Expression of recombinant human cyclooxygenase isoenzymes in transfected COS-7 cells in vitro and inhibition by tenoxicam, indomethacin and aspirin

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(5), 361-367 © Pearson ProfessionalLtd 1997

Expression of recombinant human cyclooxygenase isoenzymes in transfected COS-7 cells in vitro and inhibition by tenoxicam, indomethacin and aspirin M. Lora, ~ S. M o r i s s e t , ~ H.-A. M e n a r d , 2 R. L e d u c , ~ A. J. de B r u m - F e r n a n d e s ~,2 1Department of Pharmacology, Faculty of Medicine, Universite de Sherbrooke. 3001 12th Avenue North, Fleurimont, PQ, J1H 5N4, Canada 2Department of Medicine, Faculty of Medicine, Universit6 de Sherbrooke. 3001 12th Avenue North, Fleurimont, PQ, J1H 5N4, Canada

Summary The recent discovery of cyiooxygenase-2 (COX-2), an isoenzyme associated mainly with inflammation created the need to reevaluate cyclooxygenase inhibitors with reliable screening methods. In the present study we standardized a technique to determine the ICsos of cyclooxygenase inhibitors on recombinant human COX-1 and COX-2 expressed in mammalian cells and used it to study the compounds tenoxicam, aspirin and indomethacin. The ICa0s of aspirin, indomethacin and tenoxicam for human COX-1 were 0.41 _+0.07 gg/ml, 0.008 _+0.003 gg/ml, and 7.94 _ 3.28 gg/ml, respectively, and for human COX-2 0.64 _+0.16 gg/ml, 0.09 _+0.05 gg/ml, and 10.61 _+ 1.50 gg/ml, for aspirin, indomethacin, and tenoxicam. Tenoxicam had the lowest ICso hCOX-2/ICs0 hCOX-1 ratio (1.34), followed by aspirin (1.53) and indomethacin (10.82). The system described in the present study provides a simple and efficient way to determine the specificity of NSAID inhibition for each of the human cyclooxygenase isoenzymes separately. INTRODUCTION

Cyclooxygenase is the first enzyme involved in the synthesis of prostaglandins, catalyzing the formation of Prostaglandin H2 (PGH2) from arachidonic acid. The unstable PGH2 is the common precursor of prostaglandins and thromboxane. Cyclooxygenase is the primary target of non-steroidal anti-inflammatory drugs (NSMDs), and this action is believed to be responsible for their anti-inflammatory action. ~Unfortunately, inhibition of prostaglandin production in organs where they play a physiological role, such as stomach and kidney, can result in gastric lesions and nephrotoxicity, important side effects that limit the use of these drugs. Recent studies have shown the existence of two isoforms of cyclooxygenase. The first isoenzyme described, cyclooxygenase-1 (COX-1, PCH Synthase, E.C. 1.14.99.1),

Received 19 June 1996 Accepted 4 October 1996 Correspondence to: Artur J. de Brum-Fernandes, Fax. 00 819 564 5265; E-mail: a.fernan @courrier.usherb.ca

often referred to as a housekeeping enzyme, is expressed constitutively and is ubiquitously distributed.2-s The recently discovered isoenzyme, cyclooxygenase-2 (COX2), is usually absent or present at low levels in nonproliferating cells, but its expression is increased by mitogens and conditions that stimulate proliferation,7-8 by phorbol esters, 3 prostaglandins,9 calcium ionophore ~° and different inflammatory cytokines.1°-I4 COX-2 expression is inhibited by glucocorticoids. 2,6-7,I~,14-~5The COX-2 gene has some characteristics of an early-response gene and its transcriptional regulation is quite complex. These facts suggest that COX-1 is responsible for the basal synthesis of prostaglandins participating in physiological processes, whereas COX-2 is responsible for prostaglandin production in inflammation and pathological situations. This hypothesis has been substantiated by the demonstration that the most important isoenzyme in human rheumatoid synovial membrane 16 and in the rat air pouch model of inflammation~z is COX-2, and that compounds capable of inhibiting specifically COX-2 are anti-inflammatory but non-ulcerogenic in animal models, lz 361

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The cDNAs encoding COX-1 and COX-2 have been cloned from mouse 18-2° and human s o u r c e s 5'21-22 a n d expressed in vitro.4,s'19-21 COX-1 and COX-2 from the same species exhibit about 60% amino acid sequence identityY Specific residues of the COX-1 protein, such as Ser530, have now been identified as being important in the catalytic site. 23-25 Each of these residues is conserved in COX-2 (COX-1 Ser530 is COX-2 Set516), 23 suggesting that the enzymatic mechanisms are fundamentally the same for both isoenzymes. Nonetheless, the differences in primary structures between COX-1 and COX-2 suggest that there may be subtle differences in how the two isoenzymes behave and that these differences could lead to different affinities for fatty acid substrates and NSAIDs. It has been shown that murine COX-1 and COX-2 differ in their sensitivity to inhibition by some NSMDs 2° as do human COX-1 and COX-2.26 Selective inhibition of COX-2 would, thus, be possible, and could provide an anti-inflammatory action without the side effects of the currently available NSMDs. 2°,2z Most currently available NSMDs, however, are preferential inhibitors of COX-1.2°,26 This is not surprising since they have been screened in in vitro systems containing mostly COX-1. An ideal system to test the sensitivities of human COX isoenzymes to different inhibitors should allow the testing of each isoenzyme separately. However, COX-1 is a rather ubiquitous enzyme in differentiated mammalian cells, COX-2 is frequently expressed in huge amounts in cells undergoing proliferation, and both are frequently present in the same cells. An adequate approach to this problem would be to find mammalian cells that can be maintained in culture and do not express COX-1 or COX2 and transfect them with the specific human cDNAs, thus permitting adequate post-translational modification of the expressed enzymes. The objective of the present study was to find an immortalized mammalian cell line that does not express endogenous COX-1 or COX-2 and to standardize a transfecfion technique capable of producing functional human COX-1 or human COX-2 in sufficient amounts to allow the determination of ICs0s of different compounds on each isoenzyme separately. With this system we determined the inhibitory efficacy of tenoxicam, aspirin and indomethacin on human cyclooxygenase isoenzymes.

MATERIALS AND METHODS Chemicals

Tenoxicam (4-Hydroxy-2-methyl-N-(2-pyridyl)-2H-thieno[2,3-e]-l,2-thiazine-3 carboxamide 1,1-dioxide) was provided by F. Hoffmann-La Roche & Co., Basel, Switzerland. Acetylsalieylic acid and indomethacin were purchased from Sigma Chemical Co., St Louis, MO. A 25 mg/ml stock

solution in dimethylsulfoxide (DMSO, Fisher Chemical, New Jersey) was prepared for both tenoxicam and acetylsalicylic acid. A 10mg/ml stock solution was prepared in DMSO for indomethacin. The final concentration of DMSO on the cell cultures was never higher than 40 gl/ml. Cell culture

Cos-7 cells and NIH 3T3 cells were grown on 100 mm Petri dishes (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) in Dulbecco's modified Eagle's medium (DMEM, Sigma Chemical Co., St Louis, MO) supplemented with 10% fetal bovine serum (FBS, ICN Biomedicals, Inc., Aurora, OH), penicillin (100 units/ml), and streptomycin (100 gg/ml), pH 7.4. For transfection, Cos-7 cells were grown in 6-well plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ). Isolation of total RNA and Northern blot analysis

Total RNA was extracted from 100 mm confluent plates of Cos-7 and NIH 3T3 cells using Trizol (Gibco BRL, Burlington, Ont), according to the manufacturer's protocol. Total RNA (25 gg) was fractionated on an agaroseformaldehyde gel, and transferred to 0.45 gm Hybond-N + membrane (Amersham Life Science Inc., Oalcville, Ont) by capillarity. Human COX-1 (hCOX-1) and human COX-2 (hCOX-2) eDNA probes were prepared by random primer [32P]dCTP with an oligolabeling kit (Pharmacia Biotech Inc., Piscataway, NJ). Both cDNAs were kindly supplied by Dr Timothy Hla. The membranes were hybridized in hybridization buffer (400 mM NaH2PO4, 1 mM EDTA, 1 mg/ml BSA, 5% Sodium Lauryl Sulfate, 50% formamide, pH 7.2) overnight at 42°C with the radioactive marked probes, and then washed twice at 55°C for 20 rain in 1 mM EDTA, 0.1% Sodium Lauryl Sulfate, 1 X SSC (stock solution 20X SSC: 3 M NaC1, 0.3 M sodium citrate, pH 7.0) and then washed once at 55°C for 20 min in 1 mM EDTA, 0.1% Sodium Lauryl Sulfate, 0.2 X SSC. The membranes were then autoradiographed on Kodak XAR-5 film at -80°C with intensifying screens. GAPDH eDNA probes were used to allow comparison of the amounts of mRNA in each lane. Transient expression Once the Cos-7 cells were at 60% to 75% of m a x i m u m

confluence, they were transfected with hCOX-1 or hCOX-2 cDNAs inserted in pcDNAI plasmids (Invitrogen, San Diego, CA), a mammalian expression vector, using an optimized LipofectAMINETM Reagent (Gibco BRL) procedure. LipofectAMINETM (5 gl per well) and plasmid (2 gg per well) were incubated together in DMEM (final

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Inhibition by tenoxicam, indomethacin and aspirin of human cyclooxygenase isoenzymes

volume of 200 gl per well) for 45 min at room temperature, then added to each well already containing 800 gl of DMEM and incubated for 5 h. The medium was then replaced with DMEM containing 10% FBS.

Lane COX-2

1 ,o

363

2

:'

Inhibition assays Transfected cells were grown for 4 0 h in DMEM 10% FBS. The supernatant was removed and replaced by 1 ml of Hepes buffer (110 mM NaC1, 1.1 mM KH2PO4,4.7 mM KC1, 5 mM D-Glucose anhydrous, 21 mM Hepes, pH 7.4) containing the different concentrations of tenoxicam, indomethacin or aspirin. After a 4 5 m i n incubation, arachidonic acid (Sigma) was added to a final concentration of 40 gM and incubated for another 20 min. All incubations were carried out at 37°C. Once the incubation completed, the supernatants were removed and preserved at -80°C. Radioimmunoassay of PGE~ PGE~ production was measured by radioimmunoassay as previously described =s with minor modifications. [5,6,8,11,12,14,15(n)-3H]PGE2 was purchased from Amersham Life Science Inc., Oakville, Ont. Standard PGE= and PGE= antiserum were purchased from Sigma Chemical Co., St Louis, MO. This antiserum does not discriminate between prostaglandin E~ (PGE~) and prostaglandin E~ (PGE~). The sensitivity of our RIA is defined as the 90% intercept of a B/Bo standard curve and was found to be 2 ng/ml. Bound and free fractions were separated by using dextran-coated charcoal and centrifugation. Determination of IC~0 The IC~0 of the various compounds tested were calculated by linear regression from semi-log plots using the results comprised between 80% and 20% of PGE~ production inhibition curves. The results presented are the averages of the ICs0 calculated for each curve separately. All results are presented on average + SEM. RESULTS Native expression of cyclooxygenase isoenzymes in Cos-7 cells Several cell lInes were screened for the presence of COX1 and of COX-2 mRNA. The autoradiograph shown in Figure 1 demonstrates the absence of both COX-1 and COX-2 mRNA in Cos-7 cells when stimulated by DMEM containing 10% FBS for 2 h (lane 1), while NIH 3T3 cells under the same conditions presented detectable levels © Pearson Professional Ltd 1997

COX-1

GAPDH

' : :!:: ' .

2.8 kb

- 1.8 kb

Fig. 1 Detection of COX-1 COX-2 and GAPDH mRNA by Northern Blot. Lane 1 : Cos-7 cells stimulated by DMEM +_ 10% FBS for 2 h (25 gg total RNA). Lane 2: NIH 3T3 cells stimulated by DMEM +_ 10% FBS for 2 h (25 gg total RNA). These results are representative of several separate experiments.

of mRNA for both isoenzymes (lane 2). Cos-7 cells were chosen for the subsequent experiments. Maximum activity of cyclooxygenase isoenzymes in transfected Cos-7 cells Intact Cos-7 cells that were mock transfected did not produce detectable amounts of PGE2 when incubated with arachidonic acid (40 gM) (data not shown). Cos-7 cells transfected with h u m a n COX-1 produced 204.5 + 18.1 ng/ml (n = 32) of PGE2 when Incubated with arachidonic acid for 20 min. Under the same conditions, but when transfected with h u m a n COX-2 the cells produced 34.2 + 5.7 ng/ml (n = 20) of PGE2. Inhibition of hCOX isoenzymes by tenoxicam, aspirin and indomethacin All three compounds tested inhibited both COX isoenzymes. Figure 2 shows the inhibition curves for these compounds on human COX-1 and the Table shows calculated ICs0s for aspirin, indomethacin and tenoxicam. Indomethacin had the lowest ICs0 against COX-l, followed by aspirin and tenoxicam. Figure 3 shows the inhibition curves for these compounds on human COX-2 and Table the calculated ICsos. Once again, of the three compounds tested, indomethacin had the lowest ICso, followed by aspirin and tenoxicam. On a equimolar basis indomethacin was more potent than aspirin

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364

140

---o- Tenoxicam

120

-c-Aspirin

ratios close to 1, while indomethacin was 10 times more potent to inhibit COX-1 than COX-2.

g 100 DISCUSSION

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Concentration (ug/ml) Fig. 2 Inhibition of hCOX-1 by tenoxicam, aspirin and indomethacin. Human COX-1 activity was measured by the production of PGE2 after a challenge with exogenous arachidonic acid 40 pM for 20 min in the presence or absence of inhibitors. Data are expressed as mean _+ SEM from 6-8 determinations.

140

--o-- Tenoxicam

"~ 120

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Concentration (ug/ml) Fig. 3 Inhibition of hCOX-2 by tenoxicam, aspirin and indomethacin. Human COX-2 activity was measured by the production of PGE2 after a challenge to exogenous arachidonic acid 40 gM for 20 min in the presence or absence of inhibitors. Data are expressed as mean _+SEM from 3 - 6 determinations.

and tenoxicam for the inhibition of both hCOX-1 and hCOX-2 (Table). None of the compounds tested w a s specific for one of the isoenzymes. As can be seen in the Table, tenoxicam and aspirin had ICso COX-2/ICso COX-1

The objective of our study was to develop a system that would provide an efficient way to determine qualitative and quantitative characteristics of NSMD inhibition for each h u m a n cyclooxygenase isoenzyme separately. Since b o t h isoenzymes are generally present in different tissues and cell lines our approach was to find a m a m malian cell line that did not express either COX isoenzymes and transfect t h e m with the proper cDNAs. Using NIH 3T3 cells as positive controls for the Northern blots we determined that Cos-7 cells, among all the cell lines tested did not express either COX-1 or COX-2. The absence of cyclooxygenase activity in these cells was further confirmed by the absence of PGE2 production by either native or mock transfected Cos-7 cells (data not shown). This cell line was thus chosen for transient transfection with h u m a n COX-1 or h u m a n COX-2 cDNAs, which would allow the expression and the testing of only one isoenzyme at a time. As shown b y the production of PGE2 upon stimulation with arachidonic acid, transfection of Cos-7 cells with hCOX-1 or hCOX-2 cDNAs yielded functional enzymes. Although both cDNAs have the same length and were transfected using the same vector, a difference in the m a x i m u m production of PGE2 by the two isoenzymes was seen in our system, with hCOX-1 producing six times more PGE 2 then hCOX-2. One reason for this difference could be a decreased expression or activity of the hCOX-2 isoenzyme. In fact, the reported sequence of the cDNA used in our transfections differs from two other sequences of hCOX-2. 29,3° This punctual difference in codon 165, GAA to GGA, which replaces a glutamic acid by a glycine was shown to cause a drop in enzyme activity. 29 The low levels of PGE2 production in our essays prompted us to verify this nucleotide difference. Sequencing hCOX-2 cDNA from codon 146 to codon 208 (data not shown) we verified that the sequence in codon 165 is GAA (glutamic acid), the same as in the more recent publications, 29,3° and not GGA (glycine) as previ-

Table Determination of the ICso of Tenoxicam, Aspirin and Indomethacin on hCOX-1 and hCOX-2 Compound

ICso for hCOX-1 (gg/ml)

n

ICso for hCOX-2 (gg/ml)

n

Ratio ICso hCOX-2/hCOX-1

Tenoxicam Aspirin Indomethacin

7.94 _+3.28 0.41 _+0.07 0.008 _+0.003

8 6 6

10.61 __ 1.50 0.64_+0.16 0.09 _+0.05

6 6 3

1.34 1.53 10.82

Results represent averages _+SEM.

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ously reported. 5 A punctual mutation in the cDNA can not, thus, explain the smaller activity of hCOX-2 when compared to hCOX-1 in our system. Since our COX-2 cDNA lacks the complete consensus Kozak sequence in the 5' untranslated region it is probable that a decreased translation of COX-2 mRNA occurs, what could explain the lower activity of COX-2 per cell when compared to COX-1. This hypothesis, however, remains to be tested. The use of whole cells in our assays may present some advantages over systems using microsomal preparations of the overexpressed enzymes, for the cell structure and metabolism are closer to the conditions found in living systems. It is known that results of the inhibitory activity of NSMDs on cyclooxygenase using whole living cells assays differ from results obtained in non-li~ng systems, probably because penetration through cell membranes plays a role in NSAID activity.31 Mso, the use of exogenous arachidonic acid allows the control of the quantity of substrate in the system, a parameter that can not be controlled when whole cells are treated with substances that stimulate PGE2 production, such as phorbol esters and interleukins. The possibility of controlling substrate availability is very important when testing competitive inhibitors of cyclooxygenase22 Another important aspect to consider when testing cyclooxygenase inhibitors is the time-dependent mode of action of some NSMDs. It has been shown that the pharmacological profile of some of these drugs can change if they are pre-incubated with the enzymes instead of being added immediately before the assay, a~ Since pre-incubation in vitro with NSMDs seems to correspond better to the in vivo situation, where patients are constantly exposed to inhibitory concentrations of these drugs, we chose to pre-incubate the cells with the NSMDs tested before the addition of arachidonic acid. Using this method we determined the inhibition of hCOX-1 and hCOX-2 by aspirin, indomethacin and tenoxicam. All three compounds inhibited both isoenzymes. The values obtained for aspirin, a time dependent and irreversible NSMD which acetylates both COX-1 and COX-218,32 differ from those reported in the literature concerning COX-2. In fact, the reported ICso of aspirin for COX-2 is between 50 and 210 gg/ml, 31,33that is, 90 to 300 times higher than the result found in the present study. However, the results from the literature were obtained in other systems that used COX-2 from different species and different protocols, such as different incubation times (instantaneous inhibition and incubations of 2 min). 3~,34 Some of these studies determined cyclooxygenase activity by oxygen consumption instead of prostaglandin production24 In the case of aspirin, a system based on the consumption of oxygen would have a different ICso for COX-2 than a system based on PGE2 production. The reason is that acetylation of hCOX-2 © Pearson Professional Ltd 1997

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by aspirin does not inhibit oxygenase activity, but modifies this isoenzyme so that it produces 15-R-hydroxyeicosatetraenoic acid (15-R-HETE) instead of PGE2.2°,35,3~ So, COX-2 inhibited by aspirin still consumes oxygen but no longer produces PGE2. In the case of COX-l, acetylation by aspirin completely inhibits cyclooxygenase activity,~7,38 and both systems are supposed to lead to similar results. In fact, the results obtained in the present study are comparable to the results reported in the literature for COX-1. 3I'33'34 The different values obtained with aspirin for human COX-2 could thus be explained on the basis of differences between the technical protocols used and the different species of COX. The results obtained with indomethacin are comparable to those reported in the literature for both COX-1 and C O X - 2 . 32'33 From the three compounds tested in the present study tenoxicam presents very close ICs0s for hCOX-1 and hCOX-2. Preliminary reports from other authors using non-stimulated and lipopolysaccharide (LPS) stimulated guinea-pig macrophages as models of COX-1 and COX-2 containing cells, respectively, showed that the ICsos of tenoxicam for COX-1 and COX-2 were 0.009 gg/ml and 0.1 1 gg/ml. 39 The problems concerning the interpretation of results obtained from stimulated cells as models for COX-2, which have been discussed above, and the different species used could explain the differences from the results obtained in the present study. On a molar basis tenoxicam was the least potent of the compounds tested for both hCOX-1 and hCOX-2 inhibition, followed by aspirin and indomethacin. This finding, however, has little ff any therapeutical implication since all three compounds are capable of completely inhibiting both isoenzymes in concentrations normally found in the plasma during chronic administration. 4°,41 Preferential inhibition of COX-2 may, based on theoretical and experimental data 17,42 lead to drugs with antiinflammatory action and reduced side effects. From this point of view the ratio ICso hCOX-2/ICso hCOX-1 is more important than the absolute ICs0s. When this ratio equals 1 for a certain compound a similar inhibition of both enzymes is achieved with a given concentration of the drug. With ratios higher than 1 hCOX-1 is inhibited to a greater extent than hCOX-2 and, inversely, ratios lower than 1 indicate a preferential inhibition of hCOX-2. From this perspective, and based on the present results, tenoxicam compares advantageously with indomethacin and is slightly better than aspirin, presenting one of the lowest ICs0 hCOX-2/ICs0 hCOX-1 ratios from the NSMDs described in the literature; a¢29.39,42-44 this could explain the smaller incidence of side effects in patients taking tenoxicam.4o, 45 The need to standardize the testing of NSMDs is evident. Many factors can change the ICs0 values of a NSMD, such as the time of incubation, instantaneous inhibition

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or p r e i n c u b a t i o n s with the drug tested, use of microsomal or whole cell preparations, e x o g e n o u s or e n d o g e n o u s substrate a n d the assays u s e d to d e t e r m i n e cyclooxygenase activity. The system described i n the p r e s e n t s t u d y provides a simple a n d efficient way to d e t e r m i n e the specificities of NSAID i n h i b i t i o n for each of the h u m a n cyclooxygenase i s o e n z y m e s separately. It allows control of the a m o u n t of substrate present i n the preparation, leading to a better control of the parameters t h a n with the use of i n t e r l e u k i n - s t i m u l a t e d cells. It m a y also have a d v a n t a g e s over m i c r o s o m a l preparations since the e n z y m e s are s t u d i e d in a n e n v i r o n m e n t close to t h a t f o u n d i n vivo, w h e n the drug m u s t cross the cell m e m b r a n e to i n h i b i t cyclooxygenases. As has already b e e n stressed, d e t e r m i n a t i o n of p r o s t a g l a n d i n p r o d u c t i o n m a y also p r e s e n t some advantages c o m p a r e d to the classical oxygen c o n s u m p t i o n analysis. Since s t a n d a r d i z a t i o n of NSAID testing i n vitro will have to be d o n e in the n e a r future to allow comparisons of the relative efficiencies of NSAIDs against COX-1 a n d COX-2, we suggest that the use of whole living cells expressing the r e c o m b i n a n t h u m a n isoenzymes, p r e i n c u b a t i o n with the drugs to be tested, a n d p r o s t a g l a n d i n p r o d u c t i o n s h o u l d be required characteristics of the assay.

ACKNOWLEDGEMENTS The authors would like to thank Dr Timothy Hla for kindly supplying human cyclooxygenase-1 and human cyclooxygenase-2 cDNAs, and the F. Hoffmann-La Roche & Co. for supplying tenoxicam as well as for providing financial support. S.M. is in receipt of a studentship of the Fonds pour la Formation de Chercheurs et l'Aide a la Recherche. R.L. is a scholar of the Fonds de la Recherche en Sant4 du Qu6bec. AJ.B.F. is a scholar of the Arthritis Society.

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7. Hershman H. R. Characterization of a gene encoding a second prostaglandin synthase/cyclooxygenase (PGS/COX),whose message and protein are induced by mitogens and inhibited by glucocorticoids. The 8th International Conference on Prostaglandins and Related Compounds, Montreal, 1992: 79. 8. Raisz L. G., Voznesensky O. S., Alander C. B., Kawaguchi H., Pilbeam C. C. Auto-amplification of inducible prostaglandin synthase (cyclooxygenase) in osteoblastic MC3T3-E1 and PY1A cells. JBone Min Res 1993; 8: S161. 9. Takahashi Y., Taketani Y., Endo T., Yamamoto S., Kumegawa M. Studies on the induction of cyclooxygenase isoenzymes by various prostaglandins in mouse osteoblastic cell line with reference to signal transduce pathway. Biochem Biophys Acta 1994; 13: 217-224. 10. Ristimaki A., Garfinkel S., Wessendorf J., Maciag T., Hla T. Induction of cyclooxygenase-2 by interleukin-1 alpha. Evidence for post-transcriptional regulation. J BioI Chem 1994; 269:11769-11775.

11. de Brum-Fernandes A.J., Laporte S., Heroux M., Lora, M., Parry C., M6nard H-A., Dumais R., Leduc R., Expression of prostaglandin endoperoxide synthase-1 and prostaglandin endoperoxide synthase-2 in human osteoblasts. Biochem Biophys Res Commun 1994; 164: 1358-1365. 12. Gilbert R. S., Reddy S. T., Kujubu D. A., Xie W., Luner S., Hershman H. R. Transforming growth factor beta 1 augments mitogen-inducedprostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene both in Swiss 3T3 cells and in murine embryo fibroblasts, y Cell Bio11994; 159: 67-75. 13. Mitchell M. K., Silver R. M., Carlton D. et al. An interleukin-l[~inducible cyclooxygenase in human amnion. The 8th International Conference on Prostaglandins and Related Compounds, Montreal, 1992; 78. 14. O'Banion M. K., Wirm V. D., Young D. A. grIPGHS: a second cyclooxygenase gene responsive to glucocorticoids, growth factors, and cytokines. The 8th International Conference of Prostaglandins and Related Compounds, Montreal, 1992; 79. 15. Masferrer J. L., Zweifel B. S., Seibert K., Needleman P. Selective regulation of cellular cyclooxygenase by dexamethasone and endotoxin in mice. J Clin Invest 1990; 86: 1375-1379. 16. Crofford L.J., Wilder R. L., Ritmaski A. P., Hajime S., Returners E. E., Epps H. R., Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissues. J Clin Invest 1994; 93:1095-1101. 17. Masferrer J. L., Zweifel B. S., Manning P. T. et al. Selective inhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory and non-ulcerogenic. Proc Natl Acad Sci USA 1994; 91: 3228-3232. 18. DeWitt D. L., E1-Harith E. A., Kraemer S. A. et al. The aspirin and heine-bindingsites of ovine and routine prostaglandin endoperoxide synthases. J Biol Chem 1990; 2 6 5 : 5 1 9 2 - 5 1 9 8 . 19. Fletcher B. S., Kujubu D. A., Perrin D. M., Herschman H. R. Structure of the mitogen-inducible TISIOgene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. JBiol Chem 1992; 267: 4338-4344. 20. Meade E. A., Smith W. L., DeWitt D. L. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isoenzymes by aspirin and other non-steroidal antiinflammatory drugs. J Biol Chem 1993; 2 6 8 : 6 6 1 0 - 6 6 1 4 . 21. Funk C. D., Funk L. B., Kennedy M. E., Pong A. S., Fitzgerald G. A. Human platelet-erythroleukemia cell prostaglandin G/H synthase cDNA cloning, expression and gene chromosomal assignement. FASEBJ 1991; 5: 2304-2312. 22. Yokoyama C., Tanabe T. Cloning of human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme. Biochem Biophys Res Commun 1989; 165: 888-894.

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Inhibition by tenoxicam, indomethacm and aspirin of human cyclooxygenase isoenzymes

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Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(5), 361-367