w wz 16«1y Kor. J. Clin. Pharm., Vol. 16, No. 1. 2006 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 301-832 b Department of Pharmacy, Samsung Medical Center, Seoul 135-710 c College of Pharmacy, Catholic University of Daegu, Kyungbuk 712-702 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 0.80-1.25. Keywords aceclofenac, metabolites, LC/MS/MS, pharmacokinetics, bioequivalence Correspondence to : ƒm w w w w Tel: 053-850-3619, Fax: 053-850-3602 E-mail: 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
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, 10 12 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, 2.1 100 mm,
54 Kor. J. Clin. Pharm., Vol. 16, No. 1, 2006 3.5 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 4.0 16 l/min. Orifice voltage 80V, ring voltage 400V, collision gas (nitrogen) pressure 3.58 10-5 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 352.9 74.9 [declustering potential (DP), -55 ev; collision energy (CE), -20 ev], 296.1 251.7 (DP, -35 ev; CE, -16 ev), 311.8 267.7 (DP, -30 ev; CE, -16 ev) 279.9 235.9 (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 368.974.9; 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 12.5 25 mg z n s³ šx ƒƒ 334 588 ng/ml s³» 0.8. 12) 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±0.6 1.6±0.7 2.2±0.9 C max (µg/ml) 7.1±1.6 0.09±0.02 0.04±0.01 AUC 12h (µg h/ml) 21.0±3.9 0.26±0.07 0.37±0.13 t 1/2 (h) 1.4±0.3 1.0±0.5 7.6±4.7 MRT (h) 2.4±0.4 2.5±0.4 11.9±4.9 Cl (L/h) 4.9±1.0
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. 3 4 -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) 1.999 2.525 0.217 0.866 0.9636~1.1310 AUC t (µg h/ml) 3.794 8.051 0.005 0.829 0.9292~1.0231
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