Direct evidence of nonadherence to antiepileptic medication in refractory focal epilepsy.

The adherence to medication in drug-resistant focal epilepsy (RFE) remains largely unknown. The present work aimed to assess the frequency of recent adherence to antiepileptic drugs (AEDs) in patients with RFE. This prospective observational study screened all patients with RFE, admitted to the Nancy University Hospital between April 2006 and September 2008, for a 5-day hospitalization without AED tapering. The adherence to AEDs was assessed by measuring serum drug levels on day 1 (reflecting the recent "at home" adherence) and day 5 (reflecting the individual reference concentration when drug ingestion was supervised). A patient was considered nonadherent if at least one of their serum drug levels was different between days 1 and 5. The day-1 value was considered different from day 5 when it was at least 30% lower (underdosed) or 30% higher (overdosed). Nonadherent patients were classified as under-consumers in the case of one or more underdosed day-1 values, over-consumers in the case of one or more overdosed day-1 values, or undefined if they exhibited both underdosed and overdosed day-1 values. Forty-four of the 48 screened patients were included. Eighteen (40.9%) of 44 patients were nonadherent. Among them, 12 (66.7%) were over-consumers, 4 (22.2%) were under-consumers, and 2 (11.1%) were undefined nonadherents. The study indicates that recent adherence to antiepileptic medication in this group of patients with RFE is poor. Overconsumption is the most frequent form of nonadherence in this population and should be specifically assessed to prevent its possible consequences in terms of AEDs dose-dependent adverse events.

Interaction of the electrophilic ketoprofenyl-glucuronide and ketoprofenyl-coenzyme A conjugates with cytosolic glutathione S-transferases.

Carboxylic acid-containing drugs are metabolized mainly through the formation of glucuronide and coenzyme A esters. These conjugates have been suspected to be responsible for the toxicity of several nonsteroidal anti-inflammatory drugs because of the reactivity of the electrophilic ester bond. In the present study we investigated the reactivity of ketoprofenyl-acylglucuronide (KPF-OG) and ketoprofenyl-acyl-coenzyme A (KPF-SCoA) toward cytosolic rat liver glutathione S-transferases (GST). We observed that KPF-SCoA, but not KPF-OG inhibited the conjugation of 1-chloro-2,4-dinitrobenzene and 4-nitroquinoline N-oxide catalyzed by both purified cytosolic rat liver GST and GST from FAO and H5-6 rat hepatoma cell lines. Photoaffinity labeling with KPF-SCoA suggested that the binding of this metabolite may overlap the binding site of 4-methylumbelliferone sulfate. Furthermore, high-performance liquid chromatography and mass spectrometry analysis showed that both hydrolysis and transacylation reactions were observed in the presence of GST and glutathione. The formation of ketoprofenyl-S-acyl-glutathione could be kinetically characterized (apparent K(m) = 196.0 +/- 70.6 microM). It is concluded that KPF-SCoA is both a GST inhibitor and a substrate of a GST-dependent transacylation reaction. The reactivity and inhibitory potency of thioester CoA derivatives toward GST may have potential implications on the reported in vivo toxicity of some carboxylic acid-containing drugs.

Quantitative dynamic contrast enhanced MRI of experimental synovitis in the rabbit knee: comparison of macromolecular blood pool agents vs. Gadolinium-DOTA.

The purpose of this study was to assess 2 Gd-based macromolecular intravascular contrast agents (P792, rapid clearance blood pool agent (rBPA) and P717, slow clearance blood pool agent (sBPA)) compared to Gd-DOTA (representative extracellular non specific agent) in MR imaging of knee rabbit experimental synovitis. Quantitative dynamic contrast enhanced MRI (qDCE-MRI) after intravascular injection of a low molecular weight contrast agent of 0.56 kDa (Gd-DOTA) and 2 high-molecular-weight contrast agents of 6.47 kDa (P792) and 52 kDa (P717) was performed in rabbits with carrageenan-induced synovitis of the right knee. P792 and P717 provided a progressive and persistent enhancement of arthritic synovial tissue while Gd-DOTA provided an early and rapidly declining enhancement with a concomitant diffusion in synovial fluid, thus limitating delineation of synovial pannus. P792 allowed acquisition of high-quality MR arthrograms, due to both a better diffusion in synovial pannus (vs. P717) and a concomitant restricted diffusion into the synovial fluid (vs. Gd-DOTA). In fact, experimental rabbit synovitis represent a specific entity that favors the T1 effect of high-molecular-weight agents, and especially rBPA P792, entrapped in synovial pannus, without diffusion in the synovial fluid. Due to this lack of arthrographic effect, P792 accumulation could be specifically sequentially analyzed by qDCE-MRI for detecting, characterizing and monitoring synovial vascular permeability changes during mono- or polysynovitis.

Irreversible inhibition of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate of ketoprofen: a key to oxidative stress induced by non-steroidal anti-inflammatory drugs?

Oxidative damage by non-steroidal anti-inflammatory drugs (NSAIDs) has been considered relevant to the occurrence of gastro-intestinal side-effects. In the case of chiral arylpropionate derivatives like ketoprofen (KPF), this mechanism has been evidenced for the R-enantiomer, especially when chiral inversion was observed, and lets us suppose the involvement of CoA conjugates. Glucose-6-phosphate dehydrogenase (G6PD) is the crucial enzyme to regenerate the GSH pool and maintain the intracellular redox potential. This enzyme is known to be down-regulated by palmitoyl-CoA thioester. We hypothesised then that G6PD is the target of carboxylic NSAIDs, via their CoA metabolites. We used molecular docking to localise a putative site in the human G6PD then we chose the Yeast orthologue, as the most suitable species to study experimentally the precise molecular interaction. KPF-CoA was effectively shown to bind covalently to the unique cysteine residue of the yeast enzyme. Binding was found to occur in the same site as palmitoyl-CoA. It was decreased in the presence of an allosteric inhibitor of G6PD, phospho(enol)pyruvate, and was not detected with G6PD of Leuconostoc mesenteroides, which does not possess the allosteric site. This site is distinct from the catalytic site, and probably allosteric, explaining the observed non-competitive inhibition of its activity by KPF-CoA. KPF-CoA was shown to induce the production of reactive oxygen species in Caco-2 cells, where its inhibition of G6PD activity was observed.

Purification and cloning of two high molecular mass isoforms of peanut seed oleosin encoded by cDNAs of equal sizes.

Oleosins are small plant proteins characterized by a long hydrophobic core flanked by amphipathic N- and C-terminal domains, which act as emulsifiers for the storage of lipids in seeds. They have been sequenced in a number of oilseeds important for the food industry but not in peanuts. We purified the major isoform of peanut oleosin by preparative electrophoresis with continuous elution, in sufficient amounts to raise specific antibodies, perform circular dichroism and N-sequence tryptic fragments. The structure of the purified oleosin was dominated by alpha-helix that may be assigned to the SDS-resistant central hydrophobic stretch. A two-step RT-PCR strategy was developed to determine the cDNA sequence of this oleosin. Two cDNA variants of equal sizes encoding for isoforms of 176 amino acids each were identified. The isoforms differed by seven amino acids mainly located in the N- and C-terminal domains. The corresponding mRNAs were estimated at 0.9 kb by Northern blot and were transcribed from genes without introns. Immunoprecipitation of the in vitro-translated peanut oleosin labeled with [14C]leucine or [35S]methionine produced the full-length protein (17 kDa) and a 6-kDa peptide corresponding to the N/C-terminal domains. This peptide was able to form SDS-PAGE stable oligomers by interacting with the full-length protein. A similar peptide was released after [125I]iodination of the purified oleosin that generated intermediate-sized oligomers also visible by Western blot on a crude oleosin extract. Oligomers reflect the natural ability of oleosins to strongly interact with each other via not only their central domains but also their N- and C-terminal domains.

Elucidation of the mechanism of inhibition of cyclooxygenases by acyl-coenzyme A and acylglucuronic conjugates of ketoprofen.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isoforms which accounts for their clinical effects. The differential inhibition of COX-1 and COX-2 is not sufficient to explain the absence of a correlation between in vitro and in vivo effects, especially for 2-aryl-propionates, thus indicating the participation of metabolites. Conjugates to glucuronic acid and to coenzyme-A are mainly produced, and have been shown to be chemically reactive. Therefore, we studied the interaction of the ketoprofen metabolites with the COX enzymes. After incubation with bovine pulmonary artery endothelial cells (BPAEC), COX-1 was inhibited stereoselectively by S-ketoprofen acylglucuronide, and more significantly by CoA-thioester. After washing-out the medium, COX-1 activity was essentially recovered, indicating a reversible inhibition. In LPS-stimulated J774.2 cells, COX activity (mainly inducible COX-2) was inhibited reversibly and stereospecifically by S-ketoprofen glucuronide, whereas it disappeared totally and was not recovered after incubation with CoA-thioester. Correspondingly, inhibition of purified COX-2 with this compound was observed to be rapid and irreversible. Using an anti-ketoprofen antibody, COX immunoprecipitated from cells exhibited adduct formation for COX-2 but not for COX-1. This was observed after incubation with CoA-thioester, and, surprisingly, also with glucuronide. Molecular docking gave support to explain this discrepancy: the glucuronide was found to establish a strong interaction with Y115 located in the membrane binding domain, whereas the thioester was preferentially bound to the active site of the enzyme. Overall, our results suggest a contribution of CoA-thioester metabolites of carboxylic NSAIDs to their pharmacological action by irreversibly and selectively inhibiting COX-2.

In vitro stereoselective covalent binding of carprofen glucuronides to human serum albumin: characterization of the mechanism.

The reactivity, in terms of covalent binding, of R- and S-carprofen acylglucuronides with human serum albumin (HSA) has been investigated in vitro. The irreversible binding of these metabolites to the HSA 580 mM occurred at pH 7.4 and 37 degrees C instantaneously and stereoselectively in favour of the R-enentiomer glucuronide. The amount of carprofen adducts remained stable with time up to 48 hr, and increased with the glucuronide concentration. It was not modified by addiction of imine-trapping reagents, suggesting that the reaction is not mediated by a Schiff base mechanism. Moreover the extreme rapidity of the covalent binding supports a mechanism of nucleophilic attack. Competition studies with ligands known to bind to different sites of HSA, indicated that carprofen glucuronides interacted mainly with site II. The extent of the binding of R-carprofen glucuronide increased with pH, thus suggesting the participation of an alkaline group in the process. The modification of HSA by amino-acid directed chemicals led to the conclusion that Tyr, Lys or Arg residues in site II were mainly involved.

Multicompartmental analysis of late contrast enhancement in areas of myocardial infarction supplied by chronically occluded coronary arteries.

PURPOSE: To analyze the relationship between late contrast enhancement (LCE) and the interstitial distribution volume (V(In)) of gadolinium (Gd) tracers in the myocardial infarction (MI) areas supplied by chronically occluded arteries from patients. In animal experimental models, LCE has already been shown to correspond to an enhanced V(In) of Gd tracers and thus, to a decrease in the amount of intact cells. MATERIALS AND METHODS: A multicompartmental analysis was applied to serial MRI images encompassing both infarct and remote areas and recorded with a conventional two-dimensional (2D) segmented inversion-recovery gradient-echo (IR-GRE) sequence during a 15-minute period following Gd-diethylenetriamine pentaacetic acid (Gd-DTPA) injection in 12 patients with Q-wave MI supplied by chronically occluded coronary arteries. RESULTS: V(In) from infarct tissue was: 1) higher than V(In) from remote areas (in % of myocardial volume: 74 +/- 16% vs. 20 +/- 7%, P < 0.001); and 2) correlated with the quantification of LCE between infarct and noninfarct areas at the 15th minute (R(2) = 0.63, P = 0.002). However, the difference in V(In) between infarct and remote myocardium was a much better correlate of this quantified LCE (R(2) = 0.85, P < 0.001). CONCLUSION: Detection of LCE in the MI territories supplied by chronically occluded arteries relates to the difference in the V(In) of tracers between the infarct and the noninfarct areas.

Synthesis and biological testing of Acyl-CoA-ketoprofen conjugates as selective irreversible inhibitors of COX-2.

Ketoprofenoyl-CoA thioester 3 was synthesized by coupling ketoprofen to coenzyme A using the mixed anhydride method. Diastereoisomeric compounds 3a and 3b corresponding to the enantiomers of ketoprofen, were obtained in optically pure form by preparative HPLC. A non-acylating analogue, rac-3-(3-benzoylphenyl)-2-oxo-butanoyl-CoA (7) was also prepared. The biological evaluation suggested that 3a and 3b are reversible inhibitors of COX-1 and irreversible inhibitors of COX-2. Compound 7 appears to be a poor but selective inhibitor of COX-1.

Limitation of the in vitro whole blood assay for predicting the COX selectivity of NSAIDs in clinical use.

AIMS: To assess if the inhibitory potency of nonsteroidal anti-inflammatory drugs (NSAIDs) on cyclooxygenase (COX) isoenzymes, when given therapeutically in humans, can be predicted from their in vitro concentration-response curves using the whole blood assay. METHODS: Twenty-four healthy male volunteers aged 20--27 years were recruited. Inhibition of blood COX isoenzymes was determined in vitro before any drug intake and ex vivo after single and repeated intake of either 7.5 mg meloxicam once, 400 mg ibuprofen three times daily or 75 mg diclofenac SR once, taken in a randomized cross-over design. Production of thromboxane B2 (TXB2) during clotting and of prostaglandin E2 (PGE2) during endotoxin exposure served as indicators of platelet COX-1 and monocyte COX-2 activity, respectively. Drugs were determined in plasma by h.p.l.c., with a chiral separation of ibuprofen and free fractions after equilibrium dialysis. RESULTS: Intra-subject variation for COX-1 and COX-2 at baseline was at 26 +/- 18% and 18 +/- 13% respectively, and intersubject variation at 39% and 36%, respectively. The ratios of IC50s and, at best, of IC80s revealed diclofenac and meloxicam as selective COX-2 inhibitors and ibuprofen as a preferential COX-1 inhibitor in vitro. However, after oral intake, ibuprofen inhibited ex vivo COX-2 by 80% whereas diclofenac inhibited COX-1 by 70%. Meloxicam inhibited COX-1 from 30 to 55% depending on the repetition of the dose and increase in plasma concentrations. Using in vitro dose--response curves, the in vivo inhibitory potency of diclofenac was estimated adequately from its circulating concentration ([-0.18, 0.21] for COX-1 and [-0.13, -0.03] for COX-2) but this was not the case for ibuprofen on COX-2 ([-0.14, 0.27]) and meloxicam on COX-1 ([0.31, 1.05]). The limited predictability of the system was not improved through considering the unbound fraction of the drugs or the variable chiral inversion of ibuprofen. CONCLUSIONS: Assessment of COX-2 selectivity based on in vitro studies and pharmacological modelling has a limited clinical relevance. There is a need to investigate COX selectivity at therapeutic plasma concentrations of NSAIDs using the ex vivo whole blood assay.