Selective inhibition of cyclooxygenase-2 by enflicoxib, its enantiomers and its main metabolites in vitro in canine blood.

Enflicoxib is approved for the treatment of pain and inflammation in canine osteoarthritis. The objective of this work was to assess the mechanistic basis of enflicoxib therapy investigating the COX inhibitory activity of enflicoxib (racemate), its enantiomers and its main metabolites using the canine whole blood assay. The (R)-(+)-Enflicoxib enantiomer and metabolite M8 (hydroxylated pyrazoline) did not induce significant COX inhibition. Enflicoxib and its (S)-(-)-Enflicoxib enantiomer inhibited COX-1 and COX-2 with variable degree of preferential isoform inhibition, but no significant therapeutic effect is anticipated in vivo. The pyrazol metabolite showed the highest COX-2 inhibition and was the most selective (IC50 COX-1/ COX-2 ratio: 19.45). As the pyrazol metabolite shows saturable binding to red blood cells, its in vivo concentrations in plasma are lower than in whole blood. Accordingly, when applying the red blood cell partitioning, the respective IC50 and IC80 for COX-2 inhibition decreased from 2.8 µM (1129 ng/ml) and 13.4 µM (5404 ng/ml) to 0.2 µM (80.7 ng/ml) and 1.2 µM (484 ng/ml) and the selectivity ratio increased to close to 55. The corrected pyrazol metabolite IC50 and IC80 are well within the plasma levels described in treated dogs.

Comparative In vitro Metabolism of Enflicoxib in Dogs, Rats, and Humans: Main Metabolites and Proposed Metabolic Pathways.

BACKGROUND: Enflicoxib is a non-steroidal anti-inflammatory drug of the coxib family characterized by a long-lasting pharmacological activity that has been attributed to its active metabolite E-6132. OBJECTIVES: The aim of this work was to explore enflicoxib biotransformation In vitro in humans, rats and dogs, and to determine its metabolic pathways. METHODS: Different In vitro test systems were used, including hepatocytes and liver and non-hepatic microsomes. The samples were incubated with enflicoxib and/or any of its metabolites at 37°C for different times depending on the test system. The analyses were performed by liquid chromatography coupled with either radioactivity detection or high-resolution mass spectrometry. RESULTS: Enflicoxib was efficiently metabolized by cytochrome P-450 into three main phase I metabolites: M8, E-6132, and M7. The non-active hydroxy-pyrazoline metabolite M8 accounted for most of the fraction metabolized in all the three species. The active pyrazol metabolite E-6132 showed a slow formation rate, especially in dogs, whereas metabolite M7 was a secondary metabolite formed by oxidation of M8. In hepatocytes, diverse phase II metabolite conjugates were formed, including enflicoxib glucuronide, M8 glucuronide, E-6132 glucuronide, M7 glucuronide, and M7 sulfate. Metabolite E-6132 was most probably eliminated by a unique glucuronidation reaction at a very low rate. CONCLUSION: The phase I metabolism of enflicoxib was qualitatively very similar among rats, humans and dogs. The low formation and glucuronidation rates of the active enflicoxib metabolite E-6132 in dogs are postulated as key factors underlying the mechanism of its long-lasting pharmacokinetics and enflicoxib's overall sustained efficacy.

Pharmacokinetics of enflicoxib in dogs: Effects of prandial state and repeated administration.

The pharmacokinetics of enflicoxib were evaluated in both a bioavailability study and a multi-dose safety study in Beagle dogs. When administered at 8 mg/kg, the oral bioavailability (F) of enflicoxib was 44.1% in fasted dogs, but F increased to 63.4% under post prandial conditions. Enflicoxib is rapidly metabolised. After the first 48 h, the plasma levels of its pyrazol metabolite were much higher and persistent than those of the parent compound. Following intravenous administration, the total body plasma clearance of enflicoxib was of 140 ml/h/kg and the volume of distribution based on the terminal phase was 4 L/kg. Plasma protein binding for both compounds was approximately 99%. The blood to plasma ratio for the pyrazol metabolite showed saturable kinetics with higher blood cell affinity at lower total blood concentrations which ranged from 2.49 to 0.95 for concentrations from 1 to 15 µg/ml. Enflicoxib and its pyrazol metabolite exhibited dose-proportional pharmacokinetics for single oral doses of 8-40 mg/kg and for multiple oral doses of 4-20 mg/kg. After 7 months of repeated weekly administrations, pre-dose plasma concentrations (Cmin,ss ) remained constant throughout the study, with no trend to any significant over-accumulation. The mean terminal elimination half-life (t½ ) was 20 h for enflicoxib and 17 days for the pyrazol metabolite. The pharmacokinetic profile of enflicoxib and its pyrazol metabolite in dogs supports the proposed dosing regimen in which doses are separated by 1 week.