Hydrogen peroxide scavenging activity by non-steroidal anti-inflammatory drugs

Hydrogen peroxide (H2O2) has been shown to be formed during inflammatory processes and is implicated in its pathophysiology. Thus, a putative scavenging activity against this reactive oxygen species (ROS) by anti-inflammatory drugs may be of great therapeutical value. The present study was undertaken to evaluate the scavenging activity for H2O2 by several non-steroidal anti-inflammatory drugs (NSAIDs), namely indomethacin, acemetacin, etodolac, tolmetin, ketorolac, oxaprozin, sulindac and its metabolites sulindac sulfide and sulindac sulfone. The H2O2 scavenging assay was performed by measuring H2O2-elicited lucigenin chemiluminescence using a microplate reader. The specificity of the method was confirmed by the use of catalase, which completely prevented the H2O2-induced lucigenin chemiluminescence. The endogenous antioxidants melatonin and reduced glutathione (GSH) were used as positive controls. The obtained results demonstrated that all the studied NSAIDs display H2O2 scavenging activity, although in different extents. The ranking order of potency found was sulindac sulfone > sulindac sulfide > GSH > sulindac > indomethacin > acemetacin > etodolac > oxaprozin > ketorolac approximately melatonin > tolmetin.     

Determination of the stereochemical composition of the major metabolites of verapamil in dog urine with enantioselective liquid chromatographic techniques

The stereochemical composition of verapamil and seven of its basic-extractable metabolites, isolated from the urine of dogs administered oral racemic verapamil, was determined by HPLC, using an Ultron OVM (ovomucoid) column. One dog was given oral (R)-verapamil alone in order to discriminate the (R)- and (S)-enantiomers of the metabolites. Structure identification of the isolated verapamil metabolites was accomplished using a combination of HPLC-MS and FAB-MS-MS techniques. Six of the urinary verapamil metabolites, including verapamil, were predominantly of the (R)-configuration, whereas one of the metabolites was predominantly in the (S)-form. The remaining isolated metabolite was comprised of approximately equal amounts of the two forms.     

Determination of warfarin enantiomers and hydroxylated metabolites in human blood plasma by liquid chromatography with achiral and chiral separation

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (alpha(1) acid glycoprotein - AGP) and achiral (C(18)) column. Both methods were consistent (R.S.D.<6.9% for warfarin enantiomers and<8.9% for monohydroxylated metabolites) and linear (r>0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4'-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.     

Development and validation of a high performance liquid chromatography-tandem mass spectrometry for the determination of etodolac in human plasma

A simple and specific method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of etodolac in human plasma, using indomethacin as an internal standard (IS). Chromatographic separation was performed isocratically using a Capcellpak MGII C(18) column with 65% acetonitrile and 35% water containing 10mM ammonium formate (adjusted to pH 3.5 with formic acid). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 287.99>172.23 for etodolac and m/z 357.92>139.01 for IS. The method was validated to determine its selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. The limit of quantitation (LLOQ) was 0.1microg/mL with a relative standard deviation of less than 15%. The devised method provides an accurate, precise and sensitive tool for determining etodolac levels in plasma.     

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-β-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2μg/ml. The calibration curves are linear from 0.4 to 16 μg/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.     

Etodolac: effect on prostaglandin concentrations in gastric mucosa of rats

Etodolac is a structurally novel compound exhibiting potent analgesic and anti-inflammatory activity in laboratory animals and man, with excellent G. I. tolerance. Like other nonsteroidal anti-inflammatory drugs (NSAIDs) etodolac inhibits prostaglandin (PG) biosynthesis. In view of the cytoprotective role of PGE2, we have investigated in normal rats the effect of etodolac on the gastric mucosal concentration of PGE2 as well as of 6-keto-PGF1 alpha, the stable metabolite of prostacyclin; naproxen and piroxicam served as reference NSAIDs. The orally effective anti-inflammatory doses in the chronic arthritic rat model (3 mg/kg for etodolac and naproxen; 0.5 mg/kg for piroxicam), and their arbitrarily selected multiples of 10 were used. Rats were killed at 1, 2, 6 and 24 hr after single doses and the PG concentrations were measured by RIA. With the low dose, 2 and 6 hr after dosing, etodolac diminished the PGE2 concentration by 20-25% (vs control) while naproxen and piroxicam caused a fall of 53-65%; the difference between etodolac and the untreated control group is not statistically significant but the difference between etodolac and both piroxicam and naproxen is significant (p less than 0.001). At the high doses, the lowering in PGE2 was similar after all three drugs, i.e. about 70% at 1 and 2 hr; 50% at 6 hr, and 20-50% at 24 hr after dosing. Except for the consistently smaller reduction of concentrations after etodolac, the effects on 6-keto-PGF1 alpha concentration followed a similar pattern but the differences are not significant. The lack of the G.I. irritation of etodolac in rats and man at therapeutically effective doses may be attributed to the benefits of the relatively short-lived and slight decrease in gastric mucosal PGE2 concentrations found in this study.     

Enantioselective determination of the hydroxylated metabolites of mexiletine in human plasma

Pre-column derivatization with o-phthaldialdehyde and N-acetyl-l-cysteine was used for liquid-chromatographic diastereomeric resolution of p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM), metabolites of mexiletine formed by aromatic and aliphatic hydroxylation, respectively. The resulting diastereomeric derivatives were resolved on a C₁₈ column and monitored by fluorescence detection. The diastereomeric elution order for both metabolites was determined on the basis of the circular dichroism spectra of each eluted fraction. Plasma samples (500 μl) showed recoveries greater than 75% for both the metabolites. Calibration curves in plasma samples were linear over the concentration ranges 10–500 and 20–1,000 ng/ml for each enantiomer of PHM and HMM, respectively. The limits of quantitation were found to be 10.0 and 5.0 ng/ml for both enantiomers of PHM and HMM. The within-day and between-day coefficients of variation were less than 10%. The assay was shown to be suitable for a pharmacokinetic study performed in a patient with ventricular arrhythmias following the short-term oral treatment of 200 mg t.i.d. of racemic mexiletine hydrochloride. Chirality 9:732–738, 1997. © 1997 Wiley-Liss, Inc.     

Temporal relationships of the anti-inflammatory effect of etodolac in the adjuvant arthritic rat

Using the curative model of adjuvant arthritis, adult male Sprague-Dawley rats were treated with vehicle or etodolac (1, 3, and 8 mg/kg/day, po) for 9 days. Rats were sacrificed after 1, 2, 4, or 9 daily doses, and paw volume, PGE2 concentrations, and N-acetyl-beta-D-glucosaminidase (NAG) activity were determined in the left adjuvant-injected hindpaws. All three doses of etodolac caused a significant decrease in PGE2 concentrations after the first dose, and the decreases persisted for 2, 4, and 9 days of treatment, respectively. In rats given four daily doses of 3 and 8 mg/kg/day of etodolac, the paw volume was significantly decreased by about 50%, compared with that of the arthritic controls. A significant decrease in NAG activity was observed only after nine daily doses of 8 mg/kg/day etodolac. The sequence of anti-inflammatory events manifested following etodolac treatment would appear to be an initial inhibition of PGE2 synthesis, followed by resorption of fluid, and then by a reduction in macrophage infiltration.     

Determination of the stereoselective aspects in in-vitro and in-vivo metabolism of the analgesic meptazinol by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method to separate meptazinol and its phase I metabolites has been developed using a LiChrosper 100 CN column and a mobile phase of trimethylammoniumacetate buffer (pH 5.5)-acetonitrile-methanol. Quantification of meptazinol and N-desmethylmeptazinol in biological samples was achieved by extraction with organic solvents and chromatographic analysis (detection limit 0.4 and 0.25 μg/ml, respectively). Afterwards the enantiomeric ratio of the two compounds was determined on a Chiral AGP column with a mobile phase of phosphate buffer (pH 7.0)-acetonitrile (α = 1.29 and 1.49, respectively). In-vitro metabolism data after incubation of the racemic compound and the enantiomers with liver supernatant and microsomes of different species are presented. Finally urinary data of two volunteers after oral application of the racemic drug were determined.     

Stereoselective metabolism of two keto-pyrrol analogues of 8-hydroxy-2-dipropyl-aminotetralin by freshly isolated rat hepatocytes

In vitro metabolism models have been used to determine the relative metabolic stability of novel 2-aminotetralin analogues for the treatment of CNS diseases. Few of these new compounds had been produced as stereochemically pure materials and the achiral analytical techniques, used initially, measured the average metabolic clearance of the two enantiomers of the racemic mixtures. A chiral HPLC assay, using a Chiral AGP column, was developed for two of these racemic analogues and was used to measure the clearance of the enantiomers from suspensions of freshly isolated rat hepatocytes. Robust separations were obtained for both compounds and a number of metabolic products. The enantiomers of only one analogue were subject to different rates of metabolism. The extent of the difference was dependent upon the initial starting concentration of the incubation. The identity of certain metabolites was investigated using LC/MS. The enantioselectivity appears to have arisen from the restricted hydroxylation of one analogue compared to that of the other. © 1996 Wiley-Liss, Inc.     

Enantiopurity and absolute configuration determination of arene cis‐dihydrodiol metabolites and derivatives using chiral boronic acids

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis‐1,2‐diol metabolites, including boronate formation, using racemic or enantiopure (+) and (?)‐2‐(1‐methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono‐ and poly‐cyclic arene cis‐dihydrodiol, cyclohex‐2‐en‐1‐one cis‐diol, heteroarene cis/trans‐2,3‐diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2‐[1‐methoxy‐2,2‐dimethylpropyl]phenyl boronic acid (MDPBA) and 2‐[1‐methoxy‐1‐phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis‐diol metabolites, are presented.     

Microbial transformation of hydroxy metabolites of 1-oxohexyl derivatives of theobromine by Cunninghamella echinulata NRRL 1384

Aim:  The biotransformation of pentoxifylline (PTX), propentofylline (PPT) and their racemic hydroxy metabolites ((±)-OHPTX and (±)-OHPPT) by using the fungus Cunninghamella echinulata NRRL 1384.
Methods and Results:  A fungus Cunninghamella echinulata NRRL 1384 was used to catalyse the ( S )-selective oxidation of the racemic hydroxy metabolites: (±)-OHPTX and (±)-OHPPT and for reduction of PTX and PPT. The first oxidation step appears to be selective and relatively fast while the second reduction step is slower and more selective with PTX. Modifications involving supplementing the bioconversion with glucose give yields and enantiomeric excess (ee) values similar to those obtained without glucose.
Conclusions:  The bioconversion of (±)-OHPTX gave an ( R )-enantiomer (LSF-lisofylline) with a higher enantiopurity (maximum approximately 93% ee) compared to the bioconversion of (±)-OHPPT, when the maximum ee value for ( R )-OHPPT was recorded at 83%.
Significance and Impact of the Study:  The conversion of (±)-OHPTX and (±)-OHPPT using Cunninghamella echinulata can be recognized as a process, which may be recommended as an alternative to the methods used to obtain ( R )-OHPTX and ( R )-OHPPT.     

Stereospecific high-performance liquid chromatographic analysis of tramadol and its O-demethylated (M1) and N,O-demethylated (M5) metabolites in human plasma

A stereospecific method for simultaneous quantitation of the enantiomers of tramadol (T) and its active metabolites O-demethyl tramadol (M1) and O-demethyl-N-demethyl tramadol (M5) in human plasma is reported. After the addition of penbutolol (IS), plasma (0.5 ml) samples were extracted into methyl tert-butyl ether, followed by back extraction into an acidic solution. The separation was achieved using a Chiralpak AD column with a mobile phase of hexanes:ethanol:diethylamine (94:6:0.2) and a flow rate of 1 ml/min. The fluorescence of analytes was then detected at excitation and emission wavelengths of 275 and 300 nm, respectively. All the six enantiomeric peaks of interest plus three unknown metabolite peaks and IS peak (a total of 10 peaks) eluted within 23 min, free from endogenous interference. The assay was validated in the plasma concentration range of 2.5-250 ng/ml, with a lower limit of quantitation of 2.5 ng/ml, for all the six analytes. The extraction efficiency (n=5) was close to 100% for both T and M1 enantiomers and 85% for M5 and IS enantiomers. The application of the assay was demonstrated by simultaneous measurement of plasma concentrations of T, M1, and M5 enantiomers in a healthy volunteer after the administration of 50 mg oral doses of racemic T.     

A Model to Evaluate the Cytotoxicity of the Fungal Volatile Organic Compound 1-octen-3-ol in Human Embryonic Stem Cells

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (“mushroom alcohol”), a major fungal volatile organic compound (VOC) associated with mushroom and mold odors. Using an airborne exposure technique, human embryonic stem cells were exposed for 1 h to different concentrations (0–1,000 ppm) of racemic 1-octen-3-ol and its enantiomers, (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Cytotoxicity was assayed using both the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and the fluorescently tagged Calcein AM-mediated “live and dead” assay. Racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol exhibited greater cytotoxicity to the undifferentiated human cell line H1 than did (R)-(−)-1-octen-3-ol. The inhibition concentration 50 (IC₅₀) values assessed by the MTS assay for racemic 1-octen-3-ol, (S)-(+)-1-octen-3-ol and (R)-(−)-1-octen-3-ol were, respectively, 109, 98, and 258 ppm. These IC₅₀ values were 40–80-fold lower than that of vapor phase toluene, an industrial chemical used as a positive control in this study. Our report pioneers the modeling of human embryonic stem cells as an in vitro approach to screen the potential toxicity of fungal VOCs. Human embryonic stem cells exposed to 1-octen-3-ol, and its enantiomers in the vapor phase showed more cytotoxicity than those exposed to toluene.     

Determination of the enantiomeric composition of mexiletine and its four hydroxylated metabolites in urine by enantioselective capillary gas chromatography

The enantiomers of mexiletine and four of its hydroxylated metabolites were directly separated by capillary gas chromatography using a heptakis(6-O-tert-butyl-dimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin column. The method was applied to the analysis of urine samples from cancer patients who were treated with racemic mexiletine as part of a study of the use of mexiletine in the relief of neuropathic pain. Samples analyzed before and after deconjugation of the urine with β-glucuronidase/arylsulfatase showed a high stereoselectivity in the formation and conjugation of these compounds. © 1996 Wiley-Liss, Inc.     

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(−)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.     

Synthesis and evaluation of uterine relaxant activity for a novel series of substituted p-hydroxyphenylethanolamines

Novel racemic 1-(4-hydroxyphenyl)-2-[3-(substituted phenoxy)-2-hydroxy-1-propyl]aminopropan-1-ol hydrochlorides (9a-h) were synthesized by condensing racemic 1-(p-hydroxyphenyl)-2-aminopropan-1-ol hydrochloride (6) with substituted aryloxymethyloxiranes (8a-h) in DMF in presence of anhydrous potassium carbonate and then reacting with dry hydrogen chloride gas. They were evaluated for uterine relaxant activity in vitro on isolated rat uterus and in vivo in pregnant rats. Their cAMP releasing potential was studied using rat uterus tissue homogenates by cAMP [3H] assay and cardiac stimulant potential was evaluated in dog. All compounds exhibited potent uterine relaxant activity in vitro and produced a significant delay in the onset of labour in pregnant rats; their cAMP releasing potential was higher than isoxsuprine hydrochloride except for 9b and 9c. Finally insignificant cardiac stimulant potential was noted for these compounds when compared to isoxsuprine hydrochloride.     

High-performance liquid chromatographic determination of the enantiomers of cyclophosphamide in serum

A high-performance liquid chromatographic (HPLC) achiral-chiral coupled assay to measure the serum concentration of the enantiomers of cyclophosphamide is described. The R- and S-enantiomers of cyclophosphamide were quantified using a 5-cm-long C₁ Spherisorb 5-μm column, with switching of the eluent containing racemic cyclophosphamide onto a 10-cm-long α₁, acid glycoprotein column. The limit of determination was 1.25 mg l⁻¹ for each enantiomer and the ratio of the enantiomers over the range 2.5 to 100 mg l⁻¹ was 1. Serum enantiomer concentrations in blood samples taken from patients receiving 0.30 to 0.75 g m⁻² of intravenous racemic cyclophosphamide could be measured at least three half-lives post dose. In six patients no significant difference in the clearance of R- and S-cyclophosphamide was found.     

The effect of etodolac on bile salt and histamine-mediated gastric mucosal injury in the rat

The effect of the selective cyclo-oxygenase-type-2 (COX-2) inhibitor etodolac on gastric mucosal integrity and gastric acid secretion was investigated in the rat. Etodolac was given in doses comparable with those being used in man for therapy of rheumatic conditions. The effect of etodolac was studied in the presence of a mild barrier breaker and in the presence of increased rates of endogenous acid secretion. In conscious pylorus-ligated rats, etodolac given intragastrically in 16 or 32 mg /kg for 3 h did not by itself give rise to visible gastric mucosal injury. Etodolac, however, exacerbated gastric mucosal injury evoked by intragastric application of acidified sodium taurocholate (5 mM in 150 mM HCl) in a dose-dependent manner. This effect of edotolac was independent of changes in gastric acid secretory responses. In rats whose gastric acid secretion was stimulated by intraperitoneal histamine (5 mg/kg), and etodolac (given i.g. in doses of 16 or 32 mg/kg) also increased gastric mucosal injury caused by histamine dose-dependently in the 3-h pylorus-ligated rats. Etodolac decreased gastric mucus in the saline- and in the sodium taurocholate-treated rats. In urethane-anaesthetized acute gastric fistula rats, intragastric etodolac (32 mg/kg) did not modify basal gastric acid secretion. Our data suggest that etodolac, a selective COX-2 inhibitor, impairs gastric mucosal resistance and can exacerbate gastric mucosal injury caused by other mucosal barrier breaking agents. Cyclooxygenase type-2 thus contributes to the gastric mucosal defences.     

In Vitro and In Vivo Characteristics of a Thermogelling Rectal Delivery System of Etodolac

Rectal etodolac–Poloxamer gel systems composed of Poloxamer and bioadhesive polymers were developed and evaluated. Hydroxypropylmethyl cellulose, poly)vinyl) pyrrolidone, methyl cellulose, hydroxyethylcellulose, and carbopol were examined as mucoadhesive polymers. The characteristics of the rectal gels differed according to the properties of mucoadhesive polymers. The physicochemical properties such as gelation temperature, gel strength, and bioadhesive force of various formulations were investigated. The analysis of release mechanism showed that the release of etodolac was proportional to the square root of time, indicating that etodolac might be released from the suppositories by Fickian diffusion. The anti-inflammatory effect of etodolac–Poloxamer gel system was also studied in rats. Moreover, liquid suppository of etodolac did not cause any morphological damage to the rectal tissues. These results suggested that in situ gelling liquid suppository with etodolac and mucoadhesive polymer was a physically safe, convenient, and effective rectal dosage form for etodolac.