Weak inhibitors of cyclooxygenases may exert their antinociceptive effect by modulation of transcription factors.
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Analgesics↗
Biomedical subjects
K Brune
Publications and source records attributed to K Brune.
Peripheral noxious stimulation releases spinal PGE2 during the first phase in the formalin assay of the rat.
Injection of formalin (5%; 50 microl) into the dorsal surface of the hind paw of rats evoked a characteristic biphasic flinching behaviour of the injured paw accompanied by a significant increase in the interstitial prostaglandin E2 (PGE2) concentration of the dorsal lumbar spinal cord. Interestingly, the increase in PGE2 concentration was only observed during the first phase of the formalin behavioural response (during the 0-10 and 10-20 min microdialysis-sample). Saline paw injection did not have a significant effect on behaviour or on PGE2 concentration. These data suggest that spinal release of PGE2 is involved in nociceptive processing in the formalin-induced hyperalgesia model of the rat during the first but not second phase.
Animals↗
Elevated serum cyclophilin levels in patients with severe sepsis.
Several cytokines are considered to be important mediators in the pathophysiology of sepsis. Cyclophilins (Cyps), the main binding proteins for the immunosuppressive drug cyclosporine A, have been suggested to function as cytokines. This study was conducted to determine (i) if serum Cyp levels were elevated in critically ill patients suffering from either sepsis or other life-threatening diseases and (ii) if so, whether there was an association between Cyp levels and a certain diagnosis and/or outcome. Serum samples of 45 patients (22 severe sepsis, 23 other diagnoses) and 17 healthy controls were prospectively analyzed by an enzymatic assay using the ability of cyclophilins to catalyze cis/trans isomerisation of peptidylprolyl-peptide bonds (PPIase activity). In addition, western blotting was applied to differentiate both isoforms. PPIase activity was significantly higher in patients with severe sepsis than in patients with other diagnoses (P = 0.004) or in healthy subjects (P = 0.001). There was no difference between healthy subjects and other critically ill patients (P = 0.067). Elevated PPIase activity was associated with high mortality (P = 0.03). It is concluded that Cyps might play a role, probably as mediators in the pathophysiology of sepsis or as symptoms of diagnostic value.
Adult↗
Effects of ibuprofen enantiomers and its coenzyme A thioesters on human prostaglandin endoperoxide synthases.
1. Ibuprofen enantiomers and their respective coenzyme A thioesters were tested in human platelets and blood monocytes to determine their selectivity and potency as inhibitors of cyclo-oxygenase activity of prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2. 2. Human blood from volunteers was drawn and allowed to clot at 37 degrees C for 1 h in the presence of increasing concentrations of the test compounds (R-ibuprofen, S-ibuprofen, R-ibuprofenoyl-CoA, S-ibuprofenoyl-CoA, NS-398). Immunoreactive (ir) thromboxane B2 (TXB2) concentrations in serum were determined by a specific EIA assay as an index of the cyclo-oxygenase activity of platelet PGHS-1. 3. Heparin-treated blood from the same donors was incubated at 37 degrees C for 24 h with the same concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 microg ml[-1]). The contribution of PGHS-1 was suppressed by pretreatment of the volunteers with aspirin (500 mg; 48 h before venepuncture). As a measure of LPS induced PGHS-2 activity immunoreactive prostaglandin E2 (irPGE2) plasma concentrations were determined by a specific EIA assay. 4. S-ibuprofen inhibited the activity of PGHS-1 (IC50 2.1 microM) and PGHS-2 (IC50 1.6 microM) equally. R-ibuprofen inhibited PGHS-1 (IC50 34.9) less potently than S-ibuprofen and showed no inhibition of PGHS-2 up to 250 microM. By contrast R-ibuprofenoyl-CoA thioester inhibited PGE2 production from LPS-stimulated monocytes almost two orders of magnitude more potently than the generation of TXB2 (IC50 5.6 vs 219 microM). 5. Western blotting of PGHS-2 after LPS induction of blood monocytes showed a concentration-dependent inhibition of PGHS-2 protein expression by ibuprofenoyl-CoA thioesters. 6. These data confirm that S-ibuprofen represents the active entity in the racemate with respect to cyclo-oxygenase activity. More importantly the data suggest a contribution of the R-enantiomer to therapeutic effects not only by chiral inversion to S-ibuprofen but also via inhibition of induction of PGHS-2 mediated by R-ibuprofenoyl-CoA thioester. 7. The data may explain why racemic ibuprofen is ranked as one of the safest non-steroidal anti-inflammatory drugs (NSAIDs) so far determined in epidemiological studies.
Blood Platelets↗
Disposition and effects of flurbiprofen enantiomers in human serum and blister fluid.
AIMS: To investigate the pharmacokinetics of the enantiomers of flurbiprofen and inhibition of prostanoid production in blister fluid and serum. METHODS: Eleven healthy volunteers received 75 mg R-, 75 mg S-flurbiprofen or no medication in a randomized 3-way cross-over study. Flurbiprofen concentrations were determined by h.p.l.c. TXB2 and PGE2 were determined by enzyme immunoassay and chemiluminescence immunoassay respectively. RESULTS: S-flurbiprofen produced almost complete (> 99% vs baseline) inhibition of thromboxane B2 (TXB2) in serum in all volunteers and significant inhibition of prostaglandin E2 (PGE2) generation in blister fluid, but there was a considerable inter-individual variation in the response ranging from -78 to +190% change from control PGE2 AUC. After administration of R-flurbiprofen, there was a mean maximum TXB2 inhibition of 65.2 +/- 15.0% in serum but no significant changes of PGE2 levels in blister fluid were observed. The pharmacokinetic parameters in serum and blister fluid were not significantly different between enantiomers. R- to S-inversion did not occur to a clinically relevant extent. For R-flurbiprofen, the complex rate constant of transfer into blister fluid was greater at the u.v.-exposed site (0.110 +/- 0.050) than at the control site (0.079 +/- 0.026, P < 0.05) which corresponded to a higher AUC and Cmax of R-flurbiprofen in u.v.-exposed blister as compared with control. For inhibition of TXB2 generation after administration of S-flurbiprofen, a sigmoidal log-linear concentration-response relationship was established in all subjects (EC50: 0.123 +/- 0.092 microgram ml-1). In contrast, inhibition of PGE2 production in blister showed no clear concentration-response relationship when correlated with concentrations of S-flurbiprofen in either serum or blister fluid. After administration of R-flurbiprofen, no concentration-effect relationship could be established. CONCLUSIONS: It is concluded that the blister model may have value for studying the pharmacokinetics and pharmacodynamics of antiinflammatory drugs in humans. Interestingly, inter-individual variation in the pharmacokinetics of flurbiprofen enantiomers could not account for the variability in response observed in the blister model.
Adult↗
Host-cell cyclophilin is important for the intracellular replication of Leishmania major.
The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host-parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.
Amino Acid Isomerases↗
Lack of analgesic activity of morphine-6-glucuronide after short-term intravenous administration in healthy volunteers.
BACKGROUND: The analgesic activity of morphine-6-glucuronide (M-6-G) is well recognized for its contribution to the effects of morphine and its possible use as an opioid analgesic with a wider therapeutic range than morphine. The present study attempted to quantify the relative contribution of M-6-G to analgesia observed after systemic administration of morphine. METHODS: In a placebo-controlled, sixfold crossover study in 20 healthy men, the effects of M-6-G were assessed at steady-state plasma concentrations of M-6-G identical to and two and three times higher than those measured after administration of morphine. Morphine and M-6-G were administered as an intravenous bolus followed by infusion over 4 h. Dosage A was M-6-G-bolus of 0.015 mg/kg plus infusion of 0.0072 mg x kg(-1) x h(-1). Dosage B was M-6-G-bolus of 0.029 mg/kg plus infusion of 0.014 mg x kg(-1) x h(-1). Dosage C was M-6-G-bolus of 0.044 mg/kg plus infusion of 0.022 mg x kg(-1) x h(-1). Dosage D was a morphine bolus of 0.14 mg/kg plus infusion of 0.05 mg x kg(-1) x h(-1) for 4 h. Dosage E was M-6-G combined with morphine (doses A + D). Dosage F was a placebo. The analgesic effects of M-6-G and morphine were measured before administration of the bolus and after 3.5 h using an experimental pain model based on pain-related cortical potentials and pain ratings after specific stimulation of the nasal nociceptor with short pulses of gaseous carbon dioxide. RESULTS: Morphine significantly reduced subjective and objective pain correlates compared with placebo. In contrast, M-6-G produced no statistically significant effects. The addition of M-6-G to morphine did not increase the effects of morphine. Morphine produced significantly more side effects than M-6-G. CONCLUSION: After short-term intravenous administration at doses that produce plasma concentrations of M-6-G similar to those seen after administration of morphine, M-6-G had no analgesic effects in the present placebo-controlled study in healthy volunteers.
Adult↗
Molecular cloning and expression of a 2-arylpropionyl-coenzyme A epimerase: a key enzyme in the inversion metabolism of ibuprofen.
The 2-arylpropionic acid derivatives, including ibuprofen, are the most widely used anti-inflammatory analgesic cyclooxygenase inhibitors. The (-)-R-enantiomer, which is inactive in terms of cyclooxygenase inhibition, is epimerized in vivo via the 2-arylpropionyl-coenzyme A (CoA) epimerase to the cyclooxygenase-inhibiting (+)-S-enantiomer. The molecular biology of the epimerization pathway is largely unknown. To clarify this mechanism, the sequence of the 2-arylpropionyl-CoA epimerase was identified, and the enzyme cloned and expressed. A cDNA clone encoding the 2-arylpropionyl-CoA epimerase was isolated from a rat liver cDNA library. The nucleotide and the deduced amino acid sequence of this enzyme was determined. Significant amino acid sequence similarity was found between the rat epimerase and carnitine dehydratases from Caenorhabditis elegans (41%) and Escherichia coli (27%). A bacterial expression system (E. coli strain M15[pREP4]) was used to express the epimerase protein, representing up to 20-30% of the total cellular E. coli protein. The expression of the epimerase was confirmed with Western blots using specific anti-epimerase antibodies and by measuring the rate of inversion of (R)-ibuprofenoyl-CoA. Northern blot analysis revealed a prominent 1.9-kb mRNA transcript in different rat tissues. In addition to its obvious importance in drug metabolism, the homology of the epimerase with carnitine dehydratases from several species suggests that this protein, which up to now has only been characterized as having a role in drug transformation, has a function in lipid metabolism.
Amino Acid Sequence↗
Pharmacokinetics of imipenem-cilastatin in critically ill patients undergoing continuous venovenous hemofiltration.
The pharmacokinetics of imipenem-cilastatin were investigated in 12 critically ill patients with acute renal failure (ARF) managed by continuous veno-venous hemofiltration (CVVH) while receiving a fixed combination of 500 mg of imipenem-cilastatin intravenously three or four times daily. No adverse drug reactions were observed. Plasma and hemofiltrate samples were taken at specified times during one dosing interval, and the concentrations of imipenem and cilastatin were determined by high-performance liquid chromatography. Pharmacokinetic variables were calculated by a first-order, two-compartment pharmacokinetic model for both substances. Total clearances of imipenem and cilastatin (mean +/- standard deviations) were 122.2 +/- 28.6 and 29.2 +/- 13.7 ml/min, respectively, with hemofiltration clearances of 22.9 +/- 2.5 and 16.1 +/- 3.1 ml/min, respectively, and nonrenal, nonhemofiltration clearances of 90.8 +/- 26.3 and 13.2 +/- 13.9 ml/min, respectively. Mean imipenem dosage requirements were approximately 2,000 mg/24 h (2,111.8 +/- 493.4 mg/24 h). They were calculated in order to achieve an average steady-state concentration of 12 mg/liter to ensure that concentrations in plasma exceeded the MICs at which 90% of intermediately resistent bacteria are inhibited (8 mg/liter) during the majority of the dosing interval. By contrast, the recommended dosage for patients with end-stage renal failure (ESRF) and infections caused by intermediately resistant bacteria is 1,000 mg/24 h. This remarkable difference may be due (i) to differences in the nonrenal clearance of imipenem between patients with ARF and ESRF and (ii) to the additional clearance by the hemofilter. Since the total clearance of cilastatin was low, marked accumulation occurred, and this was particularly pronounced in patients with additional liver dysfunction. Thus, in patients with ARF managed by CVVH, rather high imipenem doses are required, and these inevitably result in a marked accumulation of cilastatin. The doses of imipenem recommended for patients with ESRF, however, will lead to underdosing and inadequate antibiotic therapy.
Acute Kidney Injury↗
Fit for life? Evolution of chaperones and folding catalysts parallels the development of complex organisms.
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Bacterial Proteins↗
Intravenous morphine-6-glucuronide (M6G) is devoid of analgesic activity in man.
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Adult↗
[Selective cyclooxygenase-2 (COX-2) inhibitors. Beliefs, reality and hope].
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Acute-Phase Reaction↗
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.
Animals↗
Up-regulation of cyclooxygenase-2 mRNA in the rat spinal cord following peripheral inflammation.
Prostaglandins (PG) have been described as mediators in spinal nociceptive processing after peripheral inflammation. Enzymes essential for PG biosynthesis, cyclooxygenase isozymes COX-1 and COX-2, have not yet been investigated in the spinal cord. In two studies on rats with adjuvant-induced peripheral inflammation levels of mRNA expression of both COX isoforms were analyzed in the lumbar section of the spinal cord using reverse transcription-polymerase chain reaction (RT-PCR) technique. We could show that mRNA of both COX isoforms is expressed constitutively in the spinal cord with COX-2 as the predominant isoform. Six hours after induction of peripheral inflammation, levels of COX-2 mRNA expression were raised significantly in respect to untreated control rats and returned to baseline within 3 days after induction of inflammation. COX-2 might therefore be regarded as the COX isozyme responsible for spinal PG release in nociceptive processing under a peripheral inflammatory stimulus.
Animals↗
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.
Anti-Inflammatory Agents, Non-Steroidal↗
Oxaceprol, an atypical inhibitor of inflammation and joint damage.
Oxaceprol, an established therapeutic agent for osteoarthritis, had no effect on macrophage prostaglandin E2 release in vitro and inhibited carrageenan paw oedema at high doses (18-150 mg/kg p.o.). In the same dose range, oxaceprol was comparable to indomethacin (3 mg/kg p.o.) as an inhibitor of yeast hyperalgaesia and at 6-50 mg/kg/day p.o. had a mild, variable inhibitory effect on cotton pellet granuloma formation. In adjuvant arthritic rats, oxaceprol (6-54 mg/kg/day p.o.) given therapeutically had no effect on the primary paw oedema response, but inhibited secondary lesions in the ears and tail. Histologically, oxaceprol markedly inhibited inflammatory cell infiltration and bone damage in the adjuvant-injected paw. In contrast to indomethacin, oxaceprol was more effective at inhibiting periarticular soft tissue inflammation but did not affect cartilage breakdown in this model. Oxaceprol has a clearly different spectrum of action to NSAIDs such as indomethacin and may act by inhibiting leucocyte infiltration and late connective tissue changes during inflammatory joint disease.
Animals↗
Pharmacokinetics of morphine and its glucuronides after intravenous infusion of morphine and morphine-6-glucuronide in healthy volunteers.
Steady-state pharmacokinetics of morphine and morphine-6-glucuronide (M-6-G) after intravenous administration of either morphine or M-6-G were determined in healthy volunteers. With a dosing regimen calculated on the basis of data obtained in a first series of experiments in four subjects (morphine: intravenous loading dose of 0.24 mg/kg for 5 minutes and an intravenous infusion of 0.069 mg.kg-1.hr-1 for 4 hours; M-6-G: loading dose of 0.011 mg/kg for 5 minutes and an infusion of 0.006 mg.kg-1.hr-1 for 4 hours), it was possible to yield plasma concentrations of morphine and M-6-G in another four subjects close to predefined targeted levels (35 and 45.5 ng/ml morphine and M-6-G, respectively). This dosing regimen may be used in further pharmacodynamic studies to compare the analgesic effects of morphine and M-6-G. In addition, metabolite kinetics of M-6-G were calculated as a function of time with use of a linear systems approach to the estimation of rate and fraction of morphine glucuronidation to M-6-G.
Adult↗
A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E2 using enhanced luminol as substrate.
A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E2 (PGE2) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE2 antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE2 conjugate. Then, after preincubating, the anti-PGE2 antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE2 conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated anti-mouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10-50,000 pg/ml. The lower limit of quantification is 100 pg/ml (2 pg/well) and 150 pg/ml (3 pg/well) for buffer and plasma, respectively. The intra-day coefficients of variation (CV) for the range of 100-50,000 pg/ml are 3.2-8.9% (buffer) and 4.2-17.7% (plasma) and inter-day CV are 2.9-19.8% (buffer) and 3.6-21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.
Animals↗