Myocardial uptake of thiopental enantiomers by the isolated perfused rat heart

Myocardial uptake of thiopental enantiomers by an isolated perfused rat heart preparation was examined after perfusion with protein-free perfusate. Outflow perfusate samples were collected at frequent intervals for 20 min during single-pass perfusion with 10 μg/ml racemic thiopental (washin phase) and for another 45 min during perfusion with drug-free perfusate (washout phase). (+)- and (−)-thiopental concentrations were assayed by chiral high-performance liquid chromatography. Heart rate, perfusion pressure, and electrocardiogram were also monitored. During the washin phase, there was no significant difference between the mean values of the equilibration rate constants of (+)- and (−)-thiopental enantiomers (0.44 ± 0.07 min⁻¹ and 0.43 ± 0.09 min⁻¹, respectively, P > 0.05). Mean volumes of distribution of (+)- and (−)-thiopental enantiomers were similar (6.34 ± 1.20 and 6.45 ± 1.29 ml/g for the washin phase and 7.22 ± 0.71 and 7.47 ± 0.81 ml/g for the washout phase, respectively, P > 0.05). This indicates that tissue accumulation of thiopental enantiomers in the isolated perfused rat heart was not stereoselective. Uptake of thiopental by the heart was perfusion flow rate-limited and independent of capillary permeability. These findings suggest that myocardial tissue concentration of racemic thiopental should be an accurate predictor of myocardial drug effect. © 1996 Wiley-Liss, Inc.     

Synthesis of the [8] gingerol enantiomers

(+)-(S)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-dodecanone 1a commonly named (+)-(S)-[8] gingerol is a natural product known to have cardiotonic activity.^1–5 A total synthesis of both enantiomers is described with details for the first time using a general synthetic scheme which was recently outlined in the literature.⁶ This synthesis relies both on the separation of the diastereoisomers 4a and 4b by simple column chromatography on silica gel and on an HPLC analysis on a chiral phase to determine the optical purity of the enantiomers 8a and 8b of protected [8] gingerol. The gingerol isomers were thus obtained in good chemical yields in greater than 96% enantiomeric excess.     

Stereoselective kinetic study of hexaconazole enantiomers in the rabbit

Hexaconazole [(RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)hexan-2-ol] is a potent triazole fungicide. The (-) isomer accounts for most of the fungicidal activity. The stereo- and/or enantioselective kinetics of hexaconazole were investigated in rabbits by intravenous injection. The concentrations of (-)- and (+)-hexaconazole in plasma, liver, and kidney tissue were determined by HPLC with a cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase and by gas chromatography-mass spectrometry. After intravenous administration of racemic hexaconazole (rac-hexaconazole) at 30 mg/kg, plasma, liver, and kidney levels of the (+)-enantiomer decreased more rapidly than those of the (-)-enantiomer. The (-)-/(+)-enantiomer ratio of the area under the concentration-time curve (AUC(0-infinity)) was 1.35. The total plasma clearance value (CL) of (+)-enantiomer was more than 1.3-fold higher than that of the (-)-hexaconazole. The enantiomeric ratio (ER) increased with time in plasma, liver, and kidney. Other pharmacokinetic parameters of the enantiomers were also different. These results indicate substantial stereoselectivity in the kinetics of hexaconazole enantiomers in rabbits.     

Stereoselective pharmacokinetics of tetrahydropalmatine after oral administration of (-)-enantiomer and the racemate

Tetrahydropalmatine (THP) is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). THP is a racemic mixture which contains 50% of the (+) and 50% of (-) enantiomer. The (-) enantiomer accounts for most of the analgesic effects. Plasma concentrations of THP enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OJ column with quantification by UV at 230 nm. The method was used to determine the pharmacokinetics of THP enantiomers in rats and dogs after oral administration of rac-THP or (-)-THP. The pharmacokinetic profiles of the two enantiomers after dosing with rac-THP were significantly different both in rats and dogs. The mean C(max) and AUC(0-infinity) values in rats were 1.93 +/- 0.36 microg/ml and 6.65 +/- 2.34 microg x h/ml for the (-) enantiomer, and 1.11 +/- 0.25 microg/ml and 2.03 +/- 0.45 microg x h/ml for the (+) enantiomer. The mean C(max) and AUC(0-infinity) in dogs were 1.60 +/- 0.81 microg/ml and 9.88 +/- 2.58 microg x h/ml for the (-) enantiomer, while 0.36 +/- 0.21 microg/ml and 1.22 +/- 0.40 microg x h/ml for the (+) enantiomer. rac-THP at 40 mg/kg and (-)-THP at 20 mg/kg had very similar plasma concentration-time profiles, and C(max), AUC(0-infinity), and t(1/2) of the (-) enantiomer in both rats and dogs, indicating that the two treatments were equivalent with respect to the pharmacokinetic properties of the (-) enantiomer.     

Separation of nicotine and nornicotine enantiomers via normal phase HPLC on derivatized cellulose chiral stationary phases

This paper describes the enantiorecognition of (±)nicotine and (±)nornicotine by high-performance liquid chromatography using two derivatized cellulose chiral stationary phases (CSPs) operated in the normal phase mode. It was found that different substituents linked to the cellulose backbone significantly influence the chiral selectivity of the derivatized CSP. The results showed that, in general, the tris(4-methylbenzoyl) cellulose CSP (Chiralcel OJ) surpasses tris(3,5-dimethylphenyl carbamoyl) cellulose CSP (Chiralcel OD). On the former column, the resolution (±)nicotine and (±)nornicotine enantiomers depended largely on mobile phase compositions. For the separation of the nicotine enantiomers, the addition of trifluoroacetic acid to a 95:5 hexane/alcohol mobile phase greatly improved the enantioresolution, probably due to enhanced hydrogen bonding interactions between the protonated analytes and the CSP. For (±)nornicotine separation, a reduction in the concentration of alcohol in the mobile phase was more effective than the addition of trifluoroacetic acid. Possible solute-mobile phase-stationary phase interactions are discussed to explain how different additives in the mobile phase and different substituents on the cellulose glucose units of the CSPs affect the separation of both pairs of enantiomers. Chirality 10:364–369, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Molecular cloning, expression and functional characterization of the chicken cation dependent mannose 6-phosphate receptor protein

Mammalian mannose 6-phosphate (M6P) receptors function in transport of lysosomal enzymes. To understand the structural and functional significance of the chicken cation dependent mannose 6-phosphate receptor (MPR) (Mr 46kDa), a full-length cDNA for the chicken protein was cloned and expressed in mpr((-/-)) MEF cells devoid of both the receptors. The stably transfected cells express the receptor that could be affinity purified by phosphomannan chromatography. The authenticity of the receptor was confirmed by its immuno-reactivity with mammalian MPR 46 antibodies and its ability to sort cathepsin D in transfected cells (92.3%) as compared to mock transfected cells (50.2%), establishing a functional role for the chicken receptor.     

Stereoselective pharmacokinetics of ornidazole after intravenous administration of individual enantiomers and the racemate

The pharmacokinetics of ornidazole (ONZ) were investigated following i.v. administration of racemic mixture and individual enantiomers in beagle dogs. Plasma concentrations of ONZ enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OB-H column with quantification by UV at 310 nm. Notably, the mean plasma levels of (-)-ONZ were higher in the elimination phase than those of (+)-ONZ. (-)-ONZ also exhibited greater t1/2, MRT, AUC(0-t) and smaller CL, than those of its antipode. The area under the plasma concentration-time curve (AUC(0-t)) of (-)-ONZ was about 1.2 times as high as that of (+)-ONZ. (+)-ONZ total body clearance (CL) was 1.4 times than its optical antipode. When given separately, there were significant differences in the values of AUC(0-infinity) and CL between ONZ enantiomers (P < 0.05), indicating that elimination of (+)-ONZ was more rapid than that of (-)-ONZ. No significant differences were found between the estimates of the pharmacokinetic parameters of (+)-ONZ or (-)-ONZ, obtained following administration as the individual and as a racemic mixture. This study demonstrates that the elimination of ONZ enantiomers is stereoselective and chiral inversion and enantiomer/enantiomer interaction do not occur when the enantiomers are given separately and as racemic mixture.     

Racemization and hydrolysis of tropic acid alkaloids in the presence of cyclodextrins

Because of the constantly increasing demand for optically pure drugs it is of great importance to elucidate factors affecting stereochemistry, in order to provide a stable formulation with a high chiral quality of the desired isomer. Therefore, the effects of cyclodextrins (CyDs) and their alkylated and hydroxyalkylated derivatives on racemization and hydrolysis of (?)-(S)-hyoscyamine and (?)-(S)-scopolamine were examined kinetically and spectroscopically (NMR). Direct methods, based on a chiral and achiral chromatographic phase system, were used to determine their degradation products and enantiomer composition during stability tests. All different CyDs, except α-CyD, retarded racemization and hydrolysis. The inclusion of the drug substances in CyDs inhibits the attack of hydroxyl ions and/or water molecules and thus retards the racemization and hydrolysis. The racemization of the tropic acid alkaloids is dependent on the pH and temperature. NMR studies were used to evidence the formation of a soluble 1:1 complex in aqueous solution. © 1993 Wiley-Liss, Inc.     

Field response of the northern spruce bark beetle Ips duplicatus (Sahlberg) (Coleoptera: Curculionidae,Scolytinae) to different combinations of synthetic pheromone with (−)‐α‐pinene and (+)‐limonene

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Production of the Inaccessible Sesquiterpene (−)‐5‐Epieremophilene by Metabolically Engineered Escherichia coli

(?)‐5‐Epieremophilene, an epimer of the versatile sesquiterpene (+)‐valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1‐3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (?)‐5‐epieremophilene production with yields of 42.4–76.0 mg/L in shake‐flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1‐FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (?)‐5‐epieremophilene were achieved. Biological assay indicated that (?)‐5‐epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC₅₀=1.25 μg/cm²), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (?)‐5‐epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology.     

The effects of (±)-, (+)-, and (−)-atenolol,sotalol, and amosulalol on the rat left atria and portal vein

The effects of (±)-, (+)-, and (?)-atenolol, sotalol, and amosulalol alone on the rat left atria and portal vein and on the respective β₁- and β₂-adrenoceptor-mediated responses to isoprenaline have been determined. (±)-Atenolol at 10^?6 M had no effect whereas high concentrations of (+)- and (?)-sotalol, 10^?5–10^?4 M, and (±)-, (+)-, and (?)-amosulalol depressed the response of the rat left atria to cardiac stimulation which indicates membrane stabilizing activity. None of the drugs tested had any effect alone on the rat portal vein. The order of potency as antagonists was (±)-amosulalol > (±)-atenolol > (±)-sotalol at β₁-adrenoceptors and (±)-amosulalol > (±)-sotalol > (±)-atenolol at β₂-adrenoceptors. (±)-Atenolol and (±)-amosulalol are β₁-selective whereas (±)-sotalol is β₂-selective. For each of the racemic β-blockers, the β₁- and β₂-adrenoceptor blocking activity was predominantly due to the (?)-enantiomer. © 1993 Wiley-Liss, Inc.     

(1′R,2′S,5′R)-Menthyl-(5R)-acetoxy-1,3-oxathiolan-(2R)-carboxylate: Synthesis and isomeric purity determination by HPLC

Chemoselective reduction of one isomer of the 1-menthylester of 1,3-oxathiolan-5-one-2-carboxylic acid produces a mixture of four lactol diastereomers from which the title compound was isolated after acylation. The isomeric purity and absolute stereochemistry were determined by spectroscopic methods, chiral HPLC techniques, and conversion to (?)-2′-deoxy-3′-thiacytidine (Lamivudine, 3TC^TM). © 1994 Wiley-Liss, Inc.     

Growth and phosphate uptake kinetics of the cyanobacterium,Cylindrospermopsis raciborskii (Cyanophyceae) in throughflow cultures

• 1 Cylindrospermopsis raciborskii occupies a rapidly expanding geographical area. Its invasive success challenges eutrophication control in many lakes. To understand better the load‐dependent behaviour of this nitrogen fixing cyanobacterium under in situ conditions, we studied P‐dependent growth of a C. raciborskii strain under continuous and pulsed P supply.
• 2 The Droop model reasonably described P‐dependent growth in the continuously supplied chemostats. Large P pulses, however, caused a delay in growth and cells subject to P pulses grew slower than their counterparts with the same P quota supplied continuously.
• 3 The kinetics of P uptake indicated that C. raciborskii is opportunistic with respect to P. Its high excess P storage capacity after a saturating P pulse (Q_ex=95 µg P [mg C]^‐1) and P‐specific uptake capacity (U_max = V_max/Q_P=150–1200) are indicative of storage adaptation. At the same time, the affinity of the P uptake system (U_max/K = 800–4000) is also high.
• 4 Rate of leakage exceeded that of the steady state net P uptake by one to two orders of magnitude. Growth affinity of C. raciborskii (µ_max/K_µ≈ 20) was relatively low, presumably due to the substantial leakage.
• 5 The dynamics of the particular water body determine which trait contributes most to competitive success of C. raciborskii. In deep lakes with vertical nutrient gradients, the cyanobacterium may rely primarily on its high P storage capacity, which is coupled to a lack of short‐term feedback inhibition and efficient buoyancy regulation. In lakes without such gradients, high P uptake affinity may be vitally important.

     

Diastereoselective [2+2] photocycloaddition of chiral cyclohexenonecarboxylates to ethylene

The diastereoselective [2+2] photocycloaddition of cyclohexenonecarboxylates containing various chiral auxiliaries to ethylene is described. The effect of the auxiliary, reaction temperature, and solvent on diastereoselectivity was examined. The (?)‐8‐(p‐methoxyphenyl)menthyl group was found to be the most effective chiral auxiliary. The photoreaction of (?)‐8‐(p‐methoxyphenyl)menthyl cyclohexenonecarboxylate in methylcyclohexane at ?78°C gave the corresponding bicyclo[4.2.0]octanone derivative in 81% diastereomeric excess (d.e.). The extent of diastereoselectivity was found to be closely related to the most stable π‐stack conformation of the starting cyclohexenones. Chirality 15:504–509, 2003. © 2003 Wiley‐Liss, Inc.     

Chirality evaluation in flavour and essential oil analysis

The simultaneous stereodifferentiation of all aromarelevant 4(5) alkylsubstituted γ(δ)-lactones is described, using enantioselective multidimensional gas chromatography (MDG), and the column combination OV 1701/octakis(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin. The method is applicated to the lactone flavour compounds of fruits, indicating the advance to the analytical differentiation between “natural” and “nature-identical” aromas. Modified cyclodextrins are also proved to be powerful tools in the chirospecific CGC analysis of monoterpenoid constituents of essential oils. Optical purity control is discussed as an indicator for their natural origin.     

Plasma membrane phosphate transport and extracellular phosphate concentration in the regulation of cellular respiration in isolated perfused rat heart

The role of extracellular P_i and transmembrane fluxes across the sarcolemma in the regulation of cellular respiration was studied in isolated Langendorff-perfused rat hearts. Extracellular phosphate did not significantly affect the oxygen consumption or cellular phosphorylation potential of the myocardium. K⁺-induced arrest was used to change the mechanical work load of the heart. Arresting the heart caused a rapid decrease in the unidirectional efflux of phosphate determined by in vitro prelabelling of the intracellular phosphate compounds with ³²P and determining the specific radioactivity of the γ-P of ATP, and the label appearance into the perfusion medium. At normal or elevated perfusate phosphate concentration there was a fairly slow net uptake of phosphate. The decrease in phosphate fluxes upon the K⁺-induced arrest was probably not due to a decrease in the transmembrane Na⁺ or K⁺ gradients because a further increase in the perfusate K⁺ concentration caused an increase in the K⁺ efflux to the levels observed in contracting hearts. The use of higher than normal concentrations of phosphate necessitated a lowering of the extracellular Ca²⁺ concentration, which caused a diminution of the oxygen consumption, accompanied by mitochondrial flavoprotein oxidation in the heart. This finding suggested that the extracellular Ca²⁺ concentration may be involved in the substrate level regulation of mitochondrial metabolism.     

Stereoselective distribution of acenocoumarol enantiomers in human plasma: Chiral chromatographic analysis of the ultrafiltrates

Stereoselectivities for the binding of rac-acenocoumarol to human serum albumin (HSA), α₁-acid glycoprotein (AGP), and human plasma were determined by chiral HPLC analysis of the ultrafiltrates on a Chiral-AGP column. The results confirmed the previously detected inverse stereoselectivities; for HSA the ratio of the enantiomeric constants was K^R/K^S ～ 2, while for AGP it was K^R/K^S ～ 0.3. In plasma the contribution of HSA dominates, although in pathological states, elevated AGP levels may compensate for stereoselective distribution. © 1993 Wiley-Liss, Inc.     

Direct separation of albuterol enantiomers in biological fluids and pharmaceutical formulations using α1-acid glycoprotein and pirkle urea type columns

A direct, isocratic, and simple chromatographic method is described for the resolution of racemic albuterol using the α₁-acid glycoprotein chiral stationary phase (AGP-CSP) under reverse phase conditions. The effect of various organic modifiers, temperature, and phosphate buffer ionic strength on the separation factor (α) and stereochemical resolution factor (R_s) has been studied. The enantiomeric separation of albuterol was also achieved using a urea-type CSP of (S)-indoline-2-carboxylic acid and (R)-1-(α-naphthyl)ethylamine, known as Chirex 3022, running in the normal phase mode. The effect of different organic acids added to the mobile phase was examined and the chiral recognition mechanism(s) is discussed. Solid phase extraction with C₁₈ Sep-Pak cartridges was applied as a clean-up step to determine the enantiomeric ratio between (?)-R and (+)-S-albuterol in pharmaceutical formulations and in human plasma. © 1995 Wiley-Liss, Inc.     

The assembly of (+)‐vincadifformine‐ and (−)‐tabersonine‐derived monoterpenoid indole alkaloids in Catharanthus roseus involves separate branch pathways

The biological activity of monoterpenoid indole alkaloids (MIAs) has led to their use in cancer treatment and other medical applications. Their biosynthesis has involved the formation of reactive intermediates by responsible enzymes to elaborate several different chemical scaffolds. Modification of scaffolds through different substitution reactions has produced chemically diverse MIAs and related biological activities. The present study characterizes the three‐step pathway involved in the formation of (+)‐echitovenine, the major O‐acetylated MIA of Catharanthus roseus roots, and differentiates it from a parallel pathway involved in the formation of hörhammericine. Separate hydrolases convert a common reactive MIA intermediate to aspidosperma skeletons of opposite specific rotations, that is (+)‐vincadifformine and (?)‐tabersonine, respectively. The formation of (+) minovincinine from (+) vincadifformine 19‐hydroxylase (V19H) is catalyzed by a root‐specific cytochrome P450 with high amino acid sequence similarity to the leaf‐specific tabersonine‐3‐hydroxylase involved in vindoline biosynthesis. Similarly, O‐acetylation of (+)‐minovincinine to form (+) echitovenine involves minovincinine‐O‐acetytransferase. The substrate specificity of V19H and MAT for their respective (+)‐enantiomers defines the separate enantiomer‐specific pathway involved in (+)‐echitovenine biosynthesis and differentiates it from a parallel (?)‐enantiomer‐specific pathway involved in the formation of hörhammericine from (?)‐tabersonine.