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*Research Department, SPA-Societa’ Prodotti Antibiotici S.p.A., Milan, Italy; and
Departments of Anatomy, Pharmacology and Forensic Medicine and
Internal Medicine, University of Turin, Turin, Italy
Address correspondence and reprint requests to Angelo Bonabello, SPA-Societa’ Prodotti Antibiotici S.p.A., Via Biella 8, Milan, Italy. Address e-mail to bonabelloa{at}libero.it
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Abstract |
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IMPLICATIONS: Ibuprofen is a nonsteroidal antiinflammatory drug often prescribed as a racemic formulation. We studied the analgesic and antiinflammatory effects of the active S(+)-isomer. The S(+)-ibuprofen was found to be more potent than the racemic formulation and produced less acute gastric damage.
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Introduction |
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Ibuprofen, a chiral 2-arylpropionic acid derivative, is an analgesic, antipyretic, and antiinflammatory drug available over the counter in many countries. In a recent collaborative metaanalysis with individual NSAIDs, ibuprofen had the smallest risk of serious gastrointestinal complications. However, several independent studies in which different in vitro approaches were used to monitor the inhibition of COX-1 and COX-2 did not suggest a preferred inhibition of COX-2 by ibuprofen (1–5). An interesting feature of ibuprofen, which is marketed in most countries as an equal mixture of R- and S-ibuprofen (racemate), is the unidirectional metabolic chiral inversion of the in vitro inactive (not prostaglandin synthesis-inhibiting) R-enantiomer to the prostaglandin synthesis-inhibiting S-form (6–8). This conversion of racemic ibuprofen to active S(+)-isomer may contribute to the variability in analgesia and may explain the poor relationship observed between plasma concentrations of ibuprofen and clinical response in acute pain and rheumatoid arthritis (9,10). The observation that selective inhibitors of COX-2 are associated with a reduced incidence of gastric erosions as compared with conventional NSAIDs suggests that the suppression of gastric COX-1 by NSAIDs is the key mechanism responsible for gastric damage (2,11). However, Wallace et al. (12) showed that the suppression of both COX-1 and COX-2 is necessary for NSAID-induced gastric erosion in rats, suggesting a distinct mechanism through which inhibition of COX-1 versus COX-2 could contribute to erosion.
Dionne and McCullagh (13) studied the analgesic effect of orally administered (PO) ibuprofen active S(+)-isomer in the clinical oral surgery model of acute pain. The administration of 200 mg of S(+)-ibuprofen resulted in an analgesic effect more than that of a racemic mixture containing approximately the same amount of active isomer. The analgesic onset was faster and the peak analgesia greater, and the authors observed a small incidence of adverse effects.
The same results were observed with the ketamine S(+)-isomer in a recent double-blinded, three-way crossover, placebo-controlled study assessing pain evoked by small-/large-area pressure stimuli. The S(+)-isomer was approximately twice as potent as the racemic mixture of ketamine in inhibiting central pain summation (14).
The aim of this study was to evaluate the analgesic and antiinflammatory actions of the active S(+)-isomer ibuprofen in comparison with the racemic formulation. We also studied the acute gastric damage of both preparations.
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Methods |
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The animals were randomly assigned to different drug-dose tests; the control animal groups received the vehicle treatment, whereas the other animal groups received the test drugs. The experiments were performed according to the Guiding Principles for the Care and Use of Laboratory Animals, the Recommendation from the Declaration of Helsinki, and the European Community legislation No. 86/605. The protocol was reviewed and approved by the Local Animal Committee.
The S(+)-ibuprofen isomer was from Societa’ Prodotti Antibiotici S.p.A. (Milan, Italy) (coded SPA-S-897). Ibuprofen (racemate) was purchased from Francis S.p.A. (Varese, Italy). Acetylsalicylic acid (ASA) and carrageenan were from Sigma (Milan, Italy).
For the Hot Plate Test, the method adopted was described by O’Callaghan and Holtzman (15). One-hundred-forty mice, 10 per group (weighing 20–25 g), or 80 rats, 10 per group (175–200 g), were placed in a stainless-steel container, thermostatically set at 52.5°C ± 0.1°C, in a precision water bath (Terzano, Milan, Italy). Reaction times were measured with a stopwatch before and 30, 60, and 120 min after the administration of the analgesic drugs. The end-point was shown by the licking of the fore or hind paws. Mice and rats scoring <12 and >18 s in the basal test were rejected (30%). An arbitrary cutoff time of 45 s was adopted to avoid serious injuries to the treated animals.
Antinociception was assessed by the tail-flick method of D’Amour and Smith (16), as modified by Dewey et al. (17). Two-hundred-ninety mice (20–25 g), 10 per group, were loosely wrapped, individually, in a thin cotton towel, with the head and the tail exposed. Each animal was then placed on a platform with the tail positioned in a special shallow groove; a focused beam of light was then directed at the tail from above, approximately 2.5 cm from the tip. Movement of the tail from the groove allowed the light beam to hit a sensor, formerly covered by the tail, which then automatically switched off the beam and stopped the timer. The duration of the time required for the tail response after exposure to the thermal stimulus was considered the tail response latency time. The maximum time allowed was 15 s, to prevent tissue damage. Mice were tested once to determine the predose tail response latency, after which they were then dosed and again tested for their tail response latency at 30 min after dosing. For each animal, we calculated the difference between postdose and predose latency time.
The abdominal constriction test was performed with 200 CD1 mice weighing 25–30 g. We used the abdominal constriction test described by Hendershot and Forsaith (18). Mice were injected intraperitoneally with acetic acid (10 mL/kg; 3% solution) to produce the typical reaction, which is characterized by a wave of contractions of the abdominal musculature followed by extension of the hind limbs. The mice were then placed in individual transparent containers, and the number of contractions in a 20-min period was counted from the time of injection.
The acute inflammation was induced by injection of 0.05 mL (0.5 mg) of carrageenan, dissolved in NaCl 0.9%, into the plantar tissue of the right rat hind paw (70 animals), as described by Winter et al. (19). The extent of inflammation was recorded by measuring the volume of the right hind paw (Plethysmometer 7150; Ugo Basile) before and after carrageenan administration. The percentage of antiinflammatory action is considered as the reduction in edema of treated animals compared with controls (20).
Gastric damage was evaluated in groups of at least 10 rats each, which were given one of the following drugs PO: ASA (100 mg/kg), racemic ibuprofen, and S(+)-isomer ibuprofen (25, 50, or 100 mg/kg). Control rats received an equal volume of the vehicle of 1% carboxymethylcellulose. Six hours later, the animals were anesthetized with halothane, and the stomach was removed and scored for hemorrhagic damage by an observer unaware of the treatments the rats had received. The scoring involved measuring the lengths of the lesions, in millimeters, and summing the values to give an overall gastric ulceration index for each rat (13). The observers of all the tests were blinded to the drug injected when making the efficacy measurements.
The data were analyzed with a software package (SPSS 6.1 for Windows; SPSS Inc., Chicago, IL). The following statistical tests were performed: analysis of variance followed by Bonferroni’s test; the Kruskal-Wallis test followed by the distribution-free multiple comparisons test; the paired Student’s t-test and probit analysis.
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Results |
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Rats were given PO S(+)-ibuprofen at doses of 50, 100, and 125 mg/kg and racemic ibuprofen at 50, 100, 200, and 300 mg/kg. The hot-plate test was performed 60 min after drug administration. We observed a dose-dependent analgesic effect for the drugs studied, with an ED50 of 128 mg/kg for S(+)-ibuprofen and an ED50 >300 mg/kg for ibuprofen. Also, in this test, no statistically significant difference was seen between S(+)-ibuprofen at the dose of 50 mg/kg and the racemic formulation at the dose of 100 mg/kg (Fig. 1C).
In the tail-flick test, we studied mice 30 min after treatment with S(+)-ibuprofen, given IV at the doses of 12.5, 25, 50, 75, and 100 mg/kg, and the ibuprofen racemic solution, given at the doses of 12.5, 25, 50, 75, 100, and 150 mg/kg. The analgesic effect of the S(+)-isomer was significantly more potent than that of the racemic formulation; the ED50 values were 54 and 84 mg/kg, respectively (Fig. 2A).
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We performed the tail-flick test in mice 60 min after PO S(+)-ibuprofen (25, 50, and 100 mg/kg) and ibuprofen racemic solution (25, 50, 100, and 200 mg/kg). Also, after PO administration, S(+)-ibuprofen was more potent than ibuprofen, with an ED50 of 37 versus 177 mg/kg, respectively. S(+)-ibuprofen 25 mg/kg was more potent than ibuprofen 50 mg/kg (P < 0.05) (Fig. 2C).
The antiinflammatory action was evaluated by the abdominal constriction test, and the inflammation of the rat right hind paw was induced by carrageenan and measured with the plethysmometer. For the abdominal constriction test, S(+)-ibuprofen 12.5, 25, and 50 mg/kg and ibuprofen racemic solution 25, 50, and 75 mg/kg were administered IV 30 min before the intraperitoneal injection of acetic acid. Both formulations showed a significant antinociceptive action compared with the controls (P < 0.05). S(+)-Ibuprofen (ED50, 32 mg/kg) was significantly more potent than the racemic formulation (ED50, 52 mg/kg) (Fig. 3A).
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In carrageenan-induced rat paw edema, S(+)-ibuprofen 30, 60, and 100 mg/kg and ibuprofen 30, 60, and 120 mg/kg were administered IV, and the effect was evaluated 1, 3, and 5 h after the administration of the inflammatory drug. S(+)-Ibuprofen (ED50 of 91 mg/kg after 3 h) and racemic ibuprofen (ED50 of 88 mg/kg after 3 h) showed a significant dose-dependent antiinflammatory action compared with the control (P < 0.05) (Table 1). In the same test after PO administration, at the doses of 30, 60, and 100 mg/kg for the S(+) product and 30, 100, and 130 mg/kg for the racemic compound, the S(+)-isomer was significantly more potent than the ibuprofen racemic formulation (ED50, 57 versus 124 mg/kg) (Table 2).
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Discussion |
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In a recent clinical study, 200 mg of S(+)-ibuprofen PO resulted in more analgesic effect than the racemic mixture (14). The 400 mg of the S(+)-isomer also provided larger peak effects and a duration comparable to that of racemic ibuprofen, with residual activity detectable for a long period. Analgesic onset was also faster for S(+)-ibuprofen than for the racemic formulation. In the same study, the incidence of adverse reactions reported by the subjects given the active treatments was similar to the incidence after placebo.
In a pharmacokinetic study, Geisslinger et al. (21) showed that S(+)-ibuprofen given PO to healthy volunteers produced a larger plasma concentration more rapidly than the same amount of S(+)-ibuprofen contained in the racemic compound. These data could explain, in part, our animal results in efficacy after PO administration of the enantiomer compared with the racemic formulation.
Many studies indicate that the S(+)-enantiomers of arylpropionic NSAIDs are the pharmacologically active enantiomers. The greatest efficacy of arylpropionic S(+)-enantiomers was observed in animal analgesia models. For example, in a rat formalin model, intrathecal injection of S(+)-ibuprofen resulted in a potent suppression of the flicking behavior in the second phase of this test (22). Subsequently, S-flurbiprofen was found to be significantly more potent than R-flurbiprofen in the same model (23). After IV administration, S(+)-flurbiprofen was also more potent than the R(-)-isomer in reducing inflammation-evoked activity in nociceptive rat spinal neurons. When injected locally into the inflamed knee, S(+)-flurbiprofen, but not R(-)-flurbiprofen, reduced the hyperexcitability of spinal cord neurons (21). Similar data were obtained by studying antinociceptive actions of spinal NSAIDs in the formalin test in rats (24).
In an animal model after PO and IV administration, S(+)-ketoprofen showed an increased antinociceptive effect in phenylbenzoquinone-induced writhing in mice. With a similar test in rats, S(+)-ketoprofen was more potently analgesic than diclofenac by both IV and PO administration. There was no significant difference between the analgesic effect of S(+)-ketoprofen treatment and the twofold dose of the racemic form in mice and rats. Similar results were observed with the carrageenan-induced paw edema model in the rat.
Our results do not support the hypothesis that R(-)-ibuprofen can contribute to the analgesic and antiinflammatory action of the racemate formulation, and this can be due to differences in the models used. In our experiments, the racemate formulation produced more gastric lesions than S(+)-ibuprofen.
As with other NSAIDs, gastrointestinal intolerance, including peptic ulceration, is the most common adverse reaction to ibuprofen. This adverse event seems to be related to inhibition of the synthesis of mucosal protective prostaglandins; it appears, therefore, that the S(+)-enantiomer, which is the active isomer, could be more ulcerogenic than its antipode. From several animal studies, Mayer and Testa (25) concluded that pure S(+)-ibuprofen does not produce more gastric toxicity than the racemic formulation. The observation that selective inhibition of COX-2 spares prostaglandin synthesis and is associated with a greatly reduced incidence compared with conventional NSAIDs led to the hypothesis that the suppression of gastric COX-1 by NSAIDs is the key mechanism responsible for erosion formation. In two experimental models in rats, using ulcerogenic doses of different NSAIDs, Laudano et al. (26) observed that the higher inhibitory COX-1 selectivity of NSAIDs led to the production of a more marked gastrointestinal injury. However, this remains an unproven hypothesis. Mice in which the gene for COX-1 was disrupted exhibited reduced gastric prostaglandin synthesis but no gastric injury. Several studies in recent years have suggested that COX-2 can contribute to gastric mucosal defense, at least in some circumstances. Using an in vivo rat model, Wallace et al. (12) showed that inhibition of both COX-1 and COX-2 is required for the development of gastric erosion after NSAID administration. Neither a COX-1 inhibitor nor a COX-2 inhibitor caused macroscopically or histologically detectable gastric damage when given at doses known to be effective at selectively inhibiting the target enzyme in vivo. However, the administration of both inhibitors invariably resulted in the development of gastric erosion.
Our results are in agreement with a previous study in which repeated administration of S(+)-enantiomer and racemic ibuprofen in rats caused the expected dose-dependent gastrointestinal toxicity. The extent of the mucosal lesions tended to be slightly more marked in animals exposed to the racemate than in rats treated with the S(+)-enantiomer (26).
In conclusion, the S(+)-ibuprofen isomer was found to be more potent than the racemic formulation of ibuprofen with respect to its analgesic and antiinflammatory properties, and it produced less acute gastric damage. Therefore, administration of the racemic formulation should be avoided if it is not essential for the therapeutic activity expected.
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Acknowledgments |
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This article has been cited by other articles:
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  P. F. White Can the Use of Specific Isomers Improve the Safety and Efficacy of Nonsteroidal Antiinflammatory Drugs? Anesth. Analg., August 1, 2003; 97(2): 309 - 310. [Full Text] [PDF]
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s = difference between postdose and predose latency time.
