Cyclooxygenase-2 up-regulation after FLAP transfection in human adenocarcinoma cell line HT29 cl.19A.

Five-lipoxygenase-activating protein (FLAP) is usually described as an essential protein to activate the leukotriene (LTs) synthesis via the 5-lipoxygenase pathway. In the enterocyte model HT29 cl.19A cell line, 5-lipoxygenase metabolism was found despite the lack of FLAP expression. Therefore HT29 cl.19A represents an original mammalian model to study FLAP-dependent leukotriene synthesis. In FLAP cDNA transfected HT29 cl.19A cells, FLAP expression led to an increase in cyclooxygenase pathway products (mainly PGE2) without an increase in 5-lipoxygenase metabolism. This increase in PGE2 synthesis was associated with a cyclooxygenase-2 upregulation in comparison to untransfected HT29 cl.19A cells. These results suggest a possible interaction between the two major pathways of arachidonic acid metabolism.

COX-2 selectivity and inflammatory processes.

Increasing amounts of experimental and clinical data support the role of selective cyclooxygenase (COX)-2 inhibition in anti-inflammatory processes and the involvement of COX-1 inhibition in the side effects associated with non steroidal anti-inflammatory drug use. This review will focus on the differences in the structure of the COX-1 and COX-2 molecules, particularly the active site and how they are bound by various NSAIDs to achieve COX-2 selectivity. This COX-2 selectivity will then be characterized in pharmacological assays in vitro and in animal models in vivo. Finally, clinical information available for this new class of selective inhibitors will be discussed.

Effects of caffeine and paracetamol alone or in combination with acetylsalicylic acid on prostaglandin E(2) synthesis in rat microglial cells.

Paracetamol has mild analgesic and antipyretic properties and is, along with acetylsalicylic acid, one of the most popular "over the counter" analgesic agents. However, the mechanism underlying its clinical effects is unknown. Another drug whose mechanism of action is unknown is caffeine, which is often used in combination with other analgesics, augmenting their effect. We investigated the inhibitory effect of paracetamol and caffeine on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)- and prostaglandin (PG)E(2)-synthesis in primary rat microglial cells and compared it with the effect of acetylsalicylic acid, salicylic acid, and dipyrone. Furthermore, combinations of these drugs were used to investigate a possible synergistic inhibitory effect on PGE(2)-synthesis. Both paracetamol (IC(50)=7.45 microM) and caffeine (IC(50)=42.5 microM) dose-dependently inhibited microglial PGE(2) synthesis. In combination with acetylsalicylic acid (IC(50)=3.12 microM), both substances augmented the inhibitory effect of acetylsalicylic acid on LPS-induced PGE(2)-synthesis. Whereas paracetamol inhibited only COX enzyme activity, caffeine also inhibited COX-2 protein synthesis. These results are compatible with the view that the clinical activity of paracetamol and caffeine is due to inhibition of COX. Furthermore, these results may help explain the clinical experience of an adjuvant analgesic effect of caffeine and paracetamol when combined with acetylsalicylic acid.

The novel 5-HT4 receptor antagonist DAU 6285 antagonizes 5-hydroxytryptamine-induced tachycardia in pigs.

The 5-HT4 receptor antagonist action of DAU 6285 was investigated in vivo in anesthetized pigs. DAU 6285 (0.3-3 mg/kg i.v.) dose dependently antagonized 5-hydroxytryptamine (5-HT)-induced tachycardiac responses. In contrast, the 5-HT3 receptor antagonist, ondansetron (0.3-3 mg/kg i.v.) did not influence the tachycardia induced by 5-HT. These results indicate that DAU 6285 is a potent antagonist of 5-HT4 receptor-mediated responses in vivo.

Continuous assessment of multiple vital physiological functions in conscious freely moving rats using telemetry and a plethysmography system.

The general pharmacologist in the pharmaceutical industry is challenged to generate physiologically relevant data on possible safety liabilities or on secondary therapeutic uses as early as possible in drug development. This implies the need for efficient use of usually only small supplies of test article. For this reason, we have developed a new animal model combining various elements to provide a broad spectrum of data focussing on the so-called vital physiological functions: cardiovascular, respiratory, and central nervous system. This system uses rats chronically implanted with transmitters for the measurement of arterial pressure, ECG, and body temperature. Modification of the transmitters also allows for the simultaneous assessment of locomotor activity. Studies are performed with these rats in plethysmographs placed directly over the antenna units thus allowing for the additional assessment of respiratory function in the same studies. Using this system, we can generate simultaneously a wide range of relevant physiological parameters in conscious rats with a modest requirement for test article. Such an approach is highly useful for getting early safety readouts of potential drug development candidates as well as for detecting possible secondary therapeutic actions of a drug.

Opioid receptor agonists in the rabbit colon: comparison of in vivo and in vitro studies.

Myoelectrical activity was recorded in the proximal and distal colon of rabbits using chronically implanted electrodes. The motility in both the proximal and distal colon was inhibited by the intravenous (IV) administration of the following opioid agonists for mu receptors: morphine and fentanyl, kappa receptors: ethylketazocine (EKC) and U 50 488 H, and delta receptors: D-Ala2 D-Leu5-enkephalin (DADLE) and D-Ser2 Leu-enkephalin-Thr6 (DSLET). In contrast, the myoelectric activity in the distal colon was increased during the infusion of an endogenous kappa opioid agonist, dynorphin (DYN). All of these effects were prevented by naloxone pretreatment. During in vitro studies using extraluminal force transducers, fentanyl, U 50 488 H and DSLET inhibited spontaneous contractions of the proximal colon, but U 50 488 H and DSLET caused a substantial increase in the motility of the distal colon. The observed motor responses in the proximal and distal colon following opioid agonist administration indicate that the control of these two intestinal segments may be different. It is suggested that the stimulatory effect of dynorphin on the distal colon is peripherally-mediated while inhibition of the whole colon by opioid agonists regardless of subtypes seems to be centrally-mediated.

The antioxidative activity of the mucoregulatory agents: ambroxol, bromhexine and N-acetyl-L-cysteine. A pulse radiolysis study.

Ambroxol and bromhexine are shown to be scavengers of both superoxide and hydroxyl radicals as determined by pulse radiolysis experiments. The dismutation of superoxide was accelerated 3-fold by bromhexine and 2.5-fold by ambroxol over the rate of spontaneous dismutation. The reaction constants of hydroxyl radicals with bromhexine and ambroxol were determined by competition kinetics to be 1.58 +/- 0.15 x 10(10) M-1S-1 and 1.04 +/- 0.1 x 10(10) M-1S-1, respectively. N-acetyl-L-cysteine also reacted with hydroxyl radicals (1.28 +/- 0.14 x 10(10) M-1S-1) but not with superoxide radical. These effects may be clinically relevant in the treatment of oxidant-associated lung damage induced by inflammatory agents and/or environmental pollutants.

Opioid control of the ruminant stomach motility: functional importance of mu, kappa and delta receptors.

In sheep, the subcutaneous (SC) or intracerebroventricular (ICV) administration of the mu-type opioid agonists, fentanyl and morphine, evokes a blockade of the cyclic contractions of the reticulum. A similar inhibition of forestomach motility was recorded following the administration of the two enkephalin analogs, D-Ala2-Met5-enkephalinamide (DAMA) and D-Ala2-D-Leu5-enkephalin (DADLE) which are mixed mu - delta opioid agonists. In contrast, the reticular contractions were enhanced by the SC or ICV administration of the kappa type agonist, ethylketazocine (EKC) and U - 50 488 H. The proximal duodenum motor activity was transiently increased resulting in the occurrence of a phase III-like activity by these opioid agonists, regardless of the subtypes. The effects of the opioid agonists on reticular motility were prevented by the injection of naloxone but not by the quaternary parent compound methylnaloxone which does not cross the blood-brain barrier. The duodenal motor effects elicited by the opioid agonists were antagonized by both naloxone and methylnaloxone. The results suggest that the inhibition of the ruminant stomach motility is centrally mediated by mu - delta type opioid agonists and are consistent with opposite effects from kappa type opioid agonists. The stimulatory effect of peptide and non-peptide opioid agonists on the duodenum may result in part from direct opioid receptor-mediated actions on smooth muscle.

Local release of monoamines in the gastrointestinal tract: an in vivo study in rabbits.

Dialysis fibers chronically implanted into the gastric submucosa of rabbits allowed us to collect an interstitial fluid (I.S.F.) dialysate in which biogenic amine concentrations were measured, and compared with those obtained from plasma and tissue samples. The results suggest that I.S.F. concentrations represent a good assessment of the local release of the amines by enteric nerves and/or paracrine cells, under basal conditions. The fact that acetylcholine and neostigmine, when perfused through the dialysis system, increased I.S.F. serotonin (5-HT) concentrations, supports a cholinergic modulation of the release of 5-HT within the gastrointestinal wall, and validates the dialysis method as a powerful tool to monitor, in vivo, dynamic changes in I.S.F. monoamine concentrations.

Distinct isoforms (COX-1 and COX-2) of cyclooxygenase: possible physiological and therapeutic implications.

The discovery of an inducible isoform of cyclooxygenase (COX-2) requires a refinement of the theory that inhibition of cyclooxygenase activity explains both therapeutic and side effects of non-steroidal anti-inflammatory drugs (NSAIDs). Indeed, new pharmacological results suggest that COX-2 inhibition provides the therapeutic (ie, anti-inflammatory) activity of NSAIDs, whereas inhibition of constitutive COX-1 is responsible for their gastric and renal side effects as well as for their antithrombotic activity. However, a role of COX-1 in inflammation cannot be excluded. Furthermore, the functional relevance of COX-2 expression and induction in various tissues warrants further investigation. These studies should help in predicting potential adverse effects as well as new indications for selective COX-2 inhibitors.

On the relevance of non-steroidal anti-inflammatory drugs in the prevention of paralytic ileus in rodents.

In the mouse, the gastrointestinal transit of a charcoal marker, halved following the intraperitoneal administration of acetic acid, was no longer inhibited after pretreatment with the non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin, ketoprofen, piroxicam or ximoprofen (0.25-2.5 mg kg-1 orally). In the fasted rat, the migrating myoelectric complex pattern of the small intestine which was disrupted for about one hour by acetic acid was unaltered by pretreatment with indomethacin or ximoprofen (0.5 mg kg-1 i.p.). In the anaesthetized rat, the inhibition by about 50% of the gastrointestinal transit due to laparotomy, did not occur following treatment with NSAIDs. It is concluded that NSAIDs prevent the occurrence of chemically-induced and postoperative ileus in rodents, an effect probably related to the analgesic properties of NSAIDs.

Ambroxol improves the broncho-spasmolytic activity of clenbuterol in the guinea-pig.

The effects of ambroxol on the spasmolytic action of clenbuterol were investigated on acetylcholine-induced bronchospasm in guinea-pigs. Ambroxol (50 mg kg-1 day-1) or vehicle was administered orally for 14 days. Approximately 45 min after the final dose on day 14, the animals were anaesthetized and the spasmolytic effects of clenbuterol (3, 6 or 12 micrograms kg-1 injected intravenously) were determined by use of acetylcholine (40 micrograms kg-1, i.v.)-induced bronchoconstriction. For both vehicle- and ambroxol-treated animals, a positive linear relationship was observed between the log-dose of clenbuterol and the percent inhibition of bronchospasm. The calculated ED25 of clenbuterol (i.e., the dose producing 25% inhibition of the acetylcholine-induced bronchospasm) was 3.98 micrograms kg-1 (3.29 to 4.82 micrograms kg-1, 95% confidence interval) in the presence of ambroxol and 5.81 micrograms kg-1 (4.98 to 6.79 micrograms kg-1) in the absence of ambroxol. The linear regressions with or without ambroxol differed from each other (P < 0.001) but ran parallel (covariance analysis), enabling us to calculate a relative potency, the value of which was 1.46 (1.16 to 1.84). These results demonstrate that the spasmolytic activity of clenbuterol is significantly improved in animals pretreated with ambroxol.

Effects of telmisartan on renal excretory function in conscious dogs.

The present study investigated the effects of telmisartan, a selective AT1 receptor antagonist, on renal function in dogs. Conscious female dogs were treated with (i) vehicle (controls) and three doses of telmisartan (0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg) administered intravenously; (ii) vehicle and three doses of telmisartan (0.3 mg/kg, 1.0 mg/kg and 3.0 mg/kg) administered orally; or (iii) 1.0 mg/kg per day telmisartan orally for 12 days. Eight dogs were used for each experiment. Each of the four treatments in (i) and (ii) was administered 7 days apart. During the 6 h after intravenous administration, urine volume was significantly higher in dogs treated with telmisartan 0.1 mg/kg (8.5 +/- 1.6 ml/kg) and 0.3 mg/kg (7.0 +/- 0.9 ml/kg) than controls (2.7 +/- 0.3 ml/kg; P < 0.05), and renal sodium excretion was increased significantly with telmisartan 0.03 mg/kg (803 +/- 124 micromol/kg), 0.1 mg/kg (1039 +/- 213 micromol/kg) and 0.3 mg/kg (966 +/- 161 micromol/kg) versus controls (159 +/- 21 micromol/kg; P < 0.05). Oral telmisartan at doses of 1.0 mg/kg and 3.0 mg/kg also produced significant increases in urine volume (7.2 +/- 1.1 ml/kg and 6.6 +/- 1.2 ml/kg, respectively) and renal sodium excretion (599 +/- 146 micromol/kg and 555 +/- 131 micromol/kg, respectively) compared with controls (2.8 +/- 0.5 ml/kg and 80 +/- 33 mciromol/kg; P < 0.05) over the 6-h post-dose period. Telmisartan at all intravenous doses and at 3.0 mg/kg orally increased the urinary excretion of chloride significantly over the 6-h post-dose period compared with vehicle alone. The excretion of potassium and creatinine were unchanged by any treatment. Telmisartan 1.0 mg/kg administered orally for 12 days produced similar results. In conclusion, acute intravenous or oral as well as subchronic oral administration of telmisartan to conscious dogs promotes diuresis and natriuresis without affecting potassium or creatinine excretion.

[Selective cyclooxygenase-2 (COX-2) inhibitors: importance and limitations]. / Inhibiteurs sélectifs de la cyclooxygénase de type 2 (COX-2): intérêts et limites.

The discovery of an inducible form of cyclooxygenase (COX-2) requires a refinement of the theory that inhibition of cyclooxygenase activity explains both therapeutic effects and side-effects of non-steroidal anti-inflammatory drugs (NSAIDs). Selective COX-2 inhibitors have demonstrated in clinical trials a significantly better gastrointestinal tolerability than classical NSAIDs, for the same anti-inflammatory activity. Their tolerability in patients with active ulcer or with a recent history of ulcer as well as in patients suffering from cardiovascular or renal diseases has still to be investigated in detail. Their therapeutic potential in several new indications, including pre-term labour, colorectal cancer and Alzheimer's disease, is currently being investigated.

[Academic power versus feminine authority: the Paris Medical School against Jacoba Felicie (1322)]. / Poder academico versus autoridad femenina: la Facultad de Medicina de Paris contra Jacoba Felicie (1322).

This article analyzes the trial that the Faculty of Medicine at Paris pursued against Jacoba Felicie in 1322. Drawing on her parents' narratives, it attempts to interpret Jacoba's medical practice and the particular nature of the relationship that she established with her parents. In order to identify and describe this relationship, we use a basic distinction between power and authority.

Inhibition of cyclooxygenase-1 and cyclooxygenase-2 analysis of in vitro test systems and their clinical relevance.

Numerous in vitro assay systems have been developed for testing and comparing the relative inhibitory activities of nonsteroidal anti-inflammatory drugs (NSAIDs) against cyclooxygenase (COX)- 1 and COX-2, the two COX isoforms responsible for prostaglandin biosynthesis. Despite variability among these systems, which precludes direct comparison of data, analysis of the ratio of inhibition of COX-2 to COX-1 by NSAIDs suggests that inhibitors can be classified based on their COX selectivity. Standard NSAIDs can be classified as preferential COX-2 inhibitors; other compounds currently under development are highly specific for COX-2. Although in vitro systems are important in characterizing activity, the clinical relevance of these data should be carefully considered. Models based on in vitro data can be constructed and maybe compared with, but are no substitute for, in vivo results when they become available.

Pharmacologic characterization of meloxicam.

Meloxicam provides a case study in the evolution of the cyclooxygenase (COX) hypothesis. Meloxicam was initially characterized in standard animal models of inflammation at a time when its improved pharmacologic profile in relation to other nonsteroidal anti-inflammatory drugs (NSAIDs) could not be explained. The subsequent discovery of COS-2 provided a reasonable working hypothesis. Meloxicam was investigated in several in vitro test systems in which it consistently demonstrated preferential COX-2 inhibition. These observations suggested that meloxicam would have a COX-1-sparing effect in vivo. Pharmacologic studies have confirmed the COX-1-sparing effect in animal models and in human subjects and suggest that the improved gastrointestinal and renal safety profile of this drugs may be related to this COX-2-sparing effect. The pharmacologic studies, taken together with the in vitro data, suggest the validity of the COX hypothesis as an estimator of NSAID efficacy and safety. Although further studies have suggested that meloxicam could affect inflammatory processes in other ways than by COX-2 inhibition, the clinical significance of these in vitro findings has yet to be determined. Moreover, accumulating evidence suggests a role for COX-2 in Alzheimer's disease and colorectal cancer. NSAIDs such as meloxicam could therefore be of potential benefit in the prevention or treatment of these diseases.

Measurement of differential inhibition of COX-1 and COX-2 and the pharmacology of selective inhibitors.

Since the discovery of a second isoenzyme of cyclooxygenase (COX)-2, it has been hypothesized that the antiinflammatory effects of non-steroidal antiinflammatory drugs (NSAIDs) are dependent on their inhibition of COX-2, whereas inhibition of constitutive COX-1 is responsible for their gastric and renal side effects as well as for inhibition of platelet activation. Consequently, a large number of in vitro assays have been developed to characterize the COX-1 and COX-2 inhibitory activities of NSAIDs in order to look for compounds with preferential inhibition of COX-2. Depending on the test system, however, the experimental conditions may vary greatly, thereby affecting the outcome. These important variables include the source of enzymes COX-1 and COX-2 (human or animal); the cell system used (intact normal cells or transfected cell lines); the method of enzyme preparation (purified enzymes, microsomal or whole cell assays); the COX-2-inducing agent; the source of arachidonic acid and its concentration; the incubation time with drug, inducing agent or arachidonic acid; and the protein concentration in the medium. Depending on the test system employed for defining the IC(50) for COX-1 and COX-2 inhibition, the resultant ratio between these activities may lead to divergent and confusing comparisons. We review the various in vitro test systems available for the measurement of COX-1 and COX-2 inhibition. The use of human recombinant enzymes and the human whole blood assay are examined in detail. The relevance of these test systems to in vivo animal models of inflammation and the side effects of NSAIDs (e.g., gastric mucosal and renal damage) is assessed. Finally, the results of clinical studies on the effect of inhibiting COX-1 and COX-2 activity is summarized.

Colonic formation of soft feces in rabbits: a role for endogenous prostaglandins.

Rabbits produce hard and soft feces in a circadian rhythm. The motor activity of the haustrated proximal colon is inhibited during the formation of soft feces, whereas the spiking activity of the distal colon is stimulated. A potential role for endogenous prostaglandins (PG) in the control of the soft-feces elaboration by the rabbit colon was investigated in conscious animals by using PGE2 and PGF2 alpha and by inhibiting them with indomethacin. The infusion of both PGE2 and PGF2 alpha induced typical electromechanical events consisting of inhibition of the proximal and stimulation of the distal colon and was followed by soft-pellet defecation. Rabbits accustomed to be fed twice daily produced soft feces at fixed intervals of 252 +/- 32 min after the evening meal, with a soft-to-hard feces ratio of 1.45. After indomethacin treatment, this ratio was significantly (P less than 0.01) reduced to 0.92. These results are consistent with the concept that endogenous prostaglandins play a major role in the motor function involved in soft-feces formation by the rabbit.