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.     

Erythrocyte sodium/hydrogen exchange inhibition by (-) epicatechin

Epicatechin, a flavonoid belonging to the group of compounds collectively called catechins, have been reported to possess insulin-like properties. Besides their anti-diabetic properties, catechins also show growth inhibition. Since cytosolic pH (pHi) plays a role in cell proliferation and the Na/H exchanger (NHE) is the major pH (pHi) regulatory mechanism, we undertook in vitro studies with human erythrocytes to examine the effect of (-) epicatechin (EC) on the NHE1 isoform. NHE activity was measured in eight healthy volunteers, eight type 1 diabetics, and nine type 2 diabetics, following 30 min incubations at 37 degrees C with either 1 mM epicatechin, 10(-9) M insulin or solvent alone. NHE activity was elevated in both groups of patients (P< 0.05). Epicatechin caused a 93% decrease in Na/H antiport activity in health controls, 89 and 86% in type 1 and type 2 diabetics, respectively (P< 0.001). Insulin caused a 36% decrease in antiport activity only in the type 2 diabetic group (P< 0.05). The strong inhibition of erythrocyte NHE1 (the ubiquitously present isoform) by epicatechin may have important implications. NHE1 inhibition could be one of the major mechanisms underlying the antiproliferative effects of catechins.     

Pharmacokinetics of the active antifungal enantiomer, SCH 42427 (RR), and evaluation of its chiral inversion in animals following its oral administration and the oral administration of its racemate genaconazole (RR/SS)

Genaconazole (SCH 39304) is a potent triazole antifungal agent that is active both orally and topically. Genaconazole is a racemic mixture which contains 50% of the RR (SCH 42427) and 50% of the SS (SCH 42426) enantiomers. The RR isomer accounts for most of the antifungal activity of genaconazole. Serum concentrations of the RR and SS enantiomers were analyzed by a chiral HPLC method which involved extraction of serum with organic solvent followed by separation on a Cyclobond I column and quantification by UV absorbance at 205 nm. The bioavailability and pharmacokinetic profiles of the two enantiomers after oral administration of the racemate (genaconazole) were very similar in cynomolgus monkeys. In rats following dosing with genaconazole, the RR enantiomer had a lower C(max) and a longer t(1/2) than the SS enantiomer, while the AUC(I) values of the two enantiomers were similar. Based on chiral HPLC analysis, there was no evidence for the inversion of the RR to the SR isomer, or of the SS to the SR isomer, indicating that there was no chiral inversion of the RR or SS enantiomers in either species. Genaconazole at 20 mg/kg and the RR (SCH 42427) enantiomer at 10 mg/kg had very similar serum concentration-time profiles and C(max), AUC(I), and t(1/2) values for the RR enantiomer in both rats and monkeys, indicating that the two treatments were equivalent with respect to the bioavailability of the RR enantiomer.     

Stereoselective pharmacokinetics of clausenamide enantiomers and their major metabolites after single intravenous and oral administration to rats

The pharmacokinetics of clausenamide (CLA) enantiomers and their metabolites were investigated in Wistar rat. After intravenous and oral administration at a dose of 80 and 160 mg/kg each enantiomer, plasma concentrations of (-)- or (+)-CLA and its major metabolites were simultaneously determined by reverse-phase HPLC with UV detection. Notably, stereoselective differences in pharmacokinetics were found. The mean plasma levels of (+)-CLA were higher at almost all time points than those of (-)-CLA. (+)-CLA also exhibited greater t(max), C(max), t(1/2beta), AUC(0-12h), and AUC(0--> infinity) and smaller CL (or CL/F) and V(d) (or V(d)/F), than its antipode. The (+)/(-) isomer ratios for t(1/2beta), t(max), AUC(0-12 h), and AUC(0--> infinity), which ranged from 1.26 to 2.08. The ratio for CL (or CL/F) was about 0.5, and there were significant differences in these values between CLA enantiomers (P < 0.05), implying that the absorption, distribution, and elimination of (-)-CLA were more rapid than those of (+)-CLA. Similar findings for (-)-7-OH-CLA, the major metabolite of (-)-CLA, and (+)-4-OH-CLA, the major metabolite of (+)-CLA, can be also seen in rat plasma. The contributing factors for the differences in stereoselective pharmacokinetics of CLA enantiomers appeared to be involved in their different plasma protein binding, first-pass metabolism and interaction with CYP enzymes, especially with their metabolizing enzyme CYP 3A isoforms.     

Stereoselective protein binding of tetrahydropalmatine enantiomers in human plasma,HSA, and AGP,but not in rat plasma

Tetrahydropalmatine (THP) is one of the active alkaloid ingredients of Rhizoma Corydalis. THP has a chiral center, and the stereoselective pharmacokinetics and tissue distribution have been reported. The aim of the present article is to study the stereoselective protein binding of THP using equilibrium dialysis followed by HPLC‐UV analysis. The results showed that THP stereoselectively binds to human serum albumin (HSA), α₁‐acid glycoprotein (AGP), and proteins in human plasma. The fraction binding of (+)‐THP was significantly higher than that of (?)‐THP, whereas such stereoselectivity was not found in rat plasma. The affinity of HSA and AGP to (+)‐THP, expressed as nK_A, were 9.0 × 10³ M^?1 and 2.34 × 10⁵ M^?1, respectively, which were notablely higher than to (?)‐THP, with the nK_A of 3.4 × 10³ M^?1 and 1.44 × 10⁵ M^?1, respectively. The binding site of HSA for (?)‐THP was Site I, whereas for (+)‐THP was both Site I and Site II. The F1/S variants of AGP were proved to be the key variants (?)‐ and (+)‐THP binding to both. Finally, the AGP binding drugs, such as mifepristone, were demonstrated to reduce the fraction binding of (?)‐ and (+)‐THP with pure AGP (1 mg/ml) but did not affect the fraction binding of both (?)‐ and (+)‐THP with proteins in human plasma. It can be concluded that protein binding of THP is species dependent and stereoselective, both HSA and AGP contribute to the stereoselective binding to THP enatiomers, and AGP binding drugs may not cause the drug–drug interaction on THP in healthy human plasma. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Total synthesis and biological evaluation of (-)-pectinatone employing a methyl-branched wax ester as key building block

Unnatural (-)-pectinatone ((-)-3) was prepared in five steps starting from the highly methyl-branched wax ester 4, employing bromination of the ester enolate and subsequent base-induced elimination to the enoate 6 as the key step. Both (-)-3 and the amides 8b and 8c, which were isolated as by-products in the reaction sequence, displayed antimicrobial activity and cytotoxicity.     

Enantioselective pharmacokinetics of ethotoin in humans following single oral doses of the racemate.

Racemic ethotoin (1000 mg) was administered orally as a single dose to six healthy adult volunteers. Blood samples were collected at appropriate times for 120 h following the dose. Ethotoin was quantified enantio-selectively in plasma using a novel chiral column HPLC procedure. One of the enantiomers of the chiral metabolite, 5-phenylhydantoin, was also quantified in the HPLC method. The Cmax and AUC0-infinity values for (+)-(S)-ethotoin were significantly greater than those for (-)-(R)-ethotoin (ratio of mean AUC0-infinity values 0.88), but the elimination half-lives of the isomers were virtually identical [12.35 +/- 5.15 h for (-)-(R)-ethotoin; 12.28 +/- 5.34 h for (+)-(S)-ethotoin]. Parameters derived from AUC0-infinity (Cl0/F and V(area)/F) also differed slightly between the isomers. The data were interpreted as indicating a small difference in the absorption of the two isomers; it seemed unlikely, in terms of the identical elimination rates, that their metabolic profiles would differ greatly. The 5-phenylhydantoin was eliminated with a significantly longer half-life (18.69 +/- 6.11 h) than that of ethotoin. Enantioselectivity in the pharmacokinetics of ethotoin is therefore a minor issue.     

(-)-Epigallocatechin-3-gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti-tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblast's integrin alpha2beta1, indicating EGCG may affect the expression and affinity of integrin alpha2beta1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen-induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three-dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non-contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti-tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor-stroma interaction.     

Protein binding of indobufen enantiomers: pharmacokinetics of free fraction-studies after single or multiple doses of rac-indobufen

The binding of the enantiomers of indobufen (INDB) to human serum proteins was investigated using the racemic mixture or the pure (+)-S-enantiomer in a concentration range of 2.5-100.0 mg/L. In addition, the pharmacokinetics of free (unbound) and total INDB enantiomers were studied 1) following administration of a single 200 mg rac-INDB tablet to healthy volunteers, and 2) in obliterative atherosclerosis patients at steady state. The free fraction of INDB was obtained by ultrafiltration. Using the racemic mixture, the binding parameters of the two enantiomers were different, showing enantioselectivity in protein binding. The (-)-R-enantiomer was bound more strongly to human serum albumin, with association constant K = 11.95 +/- 0.98 x 10(5) M(-1) and n = 0.72 +/- 0.02 binding sites. The comparable data for the (+)-S-enantiomer were K = 4.65 +/- 0.02 x 10(5) M(-1), n = 0.92 +/- 0.01. When the binding of (+)-S-enantiomer was studied alone, the association constant K (2.10 +/- 0.18 x 10(5) M(-1)) was lower and the number of binding sites was increased, to n = 1.87 +/- 0.17. Competition occurred between the enantiomers, with the (-)-R-enantiomer displacing its antipode. The fraction of both enantiomers bound to serum proteins was 99.0%, which increased with decreasing initial concentration of the enantiomers. In healthy volunteers the (+)-S-enantiomer was eliminated faster than its (-)-R antipode, resulting in a lower AUC for the (+)-S-enantiomer. Significant differences were observed in the total INDB enantiomer concentrations. The mean unbound fraction of (-)-R- and (+)-S-INDB was 0.45% and 0.43%, respectively. Levels of the free (+)-S-enantiomer were higher than its (-)-R-antipode at steady state in patients with obliterative atherosclerosis who also took other drugs. The free enantiomer fraction increased to around 1% upon repeated administration. We conclude that the more rapid elimination of the (+)-S enantiomer is associated with its weaker binding to serum proteins.     

Myocardial uptake kinetics of tocainide enantiomers in the isolated perfused rabbit heart

The extent of myocardial accumulation of tocainide, administered as single enantiomers and as well as racemate, was determined in the isolated, spontaneous beating rabbit heart. The heart was retrogradely perfused at a constant rate and fractions of the perfusate were collected during and after infusion. Kinetic parameters for myocardial accumulation and disposition of tocainide were indirectly determined from drug concentration/time course in the outflow perfusate. No stereoselectivity in myocardial accumulation was observed. A two compartment model with mean half-lives for distribution and elimination of 0.60 and 3.78 min, respectively, was fitted to the accumulation and disposition data. At steady-state, tocainide enantiomers were accumulated about three times in the myocardium relative to the perfusion liquid. © 1995 Wiley-Liss, Inc.     

Synthesis of new pryridyl alcohols and their use as catalysts in the asymmetric addition of diethylzinc to aldehydes.

Optically active new pyridyl alcohols 1-4, which can be easily synthesized by the reaction of (+)-camphor or (-)-menthone with lithiated pyridine derivatives, were applied as chiral ligands in the asymmetric addition of diethylzinc to aldehydes. Good yields with up to 94.0% enantiomeric excesses were observed in these reactions.     

Stereoselective pharmacokinetics of RS-8359, a selective and reversible MAO-A inhibitor, by species-dependent drug-metabolizing enzymes

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]pyrimidine selectively and reversibly inhibits monoamine oxidase A (MAO-A). After oral administration of rac-RS-8359 to rats, mice, dogs, monkeys, and humans, plasma concentrations of the (R)-enantiomer were greatly higher than were those of the (S)-enantiomer in all species studied. The AUC((R)) to AUC((S)) ratios were 2.6 in rats, 3.8 in mice, 31 in dogs, and 238 in monkeys, and the (S)-enantiomer was almost negligible in human plasma. After intravenous administration of RS-8359 enantiomers to rats, the pharmacokinetic parameters showed that the (S)-enantiomer had a 2.7-fold greater total clearance (CL(t)) and a 70% shorter half-life (t(1/2)) than those for the (R)-enantiomer but had no difference in distribution volume (V(d)). No significant difference in the intestinal absorption rate was observed. The principal metabolites were the 2-keto form, possibly produced by aldehyde oxidase, the cis-diol form, and the 2-keto-cis-diol form produced by cytochrome P450 in rats, the cis-diol form in mice, RS-8359 glucuronide in dogs, and the 2-keto form in monkeys and humans. Thus, the rapid disappearance of the (S)-enantiomer from the plasma was thought to be due to the rapid metabolism of the (S)-enantiomer by different drug-metabolizing enzymes, depending on species.     

Modeling of the biotransformation of crotonobetaine into L-(-)-carnitine by Escherichia coli strains

A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors. Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E. coli. Cell growth and biotransformation were studied in both anaerobic and aerobic conditions. For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth. Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions. As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death. Moreover, the production of enzymes involved within the E. coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system. The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions. Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration. The proposed model was useful for describing the whole set of variables under both growing and resting conditions. Both E. coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data.     

GC-MS detection of chiral markers in cocoa beans of different quality and geographic origin

Fermented cocoa beans (Theobroma cacao L., Sterculiaceae) from different countries of origin (Ecuador, Ghana, Trinidad) and cocoa beans roasted under defined conditions (industrial roasting; 150-220 degrees C for 20 min, dry roasting in conventional oven) were analyzed for their contents of certain chiral hydroxy acids, catechins, and amino acids. Cocoa beans are fermented, dried, and industrially transformed by roasting for the production of chocolate, cocoa powders, and other cocoa-related products. Fermentation and roasting conditions influence the contents of chiral compounds such as hydroxy acids, amino acids, and polyphenols, depending on technological procedures as well as some technical parameters. The aim of this work was to check if the content and nature of the named chiral compounds present both in fermented and roasted cocoa beans could be related to the traditional parameters used to classify the variety of seeds and the degree of fermentation. The extent of racemization of amino acids in fermented cocoa beans was low while it slowly increased during roasting, depending on the temperature applied. L-lactic acid was always higher than the D-form while citric acid was generally the most abundant hydroxy acid detected in beans. A correlation was found between polyphenol content and degree of fermentation, while epimerization of (-)-epicatechin to (+)-catechin was observed during roasting. On the whole, results showed that several chiral compounds could be considered as good quality markers for cocoa seeds and cocoa-related products of different quality and geographic origin.     

Chiral HPLC determination and stereoselective pharmacokinetics of tetrahydroberberine enantiomers in rats

Tetrahydroberberine (THB), a racemic mixture of (+)‐ and (?)‐enantiomer, is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). A chiral high performance liquid chromatography method has been developed for the determination of THB enantiomers in rat plasma. The enantioseparation was carried out on a Chiral®‐AD column using methanol:ethanol (80:20, v/v) as the mobile phase at the flow rate 0.4 ml/min. The ultraviolet detection was set at 230 nm. The calibration curves were linear over the range of 0.01–2.5 μg/ml for (+)‐THB and 0.01‐5.0 μg/ml for (?)‐THB, respectively. The lower limit of quantification was 0.01 μg/ml for both (+)‐THB and (?)‐THB. The stereoselective pharmacokinetics of THB enantiomers in rats was studied after oral and intravenous administration at a dose of 50 and 10 mg/kg racemic THB (rac‐THB). The mean plasma levels of (?)‐THB were higher at almost all time points than those of (+)‐THB. (?)‐THB also exhibited greater C_max, and AUC_0–∞, smaller CL and V_d, than its antipode. The (?)/(+)‐enantiomer ratio of AUC_0–∞ after oral and intravenous administration were 2.17 and 1.43, respectively. These results indicated substantial stereoselectivity in the pharmacokinetics of THB enantiomers in rats. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Practical access to the proline analogs (S,S,S)- and (R,R,R)-2-methyloctahydroindole-2-carboxylic acids by HPLC enantioseparation

An efficient methodology for the preparation of the α‐tetrasubstituted proline analog (S,S,S)‐2‐methyloctahydroindole‐2‐carboxylic acid, (S,S,S)‐(αMe)Oic, and its enantiomer, (R,R,R)‐(αMe)Oic, has been developed. Starting from easily available substrates and through simple transformations, a racemic precursor has been synthesized in excellent yield and further subjected to HPLC resolution using a cellulose‐derived chiral stationary phase. Specifically, a semipreparative (250 mm × 20 mm ID) Chiralpak® IC column has allowed the efficient resolution of more than 4 g of racemate using a mixture of n‐hexane/tert‐butyl methyl ether/2‐propanol as the eluent. Multigram quantities of the target amino acids have been isolated in enantiomerically pure form and suitably protected for incorporation into peptides. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

P-glycoprotein inhibitory activity of two phenolic compounds, (-)-syringaresinol and tricin from Sasa borealis

(-)-Syringaresinol and tricin, isolated from the AcOEt-soluble extract of the whole plants of Sasa borealis (Gramineae), showed inhibitory effects on the P-glycoprotein in adriamycin-resistant human breast cancer cells, MCF-7/ADR.     

Neuroprotective effects of (-)-epigallocatechin-3-gallate (EGCG) on paraquat-induced apoptosis in PC12 cells

Green tea, owing to its beneficial effect on health, is becoming more and more popular worldwide. (-)-Epigallocatechin-3-gallate (EGCG), the main ingredient of green tea polyphenols, is a known protective effect on injured neurons in neurodegenerative disease, such as Alzheimer's disease and Parkinson's disease. Paraquat (PQ) is a widely used herbicide that possesses a similar structure to MPP(+) and is toxic to mesencephalic dopaminergic neurons. In the present study, PQ-injured PC12 cells were chosen as an in vitro cell model of Parkinson's disease and the neuroprotective effects of EGCG were investigated. The results showed that EGCG attenuated apoptosis of PC12 cells induced by PQ. The possible mechanism may be associated with maintaining mitochondrial membrane potential, inhibiting caspase-3 activity and downregulating the expression of pro-apoptotic protein Smac in cytosol. The present study supports the notion that EGCG could be used as a neuroprotective agent for treatment of neurodegenerative diseases.