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Selective suppression of cyclooxygenase-2 during chronic administration of nimesulide in man
226
EFA & Eicosanoids 1997- Edinburgh
Poster Presentations Monday 21 July
P49
P50
Inhibition of peroxidase and cyclooxygenase activity of prostaglandin synthase (PGHS) by hydroxamic acid derivatives of salicylic acid. O'Connor S, Le Roy S, Nolan K, Fitzgerald DJ. Depts. of Clinical Pharmacology and Chemistry, RCSI, Dublin 2.
PGE2 EP2 A N D EP4 R E C E P T O R S U B T Y P E M R N A E X P R E S S I O N IN M A C A Q U E T I S S U E S
PGHS is both a cyclooxygenase (COX) and a potent peroxidase (POX). Pox activity persists following inhibition of COX by aspirin and similar drugs. possibly limiting their effectiveness in suppressing tissue damage at sites of inflammation. We synthesized a series of hydroxamic acid (HA) derivatives of salicylic acid (SA) and tested their activity against hydrogen peroxide- and arachidonic acid-catalysed oxidation of N,N,N',N'-tetramethyl-pphenylenediamine by purified ovine COX-1. SA and aceto-HA alone had little effect on COX or POX. The HA ester of SA inhibited the POX activity by >50%, at room temperature and at 37°C. The HA ester of benzoic acid had similar potency against POX. Moreover, both SA-HA and benzo-HA but not HA or SA abolished platelet aggregation induced by arachidonic acid. To develop a compound that could inhibit both activities, we synthesized acetyI-HA derivatives ofSA (SA-HA-Ac) and ASA (ASA-HA-Ac). Both inhibited COX activity, but not POX activity. SA-HA-Ac almost completely knocked out COX activity within one minute. Their inhibitory function on COX, like aspirin was dependent on the presence of heme in the enzyme. SA-HA still inhibits POX activity by almost 50% after preincubation of PGHS with SAHA-Ac. This provides strong evidence for the existence of COX and POX activities on PGHS at two spatially distinct sites.
% inhibition Control ASA SA SA-HA SA-HA-Ac Aceto-HA Benzo-HA Phenyl acetic acid
Arachidonic acid 100 88 97 44 118 36 78
Hydrogen peroxide 100 106 100 54 92 125 68 102
P51
L i c h t e n w a l n e r A B and Patton DL: Dept. o f M e d i c i n e , Div. A l l e r g y and Infectious Disease; Dept. o f Obstetrics and G y n e c o l o g y , U n i v e r s i t y o f W a s h i n g t o n , Seattle W A USA. P r o s t a g l a n d i n E2 ( P G E 2 ) c a u s e s smooth m u s c l e r e l a x a t i o n and decreases i m m u n e r e s p o n s e s in various tissues. P G E 2 receptors m e d i a t e its v a r i o u s effects, and the activities o f the EP2 and EP4 receptor s u b t y p e s a p p e a r to be similar. W e e v a l u a t e d M a e a c a n e m e s t r i n a t i s s u e in order to (1), investigate w h e t h e r EP2 and EP4 receptor subtype m R N A are detectable u t i l i z i n g h u m a n m R N A probes, (2) to d e s c r i b e the m a c a q u e tissues e x p r e s s i n g these receptors, and (3) to test w h e t h e r c h a n g e s in receptor e x p r e s s i o n o c c u r due to chronic infection. W e utilized in situ h y b r i d i z a t i o n w i t h 3sS-labelled m R N A probes for the p r o d u c t i o n o f P G E 2 EP2 and P G E 2 E P 4 receptor subtype m R N A m a d e using h u m a n e D N A . Tissues w e r e obtained f r o m n o r m a l control a n i m a l s o b t a i n e d at n e c r o p s y i m m e d i a t e l y f o l l o w i n g e u t h a n a s i a (n= 1; m u l t i p l e tissues; n= 10; reproductive t i s s u e s only), and from a n i m a l s e x p e r i m e n t a l l y infected w i t h C. t r a c h o m a t i s at h y s t e r e c t o m y a p p r o x i m a t e l y 2 m o n t h s f o l l o w i n g a n t i b i o t i c t r e a t m e n t for chlarnydial cervicitis and p e l v i c i n f l a m m a t o r y d i s e a s e (n=10). Signal for EP4 w a s detected in heart, kidney, spleen a n d in p e r i v a s c u l a r tissues o f the control animal, and s i g n a l for EP2 and E P 4 w a s detected in r e p r o d u c t i v e tract t i s s u e s o f both control and i n f e c t e d animals. This project w a s s u p p o r t e d by PHS grant A I - 3 3 1 1 8 and b y W a s h i n g t o n R e g i o n a l P r i m a t e Research C e n t e r grant R R 0 0 1 6 6 .
P52 Selective Suppression Of Cyclooxygenase-2 During Chronic Administration Of Nimesulide Ill Man. L Cullen, L Kelly, D Coyle, R Forde and D Fitzgerald, Dept. of Clinical Pharmacology, Royal College of Surgeons, Dublin 2, Ireland.
JAbstract Withdrawn
[
Non-steroidal anti-inflammatory drugs (NSAIDs) are used in the treatment of inflammatory conditions such as arthritis, however they are potential irritants to the gastrointestinal (GI) mucosa due to inhibition of Cox-I mediated prostaglandin production. The currently commercially available NSAIDs are non-selective Cox inhibitors and are associated with peptic ulceration in the stomach. Nimesulide is a novel NSAID with selectivity towards Cox-2 in vitro. Here, we examined the effect ofnimesulide on Cox-I (serum TXB2) and Cox-2 (endotoxin-induced generation of PGE2 in whole blood) activity in patients with muscoloskeletal pain. In addition, urinary TXB 2 and 6-ketoPGF=~ levels were determined, to explore the effect of nimesulide on renal cyclooxygenase activity. Nimesulide, 100 mg bid or aspirin, 300 mg tid was administered for 14 days followed by a washout period of 10 days, with sampling on days 2, 5, 10 and 14 during treatment and 2, 5 and I0 following drug withdrawal. Ten patients received nimesulide and ten received aspirin. The mean age of the nimesulide group (5M,5 F) was 60.89 yrs and in the aspirin group (6M,4F) was 58.71 yrs. Endotoxin-induced PGE 2 formation fell markedly in the nimesulide-treated patients (34.24 _+ 8.9 ng/ml to 2.6 + 0.94 ng/ml) whereas aspirin had no effect (28.73 + 4.6 ng/ml to 27.14 + 3.8 ng/ml). The effect on endotoxin-induced PGE 2 formation recovered 24 hr following discontinuation of the nimesulide. In contrast, nimesulide had no significant effect on serum TXB2( 207.5 + 44.6 ng/ml to 188.8 + 52.43 ng/ml) which was suppressed by aspirin (187.28 +.+_20 ng/ml to 2.82 + 0.9 ng/ml). No change was detected in urinary prostaglandins in patients treated with nimesulide. Nimesulide suppressed Cox-2 in vivo with no detectable effect on platelet Cox1. Moreover, nimesulide had no effect on renal production of prostaglandins in this population. This is the first study that demonstrates the Cox-2 selectivity ofa NSAID in vivo, an approach that may lead to a safer treatment for pain and arthritis.
EFA & Eicosanoids 1997- Edinburgh
Poster Presentations Monday 21 July
P49
P50
Inhibition of peroxidase and cyclooxygenase activity of prostaglandin synthase (PGHS) by hydroxamic acid derivatives of salicylic acid. O'Connor S, Le Roy S, Nolan K, Fitzgerald DJ. Depts. of Clinical Pharmacology and Chemistry, RCSI, Dublin 2.
PGE2 EP2 A N D EP4 R E C E P T O R S U B T Y P E M R N A E X P R E S S I O N IN M A C A Q U E T I S S U E S
PGHS is both a cyclooxygenase (COX) and a potent peroxidase (POX). Pox activity persists following inhibition of COX by aspirin and similar drugs. possibly limiting their effectiveness in suppressing tissue damage at sites of inflammation. We synthesized a series of hydroxamic acid (HA) derivatives of salicylic acid (SA) and tested their activity against hydrogen peroxide- and arachidonic acid-catalysed oxidation of N,N,N',N'-tetramethyl-pphenylenediamine by purified ovine COX-1. SA and aceto-HA alone had little effect on COX or POX. The HA ester of SA inhibited the POX activity by >50%, at room temperature and at 37°C. The HA ester of benzoic acid had similar potency against POX. Moreover, both SA-HA and benzo-HA but not HA or SA abolished platelet aggregation induced by arachidonic acid. To develop a compound that could inhibit both activities, we synthesized acetyI-HA derivatives ofSA (SA-HA-Ac) and ASA (ASA-HA-Ac). Both inhibited COX activity, but not POX activity. SA-HA-Ac almost completely knocked out COX activity within one minute. Their inhibitory function on COX, like aspirin was dependent on the presence of heme in the enzyme. SA-HA still inhibits POX activity by almost 50% after preincubation of PGHS with SAHA-Ac. This provides strong evidence for the existence of COX and POX activities on PGHS at two spatially distinct sites.
% inhibition Control ASA SA SA-HA SA-HA-Ac Aceto-HA Benzo-HA Phenyl acetic acid
Arachidonic acid 100 88 97 44 118 36 78
Hydrogen peroxide 100 106 100 54 92 125 68 102
P51
L i c h t e n w a l n e r A B and Patton DL: Dept. o f M e d i c i n e , Div. A l l e r g y and Infectious Disease; Dept. o f Obstetrics and G y n e c o l o g y , U n i v e r s i t y o f W a s h i n g t o n , Seattle W A USA. P r o s t a g l a n d i n E2 ( P G E 2 ) c a u s e s smooth m u s c l e r e l a x a t i o n and decreases i m m u n e r e s p o n s e s in various tissues. P G E 2 receptors m e d i a t e its v a r i o u s effects, and the activities o f the EP2 and EP4 receptor s u b t y p e s a p p e a r to be similar. W e e v a l u a t e d M a e a c a n e m e s t r i n a t i s s u e in order to (1), investigate w h e t h e r EP2 and EP4 receptor subtype m R N A are detectable u t i l i z i n g h u m a n m R N A probes, (2) to d e s c r i b e the m a c a q u e tissues e x p r e s s i n g these receptors, and (3) to test w h e t h e r c h a n g e s in receptor e x p r e s s i o n o c c u r due to chronic infection. W e utilized in situ h y b r i d i z a t i o n w i t h 3sS-labelled m R N A probes for the p r o d u c t i o n o f P G E 2 EP2 and P G E 2 E P 4 receptor subtype m R N A m a d e using h u m a n e D N A . Tissues w e r e obtained f r o m n o r m a l control a n i m a l s o b t a i n e d at n e c r o p s y i m m e d i a t e l y f o l l o w i n g e u t h a n a s i a (n= 1; m u l t i p l e tissues; n= 10; reproductive t i s s u e s only), and from a n i m a l s e x p e r i m e n t a l l y infected w i t h C. t r a c h o m a t i s at h y s t e r e c t o m y a p p r o x i m a t e l y 2 m o n t h s f o l l o w i n g a n t i b i o t i c t r e a t m e n t for chlarnydial cervicitis and p e l v i c i n f l a m m a t o r y d i s e a s e (n=10). Signal for EP4 w a s detected in heart, kidney, spleen a n d in p e r i v a s c u l a r tissues o f the control animal, and s i g n a l for EP2 and E P 4 w a s detected in r e p r o d u c t i v e tract t i s s u e s o f both control and i n f e c t e d animals. This project w a s s u p p o r t e d by PHS grant A I - 3 3 1 1 8 and b y W a s h i n g t o n R e g i o n a l P r i m a t e Research C e n t e r grant R R 0 0 1 6 6 .
P52 Selective Suppression Of Cyclooxygenase-2 During Chronic Administration Of Nimesulide Ill Man. L Cullen, L Kelly, D Coyle, R Forde and D Fitzgerald, Dept. of Clinical Pharmacology, Royal College of Surgeons, Dublin 2, Ireland.
JAbstract Withdrawn
[
Non-steroidal anti-inflammatory drugs (NSAIDs) are used in the treatment of inflammatory conditions such as arthritis, however they are potential irritants to the gastrointestinal (GI) mucosa due to inhibition of Cox-I mediated prostaglandin production. The currently commercially available NSAIDs are non-selective Cox inhibitors and are associated with peptic ulceration in the stomach. Nimesulide is a novel NSAID with selectivity towards Cox-2 in vitro. Here, we examined the effect ofnimesulide on Cox-I (serum TXB2) and Cox-2 (endotoxin-induced generation of PGE2 in whole blood) activity in patients with muscoloskeletal pain. In addition, urinary TXB 2 and 6-ketoPGF=~ levels were determined, to explore the effect of nimesulide on renal cyclooxygenase activity. Nimesulide, 100 mg bid or aspirin, 300 mg tid was administered for 14 days followed by a washout period of 10 days, with sampling on days 2, 5, 10 and 14 during treatment and 2, 5 and I0 following drug withdrawal. Ten patients received nimesulide and ten received aspirin. The mean age of the nimesulide group (5M,5 F) was 60.89 yrs and in the aspirin group (6M,4F) was 58.71 yrs. Endotoxin-induced PGE 2 formation fell markedly in the nimesulide-treated patients (34.24 _+ 8.9 ng/ml to 2.6 + 0.94 ng/ml) whereas aspirin had no effect (28.73 + 4.6 ng/ml to 27.14 + 3.8 ng/ml). The effect on endotoxin-induced PGE 2 formation recovered 24 hr following discontinuation of the nimesulide. In contrast, nimesulide had no significant effect on serum TXB2( 207.5 + 44.6 ng/ml to 188.8 + 52.43 ng/ml) which was suppressed by aspirin (187.28 +.+_20 ng/ml to 2.82 + 0.9 ng/ml). No change was detected in urinary prostaglandins in patients treated with nimesulide. Nimesulide suppressed Cox-2 in vivo with no detectable effect on platelet Cox1. Moreover, nimesulide had no effect on renal production of prostaglandins in this population. This is the first study that demonstrates the Cox-2 selectivity ofa NSAID in vivo, an approach that may lead to a safer treatment for pain and arthritis.