Prostaglandins Leukotrienes and Medicine 10: 319-329, 1983

INHIBITION OF PROSTAGLANDIN SYNTHETASES DERIVED FROM NEURONAL AND GLIAL CELLS AND RAT RENAL MEDULLA BY ORTHO-, META- AND PARA-SUBSTITUTED AMINOPHENOLIC COM- POUNDS Joachim Baumann, Franz v. Bruchhausen and Gotthard Wurm +)

Pharmakologisches Institut der FU, Thielallee 69/‘73, D-1000 Berlin 33 +) Inst. f. Pharmazie der FU, Konigin-Luise -Str. 2 -4, D-1000 Berlin 33 (reprint requests to J. B. )

ABSTRACT

Acetophenetidines, acetamidophenols, phenetidines and aminophenols substituted in Q-, m- or p-position inhibit prostaglandin-synthetases originating from C 1300 mouse neuroblastoma cells (clone N2A), rat astrocytoma cells (clone C 6) and rat renal medulla. Desacetylated compounds were more potent inhibitors than their corresponding acetyl derivatives and many o- and m-analogues were more active than p-sub- stituted structures like paracetamol (p-acetamidophenol) or phenacetin (p-acetophenetidine). When twelve o-, m- or p-aminophenolic test com- pounds were compared to acetylsalicylic acid and indomethacin, o-, and p-phenetidine and o-aminophenol were as effective as acetylsali- cylic acid. All aminophenol derivatives which inhibited prostaglandin synthesis suppressed cultured nervous cell and kidney cycle-oxygenases to similar extents. Our results suggest that aminophenolic drugs are not more effective against prostaglandin-synthetases in the CNS than against those in the periphery.

INTRODUCTION Paracetamol (acetaminophen, N-acetyl-p-aminophenol) and phenacetin (p-acetophenetidine) are widely used analgesic-antipyretic drugs which belong to the chemical group of aminophenol derivatives. Unlike acetyl- salicylic acid (ASA) or indomethacin their anti-inflammatory activity is less pronounced, although all these compounds are known inhibitors of the prostaglandin (PG)-synthetase pathway (l-4). A first explanation of the different therapeutic actions of the aminophenol analgesics and other predominant antiphlogistic “aspirin-like drugs” was sought in terms of different enzyme inhibition potencies by Flower and Vane (5). They pos- tulated that paracetamol, the main metabolite of phenacetin in man is a stronger inhibitor of PG-synthetases in the CNS than of peripheral PG- synthetases. Recently, however, we added further evidence against this hypothesis studying the inhibitory potencies of both aminophenol analge-

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sits - paracetamol and phenacetin - and of their corresponding desace- tylated metabolites in comparison to ASA and indomethacin against PG- synthetases of central nervous and extra-central nervaus origin (6). Al.1 compounds tested inhibited neuronal and glial cycle-oxygenases as well. as kidney PG-synthetase to similar extents without revealing significant differences. We could also show that p-phenetidine, which is formed during the hepatic desacetylation of phenacetin (7, 8) and by a rather specific phenacetin-N-desacetylase in brain (9) as well as p-amino- phenol, the desacetylation product of paracetamol, are stronger PG- synthetase inhibitors than their parent compounds which are used for analgesic purposes. Based on the IC 50 -values derived from inhibition studies on three different PG-synthetases p-phenetidine was 8. 7 times and p-aminophenol 4.1 times more potent than paracetamolwhich itself was some 3 times more active than phenacetin (6). Therefore, we as- sumed that PG synthesis inhibition by the aminophenol analgesics para- cetamol and phenacetin may be strongly enhanced, if their more potent metabolites p-aminophenol and p-phenetidine are formed by enzymatic desacetylation.

In the present study we investigated, whether PG-synthetase inhibition by aminophenolic compounds strictly depends on p-substitution of these aminobenzene structures. Ortho- and meta-analogues of phenacetin, paracetamol and of their desacetylation products p-phenetidine and p-aminophenol were studied as to their PC-synthetase inhibitory effi- cacy in comparison to their corresponding p-compound% ASA and indo- methacin, which served as reference inhibitors. Since there might be differences in the degree to which PG-synthetases in t,he CNS or in the periphery are inhibited by these aminophenol derivatives, each com- pound was tested against cycle-oxygenases from neuronal cells, glial cells and kidney medulla, We chose the N2A-clone of C 1300 mouse neuroblastoma and the C 6 -clone of rat astrocytoma which, in our pre - vious investigations (6), had proved to be very suitable tools for the characterization of central nervous PC synthesis and the inner medulla of rat kidney as a well-established assay system for PG-synthetase in- hibition studies.

MATERIALS AND METHODS

Chemicals

[l -14Cj -Arachidonic acid (sp. act. = 50-52 Ci/mole) was supplied by the Radiochemical Centre, Amersham, U. K. Reduced glutathione, l-adrenalin, hematin, indomethacin, paracetamol, phenacetin were purchased from Sigma (St. Louis, MO., U. S.A. ). Aminophenols (o-, m-, p-), phenetidines (o-, m-, p-), acetamidophenols (o-, m-), silica gel 60 precoated glass plates for TLC, 20 x 20 cm, 0. 5 mm thick, and all organic solvents were commercial products of the highest quality

320

available (E. Merck, Darmstadt, F. R. G.; EGA-Chemie, Steinheim, F. R. G. ), authentic prostaglandins (PG A2, E2, D2, F2a) were kindly provided by the Upjohn Comp., Kalamazoo, Mi., U. S. A., or purchas - ed from New England Nuclear Corp., Boston, Mass., U. S. A. Acetyl- salicylic acid was a gift from Bayer AC, Leverkusen, F. R. G. Aceto- phenetidines (o -, m-) were synthesised by boiling the phenetidines in acetic acid according to standard procedures, the acid was removedand the products were dissolved in ethanol and crystallised. Molar masses were verified by mass spectrometric analysis, melting points corres- ponded to those indicated in the literature. The chemical purity of all aminophenolic compounds was analysed prior to experiments by column and thin layer chromatography and was not lower than 9870 (w/w).

Prostaglandin-synthetase preparations

Rat kidney: Renal medulla tissue from male Wistar rats (250-300 g b. w.) was homogenized in 10 ~01s. 0. 1 M K2HPO4 buffer (pH 7. 4) for 4 min at O°C using a Potter-Elvehjem homogenizer (30 ml, glass-teflon) spin- ning at BOO/min (6). Enzyme preparation protein was lo-12 g/l as de- termined by the method of Lowry et al. (10).

Neuronal cells: The N2A-clone of C 1300 mouse neuroblastoma (CCL 131) was obtained from Gibco-Biocult Ltd., Glasgow, U.K. The cells were grown in plastic flasks (75 cm2) using MEM with Hanks’ salts + 10% fetal calf serum (FCS) containing 1 mM glutamine, 0. 5 ml NEAA, 0. 06 g/l penicillin and 0. 1 g/l streptomycin as previously described (6, 11, 12).

Glial cells: C 6 glial cells of rat astrocytoma (CCL 107, Gibco-Biocult, Glasgow, U. K. ) were cultivated in Ham’s F 10 nutrient mixture supple- mented with 1570 horse serum, 2. 5% FCS, glutamine, penicillin and streptomycin (6, 11, 12).

Neuronal and glial cells were taken for PG-synthetase assays when con- fluent phases were reached. After mild trypsinization (0.05% trypsin, 0. 03% EDTA), cells were washed twice in isotonic Ca-, Mg-free potas- sium phosphate buffer (pH 7. 4) and finally homogenized in 0.1 MK2HP04 (pH 7.4) as described above for the rat kidney enzyme. Cell homoge- nates containing 7.6 g protein/l (N2A) and 6 g protein/l (C 6) were used for the following PG-synthetase inhibition studies to obtain similar per- oxidation rates of arachidonic acid by both enzyme preparations (6).

Inhibition of PG -synthetas es

Central nervous cell and rat kidney cycle-oxygenase preparations were incubated with arachidonic acid in the presence of varying concentra- tions of inhibitors and total PG-formation was measured (6, 13): the re- action mixture contained potassium phosphate buffer (0.1 M, pH 7. 4),

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l-adrenalin (1 mM), hematin (1 PM), glutathione (1. 3 mM), [l-14C]- arachidonic acid (38 nCi, 2. 5 PM), 1% ethanol, enzyme preparation and inhibitor (10m6 M - 5 x 10 -3 M) in a final volume of 0. 3 ml. Enzymes and inhibitors were pre-incubated for 10 min at ambient temperature, PC synthesis was started by the addition of the fatty acid and incuba- tions at 3’7’C were stopped by acidification after 20 min, when PG-for- mation was complete. All eicosanoids were immediately extracted into ethylacetate, concentrated, spotted on silica gel TLC -plates and deve - loped (benzene/dioxane/acetic acid = 60 : 30 : 3). Radioactive precursor and total PG-products were localized by radioscanning and quantitated by standard liquid scintillation procedures as previously described (6, 13). The concentration-response curves and the IC50-values of all in- hibitors against the three cycle-oxygenases we tested were derived from 4-6 observations for each concentration of test compound.

RESULTS

The two aminophenolic analgesics paracetamol (p-acetamidophenol) and phenacetin (p-acetophenetidine) were tested against PG -synthetase pre - parations of rat kidney medulla, neuronal and glial cell origin and their concentration -dependent enzyme inhibition was compared with the ac - tions of their corresponding o- and m-analogues. The results areshown in Fig. 1 (acetamidophenols) and Fig. 2 (acetophenetidines). Both fig- ures reveal that not only therapeutically used paracetamol and phenace- tin inhibit PG synthesis. In comparison to phenacetin its o- and especi- ally its m-analogue are more potent inhibitors (Fig. 2) and among the acetamidophenols paracetamol is only about half as active as is o-sub- stituted acetamidophenol, whereas m -acetamidophenol does not possess any anti -cycle -oxygenase activity (Fig. 1).

As previously reported for p-substituted aminophenolic compounds (6), the concentration-dependent inhibition of PG-synthetase by o- and m- analogues of acetophenetidine and acetamidophenol (Figs. 1 and 2) was almost identical regardless of whether neuronal or glial cells or rat kidney were the enzyme sources.

During the course of biotransformation phenacetin and paracetamol are largely converted to p-phenetidine or p-aminophenol, which proved to be more potent cycle-oxygenase inhibitors than their acetylated parent compounds. We, therefore, investigated, whether other phenetidines or aminophenols substituted in o- or m-position were suitable PG-synthe- tase inhibitors and measured their effects against the three cyclo-oxy- genase systems. Their inhibitory efficacies, expressed as IC50-values, are given in Fig, 3 and Table 1. Without exception the desacetylated metabolites were more potent inhibitors than the acetylated aminophe- nolic compounds, irrespective of whether they were substituted in o-, m- or p-position. PG-synthetases of different origin were inhibited to almost the same degree by these aminophenol derivatives. Among the

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As to the phenetidines, the o- and p-analogues were as potent PG-syn- thetase inhibitors as was ASA (Fig, 3, Table 1) in contrast to the aceto- phenetidines which, in general, were rather ineffective inhibitors. Al- though m-phenetidine was the least efficacious phenetidine in our hands, it still exhibited twice as strong anti-cycle-oxygenase activity when compared to clinically used paracetamol (see Fig. 3 and Table 1).

DISCUSSION

p-Substituted paracetamol and phenacetin, which were introduced as an- algesic-antipyretic drugs during the last decade of the 19th century, are not the only PC-synthetase inhibitors among the chemical group of ami- nophenol derivatives. Most of their o- a.nd m-substituted analogues were of equal or stronger efficacy against several PG-synthetases. Because of the potent enzyme inhibitory properties simple aminophenols and phe- netidines have in common, desacetylation may strongly enhance the pharmacological effects of acetamidophenols and acetophenetidines, which are rather weak PG-synthetase inhibitors. These compounds un- dergo a remarkable enzymatic desacetylation in rat brain (data to be published) and other tissues (7, 8). Recently, the desacetylation of phen- acetin in monkey cerebellum was described (9).

Surveying our inhibition studies on 12 aminophenolic compounds against PG-synthetase preparations of central nervous or peripheral origin, the o- and m-analogues inhibited arachidonic acid peroxidation without sig- nificant differences regardless of the origin of the enzymes. These re- sults are in full accordance with our data recently published for p-sub- stituted paracetamol, phenacetin and their desacetylation products (6) and add further evidence against the hypothesis of Flower and Vane (5) that aminophenol analgesics selectively inhibit cerebral PG-synthetases In our opinion the rather weak antiphlogistic effects of these drugs can- not be explained solely by the degree to which PG-synthetases from dif- ferent tissues are inhibited. As shown in Table 1, all aminophenol de- rivatives as well as the classic antiphlogistics ASA and indomethacin were not more effective against neuronal or glial PC-synthetases than against kidney PG-synthetases.

Our findings further tempt us to speculate that enzymic desacetylation of acetamidophenols and acetophenetidines, which results in the forma- tion of more potent PG-synthetase inhibitors, may, at least partially, regulate the therapeutic potency of these drugs in different tissues de- pending on their ability to desacetylate aminophenolic compounds. From

327

a pharmacological point of view all acetophenetidines and especially m- acetamidophenol might be considered as pro-drugs, because they pos- sess little or no anti-cycle-oxygenase activity but are converted to strong PG-synthetase inhibitors by N-desacetylases in brain (9) and other tissues (7, 8). Most recently, we measured considerable enzyma- tic desacetylation of m-acetamidophenol in rat brain and further studies on the degradation of acetylated aminophenol derivatives in different brain regions of various species are in progress in our laboratory.

ACKNOWLEDGEMENTS

The authors thank Miss D. Oberbeck and Miss C. Schmidt for their technical assistance,

This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, F. R. G.

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