Francesc Cabré

Stereoselective Inhibition of Inducible Cyclooxygenase by Chiral Nonsteroidal Antiinflammatory Drugs

The stereoselective inhibition of inducible cyclooxygenase (COX‐2) by chiral nonsteroidal antiinflammatory drugs (NSAIDs)—ketoprofen, flurbiprofen, and ketorolac—has been investigated. The activity and inhibition of COX‐2 was assessed in three different in vitro systems: guinea pig whole blood, lipopolysaccharide (LPS)‐stimulated human monocytes, and purified preparations of COX‐2 from sheep placenta. The results were compared with the inhibition of constitutive cyclooxygenase (COX‐1) in three parallel in vitro models: clotting guinea pig blood, human polymorphonuclear leukocytes, and purified COX‐1 from ram seminal vesicles. In the whole blood model, both isoenzymes were inhibited by S‐enantiomers with equal potency but S‐ketoprofen was the most active on COX‐2 (IC50 = 0.024 μmol/L). In contrast, both isoenzymes were inhibited less than 40% by all three R‐enantiomers at high concentration (>1 μmol/L). The inhibition of COX by the R‐enantiomers may be attributed to contamination with the S‐enantiomers (approximately 0.5%). A significant degree of enantioselectivity in COX‐2 inhibition was also observed in intact cells. The S‐enantiomers inhibited COX‐2 from monocytes with IC50 values in the range of 2 to 25 nmol/L, being 100 to 500‐fold more potent than the corresponding R‐enantiomers. Finally, S‐ketoprofen inhibited COX‐2 from sheep placenta (IC50 = 5.3 μmol/L) with slightly less potency than S‐ketorolac (IC50 = 0.9 μmol/L) and S‐flurbiprofen (IC50 = 0.48 μmol/L), whereas the R‐enantiomers were found to be essentially inactive (IC50 ≥ 80 μmol/L). It is concluded that the chiral NSAIDs studied here inhibit with comparable stereoselectivity both COX‐2 and COX‐1 isoenzymes, and that the inhibition of COX‐2 previously observed for racemic NSAIDs should be attributed almost exclusively to their S‐enantiomers.

Structure-based design of cyclooxygenase-2 selectivity into ketoprofen.

We have recently described how to achieve COX-2 selectivity from the non-selective inhibitor indomethacin (1) using a combination of a pharmacophore and computer 3-D models based on the known X-ray crystal structures of cyclooxygenases. In the present study we have focused on the design of COX-2 selective analogues of the NSAID ketoprofen (2). The design is similarly based on the combined use of the previous pharmacophore together with traditional medicinal chemistry techniques motivated by the comparative modeling of the 3-D structures of 2 docked into the COX active sites. The analysis includes use of the program GRID to detect isoenzyme differences near the active site region and is aimed at suggesting modifications of the basic benzophenone frame of the lead compound 2. The resulting series of compounds bearing this central framework is exemplified by the potent and selective COX-2 inhibitor 17 (LM-1669).

Analgesic, Antiinflammatory, and Antipyretic Effects of S(+)‐Ketoprofen In Vivo

Many studies indicate that the S‐enantiomers of arylpropionic (APA) nonsteroidal antiinflammatory drugs (NSAIDs) are the pharmacologically active enantiomers. S(+)‐ketoprofen (dexketoprofen) stereoselectively inhibits cyclooxygenase (COX) in vitro but very little is known about the differential activity of ketoprofen enantiomers in vivo. We examined the analgesic, antiinflammatory, and antipyretic activities of S(+)‐ketoprofen in rats and mice. First, we measured the antinociceptive action of S(+)‐ketoprofen in abdominal pain models. After intravenous administration, 0.5 mg/kg S(+)‐ketoprofen inhibited 92.1 ± 2.2% of writhing in mice. Stereoselectivity in the activity was detected; intravenous administration of the R(−)‐enantiomer resulted in no statistically significant activity in a dose range of 0.15–1 mg/kg. Similar results were obtained after oral administration in mice. In the rat, S(+)‐ketoprofen was a more potent analgesic than diclofenac by both intravenous and oral administration. There was no significant difference between the analgesic effect of S(+)‐ketoprofen treatment and the twofold dose of the racemic form in both the mouse and rat models. Second, we measured the antiinflammatory activity of S(+)‐ketoprofen using a carrageenan‐induced paw edema model in the rat. Intravenous administration of 5 mg/kg of S(+)‐ketoprofen almost completely inhibited edema formation. After oral administration, S(+)‐ketoprofen is both more potent and effective than diclofenac. Third, we measured antipyretic activity. S(+)‐ketoprofen showed a marked antipyretic action (ED50 = 1.6 mg/kg) and was the most potent of the NSAIDs tested. S(+)‐ketoprofen is a potent antiinflammatory, analgesic, and antipyretic agent in vivo, consistent with its potent anti‐COX activity.

Occurrence and comparison of sulfite oxidase activity in mammalian tissues

Tissue extracts from six mammalian species have been assayed for sulfite oxidase (sulfite: ferricytochrome c oxidoreductase, EC 1.8.3.1) activity with cytochrome c as electron acceptor. Our results show a large distribution of sulfite oxidase activity in mammalian tissues. Liver, kidney, and heart tissues exhibit high activities whereas brain, spleen, and testis show very low activities. No significant species dependence was observed for the activity of this enzyme.

Antiflammins: Anti-inflammatory activity and effect on human phospholipase A2

Two anti-inflammatory peptides (antiflammins) corresponding to a high amino acid similarity region between lipocortin I and uteroglobin were tested for their ability to inhibit purified human synovial fluid phospholipase A2 (HSF-PLA2). No inhibitory activity was observed, even at such high concentrations of peptides as 50 microM. When antiflammins were preincubated with the enzyme and/or the substrate, no HSF-PLA2 inhibition was detected. In vivo anti-inflammatory activity of these peptides was evaluated in several experimental models of inflammation induced by carrageenan, croton-oil, oxazolone and Naja naja naja venom phospholipase A2 (PLA2). In contrast to the in vitro results, anti-inflammatory activity was observed in all tests, except when inflammation was induced by snake venom PLA2. Taken together, our results do not support the hypothesis that the in vivo anti-inflammatory effect of antiflammins is directly related to inhibition of PLA2 activity.

Stereoselective metabolic pathways of ketoprofen in the rat: Incorporation into triacylglycerols and enantiomeric inversion

The enantiomeric bioinversion of ketoprofen (KP) enantiomers and their incorporation into triacylglycerols were investigated in the rat (1) in vitro, using liver homogenates, subcellular fractions, and hepatocytes, and (2) in vivo, in different tissue samples after oral administration of the radiolabelled compounds. In liver homogenates or subcellular fractions, the enantiomer (S)-ketoprofen (S-KP) was recovered unchanged, whereas (R)-ketoprofen (R-KP) was partially converted into its Coenzyme A (CoA) thioester and inverted to S-KP. Both processes occurred mainly in the mitochondrial fraction. This supports the mechanism of inversion via stereoselective formation of CoA thioester of R-KP, already described for other non-steroidal anti-inflammatory drugs. Incorporation into triacylglycerols was detected after incubation with intact hepatocytes in the presence of added glycerol. The process was stereoselective for R-KP vs. S-KP (covalently bound radioactivity 26,742 +/- 4,665 dpm/10(6) cells vs. 6,644 +/- 3,179 dpm/10(6) cells, respectively). However, no incorporation was found in liver samples after oral administration of either R-KP or S-KP. On the contrary, in adipose tissue samples a significant and stereoselective formation of hybrid triacylglycerols was observed: 11,076 +/- 2,790 dpm.g-1 for R-KP vs. 660 +/- 268 dpm.g-1 for S-KP. The incorporated R/S ratio, higher in adipose tissue (R/S = 17) than in hepatocytes (R/S = 4), indicates that fat may be the main tissue store for the xenobiotic R-KP in rats.

Identification and rejection of outliers in enzyme kinetics

A program (AICOUT) for the correct choice of the experimental value and weight for replicate enzyme kinetic determinations is given. It is based on the method of identification of outliers proposed by Kitagawa (Technometrics, 21 (1979) 193-199). The program is written in BASIC and FORTRAN77. The FORTRAN77 version of AICOUT program coupled to a FORTRAN77 version of the non-linear regression program previously published by Canela (Int J Biomed Comput, 15 (1984) 121-130) is given. This joint program leads to an improvement of precision and confidence in the estimated parameters when the suitable strategy is used. This strategy is as follows: (i) the experimental points are selected, (ii) several replicates of each point are performed, (iii) data are analyzed and outliers are rejected, (iv) normal or biweighted regression is carried out.

Antinociceptive effects of S(+)-ketoprofen and other analgesic drugs in a rat model of pain induced by uric acid.

We investigated the antinociceptive properties of dexketoprofen trometamol (S(+)‐ketoprofen tromethamine salt; SKP), a new analgesic, antiinflammatory drug, using the pain‐induced functional impairment model in the rat (PIFIR), an animal model of arthritic pain. SKP was compared with racemic ketoprofen tromethamine salt (rac‐KP), R(−)‐ketoprofen tromethamine salt (RKP), ketorolac (KET), and morphine (MOR). We also assessed the effects of flurbiprofen (rac‐FB) and its enantiomers (SFB and RFB) in the same model. Groups of six rats received either vehicle or analgesic drug and antinociception was evaluated by evaluating the dose‐response curves over time. SKP was an effective antinociceptive drug in this model and was almost equally potent by either oral or intracerebroventricular administration. The oral potency of SKP was similar to that of oral KET and greater than that of oral MOR. No significant differences were observed between racemic ketoprofen and its enantiomers when administered orally. In the rat, significant bioinversion of RKP to SKP occurs when RKP is given orally. After oral administration of RKP, SKP was detectable in 30 min and surpassed the concentration of RKP after 3 h. Nevertheless, when the compounds were given intracerebroventricularly, some stereoselectivity in favor of SKP was observed. Stereoselectivity was observed with flurbiprofen, an analogue of ketoprofen that does not undergo significant metabolic inversion. Whereas SFB was an effective antinociceptive, RFB had no antinociceptive effect at the doses tested when given either orally or intracerebroventricularly.

Intestinal ulcerogenic effect of S(+)-ketoprofen in the rat

Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit prostaglandin synthesis in the gastrointestinal mucosa, which can lead not only to stomach ulcers but also ulcers in the small and large intestines. Ulcers of the small intestine are less common than those of the stomach, but intestinal lesions are more life threatening. Although the R(−)‐enantiomers of the arylpropionic acid (APA) class of NSAIDs are assumed to lack the toxic effects of cyclooxygenase inhibition, they may contribute to the ulcerogenicity of racemates. We have examined the intestinal ulcerogenic effects of single oral doses of S(+)‐ketoprofen compared with racemic ketoprofen in the small intestine and cecum of rats. The toxicity in the small intestine was measured as the weight ratio between portions of intestine showing lesions and the total weight of the tissue. Toxicity in the cecum was evaluated by measuring the size of the ulcers. S(+)‐ketoprofen had no significant ulcerogenic effect at 10 or 20 mg/kg. However, racemic ketoprofen was clearly ulcerogenic in the small intestine and cecum at the 40 mg dose. R(−)‐ketoprofen at 20 mg/kg does not show any effect in the cecum and only limited ulcerogenesis in the small intestine: The latter effect may be the result of racemic inversion. Therefore, the ulcerogenic action of racemic ketoprofen can be interpreted as a synergism between S(+)‐ and R(−)‐ketoprofen. The mechanism of this synergism is not well understood but may be a general feature of APA NSAIDs.

A broad scope highly efficient synthesis of bis(R-thio)acetylenes

Treatment of dichloroacetylene with two equivalents of a thiol and two equivalents of potassium hydride in THF solution affords the corresponding bis(R-thio)acetylene in high yield