The potency of the fatty acid amide hydrolase inhibitor URB597 is dependent upon the assay pH

Inhibitors of the enzyme fatty acid amide hydrolase (FAAH), the principal enzyme involved in the metabolism of the endogenous cannabinoid anandamide, have potential utility in the treatment of disorders including inflammation and inflammatory pain. The carbamate compound URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) potently and selectively inhibits FAAH by forming a covalent bond with a key serine residue of the enzyme. Little is known as to the pH dependency of this inhibition. Using a preincubation time of 10min, URB597 inhibited rat brain anandamide hydrolysis with pI(50) values of 7.19+/-0.02 and 7.75+/-0.06 at pH 6 and 8, respectively. The inhibition was time-dependent, and second order rate constants of approximately 0.15x10(6)M(-1)min(-1) (pH 6) and approximately 1.2x10(6)M(-1)min(-1) (pH 8) could be estimated. In intact C6 glioma cells and using a preincubation time of 10min, URB597 inhibited the hydrolysis of 250nM [(3)H]AEA hydrolysis with pI(50) values of 5.58+/-0.07 and 6.45+/-0.07 at extracellular pH values of 6 and 8, respectively. Since tissue pH is affected by inflammation, these data would suggest that the pH selectivity of the inhibition can contribute to the potency of the compound in vivo.     

Stereoselective inhibitory effect of flurbiprofen, ibuprofen and naproxen on human organic anion transporters hOAT1 and hOAT3

Nonsteroidal anti-inflammatory drugs (NSAIDs) delay the renal excretion of antifolate methotrexate by inhibiting human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8). In this study, uptake experiments were performed using Xenopus laevis oocytes to assess stereoselectivity in the inhibitory characteristics of flurbiprofen, ibuprofen and naproxen against hOAT1 and hOAT3. Uptake of p-aminohippurate by hOAT1 was inhibited by each enantiomer of the three NSAIDs, and the inhibitory effect was superior in each (S)-enantiomer around 10 μM. The apparent 50% inhibitory concentrations were estimated to be 0.615 μM for (S)-flurbiprofen, 2.84 μM for (S)-ibuprofen and 1.93 μM for (S)-naproxen, and these values were significantly lower than those of the respective (R)-enantiomers [(R)-flurbiprofen: 2.35 μM, (R)-ibuprofen: 6.14 μM, (R)-naproxen: 5.26 μM]. Furthermore, the (S)-NSAIDs at 3 μM reduced methotrexate accumulation in hOAT1-expressing oocytes more strongly than the corresponding (R)-enantiomers. All enantiomers inhibited hOAT3-mediated transport of estrone sulfate and methotrexate, but there was no difference between both enantiomers of each NSAID in the inhibitory potencies. Eadie-Hofstee plot analysis showed that (S)-flurbiprofen and (R)-flurbiprofen inhibited hOAT1 and hOAT3 in a competitive manner. These findings represent the stereoselective inhibitory potencies of flurbiprofen, ibuprofen and naproxen on hOAT1, and the (S)-enantiomers are greater. In contrast, stereoselectivity was not recognized in their inhibitory effect on hOAT3.     

The site of inversion of R(-)-ibuprofen: studies using rat in-situ isolated perfused intestine/liver preparations.

The site of metabolic inversion of R(-)-ibuprofen to the pharmacologically active S(+)-enantiomer has been investigated using an array of in-situ rat perfused organ preparations allowing vascular perfusion (55-60 min) of the separate or combined intestine and liver. After addition of R(-)-ibuprofen (20 mg kg-1 body weight) to the closed (static) lumen of isolated 25 cm lengths of duodenum, jejunum or ileum, and single-pass vascular perfusion, both isomers were measured in the lumen and in vascular perfusate plasma (mean plasma AUC values (+/- s.d., micrograms mL-1 min, n = 5) R(-)-ibuprofen: 1669 +/- 115 (duodenum), 1687 +/- 203 (jejunum), 2061 +/- 188 (ileum); S(+)-ibuprofen: 23 +/- 6 (duodenum), 14 +/- 5 (jejunum), 26 +/- 1 (ileum]. Addition of the same dose of S(+)-ibuprofen to the jejunum (n = 5) resulted in AUC values of 1864 +/- 238 for S(+)-ibuprofen and 6 +/- 3 for R(-)-ibuprofen. After addition of R(-)-ibuprofen (30 micrograms mL-1) to the recirculating vascular perfusate (100 mL) of the entire small intestine (n = 6) AUC values were 1647 +/- 34 for R(-)-ibuprofen and 13 +/- 3 for S(-)-ibuprofen. The same dose of R(-)-ibuprofen to combined intestine/liver (n = 6) and liver only preparations (n = 6) gave AUC values of 1011 +/- 25 and 1021 +/- 49 for R(-)-ibuprofen and 220 +/- 28 and 238 +/- 22 for S(+)-ibuprofen, respectively. In all experiments, except those involving perfusion of the combined intestine/liver and the liver, the concentrations of the isomer opposite to that administered could be accounted for solely by the level of enantiomeric impurity (1.3% for R(-)-ibuprofen and 0.6% for S(+)-ibuprofen). We conclude that inversion of R(-)-ibuprofen to the S(+) antipode occurs in the liver but does not occur on either mucosal or serosal sides of the small intestine of the rat.     

Role of fatty acid amide hydrolase in the transport of the endogenous cannabinoid anandamide

A facilitated transport process that removes the endogenous cannabinoid anandamide from extracellular spaces has been identified. Once transported into the cytoplasm, fatty acid amide hydrolase (FAAH) is responsible for metabolizing the accumulated anandamide. We propose that FAAH contributes to anandamide uptake by creating and maintaining an inward concentration gradient for anandamide. To explore the role of FAAH in anandamide transport, we examined anandamide metabolism and uptake in RBL-2H3 cells, which natively express FAAH, as well as wild-type HeLa cells that lack FAAH. RBL-2H3 and FAAH-transfected HeLa cells demonstrated a robust ability to metabolize anandamide compared with vector-transfected HeLa cells. This activity was reduced to that observed in wild-type HeLa cells upon the addition of the FAAH inhibitor methyl arachidonyl fluorophosphonate. Anandamide uptake was reduced in a dose-dependent manner by various FAAH inhibitors in both RBL-2H3 cells and wild-type HeLa cells. Anandamide uptake studies in wild-type HeLa cells showed that only FAAH inhibitors structurally similar to anandamide decreased anandamide uptake. Because there is no detectable FAAH activity in wild-type HeLa cells, these FAAH inhibitors are probably blocking uptake via actions on a plasma membrane transport protein. Phenylmethylsulfonyl fluoride, a FAAH inhibitor that is structurally unrelated to anandamide, inhibited anandamide uptake in RBL-2H3 cells and FAAH-transfected HeLa cells, but not in wild-type HeLa cells. Furthermore, expression of FAAH in HeLa cells increased maximal anandamide transport 2-fold compared with wild-type HeLa cells. These results suggest that FAAH facilitates anandamide uptake but is not solely required for transport to occur.     

Design,synthesis, and biological evaluation of new inhibitors of the endocannabinoid uptake: comparison with effects on fatty acid amidohydrolase

A new series of arachidonic acid derivatives were synthesized and evaluated as inhibitors of the endocannabinoid uptake. Most of them are able to inhibit anandamide uptake with IC(50) values in the low micromolar range (IC(50) = 0.8-24 microM). In general, the compounds had only weak effects upon CB(1), CB(2), and VR(1) receptors (K(i) > 1000-10000 nM). In addition, there was no obvious relationship between the abilities of the compounds to affect anandamide uptake and to inhibit anandamide metabolism by fatty acid amidohydrolase (FAAH; IC(50) = 30-113 microM). This indicates that the compounds do not exert their effects secondarily to FAAH inhibition. It is hoped that these compounds, particularly the most potent in this series (compound 5, UCM707, with IC(50) values for anandamide uptake and FAAH of 0.8 and 30 microM, respectively), will provide useful tools for the elucidation of the role of the anandamide transporter system in vivo.     

Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study

The response of anandamide is terminated by a carrier-mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC-3 cells by a saturable and temperature-dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7+/-0.2 microm and Vmax=3.3+/-0.3 pmol min-1 (10(6) cells)-1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC-3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1-8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC-3 cells, with (9Z,12Z)-N-(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3+/-0.5 microM, respectively. In conclusion, PC-3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator.British Journal of Pharmacology (2004) 141, 457-467. doi:10.1038/sj.bjp.0705628     

Stereoselective disposition of flurbiprofen in healthy subjects following administration of the single enantiomers.

Plasma concentrations of the enantiomers of flurbiprofen were measured following oral administration of (S)-flurbiprofen 50 mg and (R)-flurbiprofen 50 mg and 100 mg to sixteen healthy subjects. Chiral inversion did not occur to a measurable extent. Significantly higher values of AUC (55.2 +/- 17.0 vs 44.6 +/- 11.2 micrograms ml-1h) elimination half-life (5.6 +/- 1.4 vs 4.0 +/- 1.0 h) and mean residence time (7.5 +/- 1.6 vs 5.7 +/- 1.2 h) were observed after 50 mg (S)-flurbiprofen as compared with 50 mg (R)-flurbiprofen. With the exception of Cmax and AUC values pharmacokinetic data for the 50 mg and the 100 mg dose of (R)-flurbiprofen did not differ significantly. The data are of clinical relevance if (R)-flurbiprofen also has analgesic activity in humans and is to be developed as an analgesic.     

Variability in the stereoselective disposition of ibuprofen in patients with rheumatoid arthritis.

1. Patients suffering from rheumatoid arthritis received oral doses of 600 mg racemic ibuprofen (n = 25; RAC) or 400 mg (S)-ibuprofen (n = 25; S-IBU) in a double-blind, randomized parallel-group study. 2. The pharmacokinetic parameters of (S)-ibuprofen were not statistically different between treatments (P > 0.05). Comparing (S)- and (R)-ibuprofen within the group receiving the racemate significantly higher Cmax (20.3 +/- 5.3 vs 17.7 +/- 4.4 micrograms ml-1; P < 0.02; 95% confidence interval for differences (CI): 0.5-4.6), AUC (86.2 +/- 23.5 vs 67.6 +/- 26.6 micrograms ml-1 h; P < 0.001; CI: 9.5-27.6), mean residence time (4.5 +/- 1.1 vs 4.1 +/- 1.2 h; P < 0.01; CI: 0.1-0.6) and renal clearance (0.8 +/- 0.6 vs 0.0 +/- 0.0 ml min-1; P < 0.001; CI: 0.5-1.0) values were observed for the (S)-enantiomer. 3. No difference was found (P > 0.05) between treatments in the percentage of the dose recovered in the urine as (R)- or (S)-ibuprofen plus metabolites (S-IBU: 80.2 +/- 8.47 vs RAC: 74.1 +/- 14.0%). 4. Interindividual variation in the pharmacokinetics of (S)-ibuprofen following administration of the racemate was similar to that following the administration of the single isomer suggesting that chiral inversion is not a major factor contributing to variability in the disposition of this drug.     

Modifications of the ethanolamine head in N-palmitoylethanolamine: synthesis and evaluation of new agents interfering with the metabolism of anandamide

The endogenous fatty acid amide anandamide (AEA) has, as a result of its actions on cannabinoid and vanilloid receptors, a number of important pharmacological properties including effects on nociception, memory processes, spasticity, and cell proliferation. Inhibition of the metabolism of AEA, catalyzed by fatty acid amide hydrolase (FAAH), potentiates the actions of AEA in vivo and therefore may be a useful target for drug development. In the present study, we have investigated whether substitution of the headgroup of the endogenous alternative FAAH substrate palmitoylethanolamide (PEA) can result in the identification of novel compounds preventing AEA metabolism. Thirty-seven derivatives of PEA were synthesized, with the C16 long chain of palmitic acid kept intact, and comprising 20 alkylated, 12 aromatic, and 4 halogenated amides. The ability of the PEA derivatives to inhibit FAAH-catalyzed hydrolysis of [(3)H]AEA was investigated using rat brain homogenates as a source of FAAH. Inhibition curves were analyzed to determine the potency of the inhibitable fraction (pI(50) values) and the maximal attained inhibition for the compound, given that solubility in an aqueous environment is a major issue for these compounds. In the alkylamide family, palmitoylethylamide and palmitoylallylamide were inhibitors of AEA metabolism with pI(50) values of 5.45 and 5.47, respectively. Halogenated derivatives (Cl and Br) exhibit pI(50) values of approximately 5.5 but rather low percentages of maximal inhibition. The -OH group of the ethyl head chain of N-palmitoylethanolamine was not necessary for interaction with FAAH. Amides containing aromatic moieties were less potent inhibitors of AEA metabolism. Compounds containing amide and ester bonds, 13 and 37, showed pI(50) values of 4.99 and 5.08, respectively. None of the compounds showed obvious affinity for CB(1) or CB(2) receptors expressed on Chinese hamster ovary (CHO) cells. It is concluded that although none of the compounds were dramatically more potent than PEA itself at reducing the metabolism of AEA, the lack of effect of the compounds at CB(1) and CB(2) receptors makes them useful templates for development of possible therapeutic FAAH inhibitors.     

The 'specific' tyrosine kinase inhibitor genistein inhibits the enzymic hydrolysis of anandamide: implications for anandamide uptake

BACKGROUND AND PURPOSE: The cellular uptake of anandamide is reduced by inhibitors of fatty acid amide hydrolase (FAAH) and by agents disrupting endocytotic mechanisms. However, it is not clear if these events occur over the same time frame and if they occur to the same extent in different cells. We have therefore investigated the effects of such compounds in three cell lines of different origins using different assay incubation times and temperatures. EXPERIMENTAL APPROACH: FAAH activity and cellular uptake of anandamide was measured using anandamide, radio-labelled either in the ethanolamine or arachidonoyl part of the molecule. KEY RESULTS: The FAAH inhibitor URB597 inhibited the uptake of anandamide into C6 glioma, RBL2H3 basophilic leukaemia cells and P19 embryonic carcinoma cells at incubation time 4 min. However, a time-dependent and temperature-sensitive residual uptake remained after URB597 treatment. The combination of progesterone and nystatin reduced the uptake, but also decreased the amount of anandamide retained by the wells. Genistein inhibited anandamide uptake in a manner that was not additive to that of URB597. However, genistein was a potent competitive inhibitor of FAAH (K(i) value 8 microM). CONCLUSIONS AND IMPLICATIONS: The reduction of anandamide uptake by genistein can be explained by its ability to inhibit FAAH with a potency which overlaps that for inhibition of tyrosine kinase. The FAAH- resistant but time-dependent uptake of anandamide is seen in all three cell lines studied and is thus presumably a generally occurring process.     

Effects of flurbiprofen and its enantiomers on the spinal c-Fos protein expression induced by noxious heat stimuli in the anaesthetized rat

We have evaluated the effects of either intravenous or intraplantar administration of racemic-, S(+)- and R(-)-flurbiprofen on the spinal c-Fos protein expression after a single noxious heat stimulation (52 degrees C for 15 s) of the rat hindpaw in urethane anaesthetized rats. Two hours after noxious heat, numerous c-Fos protein immunoreactive (c-Fos-IR) nuclei (>70 c-Fos-IR nuclei per section at the level of L4-L5 segments) were observed with essential localization in the superficial (I-II) laminae of the spinal dorsal horn, i.e. areas containing numerous neurons driven exclusively by noxious stimuli. Considering the number of c-Fos-IR nuclei in laminae I-II, the intravenous injection of racemic-flurbiprofen (0.3, 3 and 9 mg/kg) was inefficacious and S(+)-flurbiprofen had weak and non-dose-related effects. The same doses of R(-)-flurbiprofen produced dose-related effects (r=0.58, P<0.05) with weak, but significant, effects for doses of 3 and 9 mg/kg (18+/-6% and 26+/-5% reduction of the number of noxious heat-evoked c-Fos-IR nuclei in laminae I-II, P<0.05 and P<0.01, respectively). The weak effects of R(-)-flurbiprofen are probably due to the central site of action since the intraplantar injection of a relatively high dose of 30 microg is inefficacious. These results provide further evidence for weak effects of non-steroidal anti-inflammatory drugs and their enantiomers on the acute responses to nociceptive stimulus which are very efficacious upon inflammatory nociception, but not upon brief noxious heat-evoked nociception.     

Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis--a difficult issue to handle

There is considerable debate at present as to whether the uptake of anandamide (AEA) into cells is by a facilitated transport process or by passive diffusion driven by fatty acid amide hydrolase (FAAH). The possibility that both processes occur, but to different extents depending upon the cell type used, has been difficult to investigate pharmacologically since available compounds show little selectivity between inhibition of AEA uptake and inhibition of FAAH. Recently, three compounds, UCM707 [N-(Fur-3-ylmethyl)arachidonamide], OMDM-1 and OMDM-2 [the 1'-(S)- and 1'-(R)-enantiomers of the 1'-4-hydroxybenzoyl analogue of oleoylethanolamide], selective for the uptake process, have been described and we have used these compounds, together with AM404 [(N-(4-hydroxyphenyl) arachidonoyl amide)] and VDM11 [(5Z,8Z,11Z,14Z)-N-(4-Hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide]), with the initial aim of determining which mechanism of uptake predominates in C6 glioma and RBL-2H3 cells. AM404 and VDM11 were both found to decrease the uptake of 2 microM AEA into cells (IC50 values 6-11 microM), but they also inhibited rat brain FAAH (IC50 values 1-6 microM). However, when using a different FAAH assay protocol, VDM11 was a much less potent FAAH inhibitor (IC50>50 microM) regardless of the cell type and animal species used. In contrast, we confirmed that UCM707, OMDM-1 and OMDM-2 were weak inhibitors of FAAH (IC50 values >50 microM) under all conditions used. However, their potency as inhibitors of AEA cellular accumulation appears to be largely dependent on the cell type and assay conditions used. In particular, the potency of UCM707 (IC50 value > or =25 microM) was considerably lower than the submicromolar potency previously reported for U937 cells. It is concluded that the cause/effect relationship between AEA uptake and hydrolysis cannot be investigated uniquely by using supposedly selective inhibitors of each process.     

Studies on the metabolism and chiral inversion of ibuprofen in isolated rat hepatocytes

The oxidative metabolism and chiral inversion of ibuprofen in freshly isolated rat hepatocytes was studied with the aid of a stereoselective GC/MS assay procedure. Hydroxylation of the isobutyl side chain at the subterminal carbon (to give hydroxyibuprofen) proved to be the major route of metabolism of both R(-)-ibuprofen and S(+)-ibuprofen, while formation of the corresponding diastereoisomeric 2-methylpropionic acid derivatives (carboxyibuprofen) was of minor quantitative importance. Both oxidative pathways were inhibited in the presence of metyrapone, a cytochrome P-450 inhibitor. R(-)-Ibuprofen underwent metabolic chiral inversion to the S(+) enantiomer, whose formation was dependent on incubation time, cell density, and substrate concentration. S(+)-Ibuprofen, on the other hand, was not converted to R(-)-ibuprofen in rat hepatocytes. When cells were incubated with a mixture of unlabeled R(-)-ibuprofen and R(-)-[3,3,3-2H3]ibuprofen, the resultant S(+) enantiomer consisted only of unlabeled and trideutero molecules (formed in the same ratio as the corresponding species of R(-)-ibuprofen), indicating that 2,3-dehydroibuprofen did not serve as the symmetrical intermediate in the chiral inversion reaction. Collectively, these results demonstrate that freshly isolated rat hepatocytes represent a convenient and reproducible in vitro model system for studies on the metabolism and chiral inversion of ibuprofen.     

Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate

There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH.In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for anandamide. It is concluded that VDM11 is an inhibitor of FAAH under the assay conditions used here, and that the inhibition may at least in part be a consequence of the compound acting as an alternative substrate.     

An examination of the thermodynamics of fusion, vaporization, and sublimation of (R,S)- and (R)-flurbiprofen by correlation gas chromatography

The vaporization, fusion, and sublimation enthalpies of (R,S)- and (R)-flurbiprofen at T = 298.15 K are reported and compared with literature values when available. Correlation gas chromatography experiments were first performed to identify appropriate standards that could be used for materials containing a single fluorine substituent. Subsequent correlations resulted in a vaporization enthalpy for (R,S)-flurbiprofen and (R)-flurbiprofen, ΔH(vap) (298.15 K), of (127.5 ± 5.5) and (127.4 ± 4.7) kJ mol, respectively. Fusion enthalpies, ΔH(fus) (387 K), of (28.2 ± and, ΔH(fus) (381 K), (22.8 ± kJ mol(-1) were also measured by differential scanning calorimetry for the racemic and chiral forms of flurbiprofen. Adjusted to T = 298.15 K and combined with the vaporization enthalpy resulted in sublimation enthalpies, ΔH(sub) (298.15 K), of (155.6 ± 5.8) and (145.1 ± 5.7) kJ mol(-1) for (R,S)- and (R)-flurbiprofen, respectively. The fusion enthalpy measured for the racemic form was in excellent agreement with the literature value, while the sublimation enthalpy varies substantially from previous work. Two weak solid-solid phase transitions were also observed for (R)-flurbiprofen at T = 353.9 K (0.30 ± 0.1) and 363.2 K (0.21 ± 0.03) kJ · mol(-1).     

Stereoselectivity of ibuprofen metabolism and pharmacokinetics following the administration of the racemate to healthy volunteers

1. The stereoselective metabolism and pharmacokinetics of the enantiomers of ibuprofen have been investigated following the oral administration of the racemic drug (400 mg) to 12 healthy volunteers.2. The stereochemical composition of the drug in serum, both total and unbound, and drug and metabolites, both free and conjugated, in urine were determined by a combination of the direct and indirect chromatographic procedures to enantiomeric analysis. 3. The oral clearance of (S)-ibuprofen was significantly greater than that of the R-enantiomer (74.5 +/- 18.1 versus 57.1 +/- 11.7 ml min(-1); p < 0.05) and the clearance of (R)-ibuprofen via inversion was ca two fold that via alternative pathways. 4. Some 74.0 +/- 9.6% of the dose was recovered in urine over 24 h as ibuprofen, 2-hydroxyibuprofen and carboxyibuprofen, both free and conjugated with glucuronic acid. Analysis of the stereochemical composition of the urinary excretion products indicated that 68% of the dose of (R)-ibuprofen had undergone chiral inversion. 5. Metabolism via glucuronidation and both routes of oxidation, showed enantio-selectivity for (S)-ibuprofen, the enantiomeric ratios (S/R) in partial metabolic clearance being 7.1, 4.8 and 3.4 for formation of ibuprofen glucuronide, 2-hydroxyibuprofen and carboxyibuprofen respectively.6. Modest stereoselectivity was observed in the formation of (2'R, 2R)- and (2'S, 2S)-carboxyibuprofen in comparison to the alternative diastereoisomers, the ratios in formation clearance being 1.6 and 1.2 respectively.     

Inhibition of fatty acid amide hydrolase by kaempferol and related naturally occurring flavonoids

Background and purpose:Recent studies have demonstrated that the naturally occurring isoflavone compounds genistein and daidzein inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) in the low micromolar concentration range. The purpose of the present study was to determine whether this property is shared by flavonoids.Experimental approach:The hydrolysis of anandamide in homogenates and intact cells was measured using the substrate labelled in the ethanolamine part of the molecule.Key results:Twenty compounds were tested. Among the commonly occurring flavonoids, kaempferol was the most potent, inhibiting FAAH in a competitive manner with a K(i) value of 5 muM. Among flavonoids with a more restricted distribution in nature, the two most active toward FAAH were 7-hydroxyflavone (IC(50) value of 0.5-1 muM depending on the solvent used) and 3,7-dihydroxyflavone (IC(50) value 2.2 muM). All three compounds reduced the FAAH-dependent uptake of anandamide and its metabolism by intact RBL2H3 basophilic leukaemia cells.Conclusions and implications:Inhibition of FAAH is an additional in vitro biochemical property of flavonoids. Kaempferol, 7-hydroxyflavone and 3,7-dihydroxyflavone may be useful as templates for the synthesis of novel compounds, which target several systems that are involved in the control of inflammation and cancer.British Journal of Pharmacology (2008) 155, 244-252; doi:10.1038/bjp.2008.237; published online 16 June 2008.     

Renal handling and effects of S(+)-ibuprofen and R(-)-ibuprofen in the rat isolated perfused kidney.

1. The renal handling and effects of S(+)- and R(-)-ibuprofen have been studied in the isolated perfused kidney (IPK) of the rat. 2. Both ibuprofen enantiomers were extensively reabsorbed and accumulated in the kidney in a concentration-dependent manner. No pharmacokinetic differences were observed between the two enantiomers. 3. S(+)-ibuprofen concentrations ranging from 0.25 to 25 micrograms ml-1 (1.2 to 120 microM) caused a decrease in urinary flow, glomerular filtration rate (GFR) and electrolyte excretion. Urinary pH and excretion of glucose were not influenced. R(-)-ibuprofen concentrations ranging from 2.5 to 25 micrograms ml-1 (12 to 120 microM) also decreased urinary flow and electrolyte excretion. This decrease, however, was less than observed with S(+)-ibuprofen. GFR, urinary pH and glucose excretion were not affected by R(-)-ibuprofen. Prostaglandin E2 (PGE2) concentrations of 133 ng ml-1 reversed the effects on renal function of both enantiomers. 4. Very high S(+)- and R(-)-ibuprofen concentrations (greater than 400 micrograms ml-1) resulted in an increase in urinary flow and fractional excretion of sodium, chloride, potassium, glucose and calcium. 5. It is concluded that the pharmacokinetic behaviour of ibuprofen in the kidney is not stereoselective. Relatively high concentrations of both enantiomers increased the urinary flow and electrolyte excretion in a nonstereoselective manner. Lower concentrations of S(+)-ibuprofen decreased urinary flow and electrolyte excretion. The pharmacologically inactive R(-)-ibuprofen was also able to affect renal function in a similar way, but at different concentrations. These effects on renal function are probably caused by inhibition of PGE2 synthesis.     

Lack of effect of cimetidine on the pharmacokinetics of R(-)- and S(+)-ibuprofen.

1. To investigate the effect of cimetidine on the pharmacokinetics of R(-)- and S(+)-ibuprofen, six healthy male volunteers received orally 800 mg racemic ibuprofen both in the drug-free state (control phase, C) and on the second day of a 3 day course of oral cimetidine, 1 g daily (treatment phase, T). The two phases (14 days apart) were randomised in a balanced cross-over manner. 2. The plasma concentrations of R(-)- and S(+)-ibuprofen were measured by high-performance liquid chromatography (h.p.l.c.). The protein binding of the enantiomers was assessed in a selection of plasma samples from each volunteer. Following alkaline hydrolysis of glucuronide conjugates, the urinary recoveries of ibuprofen and its major metabolites were measured by h.p.l.c. 3. There was no difference (P greater than 0.05, two-tailed Student's t-test; data expressed as mean +/- s.d.) between C and T phases in the total area under the plasma concentration-time curve of R(-)-ibuprofen (C 4514 +/- 1063 mg 1(-1) min vs T 4665 +/- 1435 mg 1(-1) min) and S(+)-ibuprofen (C 6460 +/- 1063 mg 1(-1) min vs T 6886 +/- 1207 mg 1(-1) min). Similarly, for each enantiomer, there was no difference between the two phases in the terminal half-life, the maximum plasma concentration or the time of its occurrence. 4. Cimetidine treatment had no effect (P greater than 0.05) on the time-averaged percent unbound in plasma of R(-)-ibuprofen (C 0.419 +/- 0.051% vs T 0.435 +/- 0.060%) and S(+)-ibuprofen (C 0.643 +/- 0.093% vs T 0.633 +/- 0.053%). (ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic chiral inversion of anti-inflammatory 2-arylpropionates: lack of reaction in liver homogenates, and study of methine proton acidity

1. Enrichment in the (S)-enantiomers for (R)-flurbiprofen, (R)-naproxen, (R)-suprofen and (R;S)-ibuprofen was investigated in various subcellular hepatic preparations containing coenzyme A. While such preparations were able to form hippuric acid from benzoic acid, the chiral inversion was never seen. 2. Using 2-dimethylaminoethanethiol 2-phenylpropionate (DEPP) as a model acyl thioester, the acidity of the methine proton was investigated by monitoring the proton/deuterium exchange occurring in deuterated solvents using high-resolution n.m.r. The compound was inert up to 22 h in D2O at 37 degrees C and pD 7.4. In pure methanol or a methanol-water mixture, only solvolysis was seen. In contrast, competitive hydrolysis (k = 0.005 h-1) and proton/deuterium exchange (k = 0.09 h-1) were seen in a CD3CN/D2O (50:50) mixture at 37 degrees C. 3. It is speculated that the failure to characterize chiral inversion of 2-arylpropionates in subcellular preparations may be due to the absence of a microenvironment of adequately moderate polarity.