Session 15 : Antalgiques / Anti-inflammatoires /Neuropeptides

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Session 15 : Antalgiques / Anti-inflammatoires /Neuropeptides

143

S 19812: A NOVEL DUAL INHIBITOR OF CYCLOOXYGENASES AND LIPOXYGENASE WITH AN EXCELLENT GASTRIC TOLERANCE

C.Tordjman, N. André, Y. Bresson, N. Picaud, M. Droual, F. Sauveur, M. Wierzbicki and J. Bonnet

Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France

Chronic NSAI treatment is associated with the development of gastrointestinal toxicity (1). The mechanism of this adverse effect was first shown to be related to the inhibition of prostaglandin synthesis (2). Then, in the late 1980s, the contribution of leukotrienes was raised (3). More recently, the selective inhibition of inducible cyclooxygenase has been proposed as a safer therapy (4).

S19812

– (4-(4,5-bis-(4-methoxy phenyl) thiophen-2yl) N-hydroxy N-methyl butanamide) – was shown to be an inhibitor of lipoxygenase (LOX) and the two cyclooxygenase (COX) pathways, as assessed in mouse macrophages and PMNs, using the techniques described (5, 6). In pretreated mice, S19812 inhibited the production of blood PGE2 and LTB4 (65% and 84% respectively, at 10 mg/kg). In vivo, S19812 exhibited analgesic activity against phenylquinone-induced writhing.

Whereas gastric toxicity occured with indomethacin from 2.5 mg kg or with nimesulide from 100 mg/kg, no gastric erosions were observed with S19812 up to 400 mg/kg po in fasted mice and rats. In adjuvant-induced arthritis in rats, S19812, inhibited the inflammatory process (ED50 = 15 mg/kg/day p.o.).

These data show that S19812 is a dual inhibitor of cyclooxygenases and lipoxygenase with good in vivo anti-inflammatory and analgesic properties and excellent gastric tolerance. Other studies are ongoing to confirm the analgesic activity of the compound and to understand better the mechanism of its gastric tolerance.

References: (1) Rainsford K.D. Advances in Inflammation Research (Raven Press) 1984, 6: 51 (2) Whittle B.J.R. Gastroenterology 1981, 80: 94 (3) Rainsford K.D. Agents Actions 1987, 21: 316 (4) Vane J.R. Nature Bew Biology 1994, 367: 215 (5) Tordjman C. et al. Journal of Chromatography 1990, 532: 135 (6)Tordjman et al. Biochimica Biophysica Acta 1995, 1256: 249.

144

NEUROPEPTIDE FF AND OPIOID TOLERANCE AND DEPENDENCE IN MICE

A. Gelot, B. Francés and J.M. Zajac.

I.P.B.S., C.N.R.S, 205 Route de Narbonne, 31077 Toulouse, France.

Chronic administration of opioid leads to the development of tolerance to their effects and to an abstinence syndrome at the cessation of the treatment. According to the anti-opioid model1, opioid tolerance could result from a progressive compensatory increase in the activity of anti-opioid peptides which are responsible for the withdrawal syndrome. Several studies have shown that NPFF, a mammalian amidated neuropeptide, is a neuromodulator of opioid systems. The aim of our study is to evaluate the real involvement of the NPFF system in the opioid tolerance and dependence. Recently, one gene encoding for the precursor protein of neuropeptide FF has been identified in human2. We have investigated the effects of the administration of an equimolar mixture of synthetic oligodeoxynucleotides (ODNs) complementary to the starting codon of the human NPFF precursor gene and to a part of the region containing the sequence of NPFF (AS ODNs), and those of the injection of mismatch ODNs (MM), controls. Intracerebroventricular (i.c.v.) treatment for 5 days with AS ODNs did not change the analgesic activity of morphine in the tail flick test. But, i.c.v. treatment of mice with AS ODNs during 2 days followed by concurrent morphine treatment during 3 days, attenuated significantly the degree of tolerance to the antinociceptive effect of subcutaneous injected morphine as compared to i.c.v. treatment with MM ODNs. Furthermore, AS ODNs treatment decreased significantly the number of jumps elicited by injection of naloxone, a behavioural response characteristic of the opiate withdrawal syndrome.

These results demonstrate directly that NPFF play a role in the development of opioid tolerance and dependence.

1- R.B. Rothman. (1992) Synapse 12 129-138. 2- S.J. Perry et al. (1997) FEBS Letters 409 426-430.

145

A UDP-GLUCURONOSYLTRANSFERASE ACTIVITY IS INCREASED BY INFLAMMATION IN RABBIT SYNOVIAL MEMBRANE

L. Ferrari¥, A. Martin¥, J. Magdalou¥,#, P. Pottie#, P. Gillet#, F. Lapicque#, P. Netter# and A.M. Batt¥

¥Centre du Médicament, UPRES, Faculté de Pharmacie, Université Henri Poincaré – Nancy 1, 30 rue Lionnois, 54000 Nancy

#

UMR 7561 CNRS, Faculté de Médecine de Nancy, Université Henri Poincaré – Nancy 1, 54505 Vandoeuvre les Nancy

The presence of drug metabolizing enzymes in rabbit synovial membranes has been determined. Total Cytochrome P450 was not detected, whereas the NADPH cytochrome P450 reductase was slightly expressed. No conjugation to glutathione occurred. By contrast, glucuronidation of 4-methylumbelliferone, supported by several isoforms of UDP-glucuronosyltransferase (UGT) could be measured. Inflammation induced by carrageenan significantly enhanced the enzyme activity. The treatment increased the apparent Vmax of the reaction (37.23 and 56.23 nmoles/h/mg respectively), but not the Km (150.33 and 148.03 µM respectively, thus suggesting a direct induction of the UGT protein. These data are the first report of the presence of such enzyme in synovial membranes and of its sensitivity to inflammation. Their potential importance with regard to glucuronidation of NSAIDs in this tissue must be evaluated.

146

CHONDROPROTECTION BY NO SYNTHASES INHIBITORS: FROM IN VITRO SCREENING TO IN VIVO EFFICACY

C. Cipolletta, N. Presle, J-Y. Jouzeau, B. Terlain and P. Netter

Laboratoire de Pharmacologie & UMR CNRS-UHP 7561, Fac. de Médecine, Av. de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy, F

Recent experimental studies, made on cartilage slices or chondrocytes cultures, have showed that interleukin-l (IL-1) decreased cartilage anabolism when increasing nitric oxide (NO) production, further suggesting that NO may have an important role in suppression of proteoglycan (PG) synthesis. As IL-1 is a key mediator of joint damage during arthritides and several widely used drugs, as NSAIDs, are ineffective in preventing cartilage injury, we looked for chondroprotection by NOS inhibitors. In a preliminary in vitro experiment, we studied the effect of different NOS inhibitors [L-arginine analogs (L-NAME, L-NMMA), aminoguanidine, S-substituted isothioureas (SMT, AETU)] on PG synthesis by rat chondrocytes embedded in alginate beads and cartilage entities (patellæ, femoral head caps) exposed to hrIL-1

inhibited rat patellar PG synthesis by 29% when increasing stable NO end products in SF by 12 fold, ii) L-NMMA corrected the suppressive effect of IL-1 on PG synthesis by 58% when reducing NO metabolites to basal level (0.65µM). Similar results were obtained when L-NMMA was administered locally (ia) at the same time as IL-1. Taken together, ours results suggest that NOS inhibitors may have heterogeneous chondroprotective potencies but that L-NMMA might be promising for therapeutic use in joint disorders possibly through a local sustained release.

147

INHIBITORY EFFECTS OF ADENOSINE RECEPTOR AGONISTS ON THE RELEASE OF CGRP, BUT NOT SUBSTANCE P. FROM THE RAT SPINAL CORD

S. Bourgoin, A. Mauborgne, H. Polienor, M. Hamon and F. Cesselin

INSERM U288, CHU Pitié-Salpêtrière, 91, Boulevard de l’Hôpital, 75634 Paris cedex 13, France

Adenosine exerts antinociceptive effects when injected intrathecally in rats (1), but the mechanisms responsible for its action at the spinal level are still poorly known. We tested here whether adenosine could affect substance P (SP)- and/or calcitonin gene-related (CGRP)-containing primary afferent fibres that convey nociceptive messages from the periphery to the spinal cord. For this purpose, slices (0.3 mm thick) of the dorsal part of the lumbar enlargement of the rat spinal cord, which contain terminals of SP- and CGRP-ergic afferent fibres, were prepared and continuously superfused with an artificial cerebrospinal fluid (aCSF, flow rate: 1 ml / 4 min. 37°C). Superfusate fractions were then collected for the measurement of SP-like material (SP-LM) and CGRP-LM released from the slices, using specific radioimmunoassays. Under basal conditions (K+ = 5.4 mM in the aCSF), none of the adenosine receptor agonists tested significantly affected the spontaneous outflow of both SP-LM and CGRP-LM. In contrast, under K+-induced depolarization (K+ = 30 mM), the potent A1 receptor agonist CPA (N6-cyclopentyladenosine), and NECA (5′-N-ethylcarboxamidoadenosine), a mixed A1-A2A receptor agonist,were found to exert a significant inhibitory influence on CGRP-LM release (~- 20 % at 0.1-100 nM, p < 0.05) without affecting SP-LM release. In addition, a selective decrease in K+-evoked CGRP-LM overflow (~-15%, p < 0.05) was also observed in the presence of the preferential A3 receptor agonist APNEA
(N6-2-[4aminophenyl]ethyladenosine), but at concentrations (10 – 100 nM) probably too high to allow the specific stimulation of A3 receptors. These results suggest that adenosine, mainly through the stimulation of A1 receptors, can exert a presynaptic inhibitory influence on CGRP-ergic primary afferent fibres. Such a mechanism probably contributes to the analgesic action of adenosine since previous behavioural studies concluded that A1 receptors also mediate the latter action (1).

(1) Karlsten, R. et al. Neurosci. Lett. 121: 267-270,1991.

148

COULD THE QUANTITATIVE ANALYSE OF SPONTANEOUS ULTRASONIC VOCALIZATIONS IN CHRONIC PAINFUL RAT BE A PHARMACOLOGICAL PAIN TEST ?

D. Jourdan, D. Ardid, E. Chapuy, A. Eschalier and J. Lavarenne

Laboratoire de Pharmacologie Médicale (groupe NPPUA), Faculté de Médecine, 63001 Clermont-Ferrand Cédex

The greater part of the pain tests used for the assessment of the analgesic activity of drugs on animal chronic pain models are based on the measure of the responses to an additional acute stimulation (thermal, mechanical or electrical) which is not absolutely relevant. It would be more interesting to develop a quantitative analyse of spontaneous behaviours induced by the chronic pain state. Several authors have suggested that ultrasonic vocalisations (USV) emitted by rats in painful situations, might reflect expression of affective pain.

The aim of our study was to compare the spontaneous USV of normal and chronic painful rats in order to obtain a quantitative behavioural parameter for pharmacological studies. The behaviour of 48 male Sprague Dawley rats was investigated: frequency and duration of the USV, freezing, grooming, exploration and aggressive behaviour. The influence of three different parameters was studied: the duration of pain, sub chronic and chronic (inflammation induced by carragenin and polyarthritism induced by Freund’s adjuvant), the interaction with an heavier conspecific, the housing conditions (isolated or collective).

No difference in frequency of the ultrasonic emissions were observed between healthy and painful rats. Significant differences in the duration of USV were only observed during social contact among the healthy rats housing alone or by group of 6 (170

22 s respectively, p>0.01) but no change was observed between healthy and painful rats. In addition, the induction of the ultrasonic emission was correlated with the establishment of the dominance relationship.

Thus, the emission of USV seems to be correlated with social experience of the rats and not with inflammatory pain. Consequently, on a pharmacological point of view, the quantitative analyse of the USV could not represent a useful tool for investigating the emotional component of chronic pain in these models.