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K. Tanaka et al. / Bioorg. Med. Chem. Lett. 12 (2002) 623–627
weeks old) were purchased from Charles River Japan
Inc. (Yokohama, Japan).
the booster injection. The severity of arthritis was eval-
uated by scoring each paw with an integer from 0 to 4
based on increasing erythema and swelling (0: normal;
16: maximum).13
Proliferation assay
HUVEC purchased from Sankou Junyaku (Tokyo,
Japan) were suspended in RPMI-1640 medium (Nissui
Pharmaceutical Co. Ltd, Tokyo, Japan) supplemented
with 2% FBS, 100 U/mL of penicillin, 100 mg/mL of
streptomycin and an inducer: 10 mg/mL of ECGS
(including 3 ng/mL of epidermal growth factor) or 10
ng/mL of bFGF or 10 ng/mL of VEGF. Three thou-
sand cells were seeded in each well of 96-well flat plates
with or without test compounds, including 0.3% dimethyl-
sulfoxide (Nacalai Tesque, Kyoto, Japan). NHDF
(Kurabo, Osaka, Japan) and KB cells (American Type
Culture Collection, Rockville, MD, USA) were cultured
in modified MCDB-202 medium (F-BM medium; Kur-
abo, Osaka, Japan) with 20% FBS and Eagle’s minimal
essential medium (Nissui Pharmaceutical Co. Ltd,
Tokyo, Japan) with 10% FBS, respectively. Concentra-
tions of growth factors including FBS were determined
by preliminary experiments to give submaximum pro-
liferation responses. They were cultured for 96 h, and
the cultures were pulsed with 3.7 kBq of [3H]-thymidine
for the last 17 h of culture, followed by harvest on glass
filter and washing with phosphate buffered saline, 5%
trichloroacetate and ethanol. The uptake of [3H]-thymi-
dine was counted by Betaplate (Pharmacia LKB Bio-
technology, Uppsala, Sweden) and expressed as cpm.
Test compounds and reagents
Magnosalin and magnoshinin were isolated from
magnolia by Professor Tohru Kikuchi in the Institute of
Natural Medicine (Oriental Medicines) of our uni-
versity.7 They were dissolved in dimethylsulfoxide
(Nacalai Tesque, Kyoto, Japan) for in vitro use. For in
vivo administration, TAS-202, prednisolone (Nacalai
Tesque, Kyoto, Japan) and indomethacin (Sigma Chemical
Co., St. Louis, MO, USA) were suspended in soy bean
oil. Fetal bovine serum (JRH Biosciences, Lenexa, KS,
USA), ECGS (Becton Dickinson Labware, Bedford,
MO, USA), EGF and bFGF (Genzyme Co., Cam-
bridge, MA, USA), VEGF (R&D Systems, Inc., Min-
neapolis, MN, USA) were also purchased.
Statistics
Data were expressed as the meansÆSEM. Differences
between the groups were evaluated by Dunnett’s test,
Welch’s t-test or Mann–Whitney U-test when indicated.
p Values less than 0.05 were considered significant.
Acknowledgements
Gelatin sponge-angiogenesis assay
We would like to thank Prof. Emeritus Tohru Kikuchi
[Research Institute of Natural Medicine (Oriental
Medicines) at Toyama Medical and Pharmaceutical
University] for kindly supplying magnosalin and
magnoshinin. We also thank Prof. Emeritus
Masayasu Kimura (Department of Chemical Pharma-
cology, Faculty of Pharmaceutical Sciences, Toyama
Medical and Pharmaceutical University) for the kind
support.
The assay was carried out according to the method
of Pesenti et al.11 with minor modifications. Briefly,
pellets containing bFGF were prepared by mixing an
equivalent volume of bFGF dissolved in 400 mg/mL of
aluminum sucrose octasulfate (Sucralfate; Nippon
Gouseikagaku Kogyo Co., Osaka, Japan) and poly(2-
hydroxyethyl methacrylate) (Hydron1 polymer Type
Ncc; Polysciences Inc., Warrington, PA, USA) dis-
solved in ethanol.12 After drying, the pellet was placed
in the center of a piece of gelatin sponge (Gelfoam1;
Nippon Upjohn, Tokyo, Japan). The sponge containing
bFGF or solvent alone was subcutaneously implanted
into the abdomen of C57BL/6j mice (one piece/one
mouse). Seven days after implantation, the mice were
sacrificed, and the sponge was carefully removed.
Hemoglobin content (Hb) was extracted from the
sponges in 0.1M ammonia solution, and measured
using a commercial assay kit (Hemoglobin B Test-
wako1, Wako Pure Chemical Industries Ltd, Osaka,
Japan). Test compounds were administered orally from
the day of implantation for 7 days.
References and Notes
1. Forkman, J. Nature Med. 1995, 1, 27.
2. Koch, A. E. Arthritis Rheum. 1998, 41, 951.
3. Firestein, G. S. J. Clin. Invest. 1999, 103, 3.
4. Brenchley, P. E. C. Clin. Exp. Immunol. 2000, 121, 426.
5. Stupack, D. G.; Storgard, C. M.; Cheresh, D. A. Braz. J.
Biol. Res. 1999, 32, 573.
6. Kimura, M.; Kobayashi, S.; Luo, B.; Kimura, I. Int. Arch.
Allergy Appl. Immunol. 1990, 93, 365.
7. Kikuchi, T.; Kadota, S.; Yanada, K.; Tanaka, K.; Wata-
nabe, K.; Yoshizaki, M.; Yokoi, T.; Shingu, T. Chem. Pharm.
Bull. 1983, 31, 1112.
Collagen-induced arthritis in mice
8. Kimura, I.; Nagaura, T.; Kobayashi, S.; Kimura, M.
Japan. J. Pharmacol. 1992, 60, 52.
Male DBA1/j mice were intradermally injected with 0.1
mL of complete Freund’s adjuvant containing 2 mg/mL
of bovine type II collagen (Collagen Technical Service,
Cosmo Bio, Tokyo, Japan). After 21days, the same
solution was injected intradermally to the base of the
tail. Test compounds were given orally for 14 days after
9. Characterization of TAS-202: mp 110–112 ꢁC; 1H NMR
(CDCl3, ref CHCl3=7.27 ppm) d 1.29 (t, J=7.0 Hz, 6H,
CH3), 3.77 (q, J=7.0 Hz, 4H, CH2), 3.90 (s, 3H, CH3O), 3.91
(s, 3H, CH3O), 3.92 (s, 3H, CH3O), 3.95 (s, 9H, CH3O), 6.61
(s, 1H, Ar–H), 7.37 (s, 2H, Ar–H), 7.61 (s, 1H, Ar–H), 7.80 (s,
1H, Ar–H); anal. (C26H33N3O6) C, H, N.