Etodolac Enantiomers Research Articles

Enantioselective analysis of etodolac in human plasma by LC–MS/MS: Application to clinical pharmacokinetics

Etodolac is a non-steroidal anti-inflammatory drug with preferential inhibition of cyclooxigenase-2 and is widely used in the management of pain in patients with inflammatory arthritis. Etodolac is available as a racemic mixture of (−)-(R)-Etodolac and (+)-(S)-Etodolac; cyclooxigenases inhibition is attributed to (+)-(S)-Etodolac. According to our knowledge, this is the first method for determination of etodolac enantiomers in plasma using LC–MS/MS. Plasma extraction were performed with 25μL of plasma and 1mL of n-hexane:ethyl acetate (95:5); racemic ibuprofen was used as internal standard. Resolution of enantiomers were performed in a Chiralcel®OD-H column; deprotonated [M-H]− and their respective ion products were monitored at transitions of 286>242 for etodolac enantiomers and 205>161 for ibuprofen. The quantitation limit was 3.2ng/mL for both enantiomers in plasma. The method was applied to study the pharmacokinetics of etodolac enantiomers after the administration of a 300 and 400mg dose of racemic drug to a healthy volunteer. Analysis of plasma samples showed higher plasma concentration of (−)-(R)-Etodolacfor both doses (300mg dose: AUC0–∞49.80 versus 4.55ugh/mL;400mg dose: AUC0–∞ 63.90 versus 6.00ugh/mL) with an (R)-(+)/(S)-(−) ratio of approximately 11.

Determination of Competitive Adsorption Isotherm of the Etodolac Enantiomers Using Inverse Method

The competitive adsorption isotherm of etodolac enantiomers on a cellulose carbamates-based chiral stationary phase (Chiralcel OD) at 25°C was determined in this study by the inverse method (IM). An equilibrium dispersive (ED) model combined with bi-Langmuir adsorption model was used in predicting the elution profiles. The adsorption isotherm model with 5 parameters was determined by fitting the two overloaded band profiles. The non-dominated sorting genetic algorithm (NSGA-II) and Levenberg-Marquardt algorithm (LMA) were crucial tools and utilities in this process. Suitable model parameters have been obtained and the experimental results were in good agreement with the model predictions.

Enantioselective HPLC-DAD method for the determination of etodolac enantiomers in tablets, human plasma and application to comparative pharmacokinetic study of both enantiomers after a single oral dose to twelve healthy volunteers

An enantioselective high performance liquid chromatographic method with diode array detection (HPLC-DAD) was developed and validated for the determination of etodolac enantiomers in tablets and human plasma. Enantiomeric separation was achieved on a Kromasil Cellucoat chiral column (250mm×4.6mm i.d., 5µm particle size) using a mobile phase consisting of hexane: isopropanol: triflouroacetic acid (90:10:0.1v/v/v) at a flow rate of 1.0mLmin−1. The chromatographic system enables the separation of the two enantiomers and the internal standard within a cycle time of 8min. The resolution between the two enantiomers was 4.25 and the resolution between each enantiomer and the internal standard was more than 2.0. Detection was carried out at 274nm, and the purity assessment was performed using a photodiode array detector. Solid phase extraction technique using C-18 cartridge was applied to extract the analytes from the plasma samples, and the percentage recovery was more than 95% for the lower quantification limit. The method has been validated with respect to selectivity, linearity, accuracy and precision, robustness, limit of detection and limit of quantification. The validation acceptance criteria were met in all cases. The linearity range for the determination of each enantiomer in human plasma was 0.4–30.0µgmL−1 and the limits of quantification of R-etodolac and S-etodolac were 0.20 and 0.19µgmL−1, respectively. The validated method was successfully applied to the determination of etodolac enantiomers in tablets and to a comparative pharmacokinetic study of the two enantiomers after the administration of 300mg single oral dose etodolac racemate tablets to twelve healthy volunteers.

Analysis of chiral non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac binding with HSA

The protein binding of non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac with human serum albumin (HSA) was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. S-(–)-1-(1-naphthyl)-ethylamine (S-NEA) was utilized as chiral derivatization reagent and pre-column derivatization RP-HPLC method was established for the separation and assay of the three pairs of enantiomer. The method had good linear relationship over the investigated concentration range without interference. The average extraction efficiency was higher than 85% in different systems, and the intra-day and inter-day precisions were less than 15%. In serum albumin, the protein binding of etodolac enantiomers showed significant stereoselectivity that the affinity of S-enantiomer was stronger than R-enantiomer, and the stereoselectivity ratio reached 6.06; Flurbiprofen had only weak stereoselectivity in HSA, and ketoprofen had no stereoselectivity at all. Scatchard curves showed that all the three chiral drugs had two types of binding sites in HSA.

The Enantiomers of Etodolac, a Racemic Anti-inflammatory Agent, Play Different Roles in Efficacy and Gastrointestinal Safety

The anti-inflammatory agent etodolac is used worldwide and it has a good gastrointestinal safety profile. Etodolac consists of two enantiomers, S- and R-etodolac. Here, we investigated the beneficial activities of racemic etodolac and its enantiomers. First, we compared S- and R-etodolac in terms of their inhibition of cyclooxygenase (COX) activity in vitro and their suppression of paw swelling in adjuvant-induced arthritic rats. The COX-2 inhibitory and anti-inflammatory effects of etodolac were found to be due to the S-enantiomer. We previously reported that etodolac attenuates allodynia in a mouse model of neuropathic pain by a COX-2-independent mechanism [N. Inoue et al., J. Pharmacol. Sci., 109, 600-605 (2009)]. In the present study, we showed that the anti-allodynic effects of etodolac in mice were also due to the S-enantiomer. In addition, we investigated the ulcerogenic activity of racemic etodolac and its enantiomers. At high doses, racemic etodolac showed a lower gastric lesion index in rats than the equivalent dose of S-etodolac. In contrast, R-etodolac showed no ulcerogenic activity and even showed protection against HCl/ethanol-induced gastric damage in rats. In conclusion, S-etodolac exhibited anti-inflammatory effects mediated by COX-2 inhibition and anti-allodynic effects that were independent of COX-2 inhibition, while R-etodolac showed gastroprotective effects that may contribute to the low gastrointestinal toxicity of racemic etodolac. Our results show that each enantiomer plays a different role in the efficacy and gastrointestinal safety of etodolac.

Preparative resolution of etodolac enantiomers by preferential crystallization method

Pure enantiomers are of large interest for several industries. This study was aimed to establish a method for separation of etodolac enantiomers by preferential crystallization after a conglomerate formation of its derivatives. S-(+)-etodolac and R-(-)-etodolac enantiomers were both prepared by classical resolution via crystallization of diastereoisomeric salt with (-)-brucine and (-)-cinchonidine. Enantiomeric purity of etodolac was determined by HPLC method using Chiralcel OD-H column. The pure diastereomeric salt collected from repeated recrystallization was further fractionated by liquid-liquid extraction to pure enantiomers. Etodolac enantiomers were recovered with overall yield more than 20% and the purities were over 99.9%.

ChemInform Abstract: Enantioselective Synthesis of Both Enantiomers of Etodolac (V) via a Lipase‐Catalyzed Kinetic Resolution.

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Determination of enantiomeric impurity of etodolac by capillary electrophoresis using (2-hydroxypropyl)-β-cyclodextrin

A capillary electrophoresis method was developed to determine the impurity of etodolac enantiomers. (2-Hydroxypropyl)-beta-cyclodextrin (HP-beta-CD) was used as a chiral selector and ketoprofen as an internal standard to improve the peak area precision. The seperation of the etodolac enantiomers was achived within 35 min at 15 degrees C and its highest resolution was about 4.0 using phosphate buffer (0.1 M, pH 6.0) with 15 mM HP-beta-CD and UV detection at 225 nm with a reference wavelength at 360 nm. This method allowed determination of 0.2% of (R)-(-)-etodolac in (S)-(+)-etodolac and method validation showed adequate linearity over the required range.

Analysis of flurbiprofen, ketoprofen and etodolac enantiomers by pre-column derivatization RP-HPLC and application to drug–protein binding in human plasma

A stereoselective reversed-phase high-performance liquid chromatography (HPLC) assay to determine the enantiomers of flurbiprofen, ketoprofen and etodolac in human plasma was developed. Chiral drug enantiomers were extracted from human plasma with liquid–liquid extraction. Then flurbiprofen and ketoprofen enantiomers reacted with the acylation reagent thionyl chloride and pre-column chiral derivatization reagent ( S)-(−)-α-(1-naphthyl)ethylamine ( S-NEA), and etodolac enantiomers reacted with S-NEA using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) as coupling agents. The derivatized products were separated on an Agilent Zorbax C 18 (4.6 mm × 250 mm, 5 μm) column with a mixture of acetonitrile–0.01 mol·L −1 phosphate buffer (pH 4.5) (70:30, v/v) for flurbiprofen enantiomers, acetonitrile–0.01 mol·L −1 phosphate buffer (pH 4.5) (60:40, v/v) for ketoprofen enantiomers and methonal–0.01 mol·L −1 potassium dihydrogen phosphate buffer (pH 4.5) (88:12, v/v) for etodolac enantiomers as mobile phase. The flow of mobile phase was set at 0.8 mL·min −1 and the detection wavelength of UV detector was set at 250 nm for flurbiprofen and ketoprofen enantiomers and 278 nm for etodolac enantiomers. The assay was linear from 0.5 to 50 μg·mL −1 for each enantiomer. The inter- and intra-day precision (R.S.D.) was less than 10% and the average extraction recovery was more than 87% for each enantiomer. The limit of quantification for the method was 0.5 μg·mL −1 (R.S.D. < 10%, n = 5). The method developed was used to study the drug–protein binding of flurbiprofen, ketoprofen and etodolac enantiomers in human plasma. The results showed that the stereoselective binding of etodolac enantiomer was observed and flurbiprofen and ketoprofen enantiomers were not.

Enantioselective Synthesis of Both Enantiomers of Etodolac via a Lipase-Catalyzed Kinetic Resolution

The efficient preparation of both enantiomers of etodolac 1 was achieved by the lipase-catalyzed kinetic resolution of the racemic primary alcohol (±)-2 followed by chemoselective oxidation.

Separation of etodolac enantiomers by capillary electrophoresis. Validation and application of the chiral method to the analysis of commercial formulations

Separation of etodolac enantiomers, which exhibit different biological activity and pharmacokinetic profiles, has been achieved using the randomly substituted (2-hydroxy)propyl-beta-cyclodextrin (HP-beta-CD) as chiral selector in capillary electrophoresis. The selection of this CD was made after screening of different CD derivatives of neutral and anionic nature. The effect on the enantioresolution of the buffer concentration and of the degree of substitution (DS) and concentration of the CD as well as of instrumental parameters, such as the capillary temperature and the separation voltage, were studied. The highest resolution of etodolac enantiomers was around 2.5 using 100 mM phosphate buffer (pH 7.0) with 20 mM HP-beta-CD (DS approximately 4.2) and UV detection at 225 (10) nm with a reference wavelength at 360 (50) nm. Validation of the chiral method in terms of selectivity, linearity, precision (instrumental repeatability, method repeatability, intermediate precision), and the limits of detection and quantitation allowed to evaluate its quality to the analysis of etodolac enantiomers in different pharmaceutical preparations containing racemic etodolac.

New enzymatic and chemical approaches to enantiopure etodolac

(+)- and (−)-etodolac enantiomers were prepared both by classical resolution via crystallisation of diastereoisomeric salts with (+) and (−)-α-methylbenzylamine, and by suitable manipulation of derivatives (−)- 3- and (+)- 4, obtained by lipase-catalysed kinetic resolution of racemic 3 X-ray diffraction analysis of the 4-bromobenzoate derivative of (+)- 3, obtained from enantiopure acetate (+)- 4, allowed us to determine the absolute (R) configuration of (−)-etodolac.

Albumin binding sites for etodolac enantiomers

Non-steroidal anti-inflammatory drugs (NSAIDs) are strongly bound to human serum albumin (HSA), mainly to sites I and II. The aim of this study was to characterize the binding site(s) of etodolac enantiomers under physiological conditions (580 microM HSA) using equilibrium dialysis. The protein binding of etodolac enantiomers, alone or in various ratios, was studied in order to evaluate the potential competition between them. Our results showed that (S)-etodolac was more strongly bound to HSA than (R)-etodolac. The displacement of one enantiomer by its antipode was observed only at high concentrations of the competitor, and was more pronounced for (S)-form. Displacement studies of the enantiomers by specific probes of sites I and II of albumin, dansylamide, and dansylsarcosine, respectively, showed that (R)-etodolac was slightly displaced by both these probes whereas the free concentration of (S)-etodolac increased markedly in the presence of dansylsarcosine. Moreover, the binding of ligands to sites I and II is usually affected by alkaline pH, by chloride ions, and by fatty acids. For etodolac, the presence of 0.1 and 1 M chloride ions and increasing pH (5.5-9) decreased the binding of both enantiomers. The same result was obtained with addition of octanoic acid. Conversely, the addition of oleic, palmitic, or stearic acid to the protein solution increased the binding of (R)-etodolac, but decreased that of its antipode. All these findings suggest (R)- and (S)-etodolac interact mainly with site II of HSA, and that the (R)-isomer is also bound to site I under physiological conditions.

Pharmacokinetics of etodolac enantiomers in the rat after administration of phenobarbital or cimetidine.

The influences of phenobarbital and cimetidine on the pharmacokinetics of etodolac enantiomers were studied in male Sprague-Dawley rats. Phenobarbital caused significant reductions in the AUC of both the active S-enantiomer (24%) and the inactive R-enantiomer of etodolac (26%), and an increase in the urinary excretion of glucuronidated S-etodolac. In bile duct-cannulated rats the initial biliary recoveries of the acyl-glucuronidated etodolac enantiomers were not affected by phenobarbital. In vitro, phenobarbital caused no changes in the hepatic microsomal net glucuronidation of etodolac enantiomers after phenobarbital, although it did result in significant increases in the apparent oxidative metabolism of both enantiomers. Therefore, phenobarbital seems to cause an enhanced CL of etodolac by induction of hepatic non-conjugative metabolism. Cimetidine had no discernible effect on the pharmacokinetics of etodolac enantiomers.

Stereoselective binding of etodolac to human serum albumin.

The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1 to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the (R)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations, S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and II, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site II.

Enantioselective Pharmacokinetics of Etodolac in the Rat: Tissue Distribution, Tissue Binding, and In Vitro Metabolism

The nonsteroidal anti-inflammatory agent etodolac (ET) exhibits stereoselectivity in its pharmacokinetics following administration to humans and rats. To underline the factors responsible for this stereoselectivity, the tissue distribution, in vitro tissue binding, and microsomal metabolism of ET enantiomers were studied in the rat. Following iv administration of racemic ET, the S:R AUC ratios in tissues were stereoselective, and different from that in plasma. Binding of enantiomers to tissues was stereoselective, although it did not relate well with in vivo tissue distribution. Rather, the tissue distribution of enantiomers appeared to be better explained by the unbound fractions of enantiomers in plasma. With respect to in vitro glucuronidation by liver microsomes, the Vmax of S-ET was 3.4-fold greater than that of R-ET; the enantiomers possessed similar Km. There appeared to be stereoselectivity in the oxidative metabolism of ET enantiomers by liver and kidney microsomes, in favor of the R-enantiomer. The lower AUC in rat plasma of pharmacologically active S-ET as compared with its antipode is due to its relatively greater distribution to tissues, owing to a lesser degree of binding to plasma proteins, and to its higher rate of glucuronidation.

Stereoselective Disposition of Etodolac Enantiomers in Synovial Fluid

The synovial fluid (SF) uptake of the chiral nonsteroidal anti-inflammatory drug, etodolac, was studied in six arthritic patients, 2 hours (n = 1) or 12 hours (n = 5) after a single 200 mg dose of racemate. Marked stereoselectivity was seen in both SF and plasma; concentrations of pharmacologically inactive R-etodolac were up to 10-fold greater than active S-etodolac. Concentrations of S-etodolac were greater in SF than in plasma (SF:plasma ratio = 1.98 +/- 0.8): No such difference was seen for R-etodolac (SF:plasma = 0.91 +/- 0.3). Considerable concentrations of conjugated enantiomers were present in SF. In vitro equilibrium dialysis studies in drug-spiked samples showed that the unbound fraction of both enantiomers in SF was greater than in plasma; both fluids bound R more extensively than S etodolac. Therapeutically active S-etodolac has greater concentrations in synovial fluid than plasma during the post-distributive phase, which may be of possible clinical relevance.

Application of a stereospecific high-performance liquid chromatography assay to a pharmacokinetic study of etodolac enantiomers in humans.

An HPLC assay suitable for pharmacokinetic analysis of enantiomers of etodolac [(+/-)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b] indole-1-acetic acid] was developed. Following addition of internal standard (IS), (+/-)-2-(4-benzoylphenyl)butyric acid, the constituents were extracted from the specimen into a mixture of isooctane:isopropanol (95:5). The organic layer was evaporated and the drug and IS were sequentially derivatized with ethyl chloroformate and iota(-)-alpha-phenylethylamine. The diastereoisomers thus formed were extracted and chromatographed on a normal-phase column, with a mobile phase consisting of hexane:ethyl acetate:isopropanol (85:15:0.2) at a flow rate of 2 mL/min. The etodolac diastereoisomers were separated with a resolution factor of 6.4 and detected at a wavelength of 280 nm. Excellent linear relationships were found between the peak area ratios (etodolac:IS) and the plasma and urine concentrations (0.2-20 mg/L), with intra- and interday variations of less than 10.1%. The assay was applied to a preliminary pharmacokinetic study following seven repeated oral administrations of 200 mg/12 h of racemic etodolac to two healthy subjects. The plasma concentrations of the active S-(+)-enantiomer were considerably less than those of the inactive antipode (AUC S:R, 2.5:30.9 mg.L-1.h-1) due to a greater volume of distribution of the latter (S, 101 and 135 L versus R, 24 and 17 L). Considerable concentrations of conjugated enantiomers were also found in plasma (AUC conjugated: intact: S, 1.1; R, 0.23).

Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities.

The peroxidase activity of prostaglandin H (PGH) synthase catalyzes reduction of 5-phenyl-4-pentenyl hydroperoxide to 5-phenyl-4-pentenyl alcohol with a turnover number of approximately 8000 mol of 5-phenyl-4-pentenyl hydroperoxide/mol of enzyme/min. The kinetics and products of reaction establish PGH synthase as a classical heme peroxidase with catalytic efficiency similar to horseradish peroxidase. This suggests that the protein of PGH synthase evolved to facilitate peroxide heterolysis by the heme prosthetic group. Comparison of an extensive series of phenols, aromatic amines, beta-dicarbonyls, naturally occurring compounds, and nonsteroidal anti-inflammatory drugs indicates that considerable differences exist in their ability to act as reducing substrates. No correlation is observed between the ability of compounds to support peroxidatic hydroperoxide reduction and to inhibit cyclooxygenase. In addition, the resolved enantiomers of MK-410 and etodolac exhibit dramatic enantiospecific differences in their ability to inhibit cyclooxygenase but are equally potent as peroxidase-reducing substrates. This suggests that there are significant differences in the orientation of compounds at cyclooxygenase inhibitory sites and the peroxidase oxidation site(s). Comparison of 5-phenyl-4-pentenyl hydroperoxide reduction by PGH synthase and horseradish peroxidase reveals considerable differences in reducing substrate specificity. Both the cyclooxygenase and peroxidase activities of PGH synthase inactivate in the presence of low micromolar amounts of hydroperoxides and arachidonic acid. PGH synthase was most sensitive to arachidonic acid, which exhibited an I50 of 0.6 microM in the absence of all protective agents. Inactivation by hydroperoxides requires peroxidase turnover and can be prevented by reducing substrates. The I50 values for inactivation by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid are 4.0 and 92 microM, respectively, in the absence and presence of 500 microM phenol, a moderately good reducing substrate. The ability of compounds to protect against hydroperoxide-induced inactivation correlates directly with their ability to act as reducing substrates. Hydroquinone, an excellent reducing substrate, protected against hydroperoxide-induced inactivation when present in less than 3-fold molar excess over hydroperoxide. The presence of a highly efficient hydroperoxide-reducing activity appears absolutely essential for protection of the cyclooxygenase capacity of PGH synthase. The peroxidase activity is, therefore, a twin-edged sword, responsible for and protective against hydroperoxide-dependent inactivation of PGH synthase.(ABSTRACT TRUNCATED AT 400 WORDS)

Stereoselective gas chromatographic analysis of etodolac enantiomers in human plasma and urine

Stereoselective gas chromatographic analysis of etodolac enantiomers in human plasma and urine