Stereoselectivity of ibuprofen metabolism andpharmacokinetics following the administrationof the racemate to healthy volunteers

S. C. TAN{}, B. K. PATEL{*, S. H. D. JACKSON{,

C. G. SWIFT{ and A. J. HUTT{{ Department of Pharmacy, King’s College London, Franklin-Wilkins Building,Stamford Street, London SE1 9NN, UK.{ Clinical Age Research Unit, Department of Health Care of the Elderly, GKT School ofMedicine and Dentistry, Denmark Hill, London, SE5 9RS, UK.

Received 14 December 2001

1. The stereoselective metabolism and pharmacokinetics of the enantiomers ofibuprofen have been investigated following the oral administration of the racemic drug(400 mg) to 12 healthy volunteers.

2. The stereochemical composition of the drug in serum, both total and unbound, anddrug and metabolites, both free and conjugated, in urine were determined by acombination of the direct and indirect chromatographic procedures to enantiomericanalysis.

3. The oral clearance of (S)-ibuprofen was signi®cantly greater than that of theR-enantiomer (74:5 § 18:1 versus 57:1 § 11:7 ml min

¡1; p < 0:05) and the clearance of(R)-ibuprofen via inversion was ca two fold that via alternative pathways.

4. Some 74:0 § 9:6% of the dose was recovered in urine over 24 h as ibuprofen, 2-hydroxyibuprofen and carboxyibuprofen, both free and conjugated with glucuronic acid.Analysis of the stereochemical composition of the urinary excretion products indicatedthat 68% of the dose of (R)-ibuprofen had undergone chiral inversion.

5. Metabolism via glucuronidation and both routes of oxidation, showed enantio-selectivity for (S)-ibuprofen, the enantiomeric ratios (S/R) in partial metabolic clearancebeing 7.1, 4.8 and 3.4 for formation of ibuprofen glucuronide, 2-hydroxyibuprofen andcarboxyibuprofen respectively.

6. Modest stereoselectivity was observed in the formation of (20R; 2R)- and (2

0S; 2S)-

carboxyibuprofen in comparison to the alternative diastereoisomers, the ratios information clearance being 1.6 and 1.2 respectively.

Introduction

The metabolism and pharmacokinetics of (R,S)-ibuprofen [(§)-(R,S)-2-(4-isobutylphenyl)propionic acid] have been extensively investigated both in vitro andin vivo, since its introduction into therapeutics. In recent years these investigations

have been primarily directed towards studies concerned with the stereoselectivityof drug disposition following the administration of the racemate and/or individualenantiomers to both healthy volunteers and patients (Lee et al. 1985, Avgerinos

and Hutt, 1990, Evans et al. 1990, Geisslinger et al. 1990, Rudy et al. 1992, Li etal. 1993, Chen and Chen 1994, Smith et al. 1994) or with the elucidation of the

xenobiotica, 2002, vol. 32, no. 8, 683±697

* Author for correspondence; e-mail : bhavesh.patel@kcl.ac.uk} Present address: Doping Control Centre, Universiti Sains Malaysia, 11800 Minden, Penang,

Malaysia.

Xenobiotica ISSN 0049±8254 print/ISSN 1366±5928 online # 2002 Taylor & Francis Ltdhttp://www.tandf.co.uk/journals

DOI: 10.1080/00498250210142994

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chemical and biochemical mechanism of the chiral inversion reaction (see below)

(Baillie et al. 1989, Knihinicki et al. 1989, 1991, Sanins et al. 1991, Knights et al.,

1992, Shieh and Chen 1993, Shieh et al. 1993, Tracy et al. 1993, Menzel et al.

1994, Roy-De Vos et al. 1996, Scheuerer et al. 1998) and its biological con-

sequences (Williams et al. 1986, Freneaux et al. 1990, Zhao et al. 1992). However,

relatively little attention has been directed towards the stereochemical composition

of the two major urinary metabolites of the drug.The metabolism of ibuprofen (I, ®gure 1) in man involves conjugation with

glucuronic acid, which is stereoselective for the S-enantiomer (Lee et al. 1985, El-

Mouelhi et al. 1987) and oxidation to yield two major metabolites, 2-hydroxyibu-

684 S. C. Tan et al.

Figure 1. Oxidative metabolism of ibuprofen (I) in man

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profen (2-[4-(2-hydroxy-2-methylpropyl)phenyl]propionic acid, II) and carboxy-ibuprofen (2-[4-(2-carboxypropyl)phenyl]propionic acid, III) (Adams et al. 1967,Mills et al. 1973, Brooks and Gilbert 1974, Kaiser et al. 1976, Petterson et al.1978). The urinary excretion of these two latter metabolites together withibuprofen, both free and conjugated with glucuronic acid, accounts for between74 and 86% of an oral dose following the administration of the racemate to man(Lockwood et al. 1983, Evans et al. 1989a, 1990, Smith et al. 1994). Conjugation ofthe regioisomeric hydroxy metabolites and carboxyibuprofen with glucuronic acidhas been shown to take place preferentially at the propionic acid moiety (Kepp etal. 1997). In addition to the above, two minor oxidation products, 1-hydroxy- (IV)and 3-hydroxyibuprofen (V) have been identi®ed in human urine (Brooks andGilbert 1974, Petterson et al. 1978, Kepp et al. 1997) and 2-(4-carboxyphenyl)-propionic acid (VI) has been identi®ed in dialysis ¯uid following administration ofthe drug to a nephrectomized patient (Petterson et al. 1978) (®gure 1). Morerecently, Shirley et al. (1994) have reported the identi®cation of a taurine conjugateof ibuprofen in urine accounting for about 1.5% of the dose following oraladministration of the racemate.

In addition to what maybe regarded as conventional routes of metabolism, theessentially inactive (R)-ibuprofen also undergoes chiral inversion to the pharma-cologically active S-enantiomer. The observation that both major urinary meta-bolites, 2-hydroxy- and carboxyibuprofen, were excreted predominantly in thedextrorotatory form, irrespective of the stereochemical composition of the admi-nistered drug, ultimately resulted in the discovery of the chiral inversion reactionfor ibuprofen (Adams et al. 1967, Mills et al. 1973, Wechter et al. 1974, Kaiseret al. 1976) and the related 2-arylpropionic acid non-steroidal antiin¯ammatorydrugs (Hutt and Caldwell 1983, Caldwell et al. 1988, Mayer 1990, Hayball 1996).

Several investigations concerned with the stereoselective plasma pharmaco-kinetics of ibuprofen have also examined the urinary recovery of the drug and thetwo major metabolites (e.g. Evans et al. 1989a, 1990, Smith et al. 1994, Rudy et al.1991, 1995). However, few of these studies have addressed the stereochemicalcomposition of the metabolic products due to methodological problems associatedwith the chromatographic resolution of the four possible stereoisomeric forms ofcarboxyibuprofen, quantitatively the most important urinary metabolite account-ing for about 40% of the dose (Lockwood et al. 1983, Baillie et al. 1989, Evans et al.1989a, 1990, Rudy et al. 1991, Smith et al. 1994). We have recently reported thechromatographic resolution and characterization of the four stereoisomers ofcarboxyibuprofen (Tan et al. 1997a), and the chromatographic methodologyemployed has been developed into a validated sequential achiral-chiral techniquefor the determination of the stereochemical composition of both major oxidationproducts in urine (Tan et al. 1997b). We now report on the application of thismethodology to an examination of the stereoselective metabolism and pharmaco-kinetics of ibuprofen following the administration of the racemic drug to healthyvolunteers.

Material and Methods

NomenclatureIn the case of carboxyibuprofen, 2-[4-(2-carboxypropyl)phenyl]propionic acid, the position of both

chiral centres are indicated as the second carbon atom in each of the two side chains. In the present

Stereoselectivity of ibuprofen disposition in man 685

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report the chiral centre introduced by metabolic oxidation of the terminal methyl groups of the isobutylmoiety in ibuprofen will be designated as the 2

0-position, whereas the original chiral centre in the

propionic acid moiety will be indicated as the 2-position.

ChemicalsThe individual enantiomers (enantiomeric purity ¶ 99:6%), (R,S)-ibuprofen and (R,S)-2-hydro-

xyibuprofen, together with (R,S)-¯urbiprofen were the generous gifts of Boots Co. Ltd (Nottingham,UK). Carboxyibuprofen was synthesized, with a chemical purity >98%, as described by Tan et al.(1997a). Radiolabelled [14C]-ibuprofen with a speci®c activity of 21.6 mCi g

¡1 and a radiochemicalpurity of >99% post-HPLC puri®cation (Tan et al. 1997c) was generously donated by KnollPharmaceuticals (Nottingham, UK). (R)-1-(Naphthen-1-yl)ethylamine and 1-(3-dimethylaminopro-pyl)-3-ethylcarbodiimide hydrochloride were purchased from Aldrich (Gillingham, UK); 1-hydroxy-benzotriazole and 4-chlorophenoxyacetic acid were obtained from Fluka Chemicals (Poole, UK) andSigma Chemicals (Poole, UK) respectively. HPLC solvents were purchased from Rathburn (Walk-erburn, UK) and all other reagents and solvents from BDH (Poole, UK). Protein assay reagents basedon the Coomassie blue method were obtained from Bio-Rad (Hemel Hempstead, UK) and dialysismembranes (Spectrapor 2) were obtained from Pierce and Warriner (Chester, UK).

Chromatography columnsThe C18 column (Waters Resolve C18), (5 mm, 150£ 3.9 mm i.d.) was obtained from Anachem Ltd

(Luton, UK) and the Partisil silica column (5 mm, 250 £ 4.6 mm i.d.) was obtained from Whatman(Maidstone, UK). Re®llable guard columns (10 £ 2.1 mm), pellicular silica (40±63 mm) and pellicularC18 (40±60 mm) were obtained from Alltech Associates (Carnforth, UK). The chiral-phase columns,amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) (10 mm, 250 £ 4.6 mm i.d.) and cellulosetris(3,5-dimethylphenylcarbamate) (Chiralcel OD) (10 mm, 250 £ 4.6 mm i.d.), together with matchingguard columns (10 mm, 50 £ 4.6 mm i.d.), were supplied by HPLC Technology Ltd (Maccles®eld, UK).

InstrumentationHigh-performance liquid chromatography (HPLC) was carried out using an LDC Constametric

3000 pump, and either an LDC Spectromonitor 3100 UV detector or Merck-Hitachi spectro¯uorom-eter, depending on the analytes under examination, linked to an LDC CI 4000 computing integrator.Samples were introduced on-column using either an Perkin-Elmer ISIS 100 or an LKB 2157autosampler ®tted with 100-ml sample loops. Liquid scintillation spectrometry was carried out usingan LKB 1209 Wallac Rackbeta liquid scintillation spectrometer. The liquid scintillation cocktail usedwas Quickscint ¯ow 302 (Zinsser Analytical, Maidenhead, UK).

Analytical methodsFull details of the analytical methodologies employed in the present investigation, including their

development, validation and preliminary application to samples of biological origin, have been reportedelsewhere (Tan et al. 1997b and c). Brief details of each method are presented below.

Ibuprofen enantiomers. The enantiomeric composition of ibuprofen in serum and urine was determinedby the indirect approach to enantiomeric analysis (Tan et al. 1997c). Following the addition of theinternal standard, (R,S)-¯urbiprofen (1 mg, 10 ml of a 100 mg ml

¡1 solution in acetonitrile) to eitherserum (0.5 ml) or urine (0.5 ml for free and 0.1 ml for total, free plus conjugated drug, following alkalinehydrolysis) the analytes were isolated by liquid±liquid extraction (serum samples, diethylether, 3ml;urine samples, hexane:isopropanol [9:1 v/v], 5 ml) at pH 3.8. Following phase separation andevaporation of the organic solvent, the residue was derivatized by the addition of (R)-1-(naphthen-1-yl)ethylamine in dichloromethane, to yield the corresponding diastereoisomeric amides using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole as coupling agents. Underthese conditions the derivatization is essentially quantitative (Tan et al. 1997c). Chromatographicresolution of the derivatives was achieved under reversed-phase conditions using the C18-stationaryphase and a mobile phase of phosphate bu� er (pH 3.5, 0.01 m):acetonitrile (1:1 v/v) at a ¯ow rate of1.5 ml min

¡1. Quanti®cation was carried out using a spectro¯uorometer with excitation and emissionwavelengths of 290 and 330 nm respectively. Under these conditions the (R)-1-(naphthen-1-yl)ethylamides of (R)- and (S)-¯urbiprofen and (R)- and (S)-ibuprofen eluted at 14.5, 17.8, 22.2 and25.6 min respectively.

Metabolite Analysis. The concentrations and stereochemical composition of 2-hydroxy- andcarboxyibuprofen in urine were determined by sequential achiral-chiral chromatography (Tan et al.1997b). Following the addition of the internal standard (4-chlorophenoxyacetic acid, 25 mg per sample)urine (0.5 ml aliquots for the determination of free and 0.1 ml aliquots for the determination of total

686 S. C. Tan et al.

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concentrations following alkaline hydrolysis) was extracted under acidic conditions withdichloromethane:ethyl acetate (14:1 v/v). Following phase separation, the organic layer was removed,evaporated to dryness and the residue reconstituted in 150 ml of the mobile phase employed for achiralchromatography and 50 ml injected on column. Achiral analysis was carried out using the Partisilcolumn with a mobile phase of hexane:ethanol (98.2:1.8 v/v) containing tri¯uoroacetic acid (TFA;0.05% v/v) at a ¯ow rate of 2.0 ml min

¡1 with UV detection at 220 nm. Under these conditions theinternal standard, carboxyibuprofen and 2-hydroxyibuprofen eluted with retention times of 4.9, 12.6and 15.8 min respectively. The HPLC eluate containing the two metabolites was separately collectedbetween 12.2±13.0 min and 15.3±16.3 min, evaporated under nitrogen and the residue dissolved in themobile phase used for chiral chromatography. Chiral-phase analysis was carried out using a ChiralpakAD CSP with a mobile phase of hexane:ethanol (92:8 v/v) containing TFA (0.05% v/v) at a ¯ow rate of1.0 ml min

¡1, and UV detection at 220 nm. Under these conditions (R)- and (S)-2-hydroxyibuprofeneluted with retention times of 13.9 and 16.1 min respectively and the retention times of the fourstereoisomers of carboxyibuprofen were 2

0S; 2R-, 11.0; 2

0R; 2R-, 12.1; 2

0R; 2S-, 16.9 and 2

0S; 2S-,

20.1 min. The stereochemical composition of both metabolites was determined by comparison of peakareas following chiral-phase chromatography and the individual concentrations were calculated from aknowledge of stereochemical composition and urinary concentration.

Protein binding of ibuprofen. Protein binding of the enantiomers of ibuprofen was carried out byequilibrium dialysis using 2.5 ml volume dialysis cells and Spectrapor 2 membranes preconditioned bysoaking overnight in phosphate bu� ered saline (PBS) (Tan et al. 1997c). Five determinations, usingserum samples obtained at 0.5, 1.0, 2.0, 3.0 and 4.0 hours post drug administration, were carried out foreach volunteer. Serum samples (1 ml) were transferred into three adjacent dialysis cells and equalvolumes of PBS were added to the opposite sides of the membranes. The cells were then placed in awater bath (378C) and allowed to equilibrate for 8 h. Analysis of the stereochemical composition ofibuprofen in the dialysate bu� er was initially carried out by HPLC using the methodology for thedetermination of the drug enantiomers in serum but employing a semi-microbore column(150 £ 2.1 mm i.d.) packed `in-house’ with Resolve C18 media obtained by emptying the packingmaterial from a Radialpak Resolve C18 cartridge (5 mm) (Anachem Ltd, Luton, UK), as neither asmaller bore column or the packing material were commercially available. The mobile phase employedwas the same as that used previously (see above) but at a ¯ow rate of 0.6 ml min

¡1 with ¯uorescencedetection as described above. During the course of this investigation a sample of racemic [14C]-ibuprofen became available and the above analytical methodology was validated against a previouslyreported procedure using radiolabelled material (Evans et al. 1989b). Both methods yielded similar data,the determined unbound enantiomer fractions showing no statistically signi®cant di� erences (Tan et al.1997c). Subsequently, the approach utilising the radiolabelled material was adopted and enantiomericresolution performed using a Chiralcel OD CSP with a mobile phase of hexane:isopropanol (100:1.1v/v) containing TFA (0.1% v/v) at a ¯ow rate of 1.0 ml min

¡1, with UV detection at 220 nm. Underthese conditions (R)- and (S)-ibuprofen eluted with retention times of 12.5 and 15.9 minutesrespectively. The eluate fractions of the drug enantiomers were collected between 12.0 to 13.2 minand 15.4 to 17.2 min into individual scintillation vials and supplemented with 4 ml scintillation cocktailfor radiochemical analysis. In this manner, higher throughput was achieved and also loss of analyte byadditional manipulation was minimized.

The unbound enantiomer fractions determined were corrected for volume shift changes occurringduring dialysis (Huang 1983), following determination of the protein concentrations both before andafter dialysis using the Coomassie blue procedure (Tan et al. 1997c).

Volunteer studyApproval for the investigation was obtained from the Local Research Ethics Committee of King’s

College Hospital. Twelve healthy volunteers (6M, 6F) between the ages of 20 and 29 years (mean § SD;M: 23 § 3 years, weight 78 § 13 kg; F: 25 § 3 years, weight 70 § 5 kg) gave written informed consentand participated in the study. Each volunteer underwent a thorough physical examination and routinebiochemical tests prior to recruitment and none of the subjects were receiving drug treatment and hadnot been involved in research studies within the last three months. The volunteers were required toabstain from any medication, alcohol and ca� eine containing beverages for 24 hours prior to theinvestigation and to fast overnight. On the morning of the study each subject swallowed a single 400 mgtablet of racemic ibuprofen (Brufen1) with approximately 150 ml of water. Blood samples (10 ml) werecollected from a cannulated forearm vein immediately prior to and at 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 6.0, 8.0 and 10.0 hours post drug administration. A 24 h blood sample was obtained byvenepuncture. The samples were collected into plain vacutainers and allowed to clot prior tocentrifugation and separation of serum. Urine samples were collected immediately prior to drugadministration and then continuously for 24 hours at the following time intervals: 0±2, 2±4, 4±6, 6±8,8±10 and 10±24 h, from which a pooled 24 h sample was prepared. All serum and urine samples werefrozen and stored at 7208C until required for analysis. All analyses were carried out in duplicate.

Stereoselectivity of ibuprofen disposition in man 687

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Data AnalysisPharmacokinetic analysis of the serum concentration-time curves for the individual enantiomers was

carried out by the non-compartmental approach using the TOPFIT program (Heinzel et al., 1993). Theterminal disposition constant (¶z) and the apparent elimination half-life (t1=2) for each pro®le werecalculated by linear regression of the log-linear portions of the serum concentration-time curves. Thearea under the concentration-time curves (AUC) from time zero to 24 hours were calculated using thetrapezoidal method and extrapolated to in®nity by dividing the last measurable serum concentration by¶z. The extrapolated areas were typically less than 2% of the total AUC. The maximum serumconcentration (Cmax) and the time to attain it (tmax) were determined by visual inspection of the data.The apparent total clearance (CL/F) and volume of distribution (VD/F) were calculated as follows:

CL=F ˆ D=AUC

VD=F ˆ D=…AUC:¶z†

where F is the fraction of the dose (D) reaching the systemic circulation.The fraction of (R)-ibuprofen undergoing inversion (Finv) was calculated from an examination of the

stereochemical composition of ibuprofen and both major oxidation products (both free and conjugated)excreted in urine over 24 hours, from:

Finv ˆ S-metabolites (%) ¡ S-dose (%)

R-dose (%)

where S-metabolites (%) is the sum of ibuprofen, and both oxidation products, excreted with theS-con®guration of the propionic acid moiety expressed as a percentage of the total drug recovery,and S- and R-dose (%) are the percentages of the administered dose in that form, i.e. 50% as a racematewas administered. The systemic exposure (i.e. the `dose’) of (S)-ibuprofen was calculated from:

`dose’ ˆ …1 ‡ Finv†:D;

where D is equal to the dose administered, i.e. 200 mg. The metabolite formation clearances werecalculated from:

CLmetab: ˆ Am=AUC

where Am is the amount of metabolite recovered in urine over 24 h and AUC corresponds to the areaunder the concentration-time curve of the drug enantiomer from which the metabolite was derived(Rowland and Tozer 1995).

Statistical comparisons of pharmacokinetic data derived from an examination of individual en-antiomers may prove problematic unless their systemic availability is known to be equivalent. In thecase of ibuprofen previous investigations, following intravenous and oral administration of the racemicdrug, have indicated that the bioavailability of the individual enantiomers is essentially equivalent andapproximates to unity (Hall et al. 1993). Statistical analysis of the data was carried out using Student’s t-test for paired and independent samples.

Results

A representative ibuprofen enantiomer serum concentration time pro®le fol-lowing oral administration of the racemic drug (400 mg) to a healthy volunteer ispresented in ®gure 2 and the pharmacokinetic parameters derived from anexamination of the individual data are presented in table 1. Peak serum concen-trations (Cmax) of both enantiomers were achieved within 3.0 h post dosing, withonly one subject showing delayed absorption for both enantiomers with a tmax at6.0 h. The mean values of Cmax for (S)- and (R)-ibuprofen were 17.5 and16.3 mg ml

¡1 respectively. The mean serum concentrations of (S)-ibuprofen weregreater than those of the R-enantiomer at all time points following four hours postdrug administration, resulting in a signi®cantly greater AUC for the S-enantiomer(table 1). The mean apparent elimination half-life of (R)-ibuprofen (1:4 § 0:3 h)was signi®cantly shorter than that of the S-enantiomer (2:3 § 0:5 h; p < 0:001)resulting in a progressive predominance of (S)-ibuprofen with respect to the totaldrug serum concentration.

Examination of protein binding indicated stereoselectivity with the unboundfraction of (S)-ibuprofen being signi®cantly greater than that of the R-enantiomer

688 S. C. Tan et al.

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(table 1) (p < 0:001) yielding an enantiomeric ratio (S/R) of the unbound fractionof 2.04.

The stereochemical composition of the drug and metabolites in urine (seebelow) was used to estimate the fraction of the dose of (R)-ibuprofen undergoingchiral inversion for each subject (table 1). Some 68% of the administered dose ofthe R-enantiomer was found to undergo inversion and the clearance via inversionwas signi®cantly greater than that via alternative routes (38:9 § 9:0 ml min

¡1

compared to 18:3 § 3:4 ml min¡1; p < 0:00001). The fraction of the dose inverted

was also used to establish the exposure of the volunteers to (S)-ibuprofen and theparameters derived for the S-enantiomer (table 1) were calculated taking thisincreased `dose’ into account.

The urinary recovery of ibuprofen, 2-hydroxy- and carboxyibuprofen, bothfree and conjugated, accounted for approximately 11%, 23% and 40% of the doserespectively during the 24 h collection period (table 2). Examination of thestereochemical composition of the drug and both metabolites in urine indicatedthat 84% of the recovered material (74% of the dose) had the S-con®guration in thepropionic acid moiety. The stereochemical composition (S/R) of ibuprofen, 2-hydroxyibuprofen and carboxyibuprofen (considering the 2-position only) being 9,

Stereoselectivity of ibuprofen disposition in man 689

Figure 2. Representative serum concentration pro®les of (R)-ibuprofen (open circles) and (S)-ibuprofen (closed circles) following the oral administration of the racemic drug (400 mg) to ahealthy female volunteer. Individual data points are the average of duplicate determinations

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6.3 and 4.3 respectively. Examination of the stereochemistry of the metabolicallyintroduced chiral centre in carboxyibuprofen, i.e. the 2

0-position, indicated pre-

ferential oxidation of the drug to yield the diastereoisomeric metabolite having thesame con®guration at the new chiral centre as that in the substrate. Thus,oxidation of …R†-ibuprofen preferentially yields the 2

0R; 2R-diastereoisomer

(ratio 20R; 2R=2

0S; 2R ˆ 1:6) whereas oxidation of (S)-ibuprofen preferentially

yields the 20S; 2S-diastereoisomer (ratio 2

0S; 2S=2

0R; 2S ˆ 1:2).

The partial metabolic clearances for each ibuprofen enantiomer are presentedin table 3. In each case the clearance of the S-enantiomer is signi®cantly greaterthan that of …R†-ibuprofen and the formation clearance of the two preferentiallyformed diastereoisomers of carboxyibuprofen are signi®cantly greater …p < 0:001†than those of the alternative stereoisomers (table 3).

The majority of the urinary ibuprofen was excreted as alkali labile conjugates,presumably the acyl glucuronides, with relatively minor quantities (<1%) of eitherenantiomer being detected in the free form. Such minor quantities of the free drugmay be due to hydrolysis of the conjugate during sample manipulation, i.e.collection, storage and analysis. In contrast, the 2-hydroxy- and carboxyibuprofenmetabolites were excreted as alkali labile conjugates in variable amounts (table 2).2-Hydroxyibuprofen showed stereoselectivity with respect to conjugation with theS-enantiomer being excreted predominantly as the conjugate (ratio conjugate/free¹2.1) whereas the R-enantiomer was excreted predominantly in the free form(ratio conjugate/free ¹0.45). In the case of the diacid all four stereoisomers werepreferentially excreted in the free form with ratios of conjugate/free varying from0.45 for the 2

0S; 2R-isomer to 0.58 for the 2

0S; 2S-diastereoisomer.

Discussion

The pharmacokinetics of ibuprofen following administration of either racemateor the R-enantiomer are complicated as a result of the metabolic chiral inversion of(R)-ibuprofen. A number of approaches have been adopted in order to determine

690 S. C. Tan et al.

Table 1. Pharmacokinetic parameters of the enantiomers of ibuprofen following the oraladministration of the racemic drug (400 mg) to healthy volunteers.

Parameter (units)

R S

Mean § SD Range Mean § SD Range

Cmax (mg ml¡1

) 16.3‹3.8 11.3±21.6 17.5‹4.3 12.6±26.2tmax (h) 2.4‹1.3 1.0±6.0 2.6‹1.4 1.0±6.0t1=2 (h) 1.4‹0.3 1.1±2.1 2.3‹0.5

¤1.9±3.6

AUC (mg.h ml¡1

) 60.6‹12.0 42.9±80.7 79.3‹19.7¤

53.6±120.0CL=F (ml min

¡1) 57.1‹11.7 41.3±77.7 74.5‹18.1

¤45.0±120.6

VD /F (L) 7.0‹1.6 5.0±9.8 15.0‹4.2¤

8.2±22.9fu (%) 0.23‹0.04 0.19±0.32 0.47‹0.11

¤0.33±0.69

Finv 0.68‹0.03 0.62±0.74 ± ±CLinv=F (ml min

¡1) 38.9‹9.0 25.6±54.1 ± ±

CLother=F (ml min¡1

) 18.3‹3.4 13.7±24.9 ± ±

Cmax, maximum observed concentration; tmax, time to Cmax; t1=2, terminal elimination half-life;AUC, area under the curve; CL/F, oral clearance; VD=F, volume of distribution; fu, fraction unbound;Finv, fraction inverted; CLinv=F, clearance via inversion; CLother=F, clearance via other metabolicroutes.

* Signi®cant di� erences between enantiomers, (p < 0:05; n ˆ 12).

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Stereoselectivity of ibuprofen disposition in man 691

Tab

le2.

Uri

nar

yre

cover

yof

ibu

pro

fen

,2-h

yd

roxyib

up

rofe

nan

dca

rboxyib

up

rofe

nst

ereo

isom

ers,

both

free

and

con

jugat

ed,

foll

ow

ing

ora

lad

min

istr

atio

nof

the

race

mic

dru

g(4

00

mg)

toh

ealt

hy

volu

nte

ers.

An

alyte

Ste

reoch

emis

try

Fre

eC

on

jugat

edT

ota

l

Mea

n§

SD

Ran

ge

Mea

n§

SD

Ran

ge

Mea

n§

SD

Ran

ge

Ibu

pro

fen

R0.0

6‹

0.0

60.0

1±0.2

11.0

‹0.3

0.8

±1.7

1.1

‹0.3

0.9

±1.8

S0.7

0‹

0.5

00.1

8±1.9

09.2

‹2.8

4.7

±13.5

9.9

‹3.0

5.0

±14.0

2-H

yd

roxyib

up

rofe

nR

2.2

‹0.6

1.4

±3.5

1.0

‹0.5

0.2

±1.7

3.1

‹0.5

2.4

±3.8

S6.4

‹2.4

3.4

±11.5

13.3

‹3.6

6.9

±18.4

19.5

‹2.9

15.7

±24.4

Car

boxyib

up

rofe

n2

0 S;2

R2.0

‹0.4

1.4

±2.8

0.9

‹0.6

0.2

±1.8

2.9

‹0.5

2.1

±3.7

20 R

;2R

3.0

‹0.6

1.5

±3.9

1.7

‹1.0

0.0

2±3.0

4.7

‹0.8

3.8

±6.3

20 R

;2S

9.7

‹2.8

5.6

±15.6

5.1

‹2.9

0.5

±9.8

14.8

‹3.2

11.3

±21.7

20 S

;2S

11.3

‹3.3

6.2

±18.1

6.5

‹3.0

1.0

±10.7

17.8

‹3.1

14.3

±23.3

Tota

lre

cover

y35.4

‹9.6

21.5

±46.3

38.6

‹11.1

21.0

±55.6

74.0

‹9.6

66.4

±91.3

Dat

aar

eth

ep

erce

nta

ge

of

the

dose

adm

inis

tere

d;

… nˆ

12† .

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the extent of chiral inversion including: administration of the individual enantio-mers and comparison of the areas under the plasma concentration time curve of(S)-ibuprofen following administration as such and that resulting from inversionfollowing dosing of the R-enantiomer (Lee et al. 1985); simultaneous administra-tion of deuterated (S)-ibuprofen with unlabelled drug followed by quanti®cationusing gas-chromatography mass spectrometry (Rudy et al. 1991); and determina-tion of the fraction of drug related material excreted in urine with the S-con®guration in the propionic acid moiety (Baillie et al. 1989, Rudy et al. 1991).Comparison of the values obtained using all three approaches have indicatedsimilar mean estimates for the fraction of the dose undergoing inversion. However,the interindividual variability using the administration of the individual enantio-mer approach was found to be greater, and in some cases results in considerableerrors in the estimate, in comparison to the alternative approaches (Rudy et al.1991). This is presumably associated with intraindividual variability in drughandling on di� erent occasions and possible enantiomer-enantiomer interactions

during drug disposition following administration of the racemate.The approach based on determination of the stereochemical composition of

material excreted in urine, i.e. that adopted in the present investigation, has beenreported to yield estimates of fraction inverted consistently greater, but withreduced interindividual variability in comparison to the approaches based onplasma determinations (Rudy et al. 1991). In one report these di� erences werefound to be signi®cantly greater, whereas in another this was not the case (Rudyet al. 1991, Hall et al. 1993). For the urinary analysis approach to be successful ahigh recovery of drug related material is essential and the observed greaterestimates of fraction inverted may be associated with incomplete drug recovery.For example in the present investigation 74 § 9:6% of the dose was accounted forwith about 84% of the material having the S-con®guration in the propionic acidmoiety (table 2). If the proportion of the 2S-con®gured material is reduced in the

692 S. C. Tan et al.

Table 3. Metabolite formation clearances of ibuprofen glucuronide,2-hydroxyibuprofen and carboxyibuprofen stereoisomers following oraladministration of the racemic drug (400 mg) to healthy volunteers

Analyte Stereochemistry

Formation clearance (ml min¡1

)

Mean § SD Range

Ibuprofen-glucuronide R 1.2‹0.4 0.7±2.0S 8.5‹2.3

a3.9±13.1

2-Hydroxyibuprofen R 3.6‹1.0 2.4±5.3S 17.3‹5.3

a11.0±29.1

Carboxyibuprofen 20S; 2R 3.3‹0.9 2.1±4.8

20R; 2R 5.4‹1.4

b3.8±7.9

20R; 2S 13.4‹5.2 7.1±26.3

20S; 2S 16.0‹5.3

b7.9±28.3

20S; 2R ‡ 2

0R; 2R 8.7‹2.1 5.9±12.4

20R; 2S ‡ 2

0S; 2S 29.3‹10.3

a15.0±54.6

Data presented as (20S; 2R ‡ 2R; 2R) and (2

0R; 2S ‡ 2

0S; 2S) for

carboxyibuprofen represent the clearances of (R)- and (S)-ibuprofen via theformation of the diacid metabolite respectively.

aSigni®cant di� erences between the drug enantiomers …p < 0:001; n ˆ 12†.

bSigni®cant di� erences between the formation clearances of the individual

diastereoisomers of the diacid arising from either enantiomer of ibuprofen…p < 0:001; n ˆ 12†.

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unrecovered fraction then the estimated extent of inversion will be reduced.However, this approach does have the advantage of not requiring single enantio-mer deuterated standards and quanti®cation by mass spectrometry or repeatedadministration of single enantiomer doses. In addition, the urinary metaboliteanalysis approach would not be susceptible to the nonlinear kinetics of the drugwith increased doses associated with concentration dependent protein binding(Evans et al. 1989b, 1990, Paliwal et al. 1993). However, an essential prerequisitefor the urinary approach to be adopted is the availability of methodology capable ofdetermining the stereochemical composition of the metabolites.

In comparison to the parent drug, relatively few studies have examined thestereochemical composition of the two major metabolic products. This is in partdue to the limited availability of the material, particularly with respect to stereo-chemically de®ned standards, and also due to methodological problems associatedwith the chromatographic resolution of the four stereoisomers of carboxyibupro-fen. Several investigations have determined the enantiomeric composition of thedrug in plasma and reported the total urinary recovery of the metabolic products(Evans et al. 1990, Geisslinger et al. 1993) and a number of investigations havecombined the data for the two carboxyibuprofen stereoisomers which are di� cultto resolve chromatographically (Baillie et al. 1989, Smith et al. 1994). Two recentpublications have successfully addressed this analytical problem using capillaryelectrophoresis (Bjùrnsdottir et al. 1998, Fanali et al. 1998), however these havenot as yet been applied to pharmacokinetic investigations. The only reports to datewhich refer to the stereochemical composition of both the drug and both majormetabolites are those of Hall and coworkers (Rudy et al. 1990, 1991, 1995, Hallet al. 1993) who determined the stereochemical composition of the metabolitesusing the indirect approach to enantiomeric analysis. However, due to the lack ofstereochemically de®ned standards of the carboxy metabolite, the chromatographicelution order could not be unequivocally assigned and was established by referenceto a previously published method by Kaiser et al. (1976), which was empirical.

In the present investigation, using our sequential achiral-chiral approach toenantiomeric analysis (Tan et al. 1997b) we were able to determine the stereo-chemical composition of both major oxidative metabolites of ibuprofen, both freeand conjugated with glucuronic acid, and estimate the fraction of (R)-ibuprofenundergoing inversion following administration of the racemate. Using this ap-proach the fraction undergoing inversion was determined to be 0:68 § 0:03 (range0.62±0.74), data in good agreement with that of Rudy et al. (1991) using theurinary analysis approach, and within the range of values 0.52±0.71 reported byothers (see Davies 1998 for review). It is noteworthy that, in comparison to otherpharmacokinetic parameters, the variability observed in fractional inversion wasrelatively low (table 1) within this group of healthy young volunteers. It would beof interest to examine, using a similar approach, the extent of inversion withinother de®ned subgroups to establish if the minimal variability observed in thepresent investigation is consistent within the population.

The signi®cance of inversion versus noninversion mechanisms in the clearanceof (R)-ibuprofen is di� cult to ascertain from the literature. Rudy et al. (1991)reported similar values for both pathways whereas in subsequent studies theseworkers, and others, have reported that clearance via inversion is the predominantpathway (Hall et al. 1993, Smith et al. 1994, Rudy et al. 1995). Our observationssupport the latter view, the about two fold greater clearance of (R)-ibuprofen via

Stereoselectivity of ibuprofen disposition in man 693

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inversion (38:9 § 9:0 ml min¡1) being signi®cantly greater than that via noninver-

sion pathways (18:3 § 3:4 ml min¡1; p < 0:00001). The values obtained being

similar to those reported by Smith et al. (1994) and lower than those obtainedby Rudy et al. (1991).

The total clearance of (S)-ibuprofen was signi®cantly greater than that of theR-enantiomer (table 1), the values obtained being similar to those of Smith et al.(1994) following administration of the individual enantiomers. The partial meta-

bolic clearance of the drug via the three alternative pathways, i.e. conjugation withglucuronic acid and oxidation at the 2- and 3- positions of the isobutyl side chain,all showed a stereo preference for (S)-ibuprofen. The most marked stereoselec-tivity being found for conjugation with glucuronic acid (ratio S/R 7.1; table 3),with oxidation at the 2

0-position yielding a ratio S/R of 4.8 and a substrate

selectivity S/R of 3.4 for the carboxy metabolite. Examination of the diastereo-meric composition of carboxyibuprofen indicated a more modest selectivity with

respect to product formation, the diastereoisomer formed in excess in each casehaving the same con®guration at the metabolically introduced centre as that in thesubstrate. Thus, the predominantly formed diastereoisomers are of the 2

0R; 2R-

and 20S; 2S- absolute con®gurations, the product diastereoselectivity being 1.6

and 1.2 fold respectively (table 3). An observation in agreement with that of Rudyet al. (1991). The signi®cance of this product selectivity is by no means clear as

obviously multiple oxidative transformations are involved in the formation ofcarboxyibuprofen each of which may exhibit its own stereoselectivity.

Examination of the in¯uence of gender on the enantiomeric disposition ofibuprofen revealed no signi®cant di� erences in either drug recovery or pharma-cokinetic parameters between males and females (data not shown), in agreementwith the observations of Knights et al. (1995) who examined the pharmacokineticsof (R)-ibuprofen following administration of the single enantiomer to male, female

and oral contraceptive-using females.In conclusion, the data presented above represents the ®rst investigation

involving the unequivocal assignment of the stereochemical composition of bothmajor urinary metabolites of ibuprofen and has con®rmed the utility of the urinarymetabolite approach as a facile means for the estimation of the fraction of (R)-ibuprofen undergoing inversion.

Acknowledgements

We gratefully acknowledge the award of a University of London TriangleTrust Postgraduate Studentship to BKP and a King’s College Research Strategy

Fund award to SHDJ, CGS and AJH.

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is U

niv.

on

11/0

8/14

For

pers

onal

use

onl

y.