Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 3

Article abstract of DOI:10.1016/j.bmcl.2010.06.122

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position o

Full text of DOI:10.1016/j.bmcl.2010.06.122

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4774–4778
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetrahydropyridine derivatives with inhibitory activity on the production
of proinflammatory cytokines: Part 3
b
c
d
d
Akira Nakao a, , Nobuyuki Ohkawa , Takayoshi Nagasaki , Takashi Kagari , Hiromi Doi ,
*
Takaichi Shimozato d, Shigeru Ushiyama e, Kazumasa Aoki a
a Lead Discovery & Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b Lead Discovery & Optimization Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Research Department I, Daiichi Sankyo RD Associe Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Frontier Research Laboratories, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
e R&D Planning Department, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cyto-
Received 20 May 2010
Revised 21 June 2010
Accepted 23 June 2010
Available online 1 July 2010
kines such as TNFa, IL-1b, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetra-
hydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological
activities were evaluated. The derivatives were found to have potent inhibitory activities on the production
of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-
(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most prom-
ising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition
Keywords:
Anti-rheumatic drug
p38 MAP kinase
of p38
tion of TNF
respectively; (iii) inhibition of LPS induced TNF
a
, p38b, p38
, IL-1b, IL-6, and IL-8 production in human whole blood: IC50 = 0.026, 0.020, 0.88, and 0.016
, IL-1b and IL-6 production in mice: ID50 = 0.93, 8.63, and
c
, and p38d MAP kinases: IC50 = 0.034, 0.572, >10, and >10
l
M, respectively; (ii) inhibi-
a
lM,
Proinflammatory cytokine
a
TNFa
0.11 mg/kg, po, respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice:
ID50 = 2.22 mg/kg, po; (v) inhibition of collagen-induced arthritis in mice: ID50 = 2.38 mg/kg, po; (vi) pro-
phylactic effect on adjuvant-induced arthritis in rats: ID50 = 3.1 mg/kg, po; (vii) therapeutic effect on adju-
vant-induced arthritis in rats: ID50 = 4.9 mg/kg, po; (viii) analgesic effect on adjuvant-induced arthritic pain
in rats: ID50 = 2.9 mg/kg, po). As a result, compound 4a was chosen as a candidate for further pre-clinical
studies.
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
p38 mitogen-activated protein (MAP) kinase is an intracellular
We have previously reported that a series of 3-pyridylpyrrole
derivatives possessing N-alkyl- or N, -dialkyltetrahydropyridine
moiety at the 4-position of the pyrrole ring potently inhibits the pro-
duction of the proinflammatory cytokine TNF
(Table 1),9 and its
in vitro and in vivo activities are suggested to be significantly af-
fected by steric hindrance around the N- and/or -position of the
tetrahydropyridine (THPy) moiety and lipophilicity of the mole-
cules, respectively. Based on these results, we attempted to gain
superior compounds which exhibit more potent pharmacological
activities both in vitro and in vivo compared with the reported deriv-
atives. Compound 1m was selected as a lead compound for further
optimization because it showed the most potent in vitro activity.
Herein, we report the results of the optimization research that
have led us to the identification of the promising compound R-
132811 (4a), which was chosen as a candidate for further pre-clin-
ical studies to develop a new class of anti-rheumatic drug.
serine/threonine (Ser/Thr) kinase that positively regulates the pro-
duction and action of several proinflammatory mediators, specifi-
a
cally the release of TNF
a
and IL-1b1 in response to stress.2 TNF
a
a
and IL-1b are associated with the onset of inflammatory diseases
and several autoimmune diseases3 including rheumatoid arthritis
(RA),4 toxic shock syndrome, osteoarthritis, and inflammatory bo-
wel disease.5,6 The recent success of anti-cytokine biological agents
has demonstrated clinical benefits in the treatment of inflamma-
tory diseases.7 However, due to the well known disadvantages
common to these protein-based therapies, such as high cost and
subcutaneous or intravenous administration, orally active small
molecules that can effectively act as anti-cytokine agents would
clearly be of additional benefit to patients.6 Small molecule inhib-
itors of p38 MAP kinase have been shown to be efficacious in clin-
ical studies as alternatives for these biological agents.8
a
First of all, we designed a series of compound 1m derivatives, in
which an N-alkyl group is linked with an
clic THPy ring to reduce the steric hindrance and lipophilicity (Fig. 1).
a-alkyl group to form a bicy-
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8609.
E-mail address: nakao.akira.g5@daiichisankyo.co.jp (A. Nakao).
0960-894X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.122

A. Nakao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4774–4778
4775
Table 1
In vitro, in vivo activities and C log P values of N-substituted or N,
a-disubstituted tetrahydropyridine derivatives
a
b
Compd
R1
R2
R3
IC50
(l
M)
ID50 (mg/kg)
C log Pc
1a
1b
1c
1d
1e
1f
1g
1h
1i
Me
Et
H
H
H
H
H
H
H
di-Me
allyl
Bn
Me
Me
Me
H
H
H
H
H
H
H
di-Me
H
H
H
H
H
3.96 (2.63–5.97)
1.21 (0.72–2.05)
0.98 (0.75–1.28)
0.52 (0.29–0.95)
1.38 (0.77–2.48
>10
2.89
6.67
6.18
6.75
45%d
3.48
4.01
4.32
4.54
7.19
4.72
5.27
5.56
4.58
5.57
3.83
4.53
5.06
i-Pr
n-Pr
n-Octyl
t-Bu
Bn
Me
Me
Me
Me
e
—
e
>30
-
8.44 (5.26–13.54)
7.27
48.7%f
—
—
e
e
1j
1k
1l
30.8%f
0.63 (0.42–0.93)
1.61 (0.94–2.75)
0.44 (0.32–0.61)
1.42
5.28
2.79
Et
n-Pr
1m
a
b
c
d
e
f
Inhibition of LPS-induced TNF
Inhibition of LPS-induced TNF
a
a
production in human whole blood. Results are given as mean and SD of three to four determinations.
production in mice N = 5.
C log P values calculated using PallasÒ (INFOCOM CORPORATION).
% Inhibition at 20 mg/kg.
Not tested.
Inhibition at 10 lM.
The derivatives with the bicyclic THPy ring (3a–d) were synthe-
sized in accordance with the established synthetic route, which has
been reported previously (Scheme 1).10 Introduction of the bicyclic
THPy group to the 4-position of the pyrrole ring was carried out by
bromine–lithium exchange of compound 2, followed by 1,2-addi-
tion with bicyclic aminoketone derivatives to form a tertiary alco-
hol. Subsequent dehydroxylation of the tertiary alcohol was carried
out by exposure to trifluoroacetic acid (TFA) concurrently with
deprotection of a triisopropylsilyl (TIPS) group with tetrabutylam-
monium fluoride (TBAF) to give the bicyclic THPy derivative 3a, b, c
or d. Regioisomers 3a and 3b were separated11 by silica gel column
chromatography and their structures were determined by mass
fragmentation studies (electron ionization method: EI-MS)
(Scheme 2). Regioisomers 3c and 3d also were separated and
determined in a similar manner.
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, À78 °C, then bicyclic
aminoketone derivatives, and then rt; (b) TFA, CH2Cl2, rt, then TBAF, THF, rt.
The bicyclic THPy derivatives 3a–d have two stereoisomers due
to an asymmetric center at the a-position of the bicyclic THPy moi-
The inhibitory activities of the bicyclic THPy derivatives on LPS-
induced TNF
a
production were evaluated in vitro and in vivo.9,10
ety. We separated the two stereoisomers of compound 3c by a pre-
parative HPLC method using a chiral column to gain the optically
pure enantiomers 4a (1st peak) and 4b (2nd peak) (Scheme 3). In
order to determine the absolute configuration of those enantio-
mers, one of them was synthesized from enantiomerically enriched
(8aS)-hexahydroindolizin-7(1H)-one 5.12 Chiral HPLC analysis indi-
cated that the synthesized compound corresponds with enantio-
mer 4a. Thus, it was confirmed that enantiomer 4a has an (S)-
configuration (Scheme 4), and therefore enantiomer 4b has an
(R)-configuration.
Their IC50s, ID50s and C log Ps13 are summarized together with
those of compound 1m in Table 2.
Compound 3a, which has a six-membered ring within the bicy-
clic THPy moiety and a lower C log P value (4.23), showed more po-
tent in vitro and in vivo activities than those of the corresponding
monocyclic THPy analog 1m (IC50: 0.31 vs 0.44 lM; ID50: 2.27 vs
2.79 mg/kg). Compound 3c with a five-membered ring and further
lowered C log P value (3.67) showed much more potent in vitro and
in vivo activities than those of the corresponding monocyclic THPy
analog, compound 1l (IC50: 0.042 vs 1.61
5.28 mg/kg). Compound 3c exceeded even compound 3a in both
in vitro and in vivo activities (IC50: 0.042 vs 0.31 M; ID50: 1.09
lM; ID50: 1.09 vs
l
vs 2.27 mg/kg). In addition, it is noteworthy that the in vitro activ-
ity of compound 3c was sevenfold as potent as that of compound
3a. This remarkable increase in the in vitro activity of compound
3c is supposed to be mainly attributed to further reduction of the
steric hindrance due to the five-membered ring formation in its
bicyclic THPy moiety. The bicyclic THPy ring of compound 3c is
thought to have a more rigid conformation than that of compound
Figure 1.

4776
A. Nakao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4774–4778
Scheme 2. Proposed pathway for generation of m/z 344 from compound 3a and generation of m/z 290 from compound 3b.
Table 2
In vitro, in vivo activities and C log P values of bicyclic tetrahydropyridine derivatives
ID50 (mg/kg) C log Pc
a
b
Compd
R
IC50
(l
M)
1m
0.44 (0.32–0.61)
0.31 (0.26–0.38)
0.81 (0.59–1.10)
1.61 (0.94–2.75)
2.79
2.27
3.55
5.28
5.06
4.23
4.23
4.53
3.67
3a
3b
1l
Scheme 3. Reagents and conditions: (a) HPLC separation. Column, CHIRALPAK AD
(
U
4.6 Â 250 mm, DAICEL CHEMICAL INDUSTRIES, LTD, Japan), eluent with n-
hexane/EtOH 80:20. The flow rate was 1.0 mL/min and the temperature was
adjusted to 40 °C. UV detection was performed at 254 nm.
3c
0.042 (0.028–0.063) 1.09
3d
0.60 (0.40–0.91)
2.85
3.67
a
Inhibition of LPS-induced TNF
a production in human whole blood. Results are
given as mean and SD of three to four determinations.
b
Inhibition of LPS-induced TNFa production in mice N = 5.
c
C log P values calculated using PallasÒ (INFOCOM CORPORATION).
Scheme 4. Reagents and conditions: (a) n-BuLi, THF, À78 °C, then (8aS)-hexahy-
droindolizin-7(1H)-one, and then rt; (b) TFA, CH2Cl2, rt, then TBAF, THF, rt.
Biological activities of compounds 3c (racemate), 4a ((S)-enan-
tiomer) and 4b ((R)-enantiomer) were examined (Table 3).
(S)-Enantiomer 4a was found to be much more active than (R)-
enantiomer 4b, that is (S)-enantiomer 4a was 40- and 7-fold as po-
tent as (R)-enantiomer 4b in vitro and in vivo, respectively.
In order to assess the potential of compound 4a for a novel type
of anti-rheumatic drug, we performed a variety of pharmacological
tests related to proinflammatory cytokine production and rheuma-
toid arthritis (Table 4).
3a because it contains a five-membered ring. That rigid conforma-
tion of the bicyclic THPy moiety might also contribute to the excel-
lent in vitro activity of compound 3c. The increase in the in vivo
activities of compound 3a and 3c is probably due to both the in-
creased in vitro activity and the improved pharmacokinetic charac-
ter brought about by the lower lipophilicity.
Compounds 3b and 3d are regioisomers of compounds 3a and
3c, respectively, and they are distinguished from their counterparts
based on the position of C–C double bond within their bicyclic
THPy rings. Both compounds 3b and 3d showed lower in vitro
and in vivo activities compared with their counterpart, as expected
based on the previous letter.9
There have been four isoforms of p38 MAP kinase, namely,
- and d-isoforms,14 that have been identified so far. Compound
4a inhibited -isoform potently (IC50 = 0.034 M) and b-isoform
weakly (IC50 = 0.572 M). On the other hand, it inhibited neither
a-,
b-,
c
a
l
l

A. Nakao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4774–4778
4777
Table 3
kg and 4.9 mg/kg, respectively). Interestingly, compound 4a also
showed analgesic effect on adjuvant-induced arthritic pain in
rats24–26 (ID50 = 2.9 mg/kg). Thus, compound 4a was found to
In vitro and in vivo activities of hexahydroindolizine derivatives 3c, 4a, and 4b
selectively inhibit p38
proinflammatory cytokines such as TNF
a
MAP kinase, block the production of the
, IL-1b, IL-6 and IL-8,
a
and exhibit the inhibitory, prophylactic, therapeutic or analgesic
effect on rheumatoid arthritis in animals.
In order to develop a new class of anti-rheumatic drug, which
inhibits the production of the proinflammatory cytokines such as
TNFa, IL-1b, IL-6, and IL-8, we synthesized a series of 3-pyridylpyr-
a
b
Compd
R
IC50
(lM)
ID50 (mg/kg)
role derivatives possessing a bicyclic THPy moiety at the 4-position
of the pyrrole ring and evaluated its pharmacological activities.
Compound3c witha specific bicyclic THPy ring, namely, the hexahy-
droindolizine ring, showed excellent in vitro and in vivo activities.
(S)-Enantiomer 4a separated from racemic compound 3c showed
much more potent inhibitory activities than those of (R)-enantiomer
4b. We evaluated compound 4a in various pharmacological assay
systems related to proinflammatory cytokine production and rheu-
matoid arthritis, and thus gained promising results. Therefore,
compound 4a (R-132811) was chosen as the candidate for further
pre-clinical studies such as pharmacological, pharmacokinetic,
toxicological, and physicochemical studies.
3c
0.042 (0.028–0.063)
0.026 (0.018–0.041)
49%c
1.09
0.93
6.59
4a
4b
a
Inhibition of LPS-induced TNF
given as mean and SD of three to four determinations.
a production in human whole blood. Results are
b
Inhibition of LPS-induced TNFa production in mice N = 5.
Inhibition at 1 lM.
c
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.122.
c
-isoform nor d-isoform even at 10
inhibited not only the production of TNF
also that of other proinflammatory cytokines such as IL-1b, IL-6,
and IL-816 in vitro (IC50 = 0.020, 0.88, and 0.016
M, respectively).
l
M.15 Compound 4a potently
(IC50 = 0.026 M), but
a
l
References and notes
l
1. (a) Adamus, J. L.; Badger, A. M.; Kumar, S.; Lee, J. C. Prog. Med. Chem. 2001, 38, 1;
(b) Lee, J. C.; Kumar, S.; Griswold, D. E.; Underwood, D. C.; Votta, B. J.; Adams, J.
L. Immunopharmacology 2000, 47, 185.
2. (a) Chen, Z.; Gibson, T. B.; Robinson, F.; Silvestro, L.; Pearson, G.; Xu, B.; Wright,
A.; Vanderbilt, C.; Cobb, M. H. Chem. Rev. 2001, 101, 2449; (b) Raingeaud, J.;
Gupta, S.; Rogers, J. S.; Dickens, M.; Han, J.; Ulevitch, R. J.; Davis, R. J. J. Biol.
Chem. 1995, 270, 7420.
Also in the in vivo activities, compound 4a potently inhibited the
production of TNFa10,17 and IL-619 (ID50 = 0.93 and 0.11 mg/kg,
respectively). Compound 4a was effective on both anti-collagen
antibody-induced arthritis20 and collagen-induced arthritis in
mice16,21 (ID50 = 2.22 and 2.38 mg/kg, respectively). Furthermore,
compound 4a demonstrated not only prophylactic effect, but also
therapeutic effect on adjuvant arthritis in rats22,23 (ID50 = 3.1 mg/
3. Vervoordeldonk Margriet, J. B. M.; Tak, P. P. Curr. Rheumatol. Rep. 2002, 4, 208.
4. Foster, M. L.; Halley, F.; Souness, J. E. Drug News Perspect. 2000, 13, 488.
Table 4
Representative results of pharmacological tests on compound 4a
Biological evaluations
Inhibition of p38 MAP kinasea IC50
(lM)
p38a15
p38b15
p38c15
p38d15
TNFa11,16
IL-1b16
IL-616
0.034 (0.028–0.042)
0.572 (0.281–1.165)
>10c
>10c
Inhibition of LPS-induced cytokine production in human whole blooda IC50
Inhibition of LPS-induced cytokine production in miceb ID50 (mg/kg)
(l
M)
0.026 (0.018–0.041)
0.020 (0.016–0.026)
0.88 (0.62–1.3)
0.016 (0.011–0.022)
0.93
8.63
0.11
2.22
2.38
3.1
IL-816
TNFa11,17
IL-1b18
IL-619
Inhibition of anti-collagen antibody-induced arthritis in miceb,20 ID50 (mg/kg)
Inhibition of collagen-induced arthritis in miceb,5,16,21 ID50 (mg/kg)
Prophylactic effect on adjuvant arthritis in ratsb,22 ID50 (mg/kg)
Therapeutic effect on adjuvant arthritis in ratsb,23 ID50 (mg/kg)
Analgesic effect on adjuvant-induced arthritic pain in ratsb,24 ID50 (mg/kg)
4.9
2.9
a
b
c
Results are given as mean and SD of three to four determinations.
N = 5.
No inhibition at 10 lM.

4778
A. Nakao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4774–4778
5. Wagner, G.; Laufer, S. Med. Res. Rev. 2006, 26, 1.
volume of normal animals. % Inhibition = {1 À (swelled foot volume of animal
on days 21)/(mean swelled foot volume of the control group on day 21)} Â 100
23. Adjuvant arthritis was induced as described above. On day 18, adjuvant
arthritis was established in most adjuvant-injected animals and the swelled
foot volume almost reached a plateau. We selected animals with prominent
swelling of the adjuvant-injected foot (foot volume P 2.75 mL). These animals
were divided into groups so that the mean value of the adjuvant-injected foot
volume in each group was about the same. The test compounds were
suspended in 0.5% CMC aqueous solution at the appropriate concentrations,
and orally administered at 5 mL/kg twice a day from day 18 to day 24 (7 days).
The volume of both feet was measured on days 18, 20, 23, and 25 (0, 2, 5, and
7 days after the first administration) by a plethysmometer (Ugo Basile) by
soaking the hind paw from the toe to the hairline in the bath of the
plethysmometer. The swelled foot volume and the ratio of the swelled foot
volume were calculated with the equations below, and the % inhibition against
control group was obtained. We used the volume of adjuvant-injected foot
(right hind foot) for the calculation because the swelling of the adjuvant-
injected foot was more prominent and stable than that of the uninjected foot.
The swelled foot volume (mL) = right hind foot volume of animals À mean
right hind foot volume of normal animals. The ratio of swelled foot
volume = {(right hind foot volume of animals on day 25 À mean right hind
foot volume of normal animal on day 25)/(right hind foot volume of animals on
day 18 À mean right hind foot volume of normal animal on day 18)} Â 100
24. The experiment was performed according to the method by Kuzuna et al.
(Kuzuna, S.; Kawai, K. Chem. Pharm. Bull. 1975, 23, 1184) with slight
modifications. Heat-killed, dried M. butyricum was ground with an agate
mortar, and was suspended in dry, sterilized liquid paraffin to make a 4.0 mg/
mL suspension. Then, the suspension was sonicated. Adjuvant arthritis was
6. Westra, J.; Limburg, P. C. Mini-Rev. Med. Chem. 2006, 6, 867.
7. (a) Papadakis, K. A. Expert Rev. Clin. Immunol. 2006, 2, 11; (b) Mease, P. J. Expert
Opin. Biol. Ther. 2005, 5, 1491; (c) Lee, S. J.; Kavanaugh, A. Therapy 2005, 2, 13.
8. (a) Gaestal, M.; Mengel, A.; Bothe, U.; Asadullah, K. Curr. Med. Chem. 2007, 14,
2214; (b) Peifer, C.; Wagner, G.; Laufer, S. A Curr. Top. Med. Chem. 2006, 6, 113;
(c) Margutti, S.; Laufer, S. A. Chem. Med. Chem. 2007, 2, 1116.
9. Nakao, A.; Ohkawa, N.; Nagasaki, T.; Doi, H.; Shimozato, T.; Ushiyama, S.; Aoki,
K. Bioorg. Med. Chem. Lett. 2010, 20, 2435.
10. Nakao, A.; Ohkawa, N.; Nagasaki, T.; Doi, H.; Shimozato, T.; Ushiyama, S.; Aoki,
K. Bioorg. Med. Chem. Lett. 2009, 16, 4607.
11. The crude ratio of regioisomers was approximately 1:1 by NMR studies. The
isolated yield is indicated in Scheme 1.
12. Harrison, J. R.; O’Brien, P.; Porter, D. W.; Smith, N. M. J. Chem. Soc., Perkin Trans.
1 1999, 24, 3623.
13. The program, PALLAS from Infocom Corporation, was used to calculate the log P
value.
14. John, M. K.; Joseph, A. Physiol. Rev. 2001, 81, 807.
15. (a) Cuenda, A.; Rouse, J.; Doza, Y. N.; Meier, R.; Cohen, P.; Gallagher, T. F.;
ˇ
´
ˇ
´
Young, P. R.; Lee, J. C. FEBS Lett. 1995, 364, 229; (b) Baranccıka, M.; Bohaccovab,
ˇ
´
´
ˇ
´
V.; Kvacckajova, J.; Hudecova, S.; Krizcanova, O.; Breierb, A. Eur. J. Pharm. Sci.
2001, 14, 29.
16. Nerad, J. L.; Griffiths, J. K.; Van der Meer, J. W. M.; Endres, S.; Poutsiaka, D. D.;
Keusch, G. T.; Bennish, M.; Salam, M. A.; Dinarello, C. A.; Cannon, J. G. J.
Leukocyte Biol. 1992, 52, 687.
17. Badger, A. M.; Bradbeer, J. N.; Votta, B.; Lee, J. C.; Adams, J. J.; Griswold, D. E. J.
Pharmacol. Exp. Ther. 1996, 279, 1453.
18. Griffiths, R. J.; Stam, E. J.; Downs, J. T.; Otterness, I. G. J. Immunol. 1995, 154,
2821.
induced in rats by injecting the adjuvant (M. butyricum, 200 lg/0.05 mL/paw)
19. Bryniarski, K.; Ptak, M.; Ptak, W. Immunopharmacology 1996, 34, 73.
20. Terato, K.; Hasty, K. A.; Reife, R. A.; Cremer, M. A.; Kang, A. H.; Stuart, J. M. J.
Immunol. 1992, 148, 2103.
21. Terato, K.; Harper, D. S.; Griffiths, M. M.; Hasty, D. L.; Ye, X. J.; Cremer, M. A.;
Seyer, J. M. Autoimmunity 1995, 22, 137.
22. The experiment was carried out according to the method described by Winder
et al. (Winder, C. V.; Lembke, L. A.; Stephens, M. D. Arthritis Rheum. 1969, 12,
472–482). Heat-killed, dried Mycobacterium butyricum was ground with an
agate mortar, and then suspended in dry-sterilized liquid paraffin to make a
4.0 mg/mL suspension. Then, this suspension was sonicated. Adjuvant arthritis
intradermally in the heel of the right hind paw on day 0. The rats with well-
established arthritis were fasted overnight on day 17. On the day of the
experiment (day 18), ‘pain-positive’ rats were selected as follows: the tarso-
tibial joint of the uninjected foot of rats was gently flexed five times at intervals
of 4–5 s, and rats squeaking at every flection were defined as ‘pain-positive’.
The pain-positive rats were then divided into groups at 0.5, 1, 2, 4, 6, 8, 24, 48,
and 72 h after administration in the same way. The pain score of each rat was
recorded by counting the number of times the animal squeaked. Statistical
analysis: IC50 and ID50 values were calculated from liner regression curves
obtained from the % inhibition and the logarithmic value of the concentrations
or dosages of the test compounds by the last-squares method. The 95%
confidence intervals (C.I.) were calculated by Fieller’s theorem. ID30 value (dose
which decreased the ratio of swelled foot volume of the control group by 30%)
was induced in rats by injecting the adjuvant (M. butyricum, 200 lg/0.05 mL/
paw) intradermally in the heel of the right hind paw on day 0. The test
compounds were suspended in 0.5% CMC aqueous solution at the appropriate
concentrations, and orally administrated at 5 mL/kg once a day from day 0
through day 20. The vehicle was administrated to the control group. The
volume of both feet was measured on days 3, 5, 7, 10, 13, 15, 18, and 21 by a
plethysmometer (Ugo Basile) by soaking the hind paw from the toe to the
hairline in the bath of the plethysmometer. The swelled foot volume and the %
inhibition of treated animals over the control on day 21 were calculated with
the equations below. We used the volume of adjuvant-injected foot (right hind
foot) for the calculation because the swelling of the adjuvant-injected foot was
more prominent and stable than that of the uninjected foot. The swelled foot
volume (mL) = right hind foot volume of animals À mean right hind foot
was calculated by the following equation: the
% inhibition = 60/[1 + exp
{Àb0 À b1 log (dose)}] (b0 and b1; parameters to be estimated). IC50 and ID50
values, and their 95% C.I. values were calculated using the SAS System for
Windows (SAS Institute Inc.).
25. Wada, Y.; Nakajima-Yamada, T.; Yamada, K.; Tsuchida, J.; Yasumoto, T.;
Shimozato, T.; Aoki, K.; Kimura, T.; Ushiyama, S. Eur. J. Pharm. Sci. 2005, 506,
285.
26. Brown, K. K.; Heitmeyer, S. A.; Hookfin, E. B.; Hsieh, L.; Buchalova, M.; Taiwo, Y.
O.; Janusz, M. J. J. Inflamm. 2008, 5, 22.