X. De Leval

New Developments on Thromboxane and Prostacyclin Modulators Part I: Thromboxane Modulators

The pathogenesis of numerous cardiovascular, pulmonary, inflammatory, and thromboembolic diseases can be related to arachidonic acid (AA) metabolites. One of these bioactive metabolites of particular importance is thromboxane A(2) (TXA(2)). It is produced by the action of thromboxane synthase on the prostaglandin endoperoxide H(2)(PGH(2)), which results from the enzymatic degradation of AA by the cyclooxygenases. TXA(2) is a potent inducer of platelet aggregation, vasoconstriction and bronchoconstriction. It is involved in a series of major pathophysiological states such as asthma, myocardial ischemia, pulmonary hypertension, and thromboembolic disorders. Therefore, TXA(2) receptor antagonists, thromboxane synthase inhibitors and drugs combining both properties have been developed by several pharmaceutical companies since the early 1980s. Several compounds have been launched on the market and others are under clinical evaluation. Moreover, the recent literature reported the interest of thromboxane modulators, which combine another pharmacological activity such as, platelet activating factor antagonism, angiotensin II antagonism, or 5-lipoxygenase inhibition. In this review, we will propose a description of the recently described thromboxane modulators of major interest from both a pharmacological and a chemical point of view.

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.

Pharmacological evaluation of the novel thromboxane modulator BM-567 (II/II). Effects of BM-567 on osteogenic sarcoma-cell-induced platelet aggregation

Evidence exists that a large number of tumor cells such as osteosarcoma cells stimulate platelet aggregation, which can be an early step in the metastatic processes of these tumors. Thromboxane A(2) (TXA(2)) is released during platelet aggregation, and it has been suggested that this release may be pathogenic for tumor metastasis for several reasons:Some tumors release large amounts of TXA(2) compared to normal tissue.TXA(2) potentiates tumor growth in culture and increases metastasis in animals.TXA(2) is a potent stimulant of platelet aggregation and causes vascular injuries that may promote implantation of tumor cell-platelet aggregates. If TXA(2) participates in tumor metastasis, it may be hypothesized that TXA(2) inhibitors should decrease tumor metastasis. So, we have evaluated the effects of the original TXA(2) synthase inhibitor and TXA(2) receptor antagonist BM-567 on platelet aggregation induced by osteosarcoma cells using MG-63 tumor cells. Results obtained showed that this drug inhibited both MG-63 tumor-cell-induced platelet aggregation and platelet TXA(2) release following the tumor cell stimulation with IC(50) values of 3.04x10(-7) and 2.51x10(-8)M, respectively.

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.

Latest discoveries in prostaglandin receptor modulators

Prostanoids (prostaglandins and the thromboxanes) are cyclooxygenase products derived from C-20 unsaturated fatty acids. Since arachidonic acid is the most abundant among these precursor fatty acids in mammals, including humans, the series 2 prostanoids are predominantly formed in the body. Thus, cyclooxygenases metabolise arachidonate to five primary prostanoids: PGE2, PGF2 α, PGI2, TXA2 and PGD2. These lipid mediators interact with specific members of a family of distinct G-protein-coupled prostanoid receptors, designated EP, FP, IP, TP and DP, respectively. As a review focused on latest discoveries and developments of novel TXA2 modulators patented from January 1997 to December 2000 has been recently published, this paper specifically focuses on newly developed IP, DP, FP and EP modulators patented for three years, from January 1998 to December 2001. Indeed, because prostaglandins are involved in a large series of pathophysiological processes, a classification of these modulators has been proposed, based on the type of receptors activated or inhibited. Their pharmacological profile is also described.

Comparison of the Effects of Nimesulide and Nimesulide‐L‐lysine on PGE2 Production by COX‐1 and COX‐2 and on Chondrocyte Metabolism In‐vitro

Nimesulide, a non-steroidal anti-inflammatory drug and one of a promising class of selective COX-2 inhibitors, has a very interesting therapeutic profile. Unfortunately, it is poorly soluble in water, which leads to important difficulties in the formulation of injectable solutions. This problem can also affect the bioavailability of nimesulide. To increase the aqueous solubility of the drug a nimesulide-L-lysine salt was synthesized in our laboratory; its aqueous solubility was greater than that of nimesulide (solubility in purified water 7.5 mg mL−1, and 0.01 mg mL-1, respectively). The aim of this study was to compare the anti-inflammatory profiles of nimesulide and nimesulide-L-lysine salt in a two-step in-vitro investigation. First, we evaluated the COX-2 selectivity of the drugs by a method using purified COX-1 and COX-2 enzymes. In a second step we evaluated the effects of the drugs on the production of prostaglandin E2 (PGE2) and proteoglycan by chondrocytes from man. The results obtained confirmed the COX-2 selectivity of the two compounds. Nimesulide-L-lysine had the same anti-inflammatory profile as nimesulide on chondrocyte cultures and better water solubility. Nimesulide-L-lysine should, therefore, be used to prepare injectable preparations and should ameliorate bioavailability after oral treatments.

Pharmacomodulation Studies of Torasemide Leading to Original Non‐carboxylic Thromboxane A2 Receptor Antagonists

Because torasemide, a loop diuretic, dose-dependently relaxes canine coronary artery precontracted with thromboxane A2 (TxA2), a series of pyridine derivatives have been investigated with the aim of developing original TxA2 receptor antagonists. A binding test showed the affinity (IC50; the dose reducing binding by 50%) of one sulphonylurea derivative (BM 27) and two sulphonylcyanoguanidine derivatives (BM 114 and BM 115) for the TxA2 receptor of washed platelets from man were 161, 1. 75 and 0.54 μM, respectively. The IC50 of torasemide was only 2.69 μM. Their antagonism was confirmed by the capacity of the compounds to prevent the aggregation of platelets, from man, induced by arachidonic acid, the biological precursor of TxA2 (IC50: BM 114, 29.3 μM; BM 115, 12.0 μM; BM 27, 234 μM; torasemide, 350 μM; sulotroban, 12.3 μM). Similar results were obtained with the best compound BM 115 (IC50 11.5 μM) when U-46619, a stable TxA2 agonist, was used as the aggregating agent. This molecule can be regarded as a template for the design of novel non-carboxylic TxA2 receptor antagonists.