David Mauleón

PHOTOCHEMICAL AND PHOTOBIOLOGICAL PROPERTIES OF KETOPROFEN ASSOCIATED WITH THE BENZOPHENONE CHROMOPHORE

Abstract Irradiation of ketoprofen in neutral aqueous medium gave rise to 3‐ethylbenzophenone as the major photoproduct. Its formation is justified via protonation of a benzylic carbanion or hydrogen abstraction by a benzylic radical. Minor amounts of eight additional compounds were isolated. Four of them are derived from the benzylic radical: 3‐(1‐hydroperoxyethyl)benzophenone, 3‐(1‐hydroxyethyl)benzophenone, 3‐acetylbenzophenone and 2,3‐bis‐(3‐benzoylphenyl)butane. The other four products involve initial hydrogen abstraction by the excited benzophenone chromophore of ketoprofen: 1,2‐bis‐(3‐ethylphenyl)‐1,2‐diphenyl‐1,2‐ethanediol, 2‐(3‐benzoylphenyl)‐1‐(3‐ethylphenyl)‐1 ‐phenylpropan‐1 ‐01,α ‐(3‐ethylphenyl)phenylmethanol, 1,2‐bis‐[3‐(2‐hydroxycarbonylethyl)phenyl]‐1,2‐di‐phenyl‐1,2‐ethanediol. The latter process was found to mediate the photoperoxidation of linoleic acid through a type I mechanism, as evidenced by the inhibition produced by the radical scavengers butylated hydroxyanisole and reduced glutathione. The major photoproduct, which contains the benzophenone moiety but lacks the propionic acid side chain, also photosensitized linoleic acid peroxidation. Because lipid peroxidation is indicative of cell membrane lysis, the above findings are highly relevant to explain the photobiological properties of ketoprofen.

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.

Preclinical and Clinical Development of Dexketoprofen

SummaryDexketoprofen trometamol is a water-soluble salt of the dextrorotatory enantiomer of the nonsteroidal anti-inflammatory drug (NSAID) ketoprofen. Racemic ketoprofen is used as an analgesic and an anti-inflammatory agent, and is one of the most potent in vitro inhibitors of prostaglandin synthesis. This effect is due to the S(+)-enantiomer (dexketoprofen), while the R(−)-enantiomer is devoid of such activity.The pharmacokinetic profile of ketoprofen and its enantiomers was assessed in several animal species and in human volunteers. In humans, the relative bioavailability of oral dexketoprofen trometamol (12.5 and 25mg, respectively) is similar to that of oral racemic ketoprofen (25 and 50mg, respectively), as measured in all cases by the area under the concentration-time curve values for S(+)-ketoprofen. Dexketoprofen trometamol, given as a tablet, is rapidly absorbed, with a time to maximum plasma concentration (tmax) of between 0.25 and 0.75 hours, whereas the tmax for the S-enantiomer after the racemic drug, administered as tablets or capsules prepared with the free acid, is between 0.5 and 3 hours. Peak plasma concentrations of 1.4 and 3.1 mg/L are reached after administration of dexketoprofen trometamol 12.5 and 25mg, respectively.From 70 to 80% of the administered dose is recovered in the urine during the first 12 hours, mainly as the acyl-glucuronoconjugated parent drug. No R(−)-ketoprofen is found in the urine after administration of dexketoprofen [S(+)-ketoprofen], confirming the absence of bioinversion of the S(+)-enantiomer in humans.In animal studies, the anti-inflammatory potency of dexketoprofen was always equivalent to that demonstrated by twice the dose of ketoprofen. Similarly, animal studies showed a high analgesic potency for dexketoprofen trometamol. The R(−)-enantiomer demonstrated a much lower potency, its analgesic action being apparent only in conditions where the metabolic bioinversion to the S(+)-enantiomer was significant.The gastric ulcerogenic effects of dexketoprofen at various oral doses (1.5 to 6 mg/kg) in the rat do not differ from those of the corresponding double doses (3 to 12 mg/kg) of racemic ketoprofen. Repeated (5-day) oral administration of dexketoprofen as the trometamol salt causes less gastric ulceration than was observed after the acid form of both dexketoprofen and the racemate. In addition, single dose dexketoprofen as the free acid at 10 or 20 mg/kg does not show a significant intestinal ulcerogenic effect in rats, while racemic ketoprofen 20 or 40 mg/kg is clearly ulcerogenic to the small intestine.The analgesic efficacy of oral dexketoprofen trometamol 10 and 20mg is superior to that of placebo and similar to that of ibuprofen 400mg in patients with moderate to severe pain after third molar extraction. The time to onset of pain relief appeared to be shorter in patients treated with dexketoprofen trometamol than in those treated with ibuprofen 400mg. Dexketoprofen trometamol was well tolerated, with a reported incidence of adverse events similar to that of placebo.

Differential binding mode of diverse cyclooxygenase inhibitors

Non-steroidal anti-inflammatory drugs (NSAIDs) are competitive inhibitors of cyclooxygenase (COX), the enzyme that mediates biosynthesis of prostaglandins and thromboxanes from arachidonic acid. There are at least two different isoforms of the enzyme known as COX-1 and -2. Site directed mutagenesis studies suggest that non-selective COX inhibitors of diverse chemical families exhibit differential binding modes to the two isozymes. These results cannot clearly be explained from the sole analysis of the crystal structures of COX available from X-ray diffraction studies. With the aim to elucidate the structural features governing the differential inhibitory binding behavior of these inhibitors, molecular modeling studies were undertaken to generate atomic models compatible with the experimental data available. Accordingly, docking of different COX inhibitors, including selective and non-selective ligands: rofecoxib, ketoprofen, suprofen, carprofen, zomepirac, indomethacin, diclofenac and meclofenamic acid were undertaken using the AMBER program. The results of the present study provide new insights into a better understanding of the differential binding mode of diverse families of COX inhibitors, and are expected to contribute to the design of new selective compounds.

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.

Clinical Comparison of Dexketoprofen Trometamol and Dipyrone in Postoperative Dental Pain

A total of 125 outpatients with moderate to severe pain after surgical removal of one impacted third molar were randomly assigned to receive dexketoprofen trometamol 12.5 or 25 mg or dipyrone 575 mg. For first‐dose assessments, patients rated their pain intensity and its relief at regular intervals. From 60 min post dose to the end of the 6‐h observation period, both doses of dexketoprofen trometamol had higher pain relief scores than dipyrone: Between 3 and 6 h the differences were statistically significant. In addition, peak measures (PIDmax and PARmax) were statistically superior after both doses of dexketoprofen trometamol compared to dipyrone. The overall efficacy assessed at the end of the first‐dose phase was rated as good or excellent by 90%, 83.3%, and 70% of patients receiving dexketoprofen trometamol 25 mg, dexketoprofen trometamol 12.5 mg, and dipyrone, respectively. The number of patients who required remedication during the 6‐h period was significantly lower in both dexketoprofen groups. Repeated‐dose data were also obtained. No significant differences were found in the efficacy after repeated doses, the number of doses taken, or the mean time elapsed between doses. The overall efficacy at the end of the repeated‐dose phase was rated as good or excellent by 84.2%, 66.7%, and 70% of patients receiving dexketoprofen trometamol 25 mg, dexketoprofen trometamol 12.5 mg, and dipyrone, respectively. The frequency of adverse events was similar for all treatments and no serious adverse events were reported during the study.

Structural basis of the dynamic mechanism of ligand binding to cyclooxygenase.

Molecular modeling studies performed on the two cyclooxygenase (COX) isozymes suggest that the cavity at the mouth of the active site on the membrane domain that may act as an actual binding site of COX ligands. Actual docking of different inhibitors at this site provides a structural basis to explain the dynamics of COX inhibition.

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.

Evaluation of ketoprofen (R,S and R/S) phototoxicity by a battery of in vitro assays.

Abstract The various enantiomers of ketoprofen (S and R) and its racemic form ( R S ) exhibited comparable phototoxicities when examined by the following in vitro test systems: (a) effects of pre-irradiated drugs on cultured hepatocytes; (b) co-irradiation of drugs with hepatocytes or fibroblasts; (c) photohaemolysis sensitized by the various ketoprofen steroisomers; (d) drug-photosensitized formation of linoleic acid hydroperoxides. Inhibition of photohaemolysis and photodynamic lipid peroxidation by butylated hydroxyanisole and reduced glutathione suggests that the phototoxicity of ketoprofen is associated with a radical chain (type I) peroxidation of membrane lipids, leading to cell lysis In view of the above results it could be advantageous to use the pharmacologically active S(+) enantiomer instead of the R/S form, since the lower doses required would result in a diminished phototoxic potential.