Enantioselective gallopamil protein binding

The protein binding of the enantiomers of gallopamil has been investigated in solutions of human serum albumin, α₁-acid glycoprotein and serum. Over the range of concentrations attained after oral gallopamil administration, the binding of both enantiomers to albumin, α₁-acid glycoprotein, and serum proteins was independent of gallopamil concentration. The binding to both human serum albumin (40 g/liter) [range of fraction bound (f_b) R: 0.624 to 0.699; S: 0.502 to 0.605] and α₁-acid glycoprotein (0.5 g/liter) (range of f_b R: 0.530 to 0.718; S: 0.502 to 0.620) was stereoselective, favoring the (R)-enantiomer (predialysis gallopamil concentrations 2.5 to 10,000 ng/ml). When the enantiomers (predialysis gallopamil concentration 10 ng/ml) were studied separately in drug-free serum samples from six healthy volunteers the fraction of (S)-gallopamil bound (f_b: 0.943 ± 0.016) was lower (P < 0.05) than that of (R)-gallopamil (f_b: 0.960 ± 0.010). The serum protein binding of both (R)- and (S)-gallopamil was unaffected by their optical antipodes (f_b R: 0.963 ± 0.011; S: 0.948 ± 0.015) indicating that at therapeutic concentrations a protein binding enantiomer–enantiomer interaction does not occur. The protein binding of (R)- and (S)-gallopamil ex vivo 2 h after single dose oral administration of 50 mg pseudoracemic gallopamil (f_b R: 0.960 ± 0.010: predialysis [R] 6.9 to 35.3 ng/ml; S: 0.943 ± 0.016: predialysis [S] 9.5 to 30.7 ng/ml) was comparable to that observed in vitro in drug-free serum. Gallopamil metabolites formed during first-pass following oral administration, therefore, do not influence the protein binding of (R)- or (S)-gallopamil. © 1993 Wiley-Liss, Inc.     

Responses Recorded from the Frog Olfactory Epithelium after Stimulation with R(+)- and S(-)- nicotine

The aim of this study was to determine whether the olfactorysystem is responsible for the discriminability of the stereoisomersof nicotine. The EOG was recorded after stimulation with differentconcentrations of undistilled S(–)-, distilled S(–)-and distilled R(–)-nicotine separately in three groupsof frogs (Xenopus laevis). The responses to all types of nicotineused in the experiments increased with increasing stimulus concentration.The responses to undistilled S(–)-nicotine were significantlylower compared to responses to distilled S(–)- and R(+)-nicotine,whereas no significant differences could be found when the purifiedstereoisomers of nicotine [distilled S(–)-nicotine, distilledR(+)-nicotine] were compared. Control measurements of time courseand peak concentration employing a UV-detection method demonstratedthat the differences between distilled and undistilled S(–)-nicotinecould not be explained by different nicotine concentrations. The fact that no differences between the pure nicotine stereoisomerscould be found for all concentrations used in our experimentsand that experiments in humans revealed similar detection thresholdsfor both stereoisomers points to a similar receptor affinityof R(+)- and S (–)-nicotine within the olfactory system.At this point, it cannot be determined whether the observeddifferences in the perception of nicotine enantiomers in humansare due to differences in quality coding by stereospecific receptorson the olfactory sensory cells or by specific receptors on thetrigeminal nerve endings. Chem. Senses 20: 337–344, 1995.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

Pharmacokinetics of reboxetine enantiomers in the dog

Reboxetine, (RS)-2-[(RS)-α-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. The pharmacokinetics of reboxetine enantiomers were determined in a crossover study in three male beagle dogs. Each animal received the following oral treatments, separated by 1-week washout period: 10 mg/kg reboxetine, 5 mg/kg (R,R)- and 5 mg/kg (S,S)-. Plasma and urinary levels of the reboxetine enantiomers were monitored up to 48 h post-dosing using an enantiospecific HPLC method with fluorimetric detection (LOQ: 1.1 ng/ml in plasma and 5 ng/ml in urine for each enantiomer). After reboxetine administration mean t_max was about 1 h for both enantiomers. C_max and AUC were about 1.5 times higher for the (R,R)- than for the (S,S)-enantiomer, mean values ± SD being 704 ± 330 and 427 ± 175 ng/ml for C_max and 2,876 ± 1,354 and 1,998 ± 848 ng.h/ml for AUC, respectively. No differences between the (R,R)- and (S,S)-enantiomers were observed in t½ (3.9 h). Total recovery of the two enantiomers in urine was similar, the Ae (0–48 h) being 1.3 ± 0.7 and 1.1 ± 0.7% of the enantiomer dose for the (R,R)- and the (S,S)-enantiomers, respectively. No marked differences in the main plasma pharmacokinetic parameters were found for either enantiomer on administration of the single enantiomers or reboxetine. No chiral inversion was observed after administration of the separate enantiomers, as already observed in humans. Chirality 9:303–306, 1997. © 1997 Wiley-Liss, Inc.     

Enantiomeric Separation and Simulation Studies of Pheniramine,Oxybutynin, Cetirizine,and Brinzolamide Chiral Drugs on Amylose‐Based Columns

Solid phase extraction ( SPE)‐chiral separation of the important drugs pheniramine, oxybutynin, cetirizine, and brinzolamide was achieved on the C₁₈ cartridge and AmyCoat (150 x 46 mm) and Chiralpak AD (25 cm x 0.46 cm id) chiral columns in human plasma. Pheniramine, oxybutynin, cetirizine, and brinzolamide were resolved using n‐hexane‐2‐PrOH‐DEA (85:15:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (80:20:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (70:30:0.2, v/v), and n‐hexane‐2‐propanol (90:10, v/v) as mobile phases. The separation was carried out at 25 ± 1 ºC temperature with detection at 225 nm for cetirizine and oxybutynin and 220 nm for pheniramine and brinzolamide. The flow rates of the mobile phases were 0.5 mLmin^‐1. The retention factors of pheniramine, oxybutynin, cetirizine and brinzolamide were 3.25 and 4.34, 4.76 and 5.64, 6.10 and 6.60, and 1.64 and 2.01, respectively. The separation factors of these drugs were 1.33, 1.18, 1.09 and 1.20 while their resolutions factors were 1.09, 1.45, 1.63 and 1.25, and 1.15, respectively. The absolute configurations of the eluted enantiomers of the reported drugs were determined by simulation studies. It was observed that the order of enantiomers elution of the reported drugs was S‐pheniramine > R‐pheniramine; R‐oxybutynin > S‐oxybutynin; S‐cetirizine > R‐cetirizine; and S‐brinzolamide > R‐brinzolamide. The mechanism of separation was also determined at the supramolecular level by considering interactions and modeling results. The reported SPE‐chiral high‐performance liquid chromatography ( HPLC) methods are suitable for the enantiomeric analyses of these drugs in any biological sample. In addition, simulation studies may be used to determine the absolute configuration of the first and second eluted enantiomers. Chirality 26:136–143, 2014. © 2014 Wiley Periodicals, Inc.     

Development and validation of a chiral liquid chromatographic method, based on Chiralpak to quantify enantiomers of (+/-)-DRF 2725 in rat plasma: lack of inversion of ragaglitazar (S(-)-DRF 2725) to its antipode in plasma

A selective, accurate and reproducible high-performance liquid chromatographic (HPLC) method for the separation of individual enantiomers of DRF 2725 [R(+)-DRF 2725 and S(-)-DRF 2725 or ragaglitazar] was obtained on a chiral HPLC column (Chiralpak). During method optimization, the separation of enantiomers of DRF 2725 was investigated to determine whether mobile phase composition, flow-rate and column temperature could be varied to yield the base line separation of the enantiomers. Following liquid-liquid extraction, separation of enantiomers of DRF 2725 and internal standard (I.S., desmethyl diazepam) was achieved using an amylose based chiral column (Chiralpak AD) with the mobile phase, n-hexane-propanol-ethanol-trifluoro acetic acid (TFA) in the ratio of 89.5:4:6:0.5 (v/v). Baseline separation of DRF 2725 enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. The eluate was monitored using an UV detector set at 240 nm. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Nominal retention times of R(+)-DRF 2725, S(-)-DRF 2725 and I.S. were 15.8, 17.7 and 22.4 min, respectively. The standard curves for DRF 2725 enantiomers were linear (R(2) > 0.999) in the concentration range 0.3-50 microg/ml for each enantiomer. Absolute recovery, when compared to neat standards, was 70-85% for DRF 2725 enantiomers and 96% for I.S. from rat plasma. The lower limit of quantification (LLOQ) for each enantiomers of DRF 2725 was 0.3 microg/ml. The inter-day precisions were in the range of 1.71-4.60% and 3.77-5.91% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. The intra-day precisions were in the range of 1.06-11.5% and 0.58-12.7% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Accuracy in the measurement of quality control (QC) samples was in the range 83.4-113% and 83.3-113% for R(+)-DRF 2725, S(-)-DRF 2725, respectively. Both enantiomers and I.S. were stable in the battery of stability studies viz., bench-top (up to 6 h), auto-sampler (up to 12 h) and freeze/thaw cycles (n = 3). Stability of DRF 2725 enantiomers was established for 15 days at -20 degrees C. The application of the assay to a pharmacokinetic study of ragaglitazar [S(-)-DRF 2725] in rats is described. It was unequivocally demonstrated that ragaglitazar does not undergo chiral inversion to its antipode in vivo in rat plasma.     

Simultaneous enantiomer determination of 20 (R)- and 20 (S)-ginsenoside-Rg2 in rat plasma after intravenous administration using HPLC method

20 (R,S)-Ginsenoside-Rg2, an anti-shock agent, is prescribed as a racemate. To analyze simultaneously the enantiomers of 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2 in plasma, a simple and reproducible high-performance liquid chromatographic (HPLC) method has been developed. The enantiomeric separation and determination were successfully achieved using a Diamonsil ODS C18 reversed-phase column (5 microm, 250 mm x 4.6 mm) with an RP18 (5 microm) guard column and a mobile phase of MeOH-aq. 4% H3PO4 (65:35, v/v, pH 5.1) with UV detection at 203 nm. Both enantiomers, 20 (R)-ginsenoside-Rg2 and 20 (S)-ginsenoside-Rg2, were well separated at 14.5 min and 13.6 min, respectively. The linear ranges of the standard curves were 2.0-250 microg/ml. The intra- and inter-day precision (R.S.D.) were 相似文献   

Pharmacokinetics and pharmacodynamics of ketoprofen enantiomers in calves

The pharmacokinetics (PK) and pharmacodynamics (PD) of (S)- and (R)- ketoprofen (KTP) enantiomers were studied in calves after intravenous administration of each enantiomer at a dose of 1.5 mg/kg. Pharmacodynamic properties were evaluated using a model of acute inflammation, comprising subcutaneously implanted tissue cages stimulated by intracaveal injection of carrageenan. Chiral inversion of (R)-KTP to the (S)-antipode occurred. The R:S ratio in plasma was 33:15 min after administration, decreasing to 1:1 at 8 h. The calculated extent of inversion was 31 ± 7%. The R:S ratio in inflammatory exudate was of the order 3:1 at all the sampling times and the ratio in transudate was approximately 2:1 for 6 h, declining to 1:1 at 30 h. Only (S)-KTP was detected in biological fluids after administration of this enantiomer. Elimination half-life was longer for the (S) (2.19 h) than the (R)-enantiomer (1.30 h) and volume of distribution was also somewhat higher for the (S)-enantiomer. Body clearance values were 0.119 1/kg/h for (S)-KTP and 0.151 1/kg/h for the (R)-antipode. For (R)-KTP effects obtained were considered as a hybrid, since they potentially reflect the actions of both enantiomers. Concentrations of LTB₄ and the cytokines interleukin-1, interleukin-6, and tumor necrosis factor alpha, in exudate were not significantly affected by either (R)- or (S)-KTP treatments. Inhibition of ex vivo thromboxane B₂ (TxB₂) synthesis, exudate prostaglandin E₂ (PGE₂) synthesis, β-glucuronidase release (β-glu), and bradykinin-induced skin swelling was significant in both treated groups. PK/PD modelling was applied to the (S)-KTP treatment only. EC₅₀ values for inhibition of serum TxB₂, exudate PGE₂ and β-glu and BK-induced swelling were 0.047, 0.042, 0.101, and 0.038 μg/ml, respectively. It is concluded that the low EC₅₀ values for inhibition of TxB₂ and PGE₂ by (S)-KTP are likely to explain the effects produced by (R)-KTP administration, since concentrations of (S)-KTP in exudate of these calves following chiral inversion were at least 5 times higher than the EC₅₀ at all sampling times. The data for β-glu and bradykinin-induced swelling inhibition indicate possible inhibitory actions of (R)-KTP as well as (S)-KTP. © 1995 Wiley-Liss, Inc.     

Enantioselective σ1 receptor binding and biotransformation of the spirocyclic PET tracer 1′‐benzyl‐3‐(3‐fluoropropyl)‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine]

It was shown that racemic (±)‐ 2 [1′‐benzyl‐3‐(3‐fluoropropyl)‐3H‐spiro[[2]benzofuran‐1,4′‐piperidine], WMS‐1813 ] represents a promising positron emission tomography (PET) tracer for the investigation of centrally located σ₁ receptors. To study the pharmacological activity of the enantiomers of 2 , a preparative HPLC separation of (R)‐2 and (S)‐2 was performed. The absolute configuration of the enantiomers was determined by CD‐spectroscopy together with theoretical calculations of the CD‐spectrum of a model compound. In receptor binding studies with the radioligand [³H]‐(+)‐pentazocine, (S)‐2 was thrice more potent than its (R)‐configured enantiomer (R)‐2 . The metabolic degradation of the more potent (S)‐enantiomer was considerably slower than the metabolism of (R)‐2 . The structures of the main metabolites of both enantiomers were elucidated by determination of the exact mass using an Orbitrap‐LC‐MS system. These experiments showed a stereoselective biotransformation of the enantiomers of 2 . Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Steric aspects of formoterol and terbutaline: Is there an adverse effect of the distomer on airway smooth muscle function?

Experiments were made on isolated tissues from guinea-pig to test the hypothesis that the distomers of rac-β₂-adrenoceptor agonists induce airway hyperreactivity. Tracheal strip preparations were contracted with carbachol. Both rac- and (R;R)-formoterol (2 and 1 μmol/1, respectively) produced an immediate relaxation, followed by a slow recovery of tone. (S;S)-Formoterol (2 μmol/1) had no effect on smooth muscle tone. Similar results were obtained with the enantiomers of terbutaline. In other strip preparations of the trachea or the main bronchi, cholinergic or nonadrenergic/noncholinergic (NANC) excitatory responses were evoked by electrical field-stimulation. The eutomers, (R;R)-formoterol and (R)-terbutaline, inhibited concentration-dependently both cholinergic and NANC-induced contractions. The distomers, (S;S)-formoterol and (S)-terbutaline, showed qualitatively the same effects but were about 1,000 times less potent than the corresponding eutomer. In a third series of experiments, either enantiomer of formoterol was administered to an electrically stimulated vagus nerve-trachea tube preparation. The nerve-induced contractions were inhibited by both enantiomers, but (S;S)-formoterol was about 1,000 times less potent than (R;R)-formoterol. For both enantiomers of formoterol, about tenfold higher concentration was required to obtain the same degree of inhibition when given intratracheally as compared with administration in the external medium. There was no indication in any of the experimental approaches that (S;S)-formoterol or (S)-terbutaline might enhance the response to cholinergic or NANC-related stimuli. Chirality 8:567–573, 1996. © 1997 Wiley-Liss, Inc.     

Marked enantioselective protein binding in humans of ketorolac in vitro: Elucidation of enantiomer unbound fractions following facile synthesis and direct chiral hplc resolution of tritium-labelled ketorolac

The protein binding of the enantiomers of the nonopiate analgesic, ketorolac, was investigated in vitro using human plasma and solutions of human serum albumin (HSA) at physiological pH and temperature. In order to detect the very low levels of unbound enantiomers in protein solutions, tritium-labelled rac-ketorolac was synthesised by regiospecific isotopic exchange of the parent drug with tritiated water as the isotope donor. Radio-chemical purification of this compound by reversed-phase HPLC followed by direct resolution using a chiral α₁-acid glycoprotein (Chiral-AGP) HPLC column afforded labelled enantiomers of high specific activity. The in vitro use of (R)- and (S)-[³H₄]ketorolac enabled reproducible radiometric detection of enantiomers in protein solution ultrafiltrate. The unbound fractions of (R)- and (S)-ketorolac [fu(R) and fu(S), respectively] were determined when drug was added to various plasma or albumin solutions as either the separate enantiomers or as the racemate. Over an enantiomeric plasma concentration range of 2.0—15.0 μg/ml, fu(S) (mean range: 1.572—1.795%) was more than 2-fold greater (P < 0.001) than fu(R) (mean range: 0.565—0.674%). Both fu(R) and fu(S) were constant over this concentration range, and each was unaffected by the presence of the corresponding antipode (P > 0.05). At a concentration of 2.0 μg/ml in 40.0 g/liter fatty acid-free HSA, fu(R) and fu(S) were approximately 0.5 and 1.1%, respectively, and both values declined with increasing concentrations of the long chain fatty acid, oleic acid. We have previously shown that the pharmacokinetics of ketorolac in humans are markedly enantioselective and suggest in this report that these differences are largely the result of substantial differences in the protein binding of ketorolac enantiomers. These findings stress the importance of monitoring the unbound concentrations of the enantiomers of chiral drugs if correct interpretations are to be made of enantioselective pharmacokinetic data. © 1994 Wiley-Liss, Inc.     

Impact of Chiral Bioanalytical Methods on the Bioequivalence of Ibuprofen Products Containing Ibuprofen Lysinate and Ibuprofen Base

The purpose was to assess the impact of the use of a chiral bioanalytical method on the conclusions of a bioequivalence study that compared two ibuprofen suspensions with different rates of absorption. A comparison of the conclusion of bioequivalence between a chiral method and an achiral approach was made. Plasma concentrations of R‐ibuprofen and S‐ibuprofen were determined using a chiral bioanalytical method; bioequivalence was tested for R‐ibuprofen and for S‐ibuprofen separately and for the sum of both enantiomers as an approach for an achiral bioanalytical method. The 90% confidence interval (90% CI) that would have been obtained with an achiral bioanalytical method (90% CI: C_max: 117.69–134.46; AUC₀^t: 104.75–114.45) would have precluded the conclusion of bioequivalence. This conclusion cannot be generalized to the active enantiomer (90% CI: C_max: 103.36–118.38; AUC₀^t: 96.52–103.12), for which bioequivalence can be concluded, and/or the distomer (90% CI: C_max: 132.97–151.33; AUC₀^t: 115.91–135.77) for which a larger difference was observed. Chiral bioanalytical methods should be required when 1) the enantiomers exhibit different pharmacodynamics and 2) the exposure (AUC or C_max) ratio of enantiomers is modified by a difference in the rate of absorption. Furthermore, the bioequivalence conclusion should be based on all enantiomers, since the distomer(s) might not be completely inert, in contrast to what is required in the current regulatory guidelines. In those cases where it is unknown if the ratio between enantiomers is modified by changing the rate of absorption, chiral bioanalytical methods should be employed unless enantiomers exhibit the same pharmacodynamics. Chirality 28:429–433, 2016. © 2016 Wiley Periodicals, Inc.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α₇-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α₇-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α₇-nAChR. The nAChR subtype selectivity was probed using α₇-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α₃β₄-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca^2 + flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α₇-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.     

The direct determination of the enantiomers of ketorolac and parahydroxyketorolac in plasma and urine using enantioselective liquid chromatography on a human serum albumin-based chiral stationary phase

An enantioselective assay has been developed for the determination of the enantiomers of ketorolac and its metabolite p-hydroxyketorolac in plasma and urine. The analytical method utilizes a coupled achiral–chiral HPLC system where the initial separation of ketorolac from p-hydroxyketorolac and matrix interferences was achieved on a C₁₈-stationary phase and the enantioselective separations of the two target solutes were accomplished on a human serum albumin-based chiral stationary phase. The two columns were attached in sequence and the assay was carried out without the necessity of column-switching techniques. The method has been validated for use in pharmacokinetic and metabolic studies and represents the initial report of the determination of ketorolac and p-hydroxyketorolac enantiomers in urine. The results of the study indicate that after the administration of racemic ketorolac there was an enantioselective distribution of ketorolac enantiomers in plasma [(R)-ketorolac: (S)-ketorolac = 3.89 ± 0.93 (n = 6) and urine (R)-ketorolac: (S)-ketorolac = 1.26 ± 0.09 (n = 7)]. The mean ratio of the p-hydroxyketorolac enantiomers was 1.77 ± 0.46 (n = 7). Both ketorolac and p-hydroxyketorolac are glucuronized in the acyl carboxyl moiety and the results of this study indicate that this process is not enantiospecific. © 1994 Wiley-Liss, Inc.     

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.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Synthesis and pKa determination of new enantiopure dimethyl‐substituted acridino‐crown ethers containing a carboxyl group: Useful candidates for enantiomeric recognition studies

New enantiopure dimethyl‐substituted acridino‐18‐crown‐6 and acridino‐21‐crown‐7 ethers containing a carboxyl group at position 9 of the acridine ring [(S,S )‐ 8 , (S,S )‐ 9 , (R,R )‐ 10 ] were synthesized. The pK _a values of the new crown ethers [(S,S )‐ 8 , (S,S )‐ 9 , (R,R )‐ 10 ] and of an earlier reported macrocycle [(R,R )‐ 2 ] were determined by UV‐pH titrations. Crown ether (S,S )‐ 8 was attached to silica gel by covalent bonds and the enantiomeric separation ability of the newly prepared chiral stationary phase [(S,S )‐CSP‐ 12 ] was studied by high‐performance liquid chromatography (HPLC). Homochiral preference was observed and the best separation was achieved for the enantiomers of 1‐NEA. Ligands (S,S )‐ 9 and (R,R )‐ 10 are precursors of enantioselective sensor and selector molecules for the enantiomers of protonated primary amines, amino acids, and their derivatives.     

Stereoselective Degradation of alpha‐Cypermethrin and Its Enantiomers in Rat Liver Microsomes

Alpha‐cypermethrin (α‐CP), [(RS)‐a‐cyano‐3‐phenoxy benzyl (1RS)‐cis‐3‐(2, 2‐dichlorovinyl)‐2, 2‐dimethylcyclopropanecarboxylate], comprises a diastereoisomer pair of cypermethrin, which are (+)‐(1R‐cis‐αS)–CP (insecticidal) and (?)‐(1S‐cis‐αR)–CP (inactive). In this experiment, the stereoselective degradation of α‐CP was investigated in rat liver microsomes by high‐performance liquid chromatography (HPLC) with a cellulose‐tris‐ (3, 5‐dimethylphenylcarbamate)‐based chiral stationary phase. The results revealed that the degradation of (?)‐(1S‐cis‐αR)‐CP was much faster than (+)‐(1R‐cis‐αS)‐CP both in enantiomer monomers and rac‐α‐CP. As for the enzyme kinetic parameters, there were some variances between rac‐α‐CP and the enantiomer monomers. In rac‐α‐CP, the V_max and CL_int of (+)‐(1R‐cis‐αS)–CP (5105.22 ± 326.26 nM/min/mg protein and 189.64 mL/min/mg protein) were about one‐half of those of (?)‐(1S‐cis‐αR)–CP (9308.57 ± 772.24 nM/min/mg protein and 352.19 mL/min/mg protein), while the K_m of the two α‐CP enantiomers were similar. However, in the enantiomer monomers of α‐CP, the V_max and K_m of (+)‐(1R‐cis‐αS) ‐CP were 2‐fold and 5‐fold of (?)‐(1S‐cis‐αR)‐CP, respectively, which showed a significant difference with rac‐α‐CP. The CL_int of (+)‐(1R‐cis‐αS)–CP (140.97 mL/min/mg protein) was still about one‐half of (?)‐(1S‐cis‐αR)–CP (325.72 mL/min/mg protein) in enantiomer monomers. The interaction of enantiomers of α‐CP in rat liver microsomes was researched and the results showed that there were different interactions between the IC₅₀ of (?)‐ to (+)‐(1R‐cis‐αS)‐CP and (+)‐ to (?)‐(1S‐cis‐αR)‐CP(IC_50(?)/(+) / IC_50(+)/(?) = 0.61). Chirality 28:58–64, 2016. © 2015 Wiley Periodicals, Inc.     

High‐Throughput Chiral LC–MS/MS Method Using Overlapping Injection Mode for the Determination of Pantoprazole Enantiomers in Human Plasma with Application to Pharmacokinetic Study

A sensitive and high‐throughput chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of R‐pantoprazole and S‐pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid–liquid extraction in 96‐well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ‐RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 μL/min. The enantiomers were quantified on a triple‐quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole‐d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r² > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R‐pantoprazole and from 92.5% to 96.5% for S‐pantoprazole and the IS‐normalized matrix factor was 0.98 to 1.07 for R‐pantoprazole and S‐pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569–575, 2016. © 2016 Wiley Periodicals, Inc.