Metabolic Chiral Inversion Research Articles

Development and application of an enantioselective liquid chromatography – tandem mass spectrometry method for the quantification of enantiomers to evaluate in vivo chiral inversion in beagle dog

To assess whether a suspected metabolic chiral inversion of a new chemical entity (NCE) with a single chiral centre occurred in an in vivo pharmacokinetic (PK) study of UCB-1, a quantitative enantioselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. Chromatographic screening of authentic standards of the two enantiomers was carried out using a range of Phenomenex Lux chiral columns under various isocratic conditions to achieve optimum chromatographic resolution. Protein precipitation was used for analyte extraction from 40 µL of dog plasma. The subjects were given a po dose of either 1, 3 or 10 mg/kg followed by blood sample collection at timepoints over a 72-hour period. For the analysis, a Shimadzu liquid chromatography system was coupled to a Sciex QTRAP 5500 mass spectrometer operated using multiple reaction monitoring (MRM) in positive ion mode. The regression for the two enantiomers was (R2 ≥ 0.999) over the range of 10 – 20,000 ng/mL while employing a quadratic fit, with a lower limit of quantification (LLOQ) of 10 ng/ml. The method was validated for intra- and inter-day precision and accuracy, which were within +/- 20%. The enantiomers were stable in dog plasma at room temperature for 4 h and after three freeze-thaw cycles. The method was successfully applied to a pharmacokinetic study in dog, after po administration of UCB-1 where a significant level of chiral inversion was observed in plasma.

Metabolic chiral inversion of 2-arylpropionic acid derivatives (profens)

2-arylpropionic acid derivatives (profens) are one of the most popular anti-inflammatory, analgesic, and antipyretic drugs. They belong to a group of nonsteroidal anti-inflammatory drugs (NSAID) and exhibit metabolic chiral inversion. Enantiomers of these chiral drugs are often characterised by different pharmacological activity. It is estimated that the values of metabolic chiral inversion of ( R )-ibuprofen in humans are between 35 and 70%, depending on the condition of the liver and the intake of other medicines, while ( R )-flurbiprofen undergoes chiral metabolic inversion to its opposed ( S ) form only in small range. The described phenomenon in the case of ( R )-ketoprofen is limited to a maximum of around 10%. The metabolic chiral inversion is associated with potentially important pharmacotherapeutic and toxicological consequences, and so an attempt was made to analyse this phenomenon for the most commonly used drugs from the profens group.

Stereoselective hepatic disposition of ibuprofen in the perfused liver of rat with adjuvant-induced arthritis

1. The effects of adjuvant-induced arthritis (AA) on the stereoselective hepatic disposition and chiral inversion of “profens” have scarcely been investigated. Ibuprofen (IB) undergoes unidirectional chiral inversion from R-IB to S-IB and is metabolized to IB-glucuronide (IB-Glu).2. We used an in situ perfused rat liver system to clarify the effects of inflammation on the metabolic activities and chiral inversion of IB without protein binding.3. After dosing of R-IB, AA had minimal effect on the elimination of R-IB from the perfusate. Larger amounts of S-IB-Glu than R-IB-Glu were observed in the bile at the dose of 2.4 and 4.8 μmol. However, after dosing of S-IB, the elimination of S-IB from the perfusate in AA rats was delayed, indicating a significant decrease in the hepatic clearance in AA rats. The cumulative biliary excretion of S-IB-Glu in AA rats was promoted after dosing with S-IB. There was little difference between the chiral inversion ratios of the control and AA rats.4. The present study demonstrated that AA results in the delayed elimination of S-IB, the active form, without changes to the chiral inversion ratio. Thus, further attention to the altered stereoselective pharmacokinetics of IB during inflammation is required.

Evaluation of the metabolic chiral inversion of d-selenomethionine in rats by stable isotope dilution gas chromatography–mass spectrometry

The stereoselective pharmacokinetics of selenomethionine enantiomers in rats has been studied to evaluate the chiral inversion of d-selenomethionine to the l-enantiomer. After bolus intravenous administration of d- or l-selenomethionine to rats, the plasma concentrations of these two enantiomers were determined by stereoselective gas chromatography–mass spectrometry with selected ion monitoring. This method involved derivatization of selenomethionine enantiomers with HCl in methanol to form methyl ester followed by N-acylation with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form the diastereomeric amide, and separation of the diastereomer on GC with an achiral column. Plasma concentrations of administered d- and l-selenomethionine declined with terminal half-lives of 96±17min and 91±6min, respectively. l-Selenomethionine appeared rapidly in plasma after administration of d-selenomethionine, whereas d-selenomethionine was not detected in plasma after administration of l-selenomethionine. The fraction of conversion of d-selenomethionine to l-selenomethionine was estimated to be 61.3±14.5%. The present method evaluates the stereoselective pharmacokinetics of selenomethionine enantiomers, including the estimation of the metabolic chiral inversion.

Hydrolysis of ibuprofenoyl-CoA and other 2-APA-CoA esters by human acyl-CoA thioesterases-1 and -2 and their possible role in the chiral inversion of profens

Ibuprofen and related 2-arylpropanoic acid (2-APA) drugs are often given as a racemic mixture and the R-enantiomers undergo activation in vivo by metabolic chiral inversion. The chiral inversion pathway consists of conversion of the drug to the coenzyme A ester (by an acyl-CoA synthetase) followed by chiral inversion by α-methylacyl-CoA racemase (AMACR; P504S). The enzymes responsible for hydrolysis of the product S-2-APA-CoA ester to the active S-2-APA drug have not been identified. In this study, conversion of a variety of 2-APA-CoA esters by human acyl-CoA thioesterase-1 and -2 (ACOT-1 and -2) was investigated. Human recombinant ACOT-1 and -2 (ACOT-1 and -2) were both able to efficiently hydrolyse a variety of 2-APA-CoA substrates. Studies with the model substrates R- and S-2-methylmyristoyl-CoA showed that both enzymes were able to efficiently hydrolyse both of the epimeric substrates with (2R)- and (2S)- methyl groups. ACOT-1 is located in the cytosol and is able to hydrolyse 2-APA-CoA esters exported from the mitochondria and peroxisomes for inhibition of cyclo-oxygenase-1 and -2 in the endoplasmic reticulum. It is a prime candidate to be the enzyme responsible for the pharmacological action of chiral inverted drugs. ACOT-2 activity may be important in 2-APA toxicity effects and for the regulation of mitochondrial free coenzyme A levels. These results support the idea that 2-APA drugs undergo chiral inversion via a common pathway.

Metabolic Chiral Inversion of Brivanib and Its Relevance to Safety and Pharmacology

Brivanib alaninate is an orally administered alanine prodrug of brivanib, a dual inhibitor of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. It is currently in clinical trials for the treatment of hepatocellular carcinoma and colorectal cancer. Brivanib has a single asymmetric center derived from a secondary alcohol. The potential for chiral inversion was investigated in incubations with liver subcellular fractions and in animals and humans after oral doses of brivanib alaninate. Incubations of [¹⁴C]brivanib alaninate with liver microsomes and cytosols from rats, monkeys, and humans followed by chiral chromatography resulted in two radioactive peaks, corresponding to brivanib and its enantiomer. The percentage of the enantiomeric metabolite relative to brivanib in microsomal and cytosolic incubations of different species in the presence of NADPH ranged from 11.6 to 15.8 and 0.8 to 3.1%, respectively. The proposed mechanism of inversion involves the oxidation of brivanib to a ketone metabolite, which is subsequently reduced to brivanib and its enantiomer. After oral doses of brivanib alaninate to rats and monkeys, the enantiomeric metabolite was a prominent drug-related component in plasma, with the percentages of area under the curve (AUC) at 94.7 and 39.7%, respectively, relative to brivanib. In humans, the enantiomeric metabolite was a minor circulating component, with the AUC <3% of brivanib. Pharmacological studies indicated that brivanib and its enantiomer had similar potency toward the inhibition of VEGF receptor-2 and FGF receptor-1 kinases. Because of low plasma concentration in humans, the enantiomeric metabolite was not expected to contribute significantly to target-related pharmacology of brivanib. Moreover, adequate exposure in the toxicology species suggested no specific safety concerns with respect to exposure to the enantiomeric metabolite.

The metabolic chiral inversion of 2-arylpropionic acids--a novel route with pharmacological consequences.

The metabolic chiral inversion of 2-arylpropionic acids--a novel route with pharmacological consequences.

Chiral inversion of 2-arylpropionyl-CoA esters by human α-methylacyl-CoA racemase 1A (P504S)—a potential mechanism for the anti-cancer effects of ibuprofen

Metabolic chiral inversion of 2-arylpropanoic acids (2-APAs;'profens'), such as ibuprofen, is important for pharmacological activity. Several 2-APA-CoA esters were good racemisation substrates for human AMACR 1A, suggesting a common chiral inversion pathway for all 2-APAs and an additional mechanism for their anti-cancer properties.

Pharmacokinetics and synovial fluid concentrations of flurbiprofen enantiomers in horses: chiral inversion

Flurbirpofen (FBP), a member of the 2-aryl propionate nonsteroidal anti-inflammatory drug class, has potent anti-inflammatory and analgesic properties. The commercial preparation is a racemic mixture of the R(-) and S(+) enantiomers of FBP. In this study, R(-) and S(+) FBP were used to investigate the metabolic chiral inversion. Each enantiomer was administered separately (0.25 mg/kg) and in a racemic mixture (0.5 mg/kg) intravenously to horses. Plasma and synovial concentration of each enantiomer was determined and the disposition of each was analyzed. After intravenous administration of R(-) FBP and S(+) FBP to horses no chiral inversion was detected. After the administration of the FBP racemate and individual enantiomers no differences were observed between pharmacokinetic parameters [t(1/2beta) (h), Cl (L/h.kg), AUC (microg.h/mL), Vss (L/kg) and MRT (h)] for R(-) and S(+) FBF. Synovial fluid concentrations of both FBP enantiomers were lower than plasma concentrations and no stereoselective differences were detected. These data indicate that the disposition of FBF in horses is not enantioselective and demonstrate a difference in the pharmacokinetic behavior of the enantiomers as compared with other 2-aryl-propionic acids, such as carprofen, ketoprofen and vedaprofen in the horse.

Enantioselective Pharmacokinetics of Ibuprofen and Involved Mechanisms

Although dexibuprofen (S-ibuprofen) was marketed in Austria and Switzerland, the racemate at various formulations is still extensively used worldwide, and there are no indications that the racemate will be replaced by the single enantiomer. Thus, elucidation of the characteristics and involved mechanisms of the chiral pharmacokinetics of racemic ibuprofen is of special importance for the understanding of the pharmacological and toxicological consequences, and for prediction of the clinically potential drug interactions and influence of the pathological states. Stereoselective pharmacokinetics and metabolism are common features for chiral nonsteroidal antiinflammatory drugs (NSAIDs) and especially for 2-arylpropionic acid derivatives characterized with a chiral center adjacent to the carboxyl group. Although the enantioselective pharmacokinetic characteristics of different NSAIDs should be treated case by case, they share similar mechanisms underlying the protein binding, metabolism and chiral inversion. Ibuprofen was the most extensively researched drug in terms of chiral characteristics and mechanisms. Therefore, elucidation of the mechanisms derived from research on ibuprofen may provide better understanding and prediction of other chiral drugs. This article attempts to elucidate the chiral pharmacokinetics and involved mechanisms of ibuprofen in comparison with other NSAIDs based on recent developments. Topics on history of ibuprofen, enantioselective analysis method, absorption, protein binding, conventional metabolism, metabolic chiral inversion, gene polymorphism, and biochemical developments were included. It is worth mentioning that some underlying biochemical mechanisms, especially for the metabolic chiral inversion and ethnic differences still remain to be seen. Further research is required to develop human-resourced researching model and to provide more evidence concerning the site of inversion, species variation, CYP450 gene polymorphisms, and biochemical mechanisms.

Enantioselective Pharmacokinetics of Ibuprofen and Involved Mechanisms

Although dexibuprofen (S-ibuprofen) was marketed in Austria and Switzerland, the racemate at various formulations is still extensively used worldwide, and there are no indications that the racemate will be replaced by the single enantiomer. Thus, elucidation of the characteristics and involved mechanisms of the chiral pharmacokinetics of racemic ibuprofen is of special importance for the understanding of the pharmacological and toxicological consequences, and for prediction of the clinically potential drug interactions and influence of the pathological states. Stereoselective pharmacokinetics and metabolism are common features for chiral nonsteroidal antiinflammatory drugs (NSAIDs) and especially for 2-arylpropionic acid derivatives characterized with a chiral center adjacent to the carboxyl group. Although the enantioselective pharmacokinetic characteristics of different NSAIDs should be treated case by case, they share similar mechanisms underlying the protein binding, metabolism and chiral inversion. Ibuprofen was the most extensively researched drug in terms of chiral characteristics and mechanisms. Therefore, elucidation of the mechanisms derived from research on ibuprofen may provide better understanding and prediction of other chiral drugs. This article attempts to elucidate the chiral pharmacokinetics and involved mechanisms of ibuprofen in comparison with other NSAIDs based on recent developments. Topics on history of ibuprofen, enantioselective analysis method, absorption, protein binding, conventional metabolism, metabolic chiral inversion, gene polymorphism, and biochemical developments were included. It is worth mentioning that some underlying biochemical mechanisms, especially for the metabolic chiral inversion and ethnic differences still remain to be seen. Further research is required to develop human-resourced researching model and to provide more evidence concerning the site of inversion, species variation, CYP450 gene polymorphisms, and biochemical mechanisms.

Chiral Inversion of Drugs: Coincidence or Principle?

2-arylpropionic acid derivatives are probably the most frequently cited drugs exhibiting the phenomenon that is best known as chiral inversion. One enantiomer of drug is converted into its antipode either in the presence of a solvent or more often in inner environment of an organism. Mechanistic studies of the metabolic chiral inversion were carried out for several drugs from NSAIDs, and a model of this inversion was suggested and subsequently confirmed. The chiral inversion of NSAIDs has been intensively studied in the context of the pharmacological and toxicological consequences. However, the group of NSAIDs is not the sole group of drugs in which the inversion phenomenon can be observed. There exist several other drugs that also display chiral inversion of one or even both of their enantiomers. These drugs belong to different pharmacotherapeutic groups as monoamine oxidase inhibitors, antiepileptic drugs, drugs used in the treatment of hyperlipoproteinemia or drugs that are effective in the treatment of leprosy. Moreover, some chiral or prochiral drugs are metabolized to give chiral metabolites that undergo chiral inversion too, which can have direct impact on pharmacological properties or toxicity of the drug. As the process of chiral inversion is affected by several factors, so the intensity of chiral inversion of individual substances and at different conditions can differ considerably. Interspecies differences and types of tissue are reported to be the main factors that were recognized to play the key role in the process of chiral inversion. Some of more recent studies have revealed that several other factors, such as the route of administration or interaction with other xenobiotics, can influence the enantiomeric conversion, too. Chiral inversion does not seem to be a phenomenon connected with only several drugs from some unique group of 2-arylpropionic acid derivatives: it is also observed in drugs with rather different chemical structures and is much more frequent than it can be realized.

Stereoselective disposition of tiaprofenic acid enantiomers in rats.

The pharmacokinetics and metabolic chiral inversion of the S(+)- and R(-)-enantiomers of tiaprofenic acid (S-TIA, R-TIA) were assessed in vivo in rats, and in addition the biochemistry of inversion was investigated in vitro in rat liver homogenates. Drug enantiomer concentrations in plasma were investigated following administration of S-TIA and R-TIA (i.p. 3 and 9 mg/kg) over 24 hr. Plasma concentrations of TIA enantiomers were determined by stereospecific HPLC analysis. After administration of R-TIA it was found that 1) there was a time delay of peak S-TIA plasma concentrations, 2) S-TIA concentrations exceeded R-TIA concentrations from approximately 2 hr after dosing, 3) Cmax and AUC(0-infinity) for S-TIA were greater than for R-TIA following administration of S-TIA, and 4) inversion was bidirectional but favored inversion of R-TIA to S-TIA. Bidirectional inversion was also observed when TIA enantiomers were incubated with liver homogenates up to 24 hr. However, the rate of inversion favored transformation of the R-enantiomer to the S-enantiomer. In conclusion, stereoselective pharmacokinetics of R- and S-TIA were observed in rats and bidirectional inversion in rat liver homogenates has been demonstrated for the first time. Chiral inversion of TIA may involve metabolic routes different from those associated with inversion of other 2-arylpropionic acids such as ibuprofen.

Stereoselective disposition of tiaprofenic acid enantiomers in rats

The pharmacokinetics and metabolic chiral inversion of the S(+)- and R(−)-enantiomers of tiaprofenic acid (S-TIA, R-TIA) were assessed in vivo in rats, and in addition the biochemistry of inversion was investigated in vitro in rat liver homogenates. Drug enantiomer concentrations in plasma were investigated following administration of S-TIA and R-TIA (i.p. 3 and 9 mg/kg) over 24 hr. Plasma concentrations of TIA enantiomers were determined by stereospecific HPLC analysis. After administration of R-TIA it was found that 1) there was a time delay of peak S-TIA plasma concentrations, 2) S-TIA concentrations exceeded R-TIA concentrations from ∼2 hr after dosing, 3) Cmax and AUC(0-∞) for S-TIA were greater than for R-TIA following administration of S-TIA, and 4) inversion was bidirectional but favored inversion of R-TIA to S-TIA. Bidirectional inversion was also observed when TIA enantiomers were incubated with liver homogenates up to 24 hr. However, the rate of inversion favored transformation of the R-enantiomer to the S-enantiomer. In conclusion, stereoselective pharmacokinetics of R- and S-TIA were observed in rats and bidirectional inversion in rat liver homogenates has been demonstrated for the first time. Chiral inversion of TIA may involve metabolic routes different from those associated with inversion of other 2-arylpropionic acids such as ibuprofen. Chirality 11:103–108, 1999. © 1999 Wiley-Liss, Inc.

Relationship between the metabolic chiral inversion and chemical structure of 4-phenyl-4-oxobutanoic acids, derivatives of anti-rheumatic agent KE-298.

The relationship between metabolic chiral inversion and chemical structure of various 4-phenyl-4-oxobutanoic acids (4-OBA), derivatives of anti-rheumatic agent KE-298 [2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid], was investigated in rats. Chiral inversion occurred with the thio-alkyl group, whereas the thio-acyl group played no role in the inversion of 4-OBA. A 2-methylene moiety was required for the inversion. When the 4-carbonyl moiety was removed, chiral inversion was significantly decreased, which provided an affinity for the intramitochondrial medium chain fatty acid CoA ligase. In addition, the distance between the chiral center and the carbonyl moiety was also an important factor for chiral inversion. While a sulfur atom was not indispensable for the chiral inversion, the existence of the sulfur atom influenced its affinity to the long chain fatty acid CoA ligase.

Stereoselective disposition and chiral inversion of KE-298, a new antirheumatic drug, in rats.

The present study was an attempt to elucidate the relationship between stereoselective pharmacokinetics and protein binding of KE-298 and its active metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2). Metabolic chiral inversion was also investigated. The levels of unchanged KE-298 in plasma after oral administration of (+)-(S)-KE-298 to rats were lower than those of (-)-(R)-KE-298, whereas the levels of M-1 and M-2 after administration of (+)-(S)-KE-298 were higher than after (-)-(R)-KE-298. In vitro, rat plasma protein binding of (+)-(S)-KE-298 was lower than that of (-)-(R)-KE-298. In contrast, the binding of (+)-(S)-M-1 and (+)-(S)-M-2 was higher than that of (-)-(R)-M-1 and (-)-(R)-M-2. Displacement studies revealed that the (+)-(S) and (-)-(R)- enantiomers of KE-298 and their metabolites bound to the warfarin binding site on rat serum albumin. These results suggested that the stereoselective plasma levels in KE-298 and its metabolites were closely related to enantiomeric differences in protein binding attributed to quantitative differences in binding to albumin rather than to the different binding sites. Unidirectional chiral inversion was detected after oral administration of either (-)-(R)-KE-298 or (-)-(R)-M-2 to rats both yielding (+)-(S)-M-2.

Bi-directional chiral inversion of ketoprofen in CD-1 mice.

The R enantiomers of some of the 2-arylpropionic acid non-steroidal antiinflammatory drugs (NSAIDs) are known to undergo metabolic chiral inversion to their more pharmacologically active antipodes. This process is drug and species dependent and usually unidirectional. The S to R chiral inversion, on the other hand, is rare and has been observed, in substantial extents, only for ibuprofen in guinea pigs and 2-phenylpropionic acid in dogs. After i.p. administration of single doses of racemic ketoprofen or its optically pure enantiomers to male CD-1 mice and subsequent study of the concentration time-course of the enantiomers, we noticed substantial chiral inversion in both directions. Following racemic doses, no stereoselectivity in the plasma-concentration time courses was observed. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated during the absorption phase. During the terminal elimination phase, however, the enantiomers had the same concentrations. Our observation is suggestive of a rapid and reversible chiral inversion for ketoprofen enantiomers in mice.

Mechanistic studies on metabolic chiral inversion of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of the new anti-rheumatic agent 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), in rats

The chiral inversion properties of 4-(4-methylphenyl)-2-methylthiomethyl-4-oxobutanoic acid (KE-748), an active metabolite of 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), were compared with those of ibuprofen in rats. After administration of R(−)-[2 α- 2 H]KE-748, S(+)-KE-748 was present in the rat plasma, and the deuterium atoms of the S(+)-enantiomer were almost all replaced by hydrogen atoms. After administration of S(+)-[2 α- 2 H]KE-748, the deuterium content of S(+)-KE-748 in the plasma remained intact. In the in vitro study, using a cell-free system and rat liver homogenates, the chiral inversion of ibuprofen was apparent when both CoA and ATP were present; however, KE-748 was not inverted. In the study on isolated rat hepatocytes, the unidirectional chiral inversion from R(−)-to S(+)-enantiomet was observed for both ibuprofen and KE-748. When R(−)-ibuprofen was incubated with medium and long chain fatty acids (carbon chain length C 6 to C 16), using isolated hepatocytes, the chiral inversion decreased significantly. On the other hand, when R(−)-KE-748 was incubated with short and medium chain fatty acids (carbon chain length C 3 to C 8), chiral inversion was inhibited markedly. To induce hepatic microsomal long chain fatty acid CoA ligase, rats were treated with clofibric acid (CF rats). In both in vitro and in vivo experiments on CF rats, chiral inversion from R(−)-to S(+)-ibuprofen was enhanced significantly compared with that in controls, whereas the enhancement was not observed in the case of R(−)-KE-748. There was no influence of benzoic acid, a typical substrate on medium chain fatty acid CoA ligase in the mitochondrial matrix, on chiral inversion of R(−)-ibuprofen, using isolated hepatocytes. In contrast, the chiral inversion from R(−)- to S(+)-KE-748 was strongly inhibited in the presence of benzoic acid. These results indicate that chiral inversion of R(−)-KE-748 may proceed via formation of the CoA-thioester intermediate with loss of the 2α-methine proton, in a manner similar to that seem with R(−)-ibuprofen. However, the enzymes needed to form CoA-thioester of R(−)-KE-748 differ from those for R(−)-ibuprofen.

2-arylpropionyl-CoA epimerase: Partial peptide sequences and tissue localization

The R-enantiomers of 2-arylpropionic acids (2-APAs) such as ibuprofen (IBU) exhibit the phenomenon of species- and substrate-dependent metabolic chiral inversion. Only R-enantiomers are activated to acyl-CoA-thioesters by an acyl-CoA-synthetase via an adenylate intermediate. The acyl-CoA-thioesters are substrates for an epimerase, which is responsible for chiral inversion. A 42 kDa epimerase from the cytosolic fraction of rat livers was isolated and purified to homogeneity. Polyclonal antibodies were raised against the epimerase in rabbits. The anti-epimerase antibodies were used for affinity column chromatography to separate homogeneous protein for amino acid sequence analysis. Sequence data analysis of 3 internal peptide sequences showed 50% and more homology with regions of enzymes involved in fatty acid metabolism. The polyclonal anti-epimerase antibodies were used to analyze the tissue distribution of the protein in guinea pigs and rats by Western blot analysis. Furthermore, the correlation of inversion enzyme activity in various tissues under comparable incubation conditions and cross-reactivity in Western blot analysis was investigated.

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.