Release of salbutamol sulphate and ketoprofen enantiomers from matrices containing HPMC and cellulose derivatives

We studied the release of salbutamol and ketoprofen enantiomers from HPMC K100M matrices containing two types of cellulose derivatives: cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (2,3-dichlorophenylcarbamate), chiral excipients used as stationary phases for liquid chromatography. These matrices provided an extended release of both drugs. Ketoprofen release from formulations elaborated with cellulose tris (2,3-dichlorophenylcarbamate) was by anomalous transport, because the value of n (release exponent of the diffusion equation) ranged between 0.60-0.68, whereas for all other formulations the value of exponent n ranged from 0.50-0.54. The drug thus diffuses through the matrix and is released following a quasi-Fickian diffusion mechanism (stereoselective process). The matrices preferentially retained R-salbutamol and S-ketoprofen and cellulose tris (3,5-dimethylphenylcarbamate) showed more capacity of chiral discrimination for both drugs than cellulose tris (2,3-dichlorophenylcarbamate). Moreover, we observed that stereoselectivity is dependent on the amount of chiral excipient in the formulation. Diffusion tests confirmed the chiral interaction between drugs and cellulose derivatives observed in the dissolution assays except for matrices elaborated with ketoprofen and cellulose tris (2,3-dichlorophenylcarbamate), where the low stereoselectivity observed with the matrices is due to the presence of HPMC K100M. We conclude that the inclusion of these cellulose derivatives in HPMC matrices does not result in a relevant stereoselectivity with respect to the two drugs studied.     

Stereoselective determination of benalaxyl in plasma by chiral high-performance liquid chromatography with diode array detector and application to pharmacokinetic study in rabbits

A chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of benalaxyl (BX) in rabbit plasma. Good separation was achieved at 20 degrees C using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase, a mixture of n-hexane and 2-propanol (97:3) as mobile phase at a flow rate of 1.0 ml/min. The assay method was linear over a range of concentrations (0.25-25 microg/ml) in plasma and the mean recovery was greater than 90% for both enantiomers. The limits of quantification and detection for both enantiomers in plasma were 0.25 and 0.1 microg/ml, respectively. Intra- and interday relative standard deviations (RSDs) did not exceed 10% for three-tested concentrations. The method was successfully applied to pharmacokinetic studies of BX enantiomers in rabbits. The result suggested that the pharmacokinetics of BX enantiomers was stereoselective in rabbits.     

Stereoselective metabolism of benalaxyl in liver microsomes from rat and rabbit

Benalaxyl (BX), methyl‐N‐phenylacetyl‐N‐2,6‐xylyl alaninate, is a potent acylanilide fungicide and consist of a pair of enantiomers. The stereoselective metabolism of BX was investigated in rat and rabbit microsomes in vitro. The degradation kinetics and the enantiomer fraction (EF) were determined using normal high‐performance liquid chromatography with diode array detection and a cellulose‐tris‐(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase (CDMPC‐CSP). The t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rat liver microsomes were 22.35 and 10.66 min of rac‐BX and 5.42 and 4.03 of BX enantiomers. However, the t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rabbit liver microsomes were 11.75 and 15.26 min of rac‐BX and 5.66 and 9.63 of BX enantiomers. The consequence was consistent with the stereoselective toxicokinetics of BX in vitro. There was no chiral inversion from the (?)‐R‐BX to (+)‐S‐BX or inversion from (+)‐S‐BX to (?)‐R‐BX in both rabbit and rat microsomes. These results suggested metabolism of BX enantiomers was stereoselective in rat and rabbit liver microsomes. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Input rate-dependent stereoselective pharmacokinetics: Effect of pulsatile oral input

Computer simulation was used to test the effects of pulsatile oral input on the stereoselectivity in the area under the blood concentration–time curves (AUCs) of the enantiomers of racemic drugs. The effects of input rate determinants, namely, dose, dosage interval, and formulation on the stereoselectivity were investigated under both steady-state and nonsteady-state conditions. Simulations were carried out for drugs undergoing Michaelis–Menten hepatic metabolism with different enantiomeric maximum velocity (V_max) or constant (K_m) values. With pulsatile input, the enantiomeric AUC ratios of both types of drugs were dependent on all the determinants of input rate. However, in most cases, the direction of input rate-dependent changes in the enantiomeric AUC ratios for drugs with different enantiomeric V_max was opposite of that for drugs with different enantiomeric K_m. The direction and magnitude of changes in the enantiomeric AUC ratios were also dependent on the selected dose, dosage interval, and formulation. Further, different conclusions could be reached based on the nonsteady-state and steady-state data. Additional simulations were then performed to test the effects of input rate-dependent stereoselective pharmacokinetics on the bioequivalence of chiral drugs with nonlinear metabolism. These simulations suggested that bioequivalence studies based on the racemic drug measurement may result in erroneous conclusions for the individual enantiomers. The results of this study may be used as a tool for the design of experiments to test the input rate dependence of stereoselective pharmacokinetics and bioequivalence of racemic drugs in animals and humans. © 1994 Wiley-Liss, Inc.     

Synthesis of regioselectively substituted cellulose derivatives and applications in chiral chromatography

Various cellulose-2,3-bis-arylcarbamate-6-O-arylesters and cellulose-2,3-bis-arylester-6-O-arylcarbamates, designed to test the possible combined effects of the known tris-arylcarbamate and tris-arylester classes, were synthesized with high regioselectivity at O-C(6), and their use as CSP s in liquid chromatography for enantiomeric separations was investigated. The separations obtained with the synthesized CSP s were compared to the separations achieved on a self-packed reference column, consisting of cellulose-tris-(3,5-dimethylphenyl-carbamate) as CSP standard. Among the synthesized, regioselectively substituted cellulose derivatives, 2,3-bis-O-(3,5-dimethylphenylcarbamate)-6-O-benzoate-cellulose and 2,3-bis-O-(benzoate)-6-O-(3,5-dichlorophenylcarbamate)-cellulose gave the best CSP s for the separation of the test racemates. CSP s from regioselectively substituted cellulose derivatives seem to exhibit higher selectivities than cellulose-tris-(3,5-dimethylphenylcarbamate) for certain classes of racemic compounds. Chirality 10:294–306, 1998. © 1998 Wiley-Liss, Inc.     

Enantiomeric Separation of Original Heterocyclic Organophosphorus Compounds in Supercritical Fluid Chromatography

New and original heterocyclic α-enamido phosphine chiral solutes were prepared: four structurally similar racemates with the chirality center placed on the phosphorus atom, and four other related pairs of enantiomers with chirality borne by the carbon atoms of the phospholane ring. The structural variations were placed on an aliphatic heterocycle (six- or seven-member rings) and on the carbamate function (methyl or t-butyl). Their separation was achieved on a commercial cellulose tris-(3,5-dimethylphenylcarbamate) stationary phase (Lux Cellulose-1, Phenomenex) in supercritical fluid chromatography (SFC). The effects of molecular structure on SFC retention and enantioresolution were studied. Among these eight pairs of enantiomers, some reversal of elution order between similar compounds was observed. The effect of changing the organic solvent (methanol and ethanol) and its proportion (between 5 and 40%) in the mobile phase was investigated. Retention data were collected over the temperature range 0–50 °C, and the results interpreted from thermodynamic aspects. Chirality, 2013. © 2013 Wiley Periodicals, Inc.     

Stereoselectivity and enantiomer-enantiomer interactions in the binding of ibuprofen to human serum albumin

Binding of ibuprofen (IB) enantiomers to human serum albumin (HSA) was studied using a chiral fluorescent derivatizing reagent, which enabled the measurement of IB enantiomers at a concentration as low as 5 × 10⁻⁸ M. Scatchard analyses revealed that there were two classes of binding sites for both enantiomers. For the high affinity site, the number of the binding sites was one for both enantiomers, and the binding constant of R-IB was 2.3-fold greater than that of S-IB. The difference in the affinity at the high affinity site may result in the stereoselective binding of IB enantiomers at therapeutic concentrations. It was confirmed that the high affinity site of IB enantiomers is Site II (diazepam binding site) by using site marker ligands. Also, significant enantiomer-enantiomer interactions were observed in the binding. The binding data were quantitatively analyzed and a binding model with an assumption of competitive interactions only at the high affinity site simulated the binding characteristics of IB enantiomers fairly well. Chirality 9:643–649, 1997. © 1997 Wiley-Liss, Inc.     

Stereoselective pharmacokinetics of ibuprofen in rats: effect of enantiomer-enantiomer interaction in plasma protein binding

Stereoselective pharmacokinetics of ibuprofen (IB) enantiomers were studied in rats. Unidirectional conversion from R-ibuprofen (R-IB) to S-ibuprofen (S-IB) was observed following intravenous administration. S-IB concentrations in plasma following racemate administration were simulated according to a conventional compartmental model using the parameters obtained after the administration of individual enantiomers, and resulted in overestimation of S-IB concentrations. Binding of IB enantiomers measured in rat plasma was stereoselective, the binding of R-IB being more favorable than that of S-IB. Moreover, there are interactions between IB enantiomers in binding, which may cause the increase of distribution volumes of IB enantiomers in the presence of their antipodes. Hence simulated S-IB concentrations according to a conventional compartment model were significantly greater than those observed. Indeed, when the enantiomer-enantiomer interactions were taken into account, simulation of S-IB concentrations in plasma following racemate administration was in good agreement with observed values. Therefore, interactions between stereoisomers as well as dispositional stereoselectivity have to be considered when pharmacokinetics of stereoisomers after administration of the racemate are compared to those after administration of individual isomers. Chirality 9:354-361, 1997. © 1997 Wiley-Liss, Inc.     

Stereoselective Degradation of Chiral Fungicide Myclobutanil in Rat Liver Microsomes

Myclobutanil, (RS)‐2‐(4‐chlorophenyl)‐2‐(1H‐1, 2, 4‐triazol‐1‐ylmethyl)hexanenitrile is a broad‐spectrum systemic triazole fungicide which consists of a pair of enantiomers. The stereoselective degradation of myclobutanil was investigated in rat liver microsomes. The concentrations of myclobutanil enantiomers were determined by high‐performance liquid chromatography (HPLC) with a cellulose‐tris‐(3,5‐dimethyl‐phenylcarbamate)‐based chiral stationary phase (CDMPC‐CSP) under reversed phase condition. The t_1/2 of (+)‐myclobutanil is 8.49 min, while the t_1/2 of (–)‐myclobutanil is 96.27 min. Such consequences clearly indicated that the degradation of myclobutanil in rat liver microsomes was stereoselective and the degradation rate of (+)‐myclobutanil was much faster than (–)‐myclobutanil. In addition, significant differences between two enantiomers were also observed in enzyme kinetic parameters. The V_max of (+)‐myclobutanil was about 4‐fold of (–)‐myclobutanil and the CL_int of (+)‐myclobutanil was three times as much as (–)‐myclobutanil after incubation in rat liver microsomes. Corresponding consequences may shed light on the environmental and ecological risk assessment for myclobutanil and may improve human health. Chirality 26:51–55, 2013. © 2013 Wiley Periodicals, Inc.     

HPLC‐Fluorescence Method for the Enantioselective Analysis of Propranolol in Rat Serum Using Immobilized Polysaccharide‐Based Chiral Stationary Phase

A stereoselective high‐performance liquid chromatographic (HPLC) method was developed and validated to determine S‐(?)‐ and R‐(+)‐propranolol in rat serum. Enantiomeric resolution was achieved on cellulose tris(3,5‐dimethylphenylcarbamate) immobilized onto spherical porous silica chiral stationary phase (CSP) known as Chiralpak IB. A simple analytical method was validated using a mobile phase consisted of n‐hexane‐ethanol‐triethylamine (95:5:0.4%, v/v/v) at a flow rate of 0.6 mL min^‐1 and fluorescence detection set at excitation/emission wavelengths 290/375 nm. The calibration curves were linear over the range of 10–400 ng mL^‐1 (R = 0.999) for each enantiomer with a detection limit of 3 ng mL^‐1. The proposed method was validated in compliance with ICH guidelines in terms of linearity, accuracy, precision, limits of detection and quantitation, and other aspects of analytical validation. Actual quantification could be made for propranolol isomers in serum obtained from rats that had been intraperitoneally (i.p.) administered a single dose of the drug. The proposed method established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology. Molecular modeling studies including energy minimization and docking studies were first performed to illustrate the mechanism by which the active enantiomer binds to the β‐adrenergic receptor and second to find a suitable interpretation of how both enantiomers are interacting with cellulose tris(3,5‐dimethylphenylcarbamate) CSP during the process of resolution. The latter interaction was demonstrated by calculating the binding affinities and interaction distances between propranolol enantiomers and chiral selector. Chirality 26:194–199, 2014. © 2014 Wiley Periodicals, Inc.     

Determination of panthenol enantiomers in cosmetic preparations using an achiral-chiral–coupled column HPLC system

A new high-performance liquid chromatography (HPLC) method for separation and determination of panthenol enantiomers in hair care products was developed. Two types of detectors, low-wavelength ultraviolet (UV) and polarimetric, were used. Optimized conditions consisted of coupled achiral, amino type, and chiral, amylose tris(3,5-dimethylphenylcarbamate), stationary phases, mixture of n-hexane/ethanol (60:40, v/v) as mobile phase under isocratic conditions and flow rate 0.8 cm³ min⁻¹. The effect of column temperature on retention and resolution of enantiomers was studied. The analysis runtime was 10 minutes, and the average retention times for d - and l -panthenol were 7.10 ±0.1 minutes and 8.21 ±0.2 minutes, respectively. The resolution of enantiomers on coupled achiral-chiral columns was R_s = 2.7. The solid-phase extraction method was employed for extraction and purification of analytes. The validated method was selective, accurate, and linear (R² > .998) over the concentration range of 0.001 to 1.0 mg cm⁻³ for both enantiomeric forms. The limits of detection (LOD) and quantitation (LOQ) of each enantiomer were 0.3 and 1.0 μg cm⁻³, respectively. The results demonstrated the occurrence of d -panthenol in hair care products.     

Establishing bioequivalence of racemic venlafaxine formulations using stereoselective assay method: Is it necessary?

A bioequivalence study for venlafaxine generic formulation was conducted as an open label, balanced, randomized, two‐way crossover, single‐dose study. In this study, a comparison of various pharmacokinetic parameters of venlafaxine hydrochloride 150 mg modified release capsules of Ranbaxy and EFEXOR®‐XR 150 mg capsules of Wyeth, in healthy, adult, male, human subjects under fasting condition was performed to conclude bioequivalence. Venlafaxine and its major active metabolite O‐desmethylvenlafaxine (ODV) are racemates. The “(S)‐(+)” and “(R)‐(−)” enantiomers of venlafaxine and ODV are established as being active. Hence, subject samples were analyzed using nonstereoselective and stereoselective assay methods. Both (S)‐(+) and (R)‐(−) enantiomers of venlafaxine and ODV showed similar absorption and disposition. The 90% confidence intervals for venlafaxine, (R)‐(−)‐venlafaxine as well as (S)‐(+)‐venlafaxine were within acceptance range concluding bioequivalence. The results obtained by stereoselective assay were comparable to the nonstereoselective analysis, as sum of concentrations of (S)‐(+)‐ and (R)‐(−)‐enantiomers of venlafaxine and ODV. The mean (S)‐(+)/(R)‐(−) ratios of the enantiomers of venlafaxine and ODV at various time points were consistent in the study subjects. Therefore, the estimation of venlafaxine and ODV using nonstereoselective assay method is effective in distinguishing formulation differences (if any) in bioequivalence studies in a cost‐effective manner. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Stereoselective Interaction Between Tetrahydropalmatine Enantiomers and CYP Enzymes in Human Liver Microsomes

Tetrahydropalmatine (THP), with one chiral center, is an alkaloid that possesses analgesic and many other pharmacological actives. The aim of the present study is to investigate stereoselective metabolism of THP enantiomers in human liver microsomes (HLM) and elucidate which cytochrome P450 (CYP) isoforms contribute to the stereoselective metabolism in HLM. Additionally, the inhibitions of THP enantiomers on activity of CYP enzymes are also investigated. The results demonstrated that (+)‐THP was preferentially metabolized by HLM. Ketoconazole (inhibitor of CYP3A4/5) inhibited metabolism of (?)‐THP or (+)‐THP at same degree, whereas the inhibition of fluvoxamine (inhibitor of CYP1A2) on metabolism of (+)‐THP was greater than that of (?)‐THP; moreover, the metabolic rate of (+)‐THP was 5.3‐fold of (?)‐THP in recombinant human CYP1A2. Meanwhile, THP enantiomers did not show obvious inhibitory effect on the activity of various CYP isoforms (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2E1, and 3A4/5), whereas (?)‐THP, but not (+)‐THP, significantly inhibited the activity of CYP2D6 with the Ki value of 6.42 ± 0.38 μM. The results suggested that THP enantiomers were predominantly metabolized by CYP3A4/5 and CYP1A2 in HLM, and (+)‐THP was preferentially metabolized by CYP1A2, whereas CYP3A4/5 contributed equally to metabolism of (?)‐THP or (+)‐THP. Besides, the inhibition of CYP2D6 by (?)‐THP may cause drug–drug interaction, which should be considered. Chirality 25:43–47, 2013. © 2012 Wiley Periodicals, Inc.     

Stereoselective metabolism of propranolol glucuronidation by human UDP‐glucuronosyltransferases 2B7 and 1A9

Stereoselective metabolism of propranolol side‐chain glucuronidation was studied for two recombinant human uridine diphosphate glucuronosyltransferases (UGTs), UGT1A9 and UGT2B7. The S‐ and R‐propranolol side‐chain glucuronides produced in the incubation mixtures were assayed simultaneously by RP‐HPLC with fluorescent detector. The excitation and emission wavelengths were set at 310 nm and 339 nm, respectively. UGT1A9 prefers catalyzing S‐enantiomer to R‐enantiomer and the intrinsic clearance (CL_int) ratios of S‐enantiomer to R‐enantiomer are 3.8 times and 6.5times for racemic propranolol and individual enantiomers, respectively. UGT2B7, however, catalyzes slightly less S‐enantiomer than R‐enantiomer and the CL_int ratio of S‐enantiomer to R‐enantiomer is 0.8 times. The high concentration of racemic propranolol (>0.57 mmol/l) and individual enantiomers (>0.69 mmol/l) exhibited substrate inhibition of glucuronidation for UGT2B7, but only the S‐enantiomer (>0.44 mmol/l) in racemic propranolol exhibited substrate inhibition for UGT1A9. The substrate inhibition constants (K_si) were all similar (P > 0.05). Drug–drug interactions were also found between S‐ and R‐enantiomer glucuronidation metabolisms by UGT1A9 and UGT2B7. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Stereoselective chiral inversion of pantoprazole enantiomers after separate doses to rats

(±)-Pantoprazole ((±)-PAN), (±)-5-(difluoromethoxy)-2-[[(3.4-dimethoxy-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole is a chiral sulfoxide that is used clinically as a racemic mixture. The disposition kinetics of (+)-PAN and (−)-PAN given separately has been studied in rats. Serum levels of (+)- and (−)-PAN and its metabolites, pantoprazole sulfone (PAN-SO₂), pantoprazole sulfide (PAN-S), 4′-O-demethyl pantoprazole sulfone (DMPAN-SO₂), and 4′-O-demethyl pantoprazole sulfide (DMPAN-S) were measured by HPLC. Following single intravenous or oral administration, both enantiomers were rapidly absorbed and metabolized, resulting in similar serum concentrations, suggesting that the two enantiomers have approximately the same disposition kinetics. The major metabolite of both (+)- and (−)-PAN was PAN-SO₂, while DMPAN-SO₂ was also detected as a minor metabolite. Serum levels of PAN-S and DMPAN-S could not be quantified after intravenous or oral administration of either enantiomer. Significant chiral inversion occurred after intravenous and oral administration of (+)-PAN. The AUCs of (−)-PAN after intravenous and oral dosing of (+)-PAN were 36.3 and 28.1%, respectively of those of total [(+) + (−)] PAN. In contrast, the serum levels of (+)-PAN were below quantitation limits after intravenous or oral administration of (−)-PAN. Therefore, chiral inversion was observed only after administration of (+)-PAN, supporting the hypothesis that stereoselective inversion from (+)-PAN to (−)-PAN occurs in rats. Chirality 10:747–753, 1998. © 1998 Wiley-Liss, Inc.     

Enantiomeric separation of triazole fungicides with 3-μm and 5-μml particle chiral columns by reverse-phase high-performance liquid chromatography

This study used chiral columns packed with 3‐μm and 5‐μm particles to comparatively separate enantiomers of 9 triazole fungicides, and Lux Cellulose‐1 columns with chiral stationary phase of cellulose‐tris‐(3,5‐dimethylphenylcarbamate) were used on reverse‐phase high‐performance liquid chromatography with flow rates of 0.3 and 1.0 mL min⁻¹ for 3‐μm and 5‐μm columns, respectively. The (+)‐enantiomers of hexaconazole ( 1 ) , tetraconazole ( 4 ) , myclobutanil ( 7 ) , fenbuconazole ( 8 ) and the (−)‐enantiomers of flutriafol ( 2 ) diniconazole ( 3 ) , epoxiconazole ( 5 ) , penconazole ( 6 ) , triadimefon ( 9 ) were firstly eluted from both columns, the elution orders identified with an optical rotation detector didn't change with variety of column particles and mobile phases (acetronitrile/water and methanol/water). The plots of natural logarithms of the selectivity factors (ln α) for all fungicides except penconazole ( 6 ) versus the inverse of temperature (1/T) were linear in range of 5–40°C. The thermodynamic parameters (ΔH°, ΔS°, ΔΔH° and ΔΔS°) were calculated using Van't Hoff equations to understand the thermosynamic driving forces for enantioseparation. This work will be very helpful to obtain good enantiomeric separation and establish more efficient analytical method for triazole fungicides. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Stereoselective uptake and degradation of (±)‐o,p‐DDD pesticide stereomers in water‐sediment system

The environmental stereoselective uptake and degradation of (±)‐o,p‐DDD pesticide stereomers in water‐sediment system are described. The results were analyzed by artificial neural network model. The optimized experimental parameters were concentration of o,p‐DDD streamers (7.0 μg L^?1), experimental time (60 min), pH (6), dose (5.0 g L^?1), and temperature (25°C). The maximum uptake and degradation were 87% and 85% and 33.0% and 30.5% for (?)‐ and (+)‐stereomers of o,p‐DDD in 15‐day time. Both uptake and degraded phenomenon showed first‐order rate reaction. Thermodynamic variables indicated exothermic nature of uptake and degradation processes. The uptake and degradation were slightly higher for (?)‐stereomer than (+)‐stereomer of o,p‐DDD. It was assumed that both uptake and degradation processes are accountable for the removal of the streomers of o,p‐DDD from earth's ecosystem, but the uptake is responsible for major contribution. The magnitudes of relative errors obtained by artificial neural network model were in the range of ±0.2 to 3.5, indicating good applicability of the experimental data. The results are very useful to control the environmental contamination due to the chiral o,p‐DDD pesticide as its two enantiomers have different ecological toxicities.     

Characterization of esterases involved in the stereoselective hydrolysis of ester‐type prodrugs of propranolol in rat liver and plasma

An inhibition study showed that the stereoselective hydrolysis of butyryl propranolol (butyryl PL) in rat liver microsomes and plasma involves carboxylesterase. The hydrolysis of (S)‐butyryl PL in plasma was specifically inhibited by eserine and bis‐nitrophenyl phosphate (BNPP), compared to the (R)‐isomer, despite the non‐stereoselective hydrolysis of butyryl PL in plasma. In addition, inhibition of hydroloysis by eserine and BNPP showed little stereoselectivity for butyryl PL in liver, although liver microsomes showed an (S)‐preferential hydrolysis for butyryl PL (R/S ratio of Vmax/Km: 2.1 ± 0.2). The hydrolysis of butyryl PL was not inhibited by a polyclonal antibody against a high affinity carboxylesterase (hydrolase A, RH1). Moreover, the high Km value and the high IC₅₀ for phenylmethylsulfonyl fluoride (PMSF) against the hydrolysis of butyryl PL in rat liver microsomes suggest that a low affinity carboxylesterase (perhaps hydrolase B) might be involved in this hydrolysis in rat liver. Chirality 11:10–13, 1999. © 1999 Wiley‐Liss, Inc.     

Steady‐State Plasma Concentration of Donepezil Enantiomers and Its Stereoselective Metabolism and Transport In Vitro

The aim of the present study was to elucidate the differences in the plasma concentration of two enantiomers of donepezil in Chinese patients with Alzheimer's disease (AD) and investigate in vitro stereoselective metabolism and transport. Donepezil enantiomers were separated and determined by LC‐MS/MS using D5‐donepezil as an internal standard on a Sepax Chiralomix SB‐5 column. In vitro stereoselective metabolism and transport of donepezil were investigated in human liver microsomes and MDCKII‐MDR1 cell monolayer. Pre‐dose (Css‐min) plasma concentrations were determined in 52 patients. The mean plasma level of (R)‐donepezil was 14.94 ng/ml and that of (S)‐donepezil was 23.37 ng/ml. One patient's plasma concentration of (R)‐donepezil was higher than (S)‐donepezil and the ratio is 1.51. The mean plasma levels of (S)‐donepezil were found to be higher than those of (R)‐donepezil in 51 patients and the ratio of plasma (R)‐ to (S)‐donepezil varies from 0.34 to 0.85. In the in vitro microsomal system, (R)‐donepezil degraded faster than (S)‐donepezil. V_max of (R)‐donepezil was significantly higher than (S)‐donepezil. The P‐gp inhibition experiment shown that the P_app of the two enantiomers was higher than 200 and the efflux ratios were 1.11 and 0.99. The results of the P‐gp inhibition identification experiment showed IC50 values of 35.5 and 20.4 μM, respectively, for the two enantiomers. The results indicate that donepezil exhibits stereoselective hepatic metabolism that may explain the differences in the steady‐state plasma concentrations observed. Neither (R)‐ nor (S)‐donepezil was a P‐gp substance and the two enantiomers are highly permeable through the blood–brain barrier. Chirality 25:498–505, 2013. © 2013 Wiley Periodicals, Inc.     

In situ evaluation of esterase stereoselectivity in two-dimensional electropherograms and tissue sections

Staining with both enantiomers of an α-naphthyl ester plus a diazonium salt and comparing the color intensities given by the two enantiomers is a convenient method to evaluate the esterase stereoselectivity for that ester in two-dimensional electropherograms and tissue sections. Application of this method for rat liver has shown that (1) several esterases, e.g., one of pI 6.4 and M_r 118 kDa, are moderately stereoselective against α-naphthyl (R)-N-acetylalaninate and (R)-N-methoxycarbonylalaninate but strictly stereoselective against α-naphthyl (S)-N-methoxycarbonylvalinate, implying that esterase stereoselectivity may be inverted by changing the ester structure; and (2) these esterases are mainly contained in the hepatocytes around central veins.