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1 w wz 16«1y Kor. J. Clin. Pharm., Vol. 16, No j rù k w y B Áy k B Á½ C Á D B w C D ƒm w w w Pharmacokinetic Study of Aceclofenac and its Metabolites, and Application to Bioequivalence Study Chun-hwa Ihm a, In-taek Hwang a, Eun-young Kim b and Wonku Kang c a Eulgi University School of medicine, Daejeon b Department of Pharmacy, Samsung Medical Center, Seoul c College of Pharmacy, Catholic University of Daegu, Kyungbuk Aceclofenac, a nonsteroidal antiinflammatory agent of a phenylacetic acid type, has been used for rheumatoid arthritis and osteoarthritis. Although the metabolic pathway of aceclofenac is relatively well-known in vitro, pharmacokinetic profiles of its three major metabolites are still unclear in human. The present study was designed to investigate pharmacokinetic profiles of the metabolites of aceclofenac, and to evaluate the bioequivalence of the generic preparation of aceclofenac 100 mg tablet. Blood samples were serially collected for a period of 12 hours following a single oral administration of 100 mg aceclofenac in 20 healthy human volunteers. A simple protein precipitation with acetonitrile was employed to purify those substances from plasma. Aceclofenac, diclofenac, 4'-hydroxyaceclofenac and 4'-hydroxydiclofenac in heparinized plasma were simultaneously measured with flufenamic acid, an internal standard, using HPLC coupled to a tandem mass spectrometer. Time courses of 4 -hydroxydiclofenac, diclofenac and aceclofenac plasma concentrations were clearly revealed, and the pharmacokinetic properties were analyzed. The 90% confidence intervals for the ratios of test/reference for log-transformed AUC and C max lie within Keywords aceclofenac, metabolites, LC/MS/MS, pharmacokinetics, bioequivalence Correspondence to : ƒm w w w w Tel: , Fax: wonkuk@cu.ac.kr j rù 2-[(2,6-dichlorophenyl)amino] phenylacetoxyacetic acid yw ƒ l m p ù w z ùküš. 1-3) m y w y y (synovial fluid)ü prostaglandin E 2 j w w, w š y xw x (polymorphonuclear leukocytes)ù w s(mononuclear cells) w prostaglandin E 2 w. 4-5) Yamazaki w j rù p y s w prostaglandin E 2 w w x. j rù j z (cytochrome P450 enzyme, CYP) CYP2C9 w 4 -hydroxyaceclofenac ƒ w 4 -hydroxy-diclofenac y. wr j rù ƒ w diclofenac y z CYP2C9 w y» w 7-8) (Fig. 1). Henrotin tw j rù cytokines cyclooxygenase(cox) w j rù 4 -hydroxy-aceclofenac yw w interlukin- 6 prostaglandin E2 w ùkû. w j rù COX-1 COX-2 w w ùkü j rù 4 hydroxy-aceclofenac COX-2 w ù kþ j rù 4 hydroxy-aceclofenac w 4.5 w z. 9) ƒ x ù j rù ü w š. w medline š x 2003 Hinz w š j rù ƒ š, 3 w j rù 52

2 j rù k w 53 Fig. 1. Metabolic pathways of aceclofenac in human (adapted from Bort et al. 7-8) ). 100mg zn w z x ƒ š. x ü j rù ƒ x (n-hexane) l (diethylether) yw» w z»d š g ww» d w. w ƒ û š l» j rù w ü k w. 10) q»(tandem mass spectrometer) w ƒ š, w mw ü j rù ƒ w w. 11) e w w j rù 100 mg z n w z x w. yw j rù x l w x w sƒw š, j rù ü q w. x j rù(aceclofenac), j rù(diclofenac) 4'- hydroxy-diclofenac t t Novartis Pharma AG(Basel, Switzerland) l œ š, ü t w flufenamic acid w formic acid Sigma l w.» Merck (Darmstadt, Germany) HPLC w. k, x t j rù ƒƒ w. x 20 w û w x w. x x w w z l v z x w š x ƒ w q w. x w x z(irb, Institutional Review Board) w x w m. x w s³ 24±3 (20~29 ) s³ 63.2±9.5 kg (45~75 kg) š j 165.4±8.4 cm (150~179 cm). x x w (hemoglobin, hematocrit, white blood cell, platelets, WBC differential counts), x yw(blood urea nitrogen, total bilirubin, cholesterol, total protein, albumin, alkaline phosphatase, glucose, AST, ALT) (specific gravity, color, ph, sugar, albumin, bilirubin, RBC, WBC, casts) ü mw w q x g. 10 ƒƒ 2 ù š, 12 z j rù 100 mg w wš x ƒƒ 1 1z n w. n n z 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, x w. x xq p þ» w 3000 rpm 10 w x š ¾ -80 o C w. 1 {» š n w z w xw x w. x j rù e ww. 11) j rù, j rù, 4'-hydroxydiclofenac flufenamic acid ƒƒ k 1 mg/ ml t w þ w w. j rù t x k w t œx t x 100 µl w. j rù t x 0.1, 0.2, 0.5, 1, 2, 5, 10 µg/ml j rù 4'-hydroxy-diclofenac t x 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.5, 1 µg/ml. t x w l x ƒ 100 µl ü t (flufenamic acid, 10 ng/ml) swwš k 400 µl ƒw z 5 yyw š, 13,200 rpm 10 w w. 10 µl HPLC w ü t v j w x v j w w. e (Zorvax eclipse XDB-C 18, mm,

3 54 Kor. J. Clin. Pharm., Vol. 16, No. 1, µm; Agilent, DE, USA) w 0.1% formic acid w wš 80% k 0.2 ml/min.» ABI Applied Biosystems/MDS SCIEX API 4000 TM LC/MS/MS System (Sciex Division of MDS Inc., Toronto, Canada) w. Turboion spray interfaceƒ 5500V, 400 o C negative mode w š v w ƒ. Nebulizing gas flow 1.04 l/min, auxiliary gas flow curtain gas ƒƒ l/min. Orifice voltage 80V, ring voltage 400V, collision gas (nitrogen) pressure Torr. w j rù, j rù, 4'-hydroxy-diclofenac ü t protonated precursor product d w multiple reaction monitoring (MRM) w. j rù, j rù, 4'-hydroxy-diclofenac flufenamic acid [M-H] - mass transition ƒƒ m/z [declustering potential (DP), -55 ev; collision energy (CE), -20 ev], (DP, -35 ev; CE, -16 ev), (DP, -30 ev; CE, -16 ev) (DP, -70 ev; CE, -26 ev),. 4'-hydroxyaceclofenac mass transition j rù w product ƒ w š DP CE j rù w w w (m/z ; DP, -55 ev; CE, -20 ev). kw m j rù x x (non-compartmental analysis) w kw parameter w. x š l šx (C max ) šx (T max ) š x š w (AUC t ) Õ e w w. (k e ) l»(t 1/2 =0.693/k e ) w. j rù w wš šx (C max ) x š w (AUC t ) yw s³ w m w (analysis of variance, ANOVA) w š 90% w. x š j rù kw w j rù 100 mg n z x j rù j rù diclofenac 4 -hydroxy-diclofenac Fig. 2, kw q l Table 1 ƒƒ ùkü. j rù s³ 1.5 (t r 0.6 ) š x 7.1±1.6 µg/ml w š 1.4±0.3 Fig. 2. Time course of the plasma aceclofenac, diclofenac, and 4 -hydroxy-diclofenac concentrations after a single 100-mg oral dose of aceclofenac in healthy volunteers. Each point represents the mean±s.d. (n=20).» ùkü 1 (1st-order elimination) w x. 12 ¾ š w 21.0±3.9 µg h/ml, üs³ 2.4±0.4 š 4.9±1.0 L/h ùkû. j rù n z j rù yw w 100 û ùkü ù x w ql ùkü. 1.6 šx 92±23 ng/ml w, j rù w» (1.0±0.5 ) ü s³ (2.5±0.4 ) ùk ü. Hinz w š w j rù mg z n s³ šx ƒƒ ng/ml s³» ) ƒ šx x ƒ ql ùkü j rù 100 mg n z j rù šx j rù 4 mg n w w w w. 4 -hydroxy-diclofenac 2.2 šx 43±14 ng/ml w 7.6±4.7» ùk ü x. ü s³ Table 1. Pharmacokinetic parameters of aceclofenac, diclofenac and 4 -hydroxy-diclofenac after an oral administration of 100mg aceclofenac in healthy volunteers Parameter aceclofenac diclofenac 4 -hydroxy-diclofenac T max (h) 1.5± ± ±0.9 C max (µg/ml) 7.1± ± ±0.01 AUC 12h (µg h/ml) 21.0± ± ±0.13 t 1/2 (h) 1.4± ± ±4.7 MRT (h) 2.4± ± ±4.9 Cl (L/h) 4.9±1.0

4 j rù k w 55 j rù ù j rù w 5 ¼ 11.9±4.9 ùkü. 4 -hydroxy-aceclofenac t w ƒ w.» w w w š û»š t y ƒ w. 4 -hydroxy-aceclofenac LC/MS/MS yw t yw j r ù d w. yw 16 ùkü w (precursor ion) w š ƒ (fragment ion) j rù w kw w w. Fig hydroxy-aceclofenac x ùkü ü t w flufenamic acid v j w 4 -hydroxy-aceclofenac v j t w. 4 -hydroxy-diclofenac 2 šx w 4.7±0.4» ùkü 1 ùkü x. j rù kw w Hinz w ƒ x ql x». 9) 4 -hydroxydiclofenac» 7.9±0.6 w ùkü ù yw» x ¼ š ( j rù, 5.0±0.4 ; j rù, 7.3±3.4 ; 4 -hydroxy-aceclofenac, 18.8±8.1 ). yw x biphasic pattern ùkü d šâ-phase»ƒ š» w. w, 3 e w x» w û w, 20 vx LC/ MS/MS w w j ùkü q. mw j rù x y y q w kw w w. w j rù y w w w» œw w. w sƒ k ( j rù 100 mg) wš t j rù ( j rù 100 mg) x w w 20 w sƒw. x j rù y Fig. 4, w sƒ w kw q l šx (C max ) 12 Fig. 3. Time course of the peak area ratio of 4 -hydroxyaceclofenac to internal standard after a single 100-mg oral dose of aceclofenac in healthy volunteers. Each point represents the mean±s.d. (n=20). Fig. 4. Time course of the plasma aceclofenac concentrations after a single 100-mg oral dose of aceclofenac in healthy volunteers. Each point represents the mean±s.d. (n=20). Table 2. Analysis of variance test (α=0.05) and 90% confidence intervals (CI) for AUC t (ln-transformed) and C max (lntransformed) for the 100 mg aceclofenac tablets Parameter ANOVA Group or sequence (4.414) Subjects/group (2.217) Period (4.414) Drug (4.414) Confidence Interval C max (µg/ml) ~ AUC t (µg h/ml) ~1.0231

5 56 Kor. J. Clin. Pharm., Vol. 16, No. 1, 2006 ¾ x š w (AUC t ) m w Table 2 ƒƒ ùkü. s³ 1.5 šx 6.4±2.2 µg/ml w w x 1 7.5±2.3 µg/ml ùk ü. x 12 ¾ x š w (AUC t ) ƒƒ 19.1± ±4.1 µg h/ml ùk ü. q l yw w w,»,. yw šx x š w s³e w 90% ƒƒ ~1.1310, ~ , x w w w sƒw. š x 1. Grau M, Guasch J, Montero JL Felipe A, Carrasco E, Julia S. Pharmacology of the potent new non-steroidal anti-inflammatory agent aceclofenac. Arzneimittel-Forschung 1999; 41: Ballesteros R, Ansoleaga JJ, Tapounet R. The efficacy and tolerance of aceclofenac in rheumatoid arthritis. A double-blind study v. placebo. Clin Trials J. 1990; 27: Ward DE, Veys EM, Bowdler JM, Roma J. Comparison of aceclofenac with diclofenac in the treatment of osteoarthritis. Clin Rheumatol 1995; 14: Cecchettin M, Cerea P, Torri G. Therapeutic efficacy of aceclofenac and diclofenac in acute knee artherosis. A study of E2-prostaglandin levels in synovial fluid and in serum. Clin Trials J. 1988; 25: Gonzalez E, del la Cruz C, de Nicolas R, Egido J, Herrero-Beaumont G. Long-term effect of nonsteroidal antiinflammatory drugs on the production of cytokines and other inflammatory mediators by blood cells of patients with osteoarthritis. Agents Actions 1994; 41: Yamazaki R, Kawai S, Matsuzaki T, Kaneda N, Hashimoto S, Yokokura T, Okamoto R, Koshino T, Mizushima Y. Aceclofenac blocks prostaglandin E2 production following its intracellular conversion into cyclooxygenase inhibitors. Eur J Pharmacol 1997; 329: Bort R, Ponsoda X, Carrasco E, Gomez-Lechon MJ, Castell JV. Metabolism of aceclofenac in humans. Drug Metabolism and Disposition 1996a; 24: Bort R, Ponsoda X, Carrasco E, Gomez-Lechon MJ, Castell JV. Cmparative metabolism of the nonsteroidal antiinflammatory drug, aceclofenac, in the rat, monkey, and human. Drug Metabolism and Disposition 1996b; 24: Henrotin Y, de Laval X, Mathy-Hartet M, Mouithys- Mickalad A, Deby-Dupont G, Dogne JM, Delarge J, Reginster JY. In vitro effects of aceclofenac and its metabolites on the production by chondrocytes of inflammatory mediators. Inflamm Res. 2001; 50: Hinz B, Auge D, Rau T, Rietbrock S, Brune K, Werner U. Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography. Biomedical Chromatography 2003; 17: Kang W. A new sensitive determination of aceclofenac and its three metabolites in human plasma using LC/MS/ MS. J Chromatogr B. in press. 12. Hinz B, Chevts J, Renner B, Wuttke H, Ran T, Schmidt A, Szelenyi I, Brune K, Werner U. Bioavailability of diclofenac potassium at low doses. Br J Clin Pharmacol 2005; 59:

16(2)-3(p ).fm

16(2)-3(p ).fm w wz 16«2y Kor. J. Clin. Pharm., Vol. 16, No. 2. 2006 k v p w w sƒ B Á z C Á½ k B Á B Á B Á x B Á x D Á x C Á ³ C Á E B w w w C w w D ƒm w w w E œw Bioequivalence Study on Two Talniflumate Preparations