SOYBEANLIPOXYGENASEINHIBITION BY NONSTEROIDAL ANTI INFLAMMATORY DRUGS
C. Sircarl, Elizabeth
Charles F. Schwender Anne Johnson.
of Chemistry, Pharmaceutical
Warner-Lambert/Parke-Davis Research Division
Company; Ann Arbor,
Abstract Eighteen known nonsteroidal antiinflammatory drugs (NSAID) were tested for their action against soybean lipoxygenase (E.C.22.214.171.124) using linoleic acid as substrate. It was found that the best inhibitors of lipoxygenase were naproxen, BW 755C, indomethacin and isoxicam. Drugs with intermediate potency were meclofenamic acid, phenylbutazone and benoxaprofen. Other drugs such as ibuprofen and zomepirac were only weakly active in the test. Introduction Arachidonic acid is metabolized by lipoxygenases in addition to cyclooxygenase (l-2). It seemed of value to examine the effects on a lipoxygenase enzyme by several drugs (3) known to inhibit cyclooxygenase. We would like to report the inhibition activity of 18 nonsteroidal antiinflammatory drugs (NSAID) on soybean lipoxygenase using linoleic acid as substrate. Soybean lipoxygenase was used for the assays since it is a commercially available enzyme source and it provides a rapid method to evaluate a large number of compounds for their effects on lipoxygenase activity. It cannot be assumed that the results reported here can be extrapolated to a mammalian lipoxygenase. There are several lipoxygenase with different specificities, and any effects of an inhibitor on one lipoxygenase cannot be extrapolated to an effect on a different lipoxygenase. Materials
Eighteen known drugs, analgesics possible inhibitory activity against (E.C.126.96.36.199) using linoleic acid ’
To whom all
and NSAID, were tested soybean lipoxygenase as substrate.
Isoxicam (4), meclofenamic acid, mefenamic acid, phenylbutazone, aminopyrine, and aspirin were obtained from chemical development of Parke-Davis and Co. Piroxicam (5) was synthesized by the literature procedure. Naproxen, BW 755C, antipyrine, ketoprofen, clozic acid, and ibuprofen were kind gifts from Syntex Laboratories, Wellcome Laboratories, Mallinckrodt Chemicals, Ives Laboratories, ICI Indomethacin, soybean Ltd, and Boots Pure Drug Co. respectively. lipoxygenase, sodium linoleate, and Trizma were purchased from Sigma Chemical Co., St. Louis, Missouri, USA. Anitrazafen and benoxaprofen, and tolmetin and zomepirac were kind gifts from Eli-Lilly and co. and McNeil Laboratories respectively. 4-Acetaminophen was purchased from Aldrich Chemical Co., Milwaukee, Wisconsin, USA. Determination of Lipoxygenase Activity The conversion of linoleic acid to 13-hydroperoxy linoleic acid was followed spectrophotometrically by the appearance of a conjugated diene at 234 nm. The enzymatic reaction was monitored using a Gilford Model 250 spectrophotometer at 24" C. Each assay had a total volume of 1 ml and contained sodium linoleate 100 nM, 0.1 M tris hydrochloride, pH 9.0, 2% ethanol, 4.2% propylene glycol and sufficient enzyme to give an easily measurable initial rate of reaction (6,7). Substrate solutions were prepared fresh prior to assays with 20% ethanol in tris buffer and inhibitors were dissolved in a propylene glycol - tris buffer solution in such a manner that an aliquot of each yielded a final concentration of 4.2% propylene glycol and 2% ethanol in each assay. The effects of inhibitors on the enzymatic reaction were compared against controls under identical conditions. The substrate concentration used for all assays was 100 PM. Results and Discussion The inhibitory activity of a series of analgesic and antiinflammatory agents varied widely against soybean lipoxygenase. This series of compounds were compared by means of their IC50, defined as the concentration of inhibitor necessary for 50 percent inhibition of the enzyme reaction. The Ki values were estimated from the relationship [IC5O/S] = Ki/Km (8) where S = substrate concentration (100 nM) and the reported Km value, 25 nM (9). The IC5O/S ratio may be used as an indication of relative affinity of inhibitor and substrate for the enzyme. The study found (Table 11, that the best inhibitors of lipoxygenase were naproxen, BW 755C, indomethacin, and isoxicam having affinity for the enzyme equal to or greater than the substrate, linoleic acid. Their estimated Ki values ranged from 4 - 22 PM.
Agents with moderate affinity for the enzyme such as meclofenamic acid, phenylbutazone, antipyrine, and benoxaprofen had Ki values between 30 - 50 uM. Compounds tested having a lower affinity or inhibition potency (Ki > 50 uM) included acetaminophen, tolmetin, aspirin, ibuprofen and zomepirac. In summary, all members of the series are reported to inhibit the PG-synthetase (12). However, they are also inhibitors of soybean lipoxygenase at somewhat higher concentration. TABLE - 1 Inhibition of Soybean Lipoxygenase By Known Antiinflammatory Analgesic Drugs. Drug
NDGA Naproxen BW755C Sulfasalazine Indomethacin Isoxicam Phenylbutazone Meclofenamic acid Mefenamic acid Antipyrine Anitrazafen Piroxicam Aminopyrine Benoxaprofen Acetaminophen Clozic acid Ketoprofen Tolmetin Aspirin Zomepirac Ibuprofen
6.lb 16.0 65.5 66.2 87.5 89.5 123 124 133 146 148 154 158 194 201 217 225 332 430 444 575
Estimated Ki (8) (nM) 4 16 16.6 (10) 22 22 31 31 33 37 37 39 40 49 50 54 56 a3 108 111 144
Enzyme reaction was followed spectrophotometrically by the appearance of conjugated diene at 234 run. IC50 values were determined graphically from at least four drug concentrations and expressed as the mean of eight determinations. The standard deviation (S.D.) of the mean was less than 10% of the mean value in all experiments. Sodium linoleate was used as substrate at 100 !.IM in all experiments. Nordihydroguaiaretic (6,111.
acid (NDGA) reported IC50 = 2.4-10 uM
MARCH 1983 VOL. 25 NO. 3
1. 2. 3.
4. 5. 6. 7. 8.
10. 11. 12.
F. A. KUEHL and R. W. EGAN, Science, 2, 978-984 (1980). J. F. BURKA, New England Sot. Allergy Proc. 1, 62-67 (1981). E. ARRIGONI-MARTELLI, Drugs of the Future, Vol-VI (91, 663-678 (1982). H. ZINNES, J.C. SIRCAR, N. LINDO, M.L. SCHWARTZ, A.C. FABIAN, J. SHAVEL, Jr., C.F. KASULANIS, J.D. GENZER, C. LUTOMSKI, and G. DiPASQUALE, J. Med. Chem., 25, 12 (1982). J. G. LOMBARDINO, E. H. WISEMAN, and J. CHIAINI, J. Med. Chem., 16, 493-496 (1973). A. L. TAPPEL, W. 0. LUNDBERG, and P. D. BOYER, Arch. Biochem. Biophys. 42, 293-304 (1953). W. L. SMITH and W. E. M. LANDS, J. Biol. Chem. 247, 1038 (1972). B.R. BAKER, Design of Active - Site - Directed Irreversible Enzyme Inhibitors, p 202-204, John Wiley and Sons, New York (1967). M. J. GIBIAN and R. A. GALAWAY, Biochemistry, 15, 4209-4214 (1976). J.C. SIRCAR, C.F. SCHWENDER, and M.E. CARETHERS, Biochem. Pharm. in press. W. P. KINGSTON, Brit. J. Pharm., 74, 919P-920P (1981). R. J. GRYGLEWSKI in 'Antiinflammatory Drugs' Editors, J. R. Vane and S. H. Ferreira, Springer-Verlag, Rerlin, Heidelberg, New York., p l-43, (1978); T.Y. SHEN ibid, p 305-347.
Editor: L. Jackson Roberts, II Received: 9-27-82 2-l-83 Accepted:
MARCH 1983 VOL. 25 NO. 3