Quantification of fenoprofen in human plasma using UHPLC-tandem mass spectrometry for pharmacokinetic study in healthy subjects.

A rapid, simple and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed to quantify fenoprofen, a nonsteroidal anti-inflammatory drug in human plasma for a pharmacokinetic study in healthy subjects. Owing to high levels of protein binding, protein precipitation followed by solid-phase extraction was employed for the extraction of fenoprofen and fenoprofen-d3 (used as internal standard) from 200 µL human plasma. Separation was performed on a BEH C18 (50 × 2.1 mm, 1.7 µm) column using methanol-0.2% acetic acid in water (75:25, v/v) under isocratic elution. Electrospray ionization was operated in the negative mode for sample ionization. Ion transitions used for quantification in the selected reaction monitoring mode were m/z 241/197 and m/z 244/200 for fenoprofen and fenoprofen-d3, respectively. Under the optimized conditions, fenoprofen showed excellent linearity in the concentration range 0.02-20 µg/mL (r2 ≥ 0.9996), adequate sensitivity, favorable accuracy (96.4-103.7%) and precision (percentage coefficient of variation ≤4.3) with negligible matrix effect. The validated method was successfully applied to a pharmacokinetic study of fenoprofen in healthy subjects. The significant features of the method include higher sensitivity, small plasma volume for processing and a short analysis time.

Albumin Supplement Affects the Metabolism and Metabolism-Related Drug-Drug Interaction of Fenoprofen Enantiomers.

The influence of albumin towards the metabolism behavior of fenoprofen enantiomers and relevant drug-drug interaction was investigated in the present study. The metabolic behavior of fenoprofen enantiomers was compared in a phase II metabolic incubation system with and without bovine serum albumin (BSA). BSA supplement increased the binding affinity parameter (Km) of (R)-fenoprofen towards human liver microsomes (HLMs) from 148.3 to 214.4 µM. In contrast, BSA supplement decreased the Km of (S)-fenoprofen towards HLMs from 218.2 to 123.5 µM. For maximum reaction velocity (Vmax), the addition of BSA increased the Vmax of (R)-fenoprofen from 1.3 to 1.6 nmol/min/mg protein. In the contrast, BSA supplement decreased the Vmax value from 3.3 to 1.5 nmol/min/mg protein. Andrographolide-fenoprofen interaction was used as an example to investigate the influence of BSA supplement towards fenoprofen-relevant drug-drug interaction. The addition of 0.2% BSA in the incubation system significantly decreased the inhibition potential of andrographolide towards (R)-fenoprofen metabolism (P < 0.001). Different from (R)-fenoprofen, the addition of BSA significantly increased the inhibition potential of andrographolide towards the metabolism of (S)-fenoprofen. BSA supplement also changed the inhibition kinetic type and parameter of andrographolide towards the metabolism of (S)-fenoprofen. In conclusion, albumin supplement changes the metabolic behavior of fenoprofen enantiomers and the fenoprofen-andrographolide interaction.

Immediate release three-layered chewing gum tablets of fenoprofen calcium: preparation, optimization and bioavailability studies in healthy human volunteers.

A three-layered gum tablet (1800 mg), containing 200 mg of Fenoprofen Calcium (Fn) with an inner core containing the drug, and two external layers containing antiadherent lubricant, has been prepared using direct compression. A 2(3) factorial plan has been designed to evaluate the effect of formulation variables namely, Pharmagum(®) M concentration, Maltodextrin type, and Co-adjuvant type on the release characteristics of Fn from the prepared tablets. The formula consisting of 65% Pharmagum(®) M, 75% Maltodextrin DE 39 and 5% Talc comparatively exhibited the highest release (66.59% ± 2.39) in the mouth after 5 min of chewing. Binary and ternary ß-cyclodextrin (ß-CD) complexation was adopted to enhance the release of Fn from the selected gum tablet. The highest significant release (p < 0.05) was achieved from the lyophilized ternary complex containing 100 mg of Fn in presence of polyvinylpyrrolidone (PVP K(25)), exhibiting a release of 88.25% ± 0.93 after 5 min of chewing. The relative bioavailability of the selected gum tablet was found to be 166.06% compared to Nalfon(®) 200 mg capsules. Reduction of the dose to 100 mg exhibited faster absorption rate than Nalfon(®) capsules. The obtained results suggest the possibility of reducing the dose of Fn in chewing gum.

Stereoselective disposition of fenoprofen in plasma and synovial fluid of patients with rheumatoid arthritis.

The simultaneous disposition of fenoprofen enantiomers in synovial fluid and plasma was studied in 11 patients with arthritis and chronic knee effusions treated with a single oral dose of 600 mg rac-fenoprofen. A plasma sample and a synovial fluid sample were collected simultaneously from each patient up to 16 h after the administration of fenoprofen. A stereospecific assay for fenoprofen using LC-MS-MS was developed and applied successfully to the analysis of the enantiomers in plasma (LOQ = 10 ng of each enantiomer/ml) and synovial fluid (LOQ = 25 ng of each enantiomer/ml). The values of the area under the curve (AUC) for the S-(+)-fenoprofen eutomer were approximately 2.5 times higher in plasma than in synovial fluid (256 vs 104 microg h/ml), while the values for the R-(-)-fenoprofen distomer were about four times higher in plasma than in synovial fluid (42.5 vs 10.5 microg h/ml). These data demonstrate accumulation of the S-(+)-fenoprofen eutomer in plasma and in synovial fluid, with concentrations versus time AUC (+)/(-) ratios of 6.0 in plasma and 9.9 in synovial fluid, suggesting a greater accumulation of the eutomer at the active site represented by synovial fluid than in plasma. This result demonstrates the importance of enantioselective methods and of analysis of synovial fluid rather than plasma in studies of the pharmacokinetics-pharmacodynamics of fenoprofen.

Pharmacodynamics, chiral pharmacokinetics, and pharmacokinetic-pharmacodynamic modeling of fenoprofen in patients with diabetes mellitus.

The objective of the present study was to assess the influence of type 1 and type 2 diabetes mellitus on the enantioselective pharmacodynamics and pharmacokinetics of fenoprofen. Patients with diabetes mellitus type 1 (n = 7) or type 2 (n = 7) and healthy volunteers (n = 13) received orally a single 600-mg dose of racemic fenoprofen. Monocompartmental analysis of (+)-(S)-fenoprofen showed a significant difference (P < .05, Kruskal-Wallis test) in area under the curve (AUC) values (153.68 vs 243.50 microg x h/mL) and oral clearance (1.95 vs 1.23 L/h) only between patients with diabetes mellitus type 2 and healthy volunteers. The inhibitory activity of cyclooxygenases was evaluated indirectly by the determination of prostaglandin E2 (COX-2) and thromboxane B2 (COX-1) using the sigmoidal inhibitory Emax model. The patients with type 2 diabetes mellitus presented lower IC50 (3.29 vs 6.0 microg/mL) and g (0.73 vs 2.01) values for COX-1 activity compared to healthy volunteers (P < .05, Kruskal-Wallis test). These results show that diabetes mellitus type 2, but not type 1, influences the pharmacokinetics and pharmacodynamics of (+)-(S)-fenoprofen.

Influence of rheumatoid arthritis in the enantioselective disposition of fenoprofen.

To investigate the influence of rheumatoid arthritis on the stereoselective disposition of fenoprofen administered as a racemic mixture, eight patients with rheumatoid arthritis receiving calcium rac-fenoprofen (200 mg/8 h) and 7 healthy volunteers given single oral dose (600 mg) were investigated. Serial blood samples and urine were collected from zero to 24 h after fenoprofen (FEN) administration. The following differences were observed between the (+)-(S) and (-)-(R)-FEN in the patients with rheumatoid arthritis (means 95% CI, Wilcoxon test, P < 0.05): C(max) 14.1 (12.5-15.8) versus 3.6 (2.5-4.7) microg/ml; AUC(ss) (0-8) 80.5 (67.3-93.7) versus 12.1 (8.8-15.4) microg.h/ml; Cl(T)/f 1.3 (1.0-1.5) versus 9.1 (6.5-11.8) l/h; and t(1/2) 3.1 (2.3-3.9) versus 1.2 (0.8-1.6) h. The Cl(T)/f of (-)-(R)-FEN was reduced in patients with rheumatoid arthritis when compared to healthy volunteers: 9.1 (6.5-11.8) versus 17.4 (13.9-20.9) l/h; P < 0.05 Mann-Whitney test. The administration of rac-FEN as a single dose to healthy volunteers or multiple doses to patients with rheumatoid arthritis resulted in lower Cl(T)/f for the (+)-(S)-FEN. The lower Cl(T)/f of (-)-(R)-FEN observed for patients with rheumatoid arthritis is consistent with lower clearance by inversion, although other metabolic pathways, drug interactions, and bioavailability of the individual enantiomers may also contribute to the difference.

Enantioselective kinetic disposition of fenoprofen in rats with experimental diabetes or adjuvant-induced arthritis.

This study was aimed to investigate the influence of diabetes or arthritis on the enantioselective metabolism and kinetic disposition of fenoprofen in rats with streptozotocin-induced diabetes or Mycobacteriumtuberculosis adjuvant-induced arthritis. Animals received i.v. 10 mg/kg racemic fenoprofen and blood samples were collected up to 24 h thereafter, with 5 animals studied at each time point. Plasma concentrations of the fenoprofen enantiomers were determined by HPLC. Diabetic and arthritic animals showed significant differences when compared with respective controls for the following pharmacokinetic variables of the (+)-(S)-fenoprofen eutomer: area under the plasma concentration time curve, total clearance and volume of distribution. The results indicate that experimental diabetes and adjuvant-induced arthritis influence the fenoprofen enantioselective metabolism.

Chiral inversion of (R)-(-)-fenoprofen in guinea-pigs pretreated with clofibrate.

The influence of clofibrate on the stereoconversion of fenoprofen (FPF) was studied in guinea pigs. This hypolipidaemic agent has been related to some biochemical changes in the liver leading to an increase in the chiral inversion process. Two groups of animals (n = 6 per group) were pretreated with oral doses of clofibrate (280 mg/kg per day) for three days and were then given (R)- or (S)-FPF (5 mg/kg, IV). The FPF enantiomers were extracted from the guinea-pigs' plasma using a solid phase procedure and analysed by HPLC with previous derivatization with L-leucinamide. Pretreatment with clofibrate increased the chiral inversion of (R)-FPF in favour of the pharmacologically active (S)-FPF enantiomer. Before this metabolic interaction can be applied to therapy with fenoprofen, the toxic effects of (S)-(+)-FPF on the gastrointestinal and renal tracts and the interference by (R)-(-)-FPF with the metabolism of lipids should be thoroughly evaluated.

Chiral inversion of R(-) fenoprofen and ketoprofen enantiomers in cats.

The chiral inversion process is a characteristic metabolic pathway for different aryl-2-propionic acids or profens. Important variations have been observed between these individual compounds as well as between animal species. In this study, R(-) fenoprofen [R(-)FPF] and R(-) ketoprofen [R(-) KTF] were used to investigate their comparative stereoconversion in cats. After intravenous (i.v.) administration of R(-) FPF, the percentage of chiral inversion was 93.20+/-13.70%. A highly significant correlation (r: 0.978) was observed between the clearance of R(-) FPF and the chiral inversion process. After i.v. administration of R(-) KTF, the percentage of inversion was only 36.73+/-2.8%. No correlation between the clearance of R(-) KTF and this process was observed. R(-) FPF was metabolized by the pathways of thioesterification - chiral inversion processes. For R(-) KTF, the competitive metabolic pathways, glucuronidation and hydroxylation may be involved. However, these metabolic steps are saturable or less functional in cats. Moreover, the thioesterification of R(-) KTF in in vitro studies has been shown to be important in carnivores. The lack of correlation between clearance and chiral inversion process of R(-) KTF may be finally explained by deviation of thioesterification to other metabolic pathways of lipids and/or aminoacid conjugation, particulary glicine derivatives.

In vitro study of fenoprofen chiral inversion in rat: comparison of brain versus liver.

The extent and the overall stereoselectivity of the combined steps involved in the chiral inversion of fenoprofen, a non-steroidal anti-inflammatory drug, was investigated in rat brain microsomes and cytosol. Results were compared with those obtained with the same liver subcellular compartments. Brain microsomes catalysed the stereoselective activation of the R(-)-enantiomer to its coenzyme A thioester with a specific activity approximately 10-fold less than that obtained with liver microsomes. Rat brain microsomes and cytosol mediated the racemization and hydrolysis of both R(-)- and S( + )-fenoprofenoyl-CoA. In brain fractions the epimerase activity was lower than in liver, whereas the hydrolysis process appeared more efficient. Thus, the data indicated that the three-step mechanism occurred in brain subcellular compartments leading to a minor chiral inversion of fenoprofen compared with that in liver.

Tissue extraction and high-performance liquid chromatographic determination of ketoprofen entantiomers.

Local transcutaneous delivery of non-steroidal anti-inflammatory drugs avoids gastrointestinal side effects and concentrates drugs in the intended tissues. An extraction and HPLC method was developed for ketoprofen in skin, fascia and muscle. Tissue samples were homogenized in NaHCO3. After methylene chloride removal of lipids, the aqueous layer was acidified with HCl and back extracted into isooctane/isopropanol. Ketoprofen was derivatized with ethylchloroformate/S-(-)-alpha-phenylethylamine in triethylamine, then detected by HPLC. Ketoprofen recovery was linear (1-33 microg/g) and was detected in these tissues following in vivo cathodic iontophoresis (160 mA*min). This represents the first non-radioactive method for determination of ketoprofen in tissues following transcutaneous iontophoresis.

Involvement of the rat gut epithelial and muscular layer, and microflora in chiral inversion and acyl-glucuronidation of R-fenoprofen.

In the presence of excised human and rat gut, the pharmacologically inactive R enantiomers of both ibuprofen and fenoprofen (FN) are bioinverted to their anti-inflammatory antipodes. In an attempt to further localize the site of inversion, we incubated R-FN, in oxygenated (O2:CO2, 95:5, v/v) Krebs-Henseleit solution (37 degrees C, pH 7.4) for 3 h in the presence of the intestinal contents, epithelium and muscular layer of upper jejunum and everted jejunum sack of antibiotic treated (500 mg/kg neomycin and erythromycin b.i.d. for 3 days) and control adult female Sprague-Dawley rats. The formation of S-FN and acylglucuronidated FN was examined in the incubation medium using a stereospecific HPLC assay. The metabolic activities are reported per g of wet tissue. The extent of inversion by the everted rat gut was substantial (30.7 +/- 5.1%) but no significant differences between the control and germ-eradicated rats was observed. The epithelial cells were found to be the major site of inversion in the intestinal wall (37.5 +/- 4.7%) with the muscular layer (7.8 +/- 2.1%) and intestinal contents (5.7 +/- 2.2%) contributing only to a small extent to the process. Both enantiomers were substantially acyl-glucuconjugated in the epithelial and muscular layers, and the intestinal content.

Serum protein binding of nonsteroidal antiinflammatory drugs: a comparative study.

The unbound fraction in serum, fu, is a critical parameter in describing and understanding the pharmacokinetics of NSAIDs. We compared fu for 6 different NSAIDs using ultrafiltration of pooled serum at pH 7.4 and 24C. Measurements covered a wide concentration range in order to define binding affinity and number of binding sites. HPLC was used to measure drug concentrations in serum and ultrafiltrate. Direct injection of ultrafiltrate and serum (diluted 250 x) permitted quantitation down to approximately 70 nM for most of the NSAIDs, i.e., approximately 15-20 ng/ml. Assuming binding only to albumin, the data were fitted to a model of two classes of binding sites with dissociation constants K1 and K2. The lowest K1 (highest affinity) was found with flurbiprofen, 0.0658 microM, the highest with ketoprofen, 5.23 microM, an 80-fold difference. At low drug concentrations, fu becomes virtually constant and approaches a lower limit, fumin. The following fumin values were calculated: diclofenac 0.21%; fenoprofen 0.25%, flurbiprofen 0.022%, ketoprofen 0.52%, naproxen 0.039%, and tolmetin 0.37%. Thus the least bound NSAID, ketoprofen, had a value 24-fold that of the most highly bound, flurbiprofen. The NSAIDs also differed widely with regard to the extent of variation in fu within the range of therapeutic concentrations, and hence with regard to their potential as displacers of other drugs.

Calculation of inversion half-lives of aryl propionic acid class nonsteroidal antiinflammatory drugs.

Although inversion of the R-enantiomers of the aryl propionic acid (APA) class of nonsteroidal antiinflammatory drugs (NSAIDs) in humans and other mammals has been known for more than 20 years, no satisfactory method has been developed for evaluating the half-life of the inversion process. This parameter is useful in assessing the pharmacodynamic contribution of the R-prodrug to the cyclooxygenase-inhibiting S-enantiomers. Further, it provides a similar means of evaluating the analgesic contributions of R-enantiomers to racemic mixtures. Using human and animal data, we have mathematically determined the inversion half-life (t1/2i) for ibuprofen, ketoprofen, and fenoprofen. The relation of the sensitivity of this value to the terminal half-life (t1/2) of the R-enantiomers of APA class drugs also is discussed.

Stereoselective reversible binding properties of the glucuronide conjugates of fenoprofen enantiomers to human serum albumin.

The stereoselective binding of fenoprofen enantiomers and fenoprofen glucuronide diastereomers to human serum albumin (HSA) was investigated using an ultrafiltration method. Fenoprofen glucuronides exhibit a considerable and stereoselective affinity to HSA, although less than seen for the parent drug. The (R)-glucuronide shows a higher affinity to HSA than the (S)-diastereomer. With the enantiomers, no significant difference could be detected. Diazepam and probenecid reduced the binding of the glucuronides, as well as that of the fenoprofen enantiomers. These results suggest that parent drug and its glucuronide metabolites occupy the same binding region on the albumin molecule. Both fenoprofen enantiomers, as well as racemic fenoprofen, are capable of reducing the extent of reversibly bound fenoprofen glucuronide.

Disposition of human drug preparations in the horse. IV. Orally administered fenoprofen.

Plasma and urinary concentrations of the non-steroidal anti-inflammatory drug fenoprofen were determined by a high-performance liquid chromatographic procedure following oral administration of a dose of 3 g to fed and fasted horses. In plasma, fenoprofen was present in detectable concentrations for 6-12 h. Free access to hay significantly reduced the peak plasma concentration and bioavailability of fenoprofen, and large interindividual differences in absorption and elimination pattern occurred. In fasted horses, fenoprofen was rapidly absorbed with a mean half-life of 0.10 h. Maximum concentrations were found 0.63 +/- 0.21 h after dosing. The elimination half-life was 0.9 h. As early as 1 h after dosage, fenoprofen could be detected in hydrolysed and unhydrolysed urine, and remained detectable up to 48 h. The maximum excretion rate and peak concentration occurred 2 h after administration, irrespective of the feeding schedule. In fed horses, a second maximum occurred after 9 h. The percentage of the dose excreted as unchanged fenoprofen in 12 h was 13.0 +/- 6.8%. A recovery of 21.9 +/- 7.4% and 42.2 +/- 7.0% of the dose was obtained after enzymatic and alkaline hydrolysis, respectively. At least three hydroxylated metabolites were detected in hydrolysed urine.

Comparative metabolism of R(-)-fenoprofen in rats and sheep.

The chiral inversion of 2-arylpropionic acids occurs in many species. It is a unique reaction specific to this group of drugs. In this study R-(-)-fenoprofen (R-(-)-FPF) was used as a model compound to investigate metabolic chiral inversion in sheep in vivo and in vitro to compare the data with the results obtained in rats. Metabolic inversion in sheep was 80%. The apparent mean values of Km and Vmax of thioester formation were: 392 microM and 2.08 nmol/min/mg in sheep and 500 microM and 22 nmol/min/mg in rats. For hydroxylation, the apparent mean values were Vmax: 0.02 nmol/min/mg in rats and 0.01 nmol/min/mg in sheep. There was no correlation between in vitro thioesterification and in vivo chiral inversion in sheep as compared to rats. In sheep most of the thioester formed underwent inversion (80%) while in rats, where in vitro thioesterification was greater, in vivo inversion was less (42%). In consequence, in rats other metabolic pathways for R(-)-FPF-CoA, such as incorporation into triacylglycerols and conjugation with amino acids, may be quantitatively more important.

Stereoselective degradation of the fenoprofen acyl glucuronide enantiomers and irreversible binding to plasma protein.

Stereoselective degradation of fenoprofen (FEN) glucuronides and irreversible binding of FEN enantiomers to human serum albumin via their glucuronides were studied. At different pH values, 37 degrees C, and in the absence of albumin, degradation half-lives were diastereomeric, resulting mainly from a combination of hydrolysis and acyl migration. Lower pH enhanced FEN glucuronide stability and reduced the extent of irreversible binding. The degradation rate of R-FEN glucuronide was greater than that of the S-glucuronide (S-FEN). When human serum albumin was added to the medium, stability was decreased as compared to protein-free buffer. FEN glucuronides were readily hydrolyzed to parent drug, indicating an esterase-like activity of the albumin molecule. In vitro irreversible binding was higher for R-FEN (1.22% +/- 0.36) than for S-FEN glucuronide (0.76% +/- 0.12), when a 0.1 mM concentration of each conjugate enantiomer was incubated under physiological conditions (pH 7.4, 37 degrees C). Incubation with unconjugated FEN did not lead to measurable irreversible binding. Analysis of plasma samples from a clinical study showed that enantioselective irreversible binding of FEN to plasma proteins also occurs in vivo. After administration of a single 600-mg dose of racemic FEN to six healthy volunteers, covalent binding of R- and S-FEN to plasma proteins was measured in all subjects. The percentage of S-FEN protein adduct was greater than that of its R-enantiomer adduct. Total amounts of FEN irreversibly bound to plasma protein in vivo were also very low (1.02 +/- 0.32 and 3.23 +/- 0.85 mol/mol protein x 10(-4) for R- and S-FEN, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro enantioselective glucuronidation of fenoprofen.

The diastereomeric glucuronic acid conjugates are major metabolites of the nonsteroidal anti-inflammatory drug fenoprofen (FEN). Glucuronidation of FEN enantiomers was investigated with liver microsomal preparations from different species (sheep, rabbit, rat and human). The formed R- and S-FEN conjugates can be separated and quantitated directly on a C18 reversed-phase HPLC column using a mixture of acetonitrile and tetrabutylammonium sulfate buffer, pH 2.5, as mobile phase. Applying this analytical procedure, it is possible to characterize enantioselective glucuronidation of FEN. For in vitro procedures, rates of glucuronide formation are substrate (FEN) and cosubstrate (UDP glucuronic acid, UDPGA) dependent with initial rates of glucuronide formation being higher for R- than for S-FEN. The R/S ratio of the formed products was independent of UDPGA (2.5-15 mmol/l) and substrate concentrations greater than or equal to 0.4 mmol/l. Enantioselective cleavage of the formed FEN conjugates by alkaline hydrolysis and hydrolytic enzymes (R greater than S-glucuronide) can be controlled during in vitro studies by pH adjustment and the addition of enzyme inhibitors.