4'-Hydroxylation of Flurbiprofen by Rat Liver Microsomes in Fasting and Feeding Conditions

Abstract

Flurbiprofen (FP) is a nonsteroidal anti-inflammatory drug (NSAID) of the 2-arylpropionic acid class. Although it possesses a chiral center, both R-(Ϫ)-and S-(ϩ)-enantiomers may possess analgesic activity, and all FP-preparations to date are marketed as the racemate. The enantiomers exhibit differences in both the level of protein binding and metabolism.1) The former difference was observed using ultracentrifugation 2) but not using equilibrium dialysis. 3,4) The latter difference was observed in diastereomeric glucuronides, but the enantiomers were undiscernibly metabolized by oxidation via the cytochrome P450 system (CYP), followed by glucuronidation.1) The major oxidative metabolites were 4Ј-hydroxy and 3Ј,4Ј-dioxygenated metabolites (Fig. 1), 5) and it was known that 4Ј-hydroxy-flurbiprofen (4Ј-HOFP) was transformed from FP involving in CYP 2C9 and not CYPs 1A2, 2C8, 2E1, or 3A4 in human. 6) Very little is known of the metabolic capabilities by CYP isoforms in rat liver microsomes.Since drugs are sometimes administered to experimental animals after overnight fasting, it is necessary to know whether the metabolism of FP is changed after the fasting. Starvation and fasting change both the composition of cytochrome P450s and the metabolic activity of rat hepatic microsomes, inducing CYPs 2B1, 2E1, and 3A2, and reducing CYPs 1A2 and 2C11.7-10) Our study describes that 4Ј-hydroxylation of FP, being a primitive metabolism, was conducted by rat microsomal CYP2C11, but not CYP2E1 induced on fasting, and also provides a possibility that the CYP2E1 might contribute to further oxidation following the 4Ј-hydroxylation. MATERIALS AND METHODSChemicals Aniline, cimetidine, disulfiram, flurbiprofen, p-aminophenol, quinine, testosterone, and troleandomycin were purchased from Wako Pure Chemicals (Tokyo). Diclofenac, furafylline, glucose-6-phosphate and SKF-525A were from Sigma Chemical Co. Kanamycin was from Meiji Seika (Tokyo), and anti-rat CYP2C11 serum and anti-rat CYP2E1 serum were from Dai-ichi Pure Chemicals Co. (Tokyo). G-6-P-D, b-NADP, and b-NADPH were from Oriental Ferm. Co. (Tokyo).Treatment of Animals Male Wistar rats (250-300 g) were obtained from Charles River Laboratories, Inc. (Japan). The animals were housed in cages under controlled conditions of temperature and humidity, and were subjected to a 12-h light/dark cycle. The rats were fed Oriental laboratory diets for the feeding condition and were fasted for 24 h for the fasting condition. They had free access to water under the both conditions.Preparation of Rat Liver Microsomes The animals were killed by decapitation, and individual liver microsomes were prepared by a general method: The liver was homogenized with 0.25 M sucrose (9 vol.) to give a supernatant by centrifugation at 1000ϫg for 10 min and then 9000ϫg for 20 min, then the supernatant was centrifuged at 105000ϫg for 60 min to give a pellet. The microsomal pellet was suspended in 0.1 M potassium phosphate buffer (pH 7.4) and stored at Ϫ30°C until use.Microsomal Protein Assay Microsomal protein conc...

Role of rat liver cytochrome P450 3A and 2D in metabolism of imrecoxib1

“…In addition, 10 mmol/L of quinine (an inhibitor specific for CYP 2D [22,27] ) significantly inhibited the rate of formation of M4 by approximately 20% compared with control activities in all types of microsomes except the microsomes obtained from dexamethasone-pretreated rats (Figure 4-6). When the concentration of quinine was increased to 50 µmol/L, the formation rate was decreased to a much lower level.…”

“…β-naphthoflavone, an inducer of CYP 1A, did not significantly increase the formation of M4 (Tables 2 and 3). Cimetidine was used to confirm the participation of CYP 2C [27,28] in imrecoxib biotransformation; however, the formation of M4 was not inhibited by cimetidine in microsomes either from control rats or from induced rats. Thus, substantial participation of CYP 2C was unlikely in this reaction.…”

Formation of 4'-carboxyl acid metabolite of imrecoxib by rat liver microsomes1

“…Quinine (a inhibitor specific for CYP 2D [11,15] ) and cimetidine (often used as an inhibitor for CYP 2C [15,16] ) did not decrease the formation rate of M2 significantly, so the involvement of CYP 2D and 2C in the formation of M2 from M4 seems unlikely. α-Naphthoflavone and methylpyrazole were used to confirm the participation of CYP 1A [11,14,15] and CYP 2E [14] in the reaction, respectively; however, the formation of M2 was not inhibited by cimetidine and methylpyrazole in microsomes either from untreated rats or from dexamethasone-treated rats. Moreover, the formation rates of M4 in untreated, β-naphthoflavone-induced, and isoniazid-induced rat liver microsomes were 6.0±1.2, 6.3±0.9, and 5.3± 0.6 pmol/ min per mg protein, respectively.…”