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Specificity of human CYP2C9 for two substrates, diclofenac and ibuprofen, was studied using chimeras and site-directed mutants of CYP2C9 and the highly related CYP2C19 expressed in Escherichia coli. Data were correlated with the presence of putative substrate recognition sites (SRS). A CYP2C19 chimera containing residues 228-340 (SRS 3 and 4) of 2C9 conferred both diclofenac hydroxylation and 2- and 3-hydroxylation of ibuprofen. The regiospecificity of this construct for metabolism of ibuprofen differed from that of CYP2C9 by favoring 2-hydroxylation over 3-hydroxylation. A CYP2C9 construct containing residues 228-340 of CYP2C19 lacked both diclofenac and ibuprofen hydroxylase activities. When residues 228-282 (containing SRS 3) of CYP2C9 were replaced by those of CYP2C19, the chimera retained appreciable activity for diclofenac and ibuprofen, and tolbutamide activity was inhibited by a specific CYP2C9 inhibitor, sulfaphenazole. This suggested that SRS 3 is not important in conferring specificity. CYP2C9 and CYP2C19 differ in five residues within the region 283-340 (within SRS 4). Mutations to analyze SRS 4 were made on a CYP2C19 chimera containing residues 228-282 of CYP2C9. A single I289N mutation conferred a dramatic increase in diclofenac hydroxylation and a small increase in ibuprofen 2-hydroxylation. A second mutation (N286S and I289N) increased diclofenac hydroxylation and conferred a dramatic increase in ibuprofen 2-hydroxylation. A V288E mutation did not increase activity toward either substrate and decreased activity toward the two substrates in combination with the I289N or the N286S, I289N mutants. Therefore residues 286 and 289 of CYP2C9 are important in conferring specificity for diclofenac and ibuprofen.

Objectives: To determine the influence of the hypolipidemic drug clofibrate on the stereoselective metabolism of ibuprofen in humans., Methods: Healthy male subjects (n = 12) ingested a dose of 400 mg pseudoracemic ibuprofen (200 mg R-ibuprofen, 160 mg S-ibuprofen, and 40 mg 13C-S-ibuprofen) on two occasions after either pretreatment with clofibrate (2 gm/day over 1 week) or no pretreatment in a randomized order., Results: When subjects were pretreated with clofibrate, clearances of R-ibuprofen and 13C-S-ibuprofen increased significantly from 55.0 and 66.4 ml/min to 186.2 and 106.7 ml/min (p < 0.01), respectively. This increase was similarly reflected in the clearance by inversion of R-ibuprofen (control, 36.0 ml/min; treated, 118.8 ml/min; p < 0.01), as well as in the clearance by noninversion (control, 19.0 ml/min; treated, 67.4 ml/min; p < 0.01). Unbound clearance values significantly increased for R-ibuprofen (control, 19.5 L/min; treated, 38.7 L/min) but not for 13C-S-ibuprofen (11.8 versus 10.6 L/min, respectively). The fractional inversion of ibuprofen calculated from the urinary metabolite data was increased after clofibrate pretreatment (clofibrate group, 66.4%; control, 53.5%; p < 0.01). However, this was not evident when fractional inversion was calculated from the plasma concentration-time data for the unmetabolized drug., Conclusions: Clofibrate altered the stereoselective disposition of ibuprofen in healthy volunteers by increased formation of R-ibuprofenoyl-coenzyme A rather than by an effect on oxidative metabolism of ibuprofen. This interaction has potential therapeutic implications.

The cytochrome P450s responsible for the regio- and stereoselectivity in the 2- and 3-hydroxylation of the chiral non-steroidal antiinflammatory drug ibuprofen were characterized in human liver microsomes. The rates of formation of both the 2- and 3-hydroxy metabolites exhibited monophasic (N = 2; N is the number of microsomal preparations) and biphasic (N = 2) substrate concentration dependence for both enantiomers of ibuprofen. The high affinity enzyme class parameters for S-ibuprofen (N = 4) were: 2-hydroxylation, Vmax = 566 +/- 213 pmol/min/mg, Km = 38 +/- 13 microM; 3-hydroxylation, Vmax = 892 +/- 630 pmol/min/mg, Km = 21 +/- 6 microM. For R-ibuprofen, the corresponding parameters were: 2-hydroxylation, Vmax = 510 +/- 117 pmol/min/mg, Km = 47 +/- 20 microM; 3-hydroxylation, Vmax = 593 +/- 113 pmol/min/mg, Km = 29 +/- 8 microM. cDNA-expressed CYP2C9 (Arg 144 and Cys 144) favored S-2- and S-3-hydroxyibuprofen formation, but CYP2C8 favored R-2-hydroxyibuprofen formation. Sulfaphenazole, retinol, and arachidonic acid competitively inhibited the rate of formation of all hydroxyibuprofens; Ki values (N = 3) for sulfaphenazole on the 2- and 3-hydroxylations of S-ibuprofen were 0.12 +/- 0.05 and 0.07 +/- 0.04 and of R-ibuprofen were 0.11 +/- 0.07 and 0.06 +/- 0.03 microM, respectively. Sulfaphenazole also competitively inhibited ibuprofen hydroxylation by cDNA-expressed CYP2C9 (Arg 144 and Cys 144) with Ki values in the range of 0.05 to 0.18 microM and CYP2C8 in the range of 0.36 to 0.55 microM. In a bank of 14 human liver microsome samples, significant correlations (r = 0.72 to 0.90; P < 0.01) were observed between the rates of formation of all four hydroxyibuprofens, and for each hydroxyibuprofen and prototypical CYP2C8/9 biotransformations. The regio- and stereoselectivities observed in vitro were consistent with those noted in vivo. The relative levels of both CYP2C8 and CYP2C9 and the expression of the corresponding variants may influence the disposition of ibuprofen in vivo.

Diabetes mellitus is associated with numerous metabolic events that may influence the elimination of R- and S-ibuprofen and the inversion of R-ibuprofen. Short (3 days) and long (14 days) term experimental type I diabetes was induced in male Sprague-Dawley rats with streptozotocin, and genetically diabetic male Zucker rats were used as a model of type II diabetes. Isolated hepatocytes from long-term streptozotocin-treated rats exhibited significantly greater rate constants for ibuprofenyl-coenzyme A (CoA) formation (1.44 +/- 0.05 vs. 0.60 +/- 0.09 hr-1) and the elimination of R-ibuprofen (0.34 +/- 0.07 vs. 0.22 +/- 0.07 hr-1) relative to control (P < or = .05). These increases were consistent with significant induction of hepatic cytochrome P450 (1.14 +/- 0.45 vs. 0.54 +/- 0.10 nmol/mg protein) and an elevated hepatic free CoA content (313.4 +/- 48.5 vs. 172.9 +/- 38.6 nmol/g) relative to control (P < or = .05). In hepatocytes from type II diabetic rats there were significant reductions (P < or = .05) in the rate constants for ibuprofenyl-CoA formation (1.02 +/- 0.12 vs. 1.22 +/- 0.12 hr-1), R-ibuprofen elimination (0.21 +/- 0.06 vs. 0.34 +/- 0.10 hr-1) and S-ibuprofen elimination (0.41 +/- 0.07 vs. 0.73 +/- 0.11 hr-1) but no change in hepatic content of cytochrome P450 or CoA relative to control. The activity of ibuprofenyl-CoA synthetase in whole liver homogenate supplemented with ATP and CoA was not influenced by experimental diabetes. In both type I and type II diabetes there was a significantly greater exposure of hepatocytes to ibuprofenyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)

By using stable isotope methodology, we studied the disposition of ibuprofen after the first and last dose of a 4-week regimen of 800 mg of racemic ibuprofen every 8 hr in three groups of subjects: 1) young healthy volunteers (n = 8); 2) healthy elderly volunteers (n = 14); and 3) elderly patients with creatinine clearance between 30 and 70 ml/min (n = 13). Stereoselective gas chromatography-mass spectrometry was used to quantify deuterated S- and nondeuterated R- and S-ibuprofen in serum up to 24 hr after the first and last doses. Urinary excretion of the stereoisomeric forms of carboxyibuprofen, hydroxyibuprofen and ibuprofen glucuronide were determined up to 24-hr postdose by stereoselective high-performance liquid chromatography. Stereoselective serum protein binding was determined by ultrafiltration. Both elderly groups had significantly decreased binding of S-ibuprofen compared to the young group. The S-ibuprofen pharmacokinetics were significantly different in the elderly patients with renal impairment compared to the young volunteers: the T1/2 was increased, the unbound clearance was decreased and the unbound concentration at steady state was increased. Fraction inverted was similar for all groups, but unbound clearances of glucuronidation and hydroxylation were reduced in the elderly patients with renal impairment. These results suggest that the disposition of ibuprofen enantiomers is altered in elderly persons with renal impairment; these changes may increase the risk for nonsteroidal anti-inflammatory drug-associated adverse effects in such patients.

R-ibuprofen undergoes chiral inversion by the formation of a coenzyme A (CoA) thioester and subsequent epimerization and hydrolysis. Using isolated rat hepatocytes, the interaction of xenobiotics with the inversion and oxidation pathways of ibuprofen enantiomers was determined from the time course of R- and S-ibuprofen and ibuprofenyl-CoA during 4-hr incubations with R- or S-ibuprofen (25 microM). By fitting a first-order model, the rate constants of the formation of ibuprofenyl-CoA (K12), oxidation of R-ibuprofen (K10), hydrolysis of ibuprofenyl-CoA (K21) and oxidation of S-ibuprofen (K30) were 1.306, 0.284, 6.858 and 0.496 hr-1, respectively. The fractional inversion of R-ibuprofen was 0.75 and the area under the curve for ibuprofenyl-CoA was 203.8 microM min. Coincubation with 50 microM of the cytochrome P450 inhibitors metyrapone and proadifen resulted in significant reductions of K10 and K30; the fractional inversion of R-ibuprofen increased to 116% and 127% and the area under the curve of ibuprofenyl-CoA to 145% and 144% of controls, respectively. Valproic acid and pivalic acid at 50 microM significantly reduced the K12 and increased the K21; the fractional inversion was unchanged but the area under the curve of ibuprofenyl-CoA was significantly reduced to 57% and 28% of controls, respectively. Valproic acid also significantly reduced K10 and K30. p-nitrobenzoic acid at 50 microM significantly increased K21 and reduced the area under the curve of ibuprofenyl-CoA to 44% of control but did not influence the fractional inversion. Selective inhibitors of ibuprofen oxidation were found to enhance significantly hepatocellular exposure to the potentially reactive ibuprofenyl-CoA intermediate.

Presystemic inversion of (R)- to (S)-ibuprofen has been proposed but not directly examined in humans. We investigated the bioavailability of the enantiomers of ibuprofen in 10 healthy volunteers. Low-dose racemic ibuprofen (400 mg) was administered orally and intravenously (60-minute infusion), in random order. There were no significant differences between oral and intravenous doses for the area under the curve values, terminal rate constants, clearances, metabolite formation clearances, and serum protein binding for (R)- and (S)-ibuprofen. The bioavailabilities of (R)-ibuprofen and total ibuprofen were 0.92 +/- 0.11 and 0.95 +/- 0.08, respectively. The fractional inversion of (R)-ibuprofen was determined by two methods (stable isotope method and from the stereochemical composition of the urinary metabolites) that gave similar estimates of inversion for oral dosing (0.56 +/- 0.12 and 0.60 +/- 0.07, respectively) and intravenous dosing (0.56 +/- 0.09 and 0.60 +/- 0.06, respectively). We conclude that the bioavailability of both enantiomers of ibuprofen is complete and find no evidence of significant presystemic inversion.

Ibuprofen [(racemic)2-(4-isobutylphenyl)propionic acid] undergoes metabolic inversion via formation, epimerization, and hydrolysis of the coenzyme A (CoA) thioester, ibuprofenyl-CoA. In this study, (R)-ibuprofen was incubated with either rat whole liver homogenate, human whole liver homogenate, rat liver mitochondria, or rat liver microsomes, and the formation of ibuprofenyl-CoA measured. Rat whole liver homogenate (Vmax/KM = 0.022 +/- 0.005 ml/min/mg protein) was approximately 4-fold more efficient at forming ibuprofenyl-CoA than human whole liver homogenate (Vmax/KM, = 0.005 +/- 0.004 ml/min/mg protein). Rat liver microsomes (Vmax/KM = 0.047 +/- 0.019 ml/min/mg protein) were approximately 2-fold more efficient than rat whole liver homogenate at forming ibuprofenyl-CoA, whereas rat liver mitochondria (Vmax/KM = 0.027 +/- 0.017 ml/min/mg protein) did not differ from whole liver homogenate. Palmitic (Ki = 0.005 mM) and octanoic acids (Ki = 0.19 mM) were capable of inhibiting ibuprofenyl-CoA formation, whereas propionic acid had no effect, suggesting the possible involvement of both long- and medium-chain fatty acyl-CoA synthetases. Of the xenobiotics tested, only bezafibrate (Ki = 0.85 mM) and (S)-ibuprofen (Ki = 0.095 mM in rats, 0.32 mM in human tissue) were capable of substantially inhibiting ibuprofenyl-CoA formation. Thus, it appears that the metabolic inversion of ibuprofen involves lipid-metabolizing pathways and may be affected by fatty acids or xenobiotics.

The pharmacokinetics of the enantiomers of ibuprofen have been investigated following oral administration of 300 or 600 mg of racemic ibuprofen four times daily to 45 patients with osteoarthritis. Fifteen of these patients also received single doses of 300 or 600 mg of racemic ibuprofen. Serum concentrations of R- and S-ibuprofen and urine concentrations of the stereoisomers of ibuprofen and its metabolites were measured by high-performance liquid chromatography. The fraction inverted (F(inv)) of the inactive R- to active S-ibuprofen was estimated by a urinary metabolite method. For the 15 patients in both the chronic and single dose studies, there were no significant differences in the clearance of R-ibuprofen or F(inv). The elimination half-lives of R- and S-ibuprofen were comparable for the single and chronic dosing studies. The area under the curve (AUC) values, 6-h trough concentrations, and average steady state concentrations of the R- and S-enantiomers were statistically different after chronic dosing. Despite considerable variability in the clearances in these patients, e.g., clearance (CL) of R-ibuprofen showed 28-49% CV, much less variability was seen in F(inv) (range 9-19% CV), implying that patients would receive similar effective doses of active S-ibuprofen in spite of large differences in kinetics.

Ibuprofen [(racemic)2-(4-isobutylphenyl)propionic acid] has been proposed but not directly demonstrated to undergo unidirectional inversion from the (R)- to the (S)-configuration via a coenzyme A (CoA) thioester intermediate. Chemically synthesized (R)- and (S)-ibuprofenyl-CoA, and rat and human liver homogenates were used to investigate the relative rates of ibuprofenyl-CoA epimerization and hydrolysis. Rat whole liver homogenate completely epimerized (R)- or (S)-ibuprofenyl-CoA, whereas hydrolysis of this intermediate occurred at a much slower rate. Rat liver mitochondria was the most efficient at both epimerizing and hydrolyzing ibuprofenyl-CoA, whereas rat liver microsomes hydrolyzed ibuprofenyl-CoA at a rate similar to whole liver homogenate but had very little epimerization activity. Rat liver cytosol was the poorest at hydrolyzing ibuprofenyl-CoA but had substantial epimerization capability. Whole liver homogenate from human tissue was less efficient at epimerizing but as efficient at hydrolyzing ibuprofenyl-CoA as rat whole liver homogenate. No stereoselectivity of either epimerization or hydrolysis was noted for any of the enzyme preparations studied. This study demonstrates that the inversion of (R)-ibuprofen occurs, at least in part, via the epimerization of the metabolic intermediate, ibuprofenyl-CoA, in both rat and human liver tissues.

Stereoselective pharmacokinetic measurements of the active enantiomer, S-ibuprofen, were correlated with clinical response in 45 participants in a randomized double blinded 4 week comparison of ibuprofen, 1200 or 2400 mg/day, for treatment of hip or knee osteoarthritis. Ibuprofen dose correlated with S-ibuprofen area under the serum concentration curve (AUC), trough and average concentration, but not with clinical outcome. AUC of S-ibuprofen correlated with improvement in disability, rest pain and in the physician's global assessment (p = 0.02, 0.08, and 0.10, respectively), and negatively with the subject's weight and creatinine clearance (p = 0.09 and 0.07, respectively). Some individual variation in responsiveness to ibuprofen (and other nonsteroidal antiinflammatory drugs) may be attributed to pharmacokinetic differences.

The possible contribution of pulmonary metabolism to the putative first-pass metabolism of 2-arylpropionic acid nonsteroidal antiinflammatory drugs has not been documented. Isolated perfused rabbit lungs, perfused with 4.5% bovine serum albumin or 5% dextran, were used to study the pulmonary elimination of (R)- and rac-ibuprofen, fenoprofen, and flurbiprofen. In the absence of protein binding, ibuprofen was metabolized via inversion and other pathways, whereas fenoprofen metabolism was essentially restricted to inversion of the (R)-enantiomer; fraction inverted (+/- SE) was 0.37 +/- 0.05 for (R)-ibuprofen and 0.85 +/- 0.03 for (R)-fenoprofen. In the presence of protein, neither ibuprofen nor fenoprofen was metabolized. Flurbiprofen did not undergo pulmonary elimination under any condition studied. This study illustrates that even though a tissue is capable of metabolism, particularly inversion of 2-arylpropionics, the quantitative importance of such elimination pathways may be minimal in the presence of the high degree of protein binding that is characteristic of these drugs.

We have studied the pharmacokinetics of methotrexate in patients with rheumatoid arthritis concurrently treated with choline magnesium trisalicylate, ibuprofen, naproxen, or a non-NSAID analgesic (control treatment). The apparent systemic clearance of methotrexate was significantly reduced by all three treatments. Trisalicylate and ibuprofen both significantly reduced methotrexate renal clearance, but only the trisalicylate significantly displaced methotrexate from protein, increasing the fraction unbound by 28%. These data show that NSAIDs can affect the disposition of methotrexate, possibly increasing the potential for toxicity and necessitating dosage adjustments. However, large inter-subject variability precludes specific dosage recommendations.

Using stable isotope methodology, we studied the effect of the enantiomeric composition of dosage form on ibuprofen metabolism. Eight healthy human subjects received racemic ibuprofen (800 mg) plus S-[aromatic-2H4]ibuprofen (10 mg), R-ibuprofen (600 mg) plus S-[aromatic-2H4]ibuprofen (10 mg) and S-ibuprofen (600 mg) orally on separate occasions in random order. Stereoselective gas chromatography-mass spectrometry was used to quantify deuterated and nondeuterated ibuprofen in serum up to 24 h postdose. Urinary excretion of the stereoisomeric forms of carboxyibuprofen, hydroxyibuprofen and ibuprofen glucuronide were determined up to 24 h postdose by stereoselective high performance liquid chromatography. The metabolism of ibuprofen enantiomers was not influenced by the enantiomeric composition of the dose. For racemic ibuprofen, the mean clearances (+/- S.D.) of S-ibuprofen, R-ibuprofen inversion and R-ibuprofen noninversion were 87.4 +/- 25.9, 57.3 +/- 31.0 and 56.3 +/- 29.0 ml/min, respectively. The fractional inversion of ibuprofen was significantly greater (P less than .05) using the stereochemical composition of the urinary metabolites (0.63 +/- 0.05) vs. the method using the clearance of deuterated S-ibuprofen (0.51 +/- 0.08) after the dose of racemate. Unreliable estimates of fractional inversion were obtained when the S-ibuprofen and racemic ibuprofen doses were combined. Metabolite formation clearances suggested that S-ibuprofen is preferred over R-ibuprofen in the formation of hydroxyibuprofen, carboxyibuprofen and ibuprofen glucuronide. Product stereoselectivity in the formation of the four diastereomers of carboxyibuprofen was modest in favor of SS- and RR-carboxyibuprofen for S- and R-ibuprofen, respectively.

A stereospecific reversed-phase high-performance liquid chromatographic (HPLC) method has been developed to simultaneously quantitate the stereoisomers of the two major metabolites of ibuprofen: hydroxyibuprofen and carboxyibuprofen. The metabolites were derivatized with S-(alpha)-methylbenzylamine to form diastereomeric amides which were separated and quantified on a C8 column. The validity of the stereoselective assay was confirmed by comparison with a non-stereoselective HPLC method. The stereoselective assay was applied to the quantification of all the stereoisomeric ibuprofen metabolites in urine from human volunteers dosed with racemic ibuprofen or the individual enantiomers of ibuprofen. Significant substrate and product stereo-selectivities were observed in the formation of carboxyibuprofen.