G. N. Ziakas et al. / Bioorg. Med. Chem. 13 (2005) 6485–6492
3. Hawkey, C. J. Gastroenterology 2000, 119, 521.
6491
fied via enzyme immunoassay using a broadly specific
antibody that binds to all the major prostaglandin com-
pounds. The inhibitory activity of the compounds was
measured at various concentrations of arachidonic acid
[0.1–1 lM]. The compounds were added to the reaction
mixture at a final concentration of 100 lM, unless other-
wise mentioned.
4. Wallace, J. L.; Carter, E.; McKnight, G. W.; Le, T.;
McCafferty, D. M.; Argentieri, D.; Capetola, R. Gastro-
enterology 1993, 104, A221.
5. Guslandi, M. Drugs 1997, 53, 1.
6. Soll, A. H.; Weinstein, W. M.; Kurata, J.; McCarthy, D.
M. Ann. Intern. Med. 1991, 114, 307.
7. Cash, J. M.; Klippel, J. H. New Engl. J. Med. 1994, 330,
1368.
8. Davies, N. M.; Wallace, J. L. J. Gastroenterol. 1997, 32,
127.
4.8. In vitro evaluation of lipoxygenase activity
9. Wallace, J. L. Gastroenterology 1997, 112, 1000.
10. Insel, P. A., 9th ed. In Goodman & billman’s The
Pharmacological Basis of Therapeutics; Hardman, J. G.,
Limbird, L. E., Eds.; McGraw-Hill: New York, 1995, pp
617–657.
11. Wallace, J. L.; Cirino, J. Trends Pharmacol. Sci. 1994, 15.
12. Xie, W.; Robertson, D. L.; Simmons, D. L. Drug Dev.
Res. 1992, 25, 249.
The reaction mixture contained (final concentration) the
test compounds, dissolved in propyleneglycol in concen-
trations of 10–300 lM, or the solvent (control), soybean
lipoxygenase, dissolved in 0.9% NaCl solution [250 u/
mL] and sodium linoleate [100 lM], in Tris/HCl buffer,
pH 9.0. The reaction was monitored for 7 min at
28 °C, by recording the absorbance at 234 nm. The per-
formance of the assay was checked using nord-
ihydroguaiaretic acid as a reference.54 under exactly
the same experimental conditions.
13. Vane, J. R. Nature 1994, 367, 215.
14. Hart, C. Modern Drug Discov. 1999, 2, 54.
15. Lawrence, R. C.; Helmick, C. G.; Arnett, F. C.; Deyo, R.
A.; Felson, D. T.; Giannini, E. H.; Heyse, S. P.; Hirsch,
R.; Hochberg, M. C.; Hunder, G. G.; Liang, M. H.;
Pillemer, S. R.; Steen, V. D.; Wolfe, F. Arthritis Rheum.
1998, 41, 778.
4.9. In vitro lipid peroxidation
The incubation mixture contained heat-inactivated rat
hepatic microsomal fraction (corresponding to 2.5 mg
of hepatic protein per milliliter or 4 mM fatty acid resi-
dues), ascorbic acid [0.2 mM] in Tris–HCl/KCl buffer
[50 mM/150 mM, pH 7.4], and the studied compounds
in dimethylsulfoxide at 1.0 mM. The peroxidation was
started with the addition of a freshly prepared FeSO4
solution [10 lM], and aliquots were taken from the incu-
bation mixture [37 °C] at various time intervals for
45 min. Lipid peroxidation was assessed by spectropho-
tometric [535 against 600 nm] determination of the 2-
thiobarbituric acid reactive material. All compounds
and solvents were tested and found not to interfere with
the assay.63
16. OÕNeil, G. P.; Ford-Hutchinson, A. W. FEBS Lett. 1993,
330, 156.
17. Peri, K. G.; Hardy, P.; Li, D. Y.; Varma, D. R.; Chemtob,
S. J. Biol. Chem. 1995, 270, 24615.
18. Gretzer, B.; Ehrlich, K.; Maricic, N.; Lambrecht, N.;
Respondek, M.; Peskar, B. M. Br J. Pharmacol. 1998, 123,
927.
19. Mc Adam, B. F.; Catella-Lawson, F.; Mardini, I. A.;
Kapoor, S.; Lawson, J. A.; FitzGerald, G. A. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 272.
20. Catella-Lawson, F.; Mc Adam, B.; Morrison, B. W.;
Kapoor, S.; Kujubu, D.; Antes, L.; Lasseter, K. C.; Quan,
H.; Gertz, B. J.; FitzGerald, G. A. J. Pharmacol. Exp.
Ther. 1999, 289, 735.
21. Belton, O.; Byrne, D.; Kearney, D.; Leahy, A.; FitzGer-
ald, D. J. Circulation 2000, 102, 840.
22. Mukherjee, D.; Nissen, S. E.; Topol, E. J. J. Am. Med.
Assoc. 2001, 286, 954.
4.10. In vitro interaction with the stable radical DPPH
23. Hennan, J. K.; Huang, J.; Barrett, T. D.; Driscoll, E. M.;
Willens, D. E.; Park, A. M.; Crofford, L. J.; Lucches, B. R.
Circulation 2001, 104, 820.
24. Dowd, N. P.; Scully, M.; Adderley, S. R.; Cunningham, A.
J.; FitzGerald, D. J. J. Clin. Invest. 2001, 108, 585.
25. Wallace, J. L.; Reuter, B.; Cicala, C.; McKnight, W.;
Grisham, M. B.; Cirino, G. Gastroenterology 1994, 107, 173.
26. Wallace, J. L.; Reuter, B.; Cicala, C.; McKnight, W.;
Grisham, M. B.; Cirino, G. Eur. J. Pharmacol. 1994, 257,
249.
Compounds, dissolved in absolute ethanol, were added
to an equal volume of an ethanolic solution of DPPH
[final concentration 0.2 mM] at room temperature
[22 2 °C]. Absorbance [517 nm] was recorded at differ-
ent time intervals for 4 h.64
Acknowledgments
27. Cuzzolin, L.; Conforti, A.; Donini, M.; Adami, A.; Del
Soldato, P.; Benoni, G. Pharmacol Res. 1994, 29, 89.
28. Reuter, B. K.; Cirino, G.; Wallace, J. L. Life Sci. 1994, 55,
PL1.
29. Feldman, P. L.; Griffith, O. W.; Stuehr, D. J. Chem. Eng.
News 1993, 26.
The authors thank ELPEN Pharmaceutical Co.
(Greece) for the donation of tolfenamic acid. G.N.Z.
wishes to thank the Greek General Secretariat of Re-
search and Technology (programme PAVET) for
financial support.
30. Bandarage, U. K.; Janero, D. R. Mini Rev. Med. Chem.
2001, 1, 57.
31. Ko, J. K.; Cho, C. H. Inflamm. Res. 1999, 48, 471.
32. Kubes, P.; Wallace, J. L. Med. Inflamm. 1995, 4, 397.
33. Alican, I.; Kubes, P. Am. J. Physiol. 1996, 270, G225.
34. Elliott, S. N.; Wallace, J. L. J. Gastroenterol. 1998, 33,
792.
References and notes
1. Garner, A. Scand. J. Gastroenterol. 1992, 27(Suppl. 193),
83.
35. Wallace, J. L. Can. J. Physiol. Pharmacol. 1993, 71,
98.
2. Smalley, W. E.; Ray, W. A.; Daugherty, J. R.; Griffin, M.
R. Am. J. Epidemiol. 1995, 141, 539.