemical Life Science
R&D Center.
Lee, E.S., Kim, Y.A., Kwon, H.J., Bang, D., Lee, S., Sohn, S., 2004. Thalidomide upregulates
macrophage inflammatory protein-1alpha in
a herpes simplex virus-induced
Behçet's disease-like animal model. Arch. Dermatol. Res. 296, 175–181.
Levälampi, T., Honkanen, V., Lahdenne, P., Nieminen, R., Hakala, M., Moilanen, E., 2007.
Effects of infliximab on cytokines, myeloperoxidase, and soluble adhesion
molecules in patients with juvenile idiopathic arthritis. Scand. J. Rheumatol. 36,
189–193.
Maus, U., Henning, S., Wenschuh, H., Mayer, K., Seeger, W., Lohmeyer, J., 2002. Role of
endothelial MCP-1 in monocyte adhesion to inflamed human endothelium under
physiological flow. Am. J. Physiol. Heart Circ. Physiol. 283, H2584–H2591.
Miles, E.A., Thies, F., Wallace, F.A., Powell, J.R., Hurst, T.L., Newsholme, E.A., Calder, P.C.,
2001. Influence of age and dietary fish oil on plasma soluble adhesion molecule
concentrations. Clin. Sci. (Lond.) 100, 91–100.
Sánchez Román, J., Castillo Palma, M.J., Torronteras Santiago, R., Varela Aguilar, J.M.,
López Checa, F., Sánchez García, F., 1992. Type I herpes virus, HLA phenotype and
Behçet disease. Med. Clin. (Barc) 98, 366–368.
Sayinalp, N., Ozcebe, O.I., Ozdemir, O., Haznedaroğlu, I.C., Dündar, S., Kirazli, S., 1996.
Cytokines in Behçet's disease. J. Rheumatol. 23, 321–322.
Schmidt, H., Rush, B., Simonian, G., Murphy, T., Hsieh, J., Condon, M., 1996. Thalidomide
inhibits TNF response and increases survival following endotoxin injection in rats. J.
Surg. Res. 63, 143–146.
Sfikakis, P.P., Theodossiadis, P.G., Katsiari, C.G., Kaklamanis, P., Markomichelakis, N.N.,
2001. Effect of infliximab on sight-threatening panuveitis in Behçet's disease.
Lancet 358, 295–296.
Shimizu, T., 1979. Behcet disease (Behcet syndrome). Semin. Arthritis Rheum. 8,
223–260.
Sohn, S., Lee, E.S., Bang, D., Lee, S., 1998. Behcet's disease-like symptoms induced by the
Herpes simplex virus in ICR mice. Eur. J. Dermatol. 8, 21–23.
References
Arnaout, M.A., 1993. Cell adhesion molecules in inflammation and thrombosis: status
and prospects. Am. J. Kidney Dis. 21, 72–76.
Bang, D., 1997. Treatment of Behçet's disease. Yonsei Med. J. 38, 401–410.
Bang, D., Yoon, K.H., Chung, H.G., Choi, E.H., Lee, E.S., Lee, S., 1997. Epidemiological and
clinical features of Behcet's disease in Korea. Yonsei Med. J. 38, 428–436.
Behcet, H., 1937. Ueber rezidivierende, aphthoese durch ein Virus verursachte Geschwuere
am Mund, am Auge und an den Genitalien. Dermatol. Wochenschr. 105, 1152–1157.
Brouckaert, P., Libert, C., Everaerdt, B., Takahashi, N., Cauwels, A., Fiers, W., 1993. Tumor
necrosis factor, its receptors and the connection with interleukin 1 and interleukin
6. Immunobiology 187, 317–329.
Cantarini, L., Tinazzi, I., Caramaschi, P., Bellisai, F., Brogna, A., Galeazzi, M., 2009. Safety
and efficacy of Etanercept in children with juvenile-onset Behcets disease. Int. J.
Immunopathol. Pharmacol. 22, 551–555.
Choi, B., Hwang, Y., Kwon, H.J., Lee, E.S., Park, K.S., Bang, D., Lee, S., Sohn, S., 2008. Tumor
necrosis factor alpha small interfering RNA decreases herpes simplex virus-induced
inflammation in a mouse model. J. Dermatol. Sci. 52, 87–97.
Direskeneli, H., Ergun, T., Yavuz, S., Hamuryudan, V., Eksioglu-Demiralp, E., 2008.
Thalidomide has both anti-inflammatory and regulatory effects in Behcet's disease.
Clin. Rheumatol. 27, 373–375.
Edwards, C.K., 2004. Drug discovery and development for inflammatroy diseases.
Expert Opin. Ther. Targets 8, 151–163.
Gerszten, R.E., Garcia-Zepeda, E.A., Lim, Y.C., Yoshida, M., Ding, H.A., Gimbrone Jr., M.A., Luster,
A.D., Luscinskas, F.W., Rosenzweig, A., 1999. MCP-1 and IL-8 trigger firm adhesion of
monocytes to vascular endothelium under flow conditions. Nature 398, 718–723.
Guglielmotti, A., D'Onofrio, E., Coletta, I., Aquilini, L., Milanese, C., Pinza, M., 2002.
Amelioration of rat adjuvant arthritis by therapeutic treatment with bindarit, an
inhibitor of MCP-1 and TNF-alpha production. Inflamm. Res. 51, 252–258.
Hamuryudan, V., Mat, C., Saip, S., Ozyazgan, Y., Siva, A., Yurdakul, S., Zwingenberger, K.,
Yazici, H., 1998. Thalidomide in the treatment of the mucocutaneous lesions of the
Sohn, S., Bang, D., Lee, E.S., Kwon, H.J., Lee, S.I., Lee, S., 2001. Experimental studies on the
antiviral agent famciclovir in Behcet's disease symptoms in ICR mice. Br. J.
Dermatol. 145, 799–804.
Van der Meide, P.H., Schellekens, H., 1996. Cytokines and the immune response.
Biotherapy 8, 243–249.
Wallach, D., Kovalenko, A., 2009. 12th international TNF conference: the good, the bad
and the scientists. Cytokine Growth Factor Rev. 20, 259–269.
Williams, L.M., Lali, F., Willetts, K., Balague, C., Godessart, N., Brennan, F., Feldmann, M.,
Foxwell, B.M., 2008. Rac mediates TNF-induced cytokine production via modula-
tion of NF-κB. Mol. Immunol. 45, 2446–2454.
Behçet syndrome.
A randomized, double-blind, placebo-controlled trial. Ann.
Intern. Med. 128, 443–450.