S. Menzel

Is the formation of R-ibuprofenyl-adenylate the first stereoselective step of chiral inversion?

Coenzyme A thioester formation is reported to be the first step of chiral inversion of R-ibuprofen. In order to investigate the mechanism of this reaction adenylate derivatives of the ibuprofen enantiomers were synthesized chemically. R- and S-ibuprofenyl-adenylates as well as free acids were incubated with rat liver mitochondria in the presence of coenzyme A, MgCl2 with or without ATP. The optical antipodes formed by inversion and the coenzyme A thioester derivatives of both enantiomers were found after incubation of both R- or S-ibuprofenyl-adenylate and R-ibuprofen. By contrast, after incubation with S-ibuprofen neither R-enantiomer nor coenzyme A thioesters were detected. These experiments suggest that the formation of R-ibuprofenyl-adenylate may be the first stereoselective step of chiral inversion.

Antinociceptive actions of R(−)-flurbiprofen - a non-cyclooxygenase inhibiting 2-arylpropionic acid - in rats

Intraperitoneal administration of R(-)- and S(+)-flurbiprofen resulted in dose dependent antinociceptive behavior in the rat paw formalin test. S(+)-flurbiprofen was significantly more potent than the non-cyclooxygenase inhibiting R(-)-enantiomer with a potency ratio of about 3 to 1. Chiral inversion was very low and does not seem to account for the action of R(-)-flurbiprofen. In a modified Randall Selitto assay also both enantiomers were active in a dose dependent manner following systemic administration. Following local administration into the inflamed paw only S(+)-flurbiprofen showed significant dose related antinociceptive effects. R(-)-flurbiprofen was unable to block prostaglandin E2 induced hyperalgesia following local administration. Consequently, a central site of action independent of prostaglandin synthesis inhibition has to be discussed with respect to antinociceptive activity following systemic administration.

Isolation and characterization of rat liver microsomal R-ibuprofenoyl-CoA synthetase

Microsomal long-chain acyl-CoA synthetase (EC 6.1.2.3.) has been suggested to be involved in the stereoselective formation of the CoA thioester of ibuprofen. In this study, we demonstrated that the microsomal enzyme from rat liver responsible for palmitoyl-CoA synthesis also catalyzes the formation of R-ibuprofenoyl-CoA in a Mg(2+)- and ATP-dependent process. Long-chain acyl-CoA synthetase from rat liver microsomes was purified to homogeneity as evidenced by SDS-gel electrophoresis. Simultaneous measurements of palmitoyl-CoA and R-ibuprofenoyl-CoA formation with HPLC in various fractions and purification steps during protein isolation revealed a high correlation between both activities. The purification procedure included solubilization of the microsomes obtained from rat livers with Triton X-100 and subsequent chromatography of the 100,000 x g supernatant on blue-sepharose, hydroxyapatite, and phosphocellulose. The purified enzyme exhibited an apparent molecular weight of 72 kDa as estimated by SDS gel electrophoresis, with specific activities of 71 nmol.min-1.mg-1 protein and 901 nmol.min-1.mg-1 protein for formation of R-ibuprofenoyl-CoA and palmitoyl-CoA, respectively. Palmitoyl-CoA formation catalyzed by the purified enzyme exhibited biphasic kinetics indicative of two isoforms, a high-affinity (KM 0.13 +/- 0.11 microM), low-capacity form and a low-affinity (KM 81 +/- 11.5 microM), high-capacity form. In contrast, measurement of R-ibuprofenoyl-CoA synthesis over a concentration range from 5 to 3000 microM showed the participation of a single CoA ligase with a KM of 184 +/- 19 microM, corresponding to the low-affinity isoform of palmitoyl-CoA synthesis with a marked enantioselectivity towards the R-form of ibuprofen. R-ibuprofenoyl-CoA formation of the enzyme preparation was inhibited by palmitic acid (KI 13.5 +/- 0.5 microM) and S-ibuprofen (KI 405 +/- 10 microM). In summary, these data give strong evidence for the identity of R-ibuprofenoyl-CoA and long-chain acyl-CoA synthetase.

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 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.

Is the inversion from R- to S-ketoprofen concentration dependent? Investigations in rats in vivo and in vitro.

The effects of dose on the pharmacokinetics of ketoprofen (KT) enantiomers were investigated in rats in vivo and in hepatoma cells in continuous culture in vitro following administration of the optically pure enantiomers and the racemate of KT. With the exception of AUC (area under the curve) no pharmacokinetic differences could be found following i.v. administration of various doses of KT enantiomers (2.5, 5 and 10 mg/kg) and of racemic KT (5, 10 and 20 mg/kg) and between single enantiomer and racemate administration in rats in vivo. Independent of the dose administered the fraction inverted was about 66%. In line with the findings in vivo good correlation between incubation concentration and AUC of R- and S-KT was found in the hepatoma cells in vitro. The ratios of AUC(S)/AUC(R) were not significantly affected by concentration after R-KT (2.5-20 micrograms/mL) and racemate incubation (5-40 micrograms/mL) in the concentration ranges investigated. However, unlike in rats in vivo enhanced inversion was observed following racemate as compared to single enantiomer incubation in vitro.

Stereospecific determination of tiaprofenic acid in plasma : problems with drug degradation

A sensitive stereospecific high-performance liquid chromatographic assay for the quantification of tiaprofenic acid in human plasma was developed. The procedure involved extraction of tiaprofenic acid from acidified plasma into hexanediethyl ether (8:2, v/v). Stereospecific separation was achieved with a prepacked alpha1-acid glycoprotein column without derivatization. The mobile phase consisted of 2% 2-propanol in 0.01 M phosphate buffer, pH 6.5. Tiaprofenic acid was detected at 317 nm. The limit of quantification was found to be 25 ng/ml for each enantiomer using a 0.5 ml plasma sample. The assay was reproducible and accurate to be applied to the stereoselective pharmacokinetic analysis of tiaprofenic acid in plasma. Because of photoinstability of tiaprofenic acid plasma sampling and sample extraction should be performed under light protection.