J. Delarge

In vitro effects of aceclofenac and its metabolites on the production by chondrocytes of inflammatory mediators.

Abstract. Objectives: To investigate the mechanisms of action underlying the anti-inflammatory action of aceclofenac in vivo, we studied in vitro the effect of aceclofenac and its main metabolite, 4′-hydroxyaceclofenac, in comparison with diclofenac, another metabolite, on cyclooxygenases activity as well as interleukin-1β, -6 and -8, nitric oxide, and prostaglandin E2 production by human osteoarthritic and normal articular chondrocytes. Methods: Enzymatically isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1β (IL-1β) or lipopolysacharride (LPS) and with or without increased amounts (1 to 30 μM) of aceclofenac or metabolites. The production of different cytokines was measured by Enzyme Amplified Sensitivity Immunoassays (EASIA). Prostaglandin E2 was quantified by a specific radioimmunoassay. Nitrite and nitrate concentrations in the culture supernatants were determined by spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2, inducible NO synthase and IL-1β gene expression were quantified by reverse transcription of mRNA followed by real time and quantitative polymerase chain reaction. Finally, cyclooxygenase inhibitory potency of the drugs was also tested in both a cell-free system using purified ovine cyclooxygenase-1 and -2 (COX-1 and COX-2) and at a cellular level using human whole blood assay. Results: We have demonstrated that aceclofenac, 4′-hydroxyaceclofenac and diclofenac significantly decreased interleukin-6 production at concentrations ranged among 1 to 30 μM and fully blocked prostaglandin E2 synthesis by IL-1β- or LPS-stimulated human chondrocytes. Aceclofenac and diclofenac had no effect on interleukin-8 production while 4′-hydroxyaceclofenac slightly decreased this parameter at the highest dose (30 μM). Aceclofenac was without effect on IL-1β- or LPS-stimulated nitric oxide production. At 30 μM, 4′-hydroxyaceclofenac inhibited both IL-1β or LPS-stimulated nitric oxide production while diclofenac inhibited only the LPS-stimulated production. Finally, at 30 μM, the three drugs significantly decreased IL-1β mRNA. In the whole blood test, aceclofenac and 4′-hydroxyaceclofenac weakly inhibited COX-1 with IC50 values superior to 100 μM, but decreased by 50% COX-2 activity at the concentration of 0.77 and 36 μM, respectively. Diclofenac strongly inhibited both COX-1 and COX-2 with IC50 values of 0.6 and 0.04 μM, respectively. On the other hand, aceclofenac and diclofenac weakly inhibited purified ovine cyclooxygenases with IC50 values superior to 100 μM, whereas 4′-hydroxyaceclofenac was without effect. Conclusions: These results suggest that aceclofenac actions are multifactorial and that metabolites could contribute to its anti-inflammatory actions.

New dibenzazepine derivatives with disinhibitory and/or antidepressant potential: neurochemical and behavioural study in the open-field and forced swimming tests.

Original bioisosteric analogues of clozapine were evaluated for potential disinhibitory and/or antidepressant effects using the open-field test in the rat and Porsolts forced swimming test in the mouse. Attempts to relate the behavioural results to the binding affinities for dopamine (D1, D2), serotonin (5-HT2) and muscarinic (M) receptors were also undertaken. In the open-field test, two main profiles were observed. The first profile corresponded to disinhibitory molecules resembling diazepam and ritanserin. The second profile corresponded to antipsychotic compounds resembling either typical (haloperidol, clothiapine) or atypical (clozapine) neuroleptics. The results obtained in the forced swimming test confirmed the neuroleptic-like activity of the second group of compounds, while two compounds of the first group (JL 3 and JL 26) showed an antidepressant-like activity, JL 3 being as active as imipramine. While it was not possible to relate the first profile to any binding interaction, a relation could be established among the second group of compounds between the typical or atypical antipsychotic behavioural profile and the 5-HT2/D2 ratio.

Advances in the field of COX-2 inhibition

Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances that are involved in several physiological processes but also in pathological conditions, such as inflammation. It is actually well known that this enzyme exists under two forms: COX-1 and COX-2. Both enzymes are sensitive to inhibition by conventional non-steroidal anti-inflammatory drugs (NSAIDs). Observations that COX-1 was involved in several homeostatic processes, while COX-2 expression was associated with inflammation and other pathologies, such as cancer proliferation, have led to the development of COX-2 selective inhibitors to improve the therapeutic potency and to reduce the classical side effects associated with the use of conventional NSAIDs. A large number of patents concerning COX inhibition are claimed each year and this article reviews patents claimed during the period January 1998 to December 2001.

Comparative study of pirlindole, a selective RIMA, and its two enantiomers using biochemical and behavioural techniques.

The interaction with monoamine oxidase A (MAO-A) and B has been shown to be sensitive to the absolute configuration of molecules. Therefore, the aim of this study was to compare the effects of the racemic pirlindole (a selective and reversible MAO-A inhibitor) and its two enantiomers using biochemical techniques (in vitro and ex vivo determination of rat brain MAO-A and MAO-B activity) and behavioural models (forced swimming test and reserpine-induced hypothermia and palpebral ptosis test). In vitro, the MAO-A IC50 of (±)-pirlindole, R-(—)-pirlindole and S-(+)-pirlindole were 0.24, 0.43 and 0.18 (j,M, respectively. Ex vivo, their ID50 were 24.4, 37.8 and 18.7 mg/kg i.p. The differences between the three compounds were not significant, with a ratio between the two enantiomers [R-(—)/S-(+)] of 2.2 in vitro and 2.0 ex vivo. MAO-B was only slightly inhibited. In the forced swimming test and the reserpine-induced hypothermia and ptosis model, the three compounds had an antidepressant profile. In the forced swimming test, the minimal effective dose ratio between the R-(-) and the S-(+) was again around 2.0. The behavioural observations were thus clearly in accordance with the biochemical data.

3-Isopropylamino-4-methyl-4H-pyrido[4,3-e][1,2,4]thiadiazine 1,1-dioxide

The title compound, C 10 H 14 N 4 O 2 S, is the 4-methyl analogue of an original potassium channel opener molecule related to diazoxide. The crystal structure determination of a compound with a methyl substituent in the 4-position provides geometric reference data which may be useful for analysing the preferential 2H- or 4H-tautomeric form adopted in unsubstituted derivatives of this class of compounds in the solid state.

N2-Cyano-N1-isopropyl-N3-[4-(3-methylphenylamino)-3-pyridylsulfonyl]guanidine

The title compound, C 17 H 20 N 6 O 2 S, is a bioisoster of torasemide, a loop diuretic whose structure has been described previously. The sulfonylurea chain of torasemide is replaced by a sulfonylcyanoguanidine function. Whereas the torasemide molecule and some sulfonylurea derivatives exhibit one of the three α, β or γ conformations, the conformation being assigned according to the torsion angles in the side chain, the title compound displays a new δ conformation. This conformation is stabilized by two intramolecular N-H...O hydrogen bonds. A prototropic form of the title compound corresponding to a zwitterion [-S-N - -C-, N + -H (pyridinium)] is observed {i.e N 2 -cyano-N 1 -isopropyl-N 3 -[4-(3-methylphenylamino)-3-pyridiniosulfonyl]guanidin-3-ide}. The crystal cohesion is the result of both van der Waals interactions and one intermolecular N + -H...N - hydrogen bond involving the N atoms of the zwitterion

4‐Ethyl‐2,3‐dihydro‐4H‐pyrido[3,2‐e]‐1,2,4‐thiadiazine 1,1‐dioxide and 4‐ethyl‐2,3‐dihydro‐4H‐pyrido[4,3‐e]‐1,2,4‐thiadiazine 1,1‐dioxide

A series of 4H-1,2,4-pyridothiadiazine 1,1-dioxides and 2,3-dihydro-4H-1,2,4-pyridothiadiazine 1,1-dioxides were tested as possible allosteric modulators of the (R/S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid receptors; the most active is 4-ethyl-2,3-dihydro-4H-pyrido[3,2-e]-1,2,4-thiadiazine 1,1-dioxide, C 8 H 11 N 3 O 2 S. Its crystal molecular geometry is compared with that of the -pyrido[4,3-e]- compound, C 8 H 11 N 3 O 2 S, a less potent analogue.

7-Chloro-(R)-3-[1-(cyclohexyl)ethylamino]-4H-1,2,4-benzothiadiazine 1,1-dioxide and 7-chloro-(S)-3-[1-(cyclohexyl)ethylamino]-4H-1,2,4-benzothiadiazine 1,1-dioxide

3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides structurally related to diazoxide are expected to present a pharmacological profile of potassium-channel openers. The influence on biological activity of the absolute configuration of the title compounds, both C 15 H 20 ClN 3 O 2 S, is being studied. The crystallographic results confirm the enantiomeric characterization of each compound.

8-Chloro-5-(4-methylpiperazin-1-yl)-11H-pyrido[2,3-b][1,5]benzodiazepine

The crystal structure determination of C 17 H 18 ClN 5 has been undertaken as part of our studies of dopamine receptors. The diazepine ring is in a boat conformation while the piperazine ring is in the normal chair conformation. The dihedral angle between the two aromatic rings that lie on the same side of the diazepine moiety is 126.4(1)°. There is one N-H...N hydrogen bond [N...N 3.184 (3) A].

3-Methyl-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1,1-dioxide

The title compound, C7H7N302S, was prepared for comparison with diazoxide, an antihypertensive agent, from a structural and pharmacological point of view. The crystal structure determination shows that the 4H (rather than 2H) tautomeric form is preferentially adopted by this pyridothiadiazine derivative in the solid state.