Metabolism of Meloxicam in human liver involves ...

x e n o b io t ic a

, 1998, v o l . 28, n o . 1, 1± 13

M etabolism of M eloxicam in hum an liver involves cytochrom es P4502C 9 and 3A 4 C . C H E SN E; ‹ *, C . G U Y OM AR D‹ , A . G U IL LOU Z OŒ , J. S CH M ID , E . LU D W IG and T. SA U TE R

Xenobiotica Downloaded from informahealthcare.com by INSERM on 07/25/14 For personal use only.

‹ Biopredic, 14± 18 rue Jean Pecker, 35000 Rennes, France Œ Inserm U 456, D e! toxication et Re! paration Tissulaire, Faculte! de Pharm acie, 35043 Rennes, France D r K . Thom ae G m bh, D ep. Pharm acokinetics and M etabolism H 91, 88397 Biberach G erm any

Received 23 April 1997 1. The m etabolism of M eloxicam (M E) and the cytochrom e(s) P450 (CYPs) involved were analysed by using prim ary hum an hepatocytes, hum an liver m icrosom es and m icrosom es from recom binant hum an B-lym phoblastoid cell lines. 2. While hum an hepatocytes were capable of converting M E to a 5-hydroxymethyl m etabolite (M 7) and then to a 5-carboxyderivative (M 5), hum an liver m icrosom es form ed m ostly only the 5-hydroxym ethylderivative. The kinetics of the form ation of M7 by hum an liver m icrosom es were biphasic with K ¯ 13± 6³ 9 ± 5 and 381³ 55± 2 l m respectively. The m corresponding V were 33± 7³ 24± 2 and 143³ 83± 9 pm ol} m in} m g protein respectively. max

3. CYP2C9 and, to a m uch lesser extent, CYP3A4 were found to convert M E to M7. The involvem ent of 2C9 was dem onstrated by inhibition of tolbutam ide hydroxylase activity in the presence of M E, inhibition of M E m etabolism by sulphap henazole, correlation betw een M E m etabolism and tolbutam ide hydroxylase activity and active m etabolism of ME by recom binant2C9. The involvem entof 3A4 was show n by inhibition of ME m etabolism by ketoconazole, correlation betw een ME m etabolism and nifedipine oxidase activity and m etabolism of M E by recom binant 3A 4. K inetics of the form ation of M 7 by the individual enzym es resulted in a K ¯ 9 ± 6 l m and V ¯ 8± 4 pm ol} m in} m g m max protein for 2C9 and a K ¯ 475 l m and V ¯ 23 pm ol} m in} m g protein for 3A4. m

max

Introduction

M eloxicam (M E ; 4-hydroxy-2-m ethyl-N-(5-m ethyl-2-thiazolyl 2-H-1, 2benzo-thiazine-3-carboxam ide-1, 1 dioxide; ® gure 1) is a new non-steroidal antiin¯ am m atorydrug structurally related to the oxicam s. It is eå ective in the treatment of rheum atoid arthritis (R eginster et al. 1996) and osteoarthritis (H osie et al. 1996) and appears to be w ell tolerated due to its preferential inhibition of cyclooxygenase(C OX)-2 (Engelhardt et al. 1995a, b). In both m an and rat M E is m ainly converted to a 5-hydroxym ethyl m etabolite (M 7), w hich is further m etabolized to a 5-carboxyderivative (M 5) (S chm id et al. 1995a, b). The occurrence of a hydroxylation pathway suggests the involvem ent of cytochrom e(s) P450 (CY Ps). The aim of this study w as to identify the hum an hepatic C YP s involved in M E m etabolism by three diå erent in vitro m odels, i.e. liver m icrosom es, prim ary hepatocytes and heterologou sly expressed CY Ps. M aterials an d m ethods Chemicals M eloxicam labelled at the CO position (® gure 1 ; 0± 51 M Bq} m g), U H -A C 110 : 2-[[(4-hyd roxy-2m ethyl-2H-1,2-benzothiazine-3yl) carbonyl] am ino]-5-thiazolecarboxylic acid S, S-dioxide (0 ± 47 Mbq} m g) and AF-U H 1 : 2-[[4-hydro xy-2-m ethyl-2H-1,2-benzothiazine-3yl)carbonyl]amino]-5 * Author for correspondence. 0049± 8254} 98 $12± 00 ’

1998 Taylor & Francis L td

Figure 1.

Metabolic pathways of M E identi® ed in prim ary hum an hepatocyte cultures and in hum an liver m icrosom es. * Position of the label. M5 and M7 correspond to the synthetic references U H -AC 110 and AF-U H 1 respectively.


2 C. ChesneU et al.

Meloxicam metabolism by cytochrome P450s

3


thiazole-m ethanol S, S-dioxide (0 ± 48 M bq} m g), both labelled at the CO position, were labelled with " % C by Thom ae G m bH (Biberach,G erm any)(® gure 1). The radiochemicalpurity was " 99 % for Meloxicam and " 98 % for the other compounds. Chem icalswere purchased from Sigm a (D eisenhofen,G erm any) except phenobarbital(B D H , Poole, U K ), glibenclam ide(Thom ae), and resoru® n and phenacetin (Aldrich Chim ie, Steinheim , G erm any).

Biological samples The use of hum an liver sam ples for scienti® c purposes was approved by the French N ational Ethics Comm ittee. H um an liver m icrosom es were obtained from patients with secondary tum ors or other liver diseases according to standard procedures. Brie¯ y, liver fragm ents were hom ogenized at 4 ° C in 50 mm Trisbuå er containing 0 ± 25 m saccharose and 1 mm ED TA, pH 7 ± 4, centrifuged at 1600g for 10 m in then at 19 000g for 20 m in. The supernata nt was ® nally centrifuged at 100 000g for 60 m in and the pellet suspende d in 50 mm Tris-H Cl and again centrifuged at 100 000g for 60 m in. M icrosom al preparations were stored at ® 80 ° C in 0± 1 m phosphate buå er, pH 7± 4. Enzym e activities were determ ined as described below. In addition, hum an liver m icrosom es from individuals (H um an Biologics Inc., Phoenix, AZ, U SA) and pooled m icrosom es were used for the enzyme kinetic studies with characterized CYP activities (20 m g protein} m l). The activities of m icrosom es of donors 2, 11, and 20 were tolbutam ide hydroxylation: 254, 63± 2 and 180 pm ol} m in} m g protein respectively; and testosteron e 6b -hydroxylation : 10 500, 5390 and 1380 pm ol} m in} m g protein respectively. H um an hepatocytes were obtained from wedge biopsies using the two-step collagenase perfusion m ethod as previously described (G uguen-G uillouzo and G uillouzo 1986). The cells were plated in W illiams’ E m edium added with 4 l g} m l bovine insulin and 10 % foetal calf serum . M icrosom es from hum an B-lym phoblastoid cell lines expressin g hum an CYP1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1 and 3A4 cDN As were obtained from G entest Corp. (W oburn, U SA).

Assays CYP- related enzyme activities. T he diå erent form s of CYPs were characterized by m easuring the following enzyme activities according to m ethods described elsewhere, i.e. ethoxyresoru® n deethylation (K lotz et al. 1984), phenacetin deethylation (D islerath et al. 1985), tolbutam ide hydroxylation (M iners et al. 1988), m ephenytoin hydroxylation (Shim ada et al. 1986), dextrom ethorphan dem ethylation (K ronbach et al. 1987), p-nitrophe nol hydroxylation (K oop 1986) and nifedipine oxidation (G uengerich et al. 1986). M ost of these assays with possible adaptation to testing of living cells are detailed in G uillouzo and Chesne! (1996). M icrosom al and cellular protein contents were estim ated using the Bradford’ s m ethod with bovine serum album in as a standard (Bradford 1976). ME solubility. ME w as ® rst dissolved in dim ethylsulphoxide (D MSO) then in the culture m edium or in the buå ers. The ® nal concentration of solvent ranged betw een 0± 1 and 1 % . ME incubations. H um an liver m icrosom es (0 ± 1± 1 m g protein} m l) were incubated at 37 ° C in 0 ± 1 m K H PO buå er, pH 7± 6, containing 1± 15 % (w } v) K Cl or in 0± 1 m Tris buå er, pH 7± 6, containing 5 mm # % M gCl . The substrate (5± 200 l m ) was preincubated with m icrosom es for 5 m in before m etabolism was # initiated by addition of N AD PH (1 or 2 mm ) or an N AD PH -generating system consistin g of 0± 6± 1± 2 mm N AD P, 0 ± 7± 1± 4 IU } m l glucose 6-phosph ate dehydrogenase and 8 mm glucose 6-phosph ate. M icrosom es from recombinant B-lym phoblastoidcells (1 m g protein} m l) were incubated at 37 ° C in 0± 1 m T ris buå er, pH 7± 6, containing 5 mm MgCl . The substrate (10 l m ) was preincubated with # m icrosom es for 2± 5 m in before m etabolism was initiated by addition of the N AD PH -generating system in the presence of 0± 93 mm N AD H . T he addition of N AD H stim ulates som e enzyme activities via cytochrom e b (recom m endation of G entest, Catalog 95} 96). & Incubations with ME lasted from 5 to 60 m in. Control incubations were perform ed with heat inactivated hum an liver m icrosom es or m icrosom es isolated from the lymphoblastoid cell line without any hum an liver CYP cDN A insert and in the absence of N AD PH or the generating system . The reactions were term inated by freezing the incubation m ixtures at ® 80 ° C in an dry ice bath until analysis. After thaw ing the sam ples were directly injected, or after protein precipitation, into the hplc system . H um an hepatocytes were used 24 h after cell seeding. ME solubilized at 25 l m in the W illiams’ E m edium without foetal calf serum and containing 5 l m hydrocortisone haemisuccinatewas incubated for 18 h w ith the cells. The m edia and dried m onolayers were then stored at ® 80 ° C until analysis. A fter thaw ing the sam ples were directly injected into the hplc system . Hplc analysis.

The chrom atographic conditions were the following (Schm id et al. 1987):

C. ChesneU et al.

4


Enrichm ent column: 20¬ 4± 6 m m i.d., Bischoå (Leonberg, G erm any) dry ® lled with Bondesil C18 (40 l m ), equilibrated with M eOH . The eluent was am m onium form ate buå er 1 %, pH 6 ± 4, with a ¯ ow rate of 1 m l} m in at room tem perature. Analytical column: 125¬ 4 ± 6 m m i.d. 17 m m guard colum n, Bischoå , slurry packed with H ypersil OD S 5 l m . The gradient was a combination of step and linear gradients from A : amm onium form ate buå er 1 % to B : m ethanol,with a ¯ ow rate of 1 m l} m in at 28 ° C. The tim e and relative amount of B were as follows: 0 m in, 0 % ; 6 m in, 0 % ; 6± 1 m in, 20 % ; 24 m in, 40 % ; 30 m in, 50 % ; 35 m in, 80 % ; 36 m in, 100 % ; 39 m in, 100 % and 40 m in, 0 %. For the recording of the m etabolic pattern of m eloxicam under the in¯ uence of inhibitors the eluent was collected in 24-well m icroplates and the radioactivity m easured in a scintillation counter (Packard, M eriden, CT, U SA). The data were processed with the C H ROM I V1 softw are and the peaks were quanti® ed as described (Schm id et al. 1995a). Enzym e kinetic experim ents (form ation of M 7, identical with the synthetic reference AF-U H 1, as show n in ® gure 1) were perform ed with U V detection at 363 nm with AF-U H 1 as synthetic reference (as external standard). Instrum ents were a hplc pum p H P1090 and a diode array detector H P1040A, (H ewlett Packard, Boeblingen, G erm any). Statistical analysis Correlation coeæ cients, r, were calculated using an AN O VA table by the least-squares regression analysis from the raw data. They were considered to be signi® cant when p ! 0± 05. K and V were obtained initiallyby graphicalanalysis of Eadie± H ofsteeplots. The resulting values m max were used as ® rst estim ates in an iterative program (Excel 4.0) based on non-linear regression analysis to calculate K and V to ® t the equation: m

max

¯

V

max "

K

m"

[ C C

V

max #

K

m#

[ C C

.

R esults

Prelim inary studies w ere perform ed with prim ary rat hepatocyte cultures to determ ine the cytotoxic concentrations of M E . C ells w ere incubated for 72 h w ith M E at concentrations ranging from 1 ± 4 l m to 1 m m . The com pound was ® rst added 4 h after cell seeding and 24 and 48 h thereafter w ith m edium renew al. M E was w ithout eå ect at 12 l m and exerted only slight m orphologicalchanges at 37 l m over the 72-h treatm ent. The inhibitory concentration 50 (IC ) w as 170 l m in the 72-h &! treated cells, using the neutral red uptake test (data not show n).

ME metabolism F or determ ination of the m etabolic pattern, 25 l m " % C-M E w ere incubated for 30 m in with m icrosom al preparations and for 18 h w ith 24-h prim ary hum an hepatocytes. M etabolites w ere identi® ed by hplc and quanti® ed by using a radioactivity detector. M 7 was the m ain m etabolite formed by m icrosomes, while alm ost all the m etabolitesfound in hepatocyte cultures w ere represented by M 5 only (® gure 1) ; its turnover w as 115 pm ol} h } m g cellular protein.

In¯ uence of various CYPs substrates on ME metabolism M E w as incubated at concentrations ranging betw een 5 and 200 l m w ith hum an hepatic m icrosom alpreparations from two or three donors in the presence of various C YP substrates, i.e. ethoxyresoru® n, phenacetin, m ephenytoin, tolbutam ide, dextrom etorphan, p-nitrophenol and nifedipine for 5± 30 m in. As shown in ® gure 2, M E did not aå ect ethoxyresoru® n and phenacetin deethylase activities supported by CY P1A , m ephenytoin hydroxylation supported by CY P2C 19, p-nitrophenol hydroxylase activity supported by CY P2E 1 and nifedipine oxidase activity supported by CY P3A . B y contrast, a 66 % inhibition of

5



Figure 2. Competitive eå ects of ME on oxidative biotransfo rm ation of ethoxyresoru® n, phenacetin, m ephenytoin, tolbutam ide, dextrom ethorphan, p-nitrophe nol and nifedipine in the presence or absence of a speci® c inhibitor in hum an liver m icrosom es. ME was incubated at concentrations ranging from 5 to 200 l m for 5± 30 m in in the presence of the diå erent substrates. Control activities were m easured without competitor. The values of control activities were the following: ethoxyresoru® n deethylase, 81³ 66 pm ol} m in} m g protein ; phenacetin deethylase, 1± 7 ³ 0± 8 nm ol} m in} m g protein; tolbutam ide hydroxylase, 110³ 28 pm ol} m in} m g protein ; m ephenytoin hydroxylase, 72³ 4 pm ol} m in} m g protein; dextrom ethorphan dem ethylase, 0 ± 09³ 0± 05 nm ol} m in} m g protein; p-nitrophenol hydroxylase, 2± 2 ³ 1± 1 nm ol} m in} m g protein ; nifedipine oxidase,5 ± 7³ 2± 8 nm ol} m in} m g protein. The values are the m ean of two or three experim ents in duplicate.


6

C. ChesneU et al.

Figure 3. Eå ects of sulphap henazole, a CYP2C9 inhibitor, and ketoconazole, a CYP3A4 inhibitor, on M E conversion to M7 m etabolite in hum an liver m icrosom es. " % C-M E was incubated (10 l m ) in 0± 1 m Tris buå er, pH 7± 4, at 37 ° C with hum an liver m icrosom es (donor 2, 1 m g protein} m l), a N AD PH -generating system (consistin g of 1 ± 2 mm N AD P, 0± 7 U } m l G 6PD H , 8 mm G 6P), N AD PH (1 ± 2 mm ) and in the presence of sulfaphenazole (10 l m ) and} or ketoconazole (5 l m ) for 30 m in. In controls, M 7 form ation was m easured in the absence of competitor. The values are expressed as percent of controls, they are the m ean of n ¯ 2.

tolbutam ide hydroxylation supported by CY P2C 9 w as obtained with 100 l m M E . In addition a slight decrease was observed for dextrom etorphan dem ethylase activity supported by C YP2D 6 w ith 100 l m M E (® gure 2).

In¯ uence of reference chemicals on ME metabolism " % C -M E at 25 l m w as incubated w ith hum an hepatic m icrosom al sam ples for 30 m in in the presence of 13 reference chem icals, i.e. ethoxyresoru® n, phenacetin, m ephenytoin, tolbutam ide, sulfaphenazole, dextrom ethorphan, p-nitrophenol, nifedipine, ketoconazole, cim etidine, m ethotrexate, paracetam ol and w arfarin. These com pounds were tested at tw o diå erent concentrations, i.e. 25 and 250 l m . A large inhibitionof M E m etabolism w as obtained in the presence of sulphaphenazole, a C YP 2C9 inhibitor and ketoconazole, a C YP 3A4 inhibitor. C onsequently these tw o inhibitors w ere tested in a second set of experim ents at lower concentrations: sulfaphenazole at 10 l m and ketoconazole at 5 l m w ere found to reduce M 7 formation to 21 and 75 % of the control values respectively. Sim ultaneousaddition of both inhibitors resulted in a 93 % decrease in M E biotransform ation (® gure 3).

Relationship between ME metabolism and 2C9 and 3A4- related enzyme activities To correlate further drug m etabolizing enzym e activities w ith hydroxylation of M E , " % C-M E was incubated at the concentration of 25 l m for 30 m in with nine or 10 diå erent m icrosom al preparations and nifedipine oxidation and tolbutam ide

7



Figure 4. Correlation betw een ME hydroxylation and tolbutam ide hydroxylation and nifedipine oxidation in hum an liver m icrosom es. " % C-M E was incubated at 25 l m for 30 m in with hum an liver m icrosom es from nine or 10 donors. Correlation coeæ cients were calculated by the leastsquares regression m ethod.

hydroxylation activities were m easured without addition of M E after a 5- and 30-m in exposure respectively. A good correlation w as observed between M 7 formation and both activities: r ¯ 0 ± 69 for tolbutam ide hydroxylation and 0 ± 93 for nifedipine oxidation (® gure 4).

Enzyme kinetics of ME metabolism in human liver microsomes M E (1 ± 25± 1000 l m ) incubation with hum an liver m icrosom es of three donors w ith diå erent CY P2C 9 and CY P3A 4 activities in the presence of an N A D P H -

C. ChesneU et al.


8

Figure 5. M ichaelis± Menten kinetics and Eadie± H ofstee plot of M E m etabolism by hum an liver m icrosom es. M E (1 ± 25± 1000 l m ) was incubated in 0± 1 m tris buå er, pH 7 ± 4, at 37 ° C with hum an liver m icrosom es (donor 20, 1 m g protein} m l) and in the presence of an N AD PH -regenerating system (consisting of 1± 2 mm N AD P, 0± 7 U } m l G 6PD H , 8 mm G 6P) for 30 m in (m eans of n ¯ 2± 4). Table 1.

K

D onor 2 11 20 M ean³

SD

m

and V

max

of ME evaluated in m icroscopes from three diå erent hum an donors calculated by non-linear regression analysis.

K m" (l m )

V max " (pm ol} m in} m g protein)

Cl i " (l l} m in} m g protein)

K m# (l m )

21 17 3

61 15 25

2± 91 0± 88 8± 33

387 434 324

13± 6³

9± 5

33± 7 ³

24± 2

4± 0 ³

The intrinsic clearance (Cl i) is described by V

max

}

3± 9

381³

55± 2

V Cl i max # # (pm ol} m in} m g (l l } m in} m protein) g protein) 206 155 42 143³

83± 9

0± 53 0± 36 0± 13 0 ± 34³

0± 20

K . m

regenerating system showed biphasicM ichaelis± M enten kinetics of M E m etabolism (® gure 5, table 1). ME metabolism by human CYP- expressed microsomes Of " % C -M E , 10 l m was incubated for 60 m in w ith m icrosom es prepared from hum an B -lym phoblastoid cells expressing recom binant hum an liver CY P values 1A 1, 1A 2, 2A6, 2B 6, 2C 9, 2D 6, 2E1 and 3A 4. Only CY P2C 9 and to a m uch lesser



9

Figure 6. Biotransfo rm ation of ME by recom binanth um an CYPs. Microsom esfrom B-lym phoblastoid cells containing hum an liver C YP1A1, 1A2, 2A6, 2C9, 2D6, 2E1 or 3A4 were incubated in 0 ± 1 m Tris-H Cl buå er, pH 7± 4, at 37 ° C with recombinanthum an m icrosom es (1 m g protein} m l) in the presence of an N AD PH -regenerating system (consistin g of 1± 2 mm N AD P, 0± 7 U } m l G 6PD H , 8 mm G 6P) and addition of N AD H (0 ± 93 mm ) for 60 m in (m ean, of n ¯ 2).

extent, C Y P3A 4 w ere found to convert M E to M 7 (® gure 6). The values slightly above controls achieved w ith C YP 2A6 were not signi® cant.

Enzyme kinetics of ME metabolism in recombinant CYP2C9 and CYP3A4 M E w as incubated w ith hum an recom binantC YP 2C9 and CY P3A 4 respectively in the presence of an N AD PH -regenerating system for 60 m in (® gure 7). B oth C YP s formed M 7 (synthetic reference A F -U H 1). K m and Vmax values were estim ated to 9 ± 6 l m and 8 ± 4 pm ol} m in} m g protein (2C9) and to 475 l m and 23 pm ol} m in} m g protein (C Y P3A 4) respectively.

D iscussion

M ost non-steroidal anti-in¯ am m atory drugs inhibit both C OX-1 and C OX-2 w ith little selectivity, leading to serious side-eå ects (M itchell et al. 1994). There is therefore a need for new com pounds that exert a selective inhibition of C OX-2. Such com pounds have potent anti-in¯ am m atoryactivity with m inim al gastric sideeå ects (S eibert et al. 1994) and M E has been show n to have these properties (Engelhardt et al. 1995b ). To predict potential drug± drug interactions w ith M E treatm ent it is of m ajor im portance to identify CY P(s) involved in its oxidative biotransform ation. W e show here that the ® rst step of M E hepatic biotransform ation is an hydroxylation m ainly supported by C YP 2C9. The involvem ent of CY P2C 9 was well dem onstrated by diå erent approaches, i.e. extensive inhibition of M E m etabolism by sulphaphenazole, a speci® c inhibitor of CY P2C 9, a selective inhibition of tolbutam ide


10

C. ChesneU et al.

Figure 7. Eadie± H ofsteeplot of M E m etabolism by hum an recom binanth um an CYP3A4and 2C9. ME was incubated in 0± 1 m tris buå er, pH 7± 4, at 37 ° C with recombinanth um an CYP3A4 or 2C9 and in the presence of an N AD PH -regenerating system (consistin g of 1 ± 2 mm N AD P, 0± 7 U } m l G 6PD H , 8 mm G 6P) for 60 m in (m eans of n ¯ 2). Concentrations: 80± 640 l m for CYP3A4 and 1± 25± 160 l m for 2C9.

hydroxylation known to be supported by CY P2C 9, a good correlation betw een M E m etabolism and tolbutam ide hydroxylase activity and a high m etabolic rate of M E by recom binant hum an CY P2C 9. In addition, the slight decrease of dextrom ethorphan dem ethylase activity by M E could be related to CY P2C 9 as it has been shown recently (Ono et al. 1996 ). H ydroxylation of M E did not appear, however, to be exclusively catalysed by C YP 2C9. Indeed, M E m etabolism was also extensively inhibited by ketoconazole, a speci® c inhibitor of 3A 4 if used in low concentrations. In addition, a high correlation w as observed between M E m etabolism and nifedipine oxidase activity and recom binant hum an C YP 3A4 was also able to convert som e M E to its M 7 m etabolite. H owever M E did not com pete with nifedipine oxidation, w hich is m ainly supported by CY P3A . The involvem ent of both C Y Ps is further supported by the biphasic biotransform ation of M E in hum an hepatic m icrosom es, indicating high- and low -aæ nity enzym es. On the basis of experim ents w ith expressed C YP s the high aæ nity could be attributed to C YP 2C9 and the low aæ nity to the C YP 3A4. Although CY P3A is present in higher am ounts than C YP 2C9 in the hum an liver (probably 2± 6-fold) our calculations of intrinsic clearances (table 1) suggest that M E hydroxylation is m ainly perform ed by C YP 2C9. K m and Vmax values w ere used to



11

calculate the contribution of C YP2C9 and C YP3A4 to M E m etabolism . M ichaelis± M enten kinetics for each isoenzym e (one-enzym e kinetics) were calculated and the sum of both set to 100 % . Then, two concentrations were selected, 1 and 17 l m , correspondingrespectivelyto m inim um and m axim um blood plasm a concentrations after single and m ultiple once daily per os M E doses adjusted to 15 m g doses. At a low M E concentration (1 l m ) with a relative low content 3A4} 2C 9 ratio (donor 20), the contribution of C YP 3A4 to the total oxidative biotransform ation was 3 % . A t a high M E concentration (17 l m ) w ith a relative high 3A4} 2C 9 ratio (donor 11), the contribution of C YP 3A4 was 40 % . M E m etabolism will not m arkedly interfere with m etabolism of other drugs that are hydroxylated by CY P3A 4 as M E has a low aæ nity for this enzym e. B y contrast, interactions with drugs w hich are m etabolized by C YP 2C9 are quite likely. It m ust be underlined that CY P2C 9 m etabolizesa wide variety of clinicallyim portant drugs such as phenytoin, tolbutam ide, warfarin and various non-steroidal anti-in¯ am m atory drugs (R ettie et al. 1992, G oldstein and de M orais 1994, M iners et al. 1995) like piroxicam , a structurally related oxicam (Zhao et al. 1992). Phenytoin is known to be a contraindication for all non-steroidal anti-in¯ am m atory drugs. The interaction betw een M E and warfarin has also been investigated (Tu$ rck et al. 1997). There was no evidence that m eloxicam altered R-warfarin pharmacokinetics ; S-warfarin showed a trend to slightly higher ( 11 % ) plasma concentrations (area under the curve). The changes were not signi® cant and not relevant for pharm acodynam ics. There are at least ® ve m em bers of the CY P2C subfam ily (R om kes et al. 1991) and three diå erent alleles appear to exist in the caucasian populations (Stubbins et al. 1996). Population studies have indicated the existence of slow m etabolizers of tolbutam idesuggesting a rare polym orphism .This slow m etabolism with a low Vmax and a high K m for C YP2C9 w as associated with a Leu359 variant (Sullivan-K lose et al. 1996) with an allele frequency of 6 % . This results in a frequency of poor m etabolizers of about 0 ± 3 % . This rare polym orphism is apparently not re¯ ected in the pharm acokinetics as M E is additionally m etabolized by CY P3A 4. In oxicam s there is an additional m etabolic cleavage of the m olecule accom plishedby leukocyte peroxidases (Ichihara et al. 1985). A nalysing the plasm a levels of M E in " 1400 patients in E urope and " 400 volunteers in w ell-controlled phase I trails in G erm any and the U K , a log-norm al distribution of C max and area under the curve (D . Tu$ rck, personal com m unication) was found. M 7 w as converted to a carboxy m etabolite by further oxidation of the m ethyl group M 5 (S chm id et al. 1995 a) only in prim ary hum an hepatocyte cultures. As the m etabolic pathw ay was not found w ith m icrosom es it m ay be concluded that C YP s are not involved in the oxidation of the alcohol function. This observation gives further support to the use of cultured hepatocytes for analysing drug m etabolic pro® les. In this m odel all the necessary enzym es are expressed and incubation w ith the drugs can be carried out for long periods. In sum m ary, the C YPs involved in the M E biotransform ation w ere clearly identi® ed: CY P2C 9 is the m ost im portant one as show n for other oxicam s; how ever, additionally C YP 3A4 was found as the m inor one and this latter enzym e is known to be involved in the m etabolism of a w ide variety of com pounds (Breim er 1995 ).

12

C. ChesneU et al.

A cknow ledge m ent s

W e thank H . Z ipp and H . Sw itek for the synthesis of the labelled com pounds, and D . Tu$ rck for helpful discussions.


R eferences Br a d f o r d , M., 1976, A rapid and sensitive m ethod for the quantitation of m icrogram quantiti es of protein utilizing the principle of protein± dye binding. Analytical Biochemistry , 72 , 248± 254. Br e i m e r , D . D ., 1995, An integrated m olecular and kinetic} dynam ic approach to m etabolism in drug developm ent.In European Cooperation in the Field of Scienti® c and Technical Research . COST B1 Conference on Variability and Speci® city in Drug Metabolism (Brussels: European Comm ission), pp. 2± 19. Di s l e r a t h , L. M., Re i l l y , P. E., Ma r t i n , M. V., Da v i s , G . G ., Wi l k i n s o n , G . R. and Gu e n g e r i c h , F. P., 1985, Puri® cation and characterization of the hum an liver cytochrom es P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polym orphism in oxidative drug m etabolism . Journal of Biological Chemistry , 260 , 9057± 9067. En g e l h a r d t , G ., Bo $ g e l , R., Sc h n i t z l e r , C . and Ut z m a n n , R ., 1995a, Meloxican: in¯ uence on arachidonic acid m etabolism . Part II : In vivo ® ndings. Biochemical Pharmacology , 51 , 21± 38. En g e l h a r d t , G ., Ho m m a , D ., Sc h l e g e l , K ., Ut z m a n n , R. and Sc h n i t z l e r , C., 1995b, Antiin¯ amm atory, analgesic, antipyretic and related properties of m eloxicam , a new non-steroid al anti-in¯ amm atory agent with favourable gastrointestinal tolerance. In¯ ammation Research , 44 , 423± 433. Go l s t e i n , J. A. and d e Mo r a i s , S. M. F., 1994, Biochemistry and m olecular biology of the hum an CYP2C subfam ily. Pharmacogenetic s, 4 , 285± 299. Gu e n g e r i c h , F. P., Ma r t i n , M . V., Be a u n e , P. H ., Kr e m e r s , P., Wo l f , T. and Wa x m a n , D . J., 1986, Characterization of rat and hum an liver m icrosom al cytochrom e P-450 form s involved in nifedipineoxidation, a prototype for genetic polym orphismi n oxidative drug m etabolism .Journal of Biological Chemistry , 261 , 5051± 5060. Gu g u e n -Gu i l l o u z o , C. and Gu i l l o u z o , A., 1986, Methods for preparation of adult and fetal hepatocytes. In Isolated and Cultured Hepatocytes , edited by A. G uillouzo and C. G uguenG uillouzo (Paris and London: INSERM and John Libbey Eurotext), pp. 1± 12. Gu i l l o u z o , A. and Ch e s n e , C., 1996, Xenobiotic m etabolism in epithelial cell cultures.. In Epithelial Cell Culture : A Practical Approach , edited by A. J. Shaw (Oxford : IRL), pp. 67± 85. Ho s i e , J., Di s t e l , M . and Bl u h m k i , E., 1996, M eloxicam in osteoarthritis: a 6-m onth double blind com parison with diclofenac sodium . British Journal of Rheumatology , 35 (suppl 1), 39± 43. Ic h i h a r a , S., To m i s a w a , H ., Fu k a z a w a , H . and Ta t e i s h i , M ., 1985, Involvem ent of leucocyte peroxidases in the m etabolism of tenoxicam . Biochemical Pharmacology , 34 , 1337± 1338. Kl o t z , A. V., St e g e m a n , J. J. and Wa l s h , C., 1984, An alternative 7-ethoxyresoru® n O-deethylase activity assay : a continuous visible spectroph otom etric m ethod for m easurem ent of cytochrom e P-450 m onooxygenase activity. Analytical Biochemistry , 140, 138± 145. Ko o p , D . R., 1986, H ydroxylation of p-nitrophenol by rabbit ethanol inducible cytochrom e P-450 isozym e IIIa. Molecular Pharmacology , 29 , 399± 404. Kr o n b a c h , T., Ma t h y s , D ., Gu t , J., Ca t i n , T. and Me y e r , U . A., 1987, H igh-perform ance liquid chrom atographic assays for bufuralo l 1-hydroxylase, debrisoq uine 4-hydroxylase, and dextrom ethorphan O-dem ethylase in m icrosom es and puri® ed cytochrom e P-450 isozym es of hum an liver. Analytical Biochemistry , 162 , 24± 32. Mi n e r s , J. O., Re e s , D . L. P., Va l e n t e , L., Ve r o n e s e , M . E. and Bi r k e t t , D . J., 1995, H um an hepatocyte cytochrom e P-450 2C9 catalyzes the rate lim iting pathw ay of torsem ide m etabolism . Journal of Pharmacology and Experimental Therapeutics , 272 , 1076± 1081. Mi t c h e l l , J. A., Ak a r a s e r e e n o n t , P., Th i e r m e r m a n n , C., Fl o w e r , R. J. and Va n e , J. R., 1994, Selectivity of nonsteroi dal antiin¯ amm atory drugs as inhibitors of constituti ve and inducible cyclooxygenase. Proceedings of the National Academy of Sciences , USA , 90 , 11693± 11697. On o , S., Ha t a n a k a , T., Ho t t a , H ., Sa t o h , T., Go n a z a l e z , F. J. and Ts u t s u i , T., 1996, Speci® city of substrate and inhibitor probes for cytochrom es P450s : evaluation of in vitro m etabolism using cDN A-expressed hum an P450s and hum an liver m icrosom es. Xenobiotica , 26 , 681± 693. Re g i n s t e r , J. Y., Di s t e l , M . and Bl u h m k i , E., 1996, A double blind three-w eek study to compare the eæ cacy and safety of m eloxicam 7± 5 m g and m eloxicam 15 m g in patients with rheum atoid arthritis. British Journal of Rheumatology , 35 (Suppl 1), 17± 21. Re t t i e , A. E., Ko r z e k w a , K . R., Ku n z e , K . L., La w r e n c e , R. F., Ed d y , A. C., Ao y a m a , T., Ge l b o i n , H . V., Go n z a l e z , F. J. and Tr a g e r , W. F., 1992, H ydroxylation of w arfarin by hum an cD N Aexpressed cytochrom e P-450 : A role for P-450C29 in the etiology of (S)-w arfarin drug interactions. Chemical Research of Toxicology , 5 , 54± 59.



13

Ro m k e s , M., Fa l e t t o , M. B., Bl a i s d e l l , J. A., Ra u c y , J. L. and Go l d s t e i n , J. A ., 1991, Cloning and expression of com plem entary D N As for m ultiple m embers of the hum an cytochrom e P-450IIC. Biochemistry , 30 , 3247± 3255. Sc h m i d , J., Bu s c h ., U ., Tr u m m l i t z , G ., Pr o x , A., Ka s c h k e , S. and Wa c h s m u t h , H ., 1995a, M eloxicam: m etabolic pro® le and biotransfo rm ation products in the rat. Xenobiotica , 25 , 1219± 1236. Sc h m i d , J., Bu s c h , U ., He i n z e l , G ., Bo z l e r , G ., Ka s c h k e , S. and Ku m m e r , M., 1995b, Meloxicam: pharm acokinetics and m etabolic pattern after intravenous infusion and oral adm inistratio n to healthy subjects. Drug Metabolism and Disposition , 23 , 1206± 1213. Sc h m i d , J. and Ro t h , W ., 1987, H PLC-procedure for drug m etabolism studies: the potential of colum n sw itching. In Drug Metabolism , From Molecules to Man , edited by D . J. Benford, J. W. Bridges and C . G . G ibson (London: T aylor & Francis), pp. 213± 216. Se i b e r t , K ., Zh a n g , Y., Le a h y , K ., Ha u s e r , S., Ma s f e r r e r , J., Pe r k i n s , W., Le e , L. and Is a k s o n , P., 1994. Pharm acological and biochem ical dem onstration of the role of cyclooxygenase 2 in in¯ amm ation and pain. Proceedings of the National Academy of Sciences , USA , 91 , 12013± 12017. Sh i m a d a , T., Mi s o n o , S. K . and Gu e n g e r i c h , F. P., 1986. H um an liver m icrosom al cytochrom e P-450 m ephenytoin 4-hydroxylase, a prototype of genetic polym orphism in oxidative drug m etabolism . Journal of Biological Chemistry , 261 , 909± 921. St u b b i n s , M . J., Ha r r i e s , L. W., Sm i t h , G ., Ta r b i t , M . H . and Wo l f , C. R., 1996, G enetic analysis of the hum an cytochrom e P-450 CYP2C9 locus. Pharmacogenetic s, 6 , 432± 439. Su l l i v a n -Kl o s e , T. H ., Gh a n a y e m , B. I., Be l l , D . A., Zh a n g , Z. Y., Ka m i n s k y , L. S., Sh e n f i e l d , G . M ., Mi n e r s , J. O., Bi r k e t t , D . J. and Go l d s t e i n , J. A., 1996. The role of CYP2C9-Leu$ & * allelic variant in the tolbutam ide polym orphism . Pharmacogenetic s, 6 , 341± 349. Tu $ r c k , D ., Su , C. A. P. F., He i n z e l , G ., Bu s c h , U ., Bl u h m k i , E. and Ho f f m a n n , J., 1997, Lack of interaction betw een m eloxicam and warfarin in healthy volunteers. European Journal of Clinical Pharmacology , 51 , 421± 425. Zh a o , J., Le e m a n n , T. and Da y e r , P., 1992, In vitro oxidation of oxicam N SAIDs by a hum an liver cytochrom e P-450. Life Sciences , 51 , 575± 581.