Stereoselective metabolic disposition of enantiomers of ofloxacin in rats

1. Stereoselective metabolic disposition of ofloxacin (OFLX) was studied in rats after oral administration of S-(-)-14C-OFLX and R-(+)-14C-OFLX at a dose of 20 mg/kg. 2. Radioactivity of the S-(-)-isomer was eliminated from blood much faster than that of the R-(+)-isomer. Marked differences in pharmacokinetic parameters exist between the enantiomers; the half life and AUC values of R-(+)-OFLX were greater than those of S-(-)-OFLX. Enantiomeric differences were also seen in the excretion of radioactivity, especially in biliary excretion. 3. 31.3 and 7.4% dose were excreted in the 8 h bile as ester glucuronides after oral administration of S-(-)- and R-(+)-OFLX, respectively. The enantiomeric difference in biliary excretion may be caused by stereoselective glucuronidation of S-(-)-OFLX to the ester glucuronide. 4. The metabolite pattern in serum and urine showed that the ester glucuronide of S-(-)-OFLX was more predominant than that of R-(+)-OFLX. 5. The stereoselective ester glucuronidation of the S-(-)-isomer in rats may induce significant differences in the pharmacokinetic parameters of S-(-)- and R-(+)-OFLX.     

Stereoselective Metabolic Disposition of Enantiomers of Ofloxacin in Rats

1. Stereoselective metabolic disposition of ofloxacin (OFLX) was studied in rats after oral administration of S-(-)-¹⁴C-OFLX and R-(+)-¹⁴C-OFLX at a dose of 20mg/kg.

2. Radioactivity of the S-(-)-isomer was eliminated from blood much faster than that of the R-(+)-isomer. Marked differences in pharmacokinetic parameters exist between the enantiomers; the half life and AUC values of R-(+)-OFLX were greater than those of S-(-)-OFLX. Enantiomeric differences were also seen in the excretion of radioactivity, especially in biliary excretion.

3. 31.3 and 7.4% dose were excreted in the 8?h bile as ester glucuronides after oral administration of S-(-)- and R-(+)-OFLX, respectively. The enantiomeric difference in biliary excretion may be caused by stereoselective glucuronidation of S-(-)-OFLX to the ester glucuronide.

4. The metabolite pattern in serum and urine showed that the ester glucuronide of S-(-)-OFLX was more predominant than that of R-(+)-OFLX.

5. The stereoselective ester glucuronidation of the S-(-)-isomer in rats may induce significant differences in the pharmacokinetic parameters of S-(-)- and R-(+)-OFLX.     

Antinociceptive effect of R-(+)-hyoscyamine on the conjunctival reflex test in rabbits

R-(+)-Hyoscyamine (1-10 microg/kg, s.c.) dose-dependently increased the local anesthetic effect of procaine (50 microg/ml) and lidocaine (50 microg/ml) in the conjunctival reflex test in the rabbit. This potentiating effect is completely prevented by the M1 antagonist dicyclomine (10 mg/kg, s.c.). The intensity of R-(+)-hyoscyamine antinociception was comparable to that induced by morphine (2 mg/kg, s.c.) and minaprine (15 mg/kg, s.c.), used as analgesic reference drugs. In the same experimental conditions, the S-(-)-enantiomer of atropine (0.1-10 microg/kg, s.c.), was completely ineffective. The present results confirm the ability of R-(+)-hyoscyamine to produce a paradoxical antinociceptive effect mediated by a cholinergic mechanism not only in rodents but also in the rabbit.     

N-methylation of nicotine enantiomers by human liver cytosol

Incubation of human liver cytosol with either R-(+)-[3H-N'CH3]nicotine or S-(-)-[3H-N'CH3]nicotine results in the formation of the corresponding N-methyl quaternary ammonium metabolite. A substrate stereoselectivity was observed in that the turnover number for the methylation of the S-(-)-isomer was 0.25 pmol mg-1 protein h-1, whereas that for the R-(+)-isomer was 2.11. The latter substrate exhibited an apparent Km value of 20.1 microM. Nicotine N-methylation appears to be species-dependent, since rat liver homogenates contained no 'nicotine N-methyltransferase' activity, whereas with guinea-pig liver homogenates, a substrate specificity for only R-(+)-nicotine was observed.     

Species-dependent stereopharmacokinetics of MK-927, a potent carbonic anhydrase inhibitor.

MK-927 [5,6-dihydro-4H-4(isobutylamino)thieno(2,3-B)thiopyran-2-sul fonamide -7,7 dioxide], a potent carbonic anhydrase inhibitor, contains a chiral center and is a mixture of two forms, R-(-)- and S-(+)-enantiomer. The latter has recently been designated as MK-417. Following iv administration of each enantiomer (0.05 mg/kg), dogs, rabbits, and rats cleared the R-(-)-enantiomer more rapidly than the S-(+)-enantiomer. The elimination clearance of the R-(-)-enantiomer was 2.01 +/- 0.34, 30.0 +/- 2.1, and 53.6 +/- 6.4 ml/hr/kg for dogs, rabbits, and rats, respectively. The corresponding values for the S-(+)-isomer were 0.0380 +/- 0.008, 1.15 +/- 0.20, and 1.29 +/- 0.09 ml/hr/kg. The ratio of the clearance of the R-(-)-enantiomer to that of the S-(+)-isomer was approximately 53 for the dog, 42 for the rat, and 26 for the rabbit, indicating that the degree of stereoselectivity in elimination kinetics of MK-927 enantiomers was species-dependent. Binding of the enantiomers to erythrocytes, presumably carbonic anhydrase, was also stereoselective and species-dependent; the S-(+)-enantiomer was bound more strongly than the R-(-)-isomer in all species. For both enantiomers, binding to carbonic anhydrase was found to be more extensive in dogs than in other species studied. The elimination clearance of the enantiomers in all species was roughly related to their binding affinity, greater Kd1 values being associated with more rapid clearance. However, binding data alone cannot quantitatively explain the degree of the species-dependent stereoselectivity in the elimination kinetics; other factors may also contribute.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metabolic chiral inversion of 2-phenylpropionic acid in rat, mouse and rabbit

The metabolic chiral inversion of the 2-arylpropionic acids has been investigated in laboratory animals, using the simplest congener, 2-phenylpropionic acid, as a model compound. The chiral inversion was found to occur after administration of the racemate to the rat and rabbit, but not in the mouse. The formation of the ester glucuronide was enantioselective for the S-(-)-isomer in the rat and mouse, but showed no stereoselectivity in the rabbit. [corrected] In the rat, the extent of inversion from R-(-) to S-(+) was greater at a dose of 30 mg/kg than at 150 or 300 mg/kg. The enantiomeric composition of the acid in urine was the same when the racemate was given orally or by i.p. injection. When the R-(-)isomer was given to rats, some 30% of the excreted acid was in the S-(+)-form, but when the S-(+)-isomer was given, the inversion was much less evident. In this case, the S/R ratio of the excreted phenylproprionic acid was ca 9:1. Following the administration of the racemate to rats, the plasma elimination half-life of the R-(-)-form was shorter (3.0 vs 4.8 hr for the S-(-)-isomer); this was due to its considerably greater plasma clearance (65.9 vs 43.6 micrograms/ml hr), since the volumes of distribution of the enantiomers were the same. The S/R ratio of 2-phenylpropionic acid in plasma rose progressively with time, from 1:1 in the dose solution to 2.1:1 at 8 hr.     

Resolution of alpha-hydroxytamoxifen; R-isomer forms more DNA adducts in rat liver cells.

The genotoxic tamoxifen metabolite alpha-hydroxytamoxifen has been resolved into R- and S-enantiomers. This was achieved by preparing its ester with S-camphanic acid, chromatographic separation into two diastereoisomers, and hydrolysis to give (+)- and (-)-alpha-hydroxytamoxifen. The configuration of the (-)-isomer was shown to be S- by degradation of an ester to a derivative of (-)-2-hydroxy-1-phenyl-1-propanone, which has already been shown to have S-configuration. Metabolism of tamoxifen by rat liver microsomes gave equal amounts of the two enantiomers. They have the same chemical properties but, on treatment of rat hepatocytes in culture, R-(+)-alpha-hydroxytamoxifen gave at least eight times as many DNA adducts as the S-(-)-isomer.     

Enantioselective metabolism during continuous administration of S-(-)- and R-(+)-nicotine isomers to guinea-pigs

The S-(-)- and R-(+)-nicotine isomers were administered subcutaneously via Alzet osmotic pumps to male Hartley guinea-pigs (n = 5 with each isomer) over a 23-day period. Estimated dosage rate throughout the experiment was 0.6 mg-1. Urine samples were collected over this time and the levels of urinary oxidative and N-methylated nicotine metabolites were measured by cation-exchange HPLC analysis. S-(-)-Nicotine formed only oxidative metabolites, whereas the R-(+)-isomer formed both oxidative and N-methylated metabolites. 3'-Hydroxycotinine and nicotine-1'-oxide were major metabolites of both enantiomers; cotinine and nornicotine were only minor metabolites. The major N-methylated metabolite of R-(+)-nicotine was N-methylnicotinium ion; N-methylcotininium ion and N-methylnornicotinium ion were also identified as metabolites of this nicotine isomer. Total N-methylated quaternary ammonium metabolites accounted for 15 to 20% of the administered dose of R-(+)-nicotine. An interesting enantioselective reduction in the percent of oxidative urinary metabolites formed S-(-)-nicotine was observed over 23 days. This may indicate the enantioselective induction of an uncharacterized metabolic pathway for this nicotine isomer.     

Stereoselective metabolic activation of alpha-hydroxy-N-desmethyltamoxifen: the R-isomer forms more DNA adducts in rat liver cells

The antiestrogenic drug tamoxifen forms DNA adducts in rat liver through two genotoxic metabolites, alpha-hydroxytamoxifen and alpha-hydroxy-N-desmethyltamoxifen. These have now each been resolved into R- and S-enantiomers. The work with alpha-hydroxytamoxifen was published earlier [Osborne, et al. (2001) Chem. Res. Toxicol. 14, 888-893]. Here, we publish results with alpha-hydroxy-N-desmethyltamoxifen. We prepared the derivative N-ethoxycarbonyl-N-desmethyltamoxifen-alpha-S-camphanate, separated it into two diastereoisomers, and hydrolyzed them to give (+)- and (-)-alpha-hydroxy-N-desmethyltamoxifen. The configuration of the (-)-isomer was shown to be S- by degradation of the above ester to a derivative of (-)-2-hydroxy-1-phenyl-1-propanone, which has already been shown to have S-configuration. The two enantiomers have the same chemical properties and were equally reactive toward DNA in vitro at pH 6. However, on treatment of rat hepatocytes in culture, R-(+)-alpha-hydroxy-N-desmethyltamoxifen gave 10 times as many DNA adducts as the S-(-)-isomer. This suggests that the R-isomer more readily undergoes sulfate conjugation to generate a reactive carbocation that attacks DNA.     

Studies on metabolism and toxicity of styrene--VI. Regioselectivity in glutathione S-conjugation and hydrolysis of racemic, R- and S-phenyloxiranes in rat liver

Rat liver cytosol converted phenyloxirane enantiomers regioselectively to glutathione S-conjugates. R-(+)-Phenyloxirane was converted to S-(1-phenyl-2-hydroxyethyl)glutathione (conjugate 1) and S-(2-phenyl-2-hydroxyethyl)glutathione (conjugate 2) (ratio 6.1:1), and S-(-)-phenyloxirane to conjugates 1 and 2 (ratio 1:32). Racemic phenyloxirane was converted to conjugates 1 and 2 (ratio 1.8:1). The conjugates were separated by HPLC on an octadecylsilicone column and identified with synthetic specimens whose structures were assigned by 13C NMR spectrometry. R-(+)-, S-(-)- and racemic phenyloxiranes were hydrolyzed to R-(-)-, S-(+)- and racemic phenylethanediols by microsomal epoxide hydrolase without inversion of absolute configurations of their benzylic carbons. R-(+)-Phenyloxirane had much smaller Km and Vmax than the S-(-)-oxirane did. The R-(+)-oxirane potentially inhibited the microsomal hydrolysis of the S-(-)-oxirane and was preferentially hydrolyzed when the racemic oxirane was used as the substrate. Microsomal monooxygenase oxidized styrene to R-(+)- and S-(-)-phenyloxiranes (ratio 1.3:1), and the ratio was little changed by the pretreatment of the animal with phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls.     

Determination of the main tropane alkaloids from transformed Hyoscyamus muticus plants by capillary zone electrophoresis

A capillary zone electrophoretic method (CZE) was developed using an uncoated fused silica capillary for the separation and determination of the main tropane alkaloids. The applicability of the developed method for analysis of plant samples was examined by analyzing samples of transgenic Egyptian henbane Hyoscyamus muticus (L.) plants. A simple 40 mM phosphate buffer at pH 7.8 using a voltage of 20 kV was found the best for this purpose. The main tropane alkaloids, atropine and scopolamine as well as nor-(−)-scopolamine, and tropic acid, the precursor of tropane alkaloids, could be separated in less than 13 min. The linear concentration range for atropine was 5.00–140 μg ml⁻¹, for scopolamine 7.50–210 μg ml⁻¹ and for tropic acid 2.50–70.0 μg ml⁻¹.     

Synthesis and structure-activity relationships of 1-substituted 4-(1,2-diphenylethyl)piperazine derivatives having narcotic agonist and antagonist activity

Racemates and enantiomers of 1-substituted 4-[2-(3-hydroxyphenyl)-1-phenylethyl]piperazine derivatives (3-18) were synthesized, and their analgesic and other pharmacological activities and structure-activity relationships were investigated. The S-(+) enantiomers of 2a, 5, 7, 9, 10, and 15-18 had a stronger analgesic activity than their R-(-) enantiomers; analgesic activity of the strongest one [(S)-(+)-10] was 105 times as potent as that of morphine. The S-(+) enantiomers of these compounds had the opposite configuration to that of morphine with respect to its (C-9) asymmetric center but the same configuration to that of the tyrosine residue of Met5-enkephalin. The R-(-) enantiomers of 16 and 18 showed narcotic antagonist activity, but the S-(+) enantiomers did not. (R)-(-)-18 had analgesic and narcotic antagonist activities comparable to pentazocine but showed no significant physical dependence liability. From these results, it is suggested that these compounds show an uncommon enantioselectivity in comparison with morphine and its surrogates, and belong to a new series of compounds having a potent analgesic activity.     

Stereoselective inhibition of renal organic cation transport in human kidney.

Amantadine was found to be concentrated by human cortical slices, with apparent Km and Vmax values of 187 +/- 11 microM and 1.37 +/- 0.28 nmol mg-1 min-1 respectively (mean +/- s.e. mean, n = 4). Addition of quinine (8S, 9R-(-)-isomer) or quinidine (8R, 9S-(+)-isomer) competitively inhibited this accumulation (apparent Ki values of 261 +/- 44 (n = 4) and 586 +/- 68 microM (n = 3), respectively). The stereoselectivity of this inhibition is the reverse of that seen with respect to the renal clearances of the diastereoisomers.     

Percutaneous absorption of the montoterperne carvone: implication of stereoselective metabolism on blood levels

The purpose of this study was to determine whether an enantioselective difference in the metabolism of topically applied R-(-)- and S-(+)-carvone could be observed in man. In a previous investigation we found that R-(-)- and S-(+)-carvone are stereoselectively biotransformed by human liver microsomes to 4R,6S-(-)- and 45,6S-(+)-carveol, respectively, and 4R,6S-(-)-carveol is further glucuronidated. We therefore investigated the metabolism and pharmacokinetics of R-(-)- and S-(+)-carvone in four healthy subjects using chiral gas chromatography as the analytical method. Following separate topical applications at a dose of 300 mg, R-(-)- and S-(+)-carvone were rapidly absorbed, resulting in significantly higher Cmax levels for S-(+)-carvone (88.0 vs 23.9 ng mL(-1)) and longer distribution half-lives (t(1/2alpha)) (19.4 vs 7.8 min), resulting in 3.4-fold higher areas under the blood concentration-time curves (5420 vs 1611 ng min mL(-1)). The biotransformation products for both enantiomers in plasma were below detection limit. Analysis of control- and beta-glucuronidase pretreated urine samples, however, revealed a stereoselective metabolism of R-(-)-carvone to 4R,6S-(-)-carveol and 4R,6S-(-)-carveol glucuronide. No metabolites could be found in urine samples after S-(+)-carvone application. These data indicate that stereoselectivity in phase-I and phase-II metabolism has significant effects on R-(-)- and S-(+)-carvone pharmacokinetics. This might serve to explain the increased blood levels of S-(+)-carvone.     

The difference between nicardipine and its enantiomers on inhibiting vasoconstriction of isolated rabbit thoracic artery

The present study was designed to study the difference effects between nicardipine and its two enantiomers on thoracic artery of rabbit. A high-performance liquid chromatographic method was used to prepare the two enantiomers of nicardipine. The thoracic artery of rabbit was removed. The vessels were cut into 3 mm in width and 15 mm in length spiral strips and immersed into tissue baths. The concentration-response curves of nicardipine and its enantiomers were obtained by cumulative administration of the vasoconstrictors. Nicardipine and the enantiomers could shift the dose-response curves of NE, KCl or CaCl2 to right in a nonparallel manner and decrease the maximum effective in a concentration-depended manner, respectively. The pD2' value of R-(-)-nicardipine showed significantly effective than that of nicardipine and S-(+)-nicardipine (P<0.01). There was not obviouse difference between the pD2' value of nicardipine and S-(+)-nicardipine (P>0.05). The results demonstrate that the stereoselective interaction between R-(-)-nicardipine and L-calcium channel receptor is more stronger than that of S-(+)-nicardipine.     

Characterization of orally active nonpeptide vasopressin V(2) receptor agonist. Synthesis and biological evaluation of both the (5R)- and (5S)-enantioisomers of 2-[1-(2-Chloro-4-pyrrolidin-1-yl-benzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepin- 5-yl]-N-isopropylacetamide

The synthesis and evaluation of both the (R)- and (S)-enantioisomers about the 5-position on a tetrahydro-1H-1-benzazepine derivative were described. The absolute configuration of the (R,R)-isomer (10) was determined by X-ray crystallographic analysis. After evaluation of both enantiomers (compounds R-2, S-2) for binding affinity, cAMP accumulation, and an in vivo study using Brattleboro rats, R-2 showed more potent activity as a V(2) receptor agonist than S-2.     

Preparative method ofR-(−)-ibuprofen by diastereomer crystallization

The economic and effective method for preparation of R-(-)-ibuprofen by diastereomer crystallization was developed. R-(-)-ibuprofen was resolved from racemic ibuprofen by forming R-(-)-ibuprofen-R-(+)-alpha-methylbenzylamine diastereomeric salt with R-(+)-alpha(-methylbenzylamine and crystallization. The purity of R-(-)-ibuprofen-R-(+)-alpha-methylbenzylamine diastereomeric salt was tested and confirmed using HPLC and 1H-NMR method. The pure diastereomeric salt collected from repeated recrystallization was further fractionated by liquid-liquid extraction to the pure enantiomer without racemization. R-(-)-ibuprofen was recovered producing overall yield of 2.4% with the purity more than 99.97%.     

Mechanistic studies on the metabolic chiral inversion of R-ibuprofen in the rat.

Deuterium labeling techniques and stereoselective GC/MS methodology have been employed to investigate the mechanism by which R-ibuprofen undergoes metabolic chiral inversion in the rat in vivo. Following oral administration of a mixture of R-ibuprofen (7.5 mg kg-1) and R-[ring-2H4; 2-2H]ibuprofen (R-[2H5]ibuprofen) (7.5 mg kg-1) to male Sprague-Dawley rats, the enantiomeric composition and deuterium excess of the drug were determined in serial plasma samples and in pooled urine collected over 10 hr. The results demonstrate that: (i) R-ibuprofen undergoes extensive inversion of configuration to its S antipode in the rat; (ii) chiral inversion of R-[2H5]ibuprofen yields S-[2H4]ibuprofen in a process that involves quantitative loss of the deuterium atom present originally at C-2; (iii) labeling of R-ibuprofen with deuterium at C-2 does not introduce a measurable kinetic deuterium isotope effect on the chiral inversion reaction; and (iv) metabolism of R-[2H5]ibuprofen leads to the appearance in plasma and urine of molecules of R-ibuprofen labeled with 4 atoms of deuterium. On the basis of these findings, a mechanism is proposed for the chiral inversion reaction that invokes the stereoselective formation of the coenzyme A thioester of R-ibuprofen as a key metabolite; conversion of this species to the corresponding enolate tautomer affords a symmetrical intermediate through which racemization of ibuprofen occurs in vivo.     

Pharmacokinetic difference between S-（＋）- and R-（-）-etodolac in rats

AIM: To study whether etodolac enantiomers have pharmacokinetic difference after oral administration. METHODS: Fourteen rats, divided into two groups randomly, were orally given S-( )- or R-(-)-etodolac at a single dose of 20 mg/kg, respectively. Blood samples were collected before and at 5, 10, 20, 30 min and 1, 3, 6, 12, 24, 48, 72 h after treatment. The plasma samples were analyzed with a high-performance liquid chromatographic method. RESULTS: The calibration curves were linear in the range of 0.5-50.0 mg/L (r=0.9999) to S-( )-etodolac and 2.0-200.0 mg/L (r=0.9999) to R-(-)-etodolac, respectively. The main pharmacokinetic parameters of S-( )- and R-(-)-etodolac were as follows: t1/2(λz)18±4 h vs 19.4±2.2 h, tmax 3.3±2.6 h vs 4±4 h; Cmax 29±6 mg/L vs97±14 mg/L, AUC0-t 706±100 h·mg·L-1 vs 2940±400 h·mg·L-1, CL(s) 0.030±0.006 L·kg-1·h-1 vs 0.0065±0.0010 L·kg-1·h-1 and V/F 0.25±0.22 L·kg-1 vs 0.03±0.05 L·kg-1. There was no significant difference in t1/2(λz) and tmax between S-( )-     

Biotransformation of sinesetin by the larvae of the common cutworm (Spodoptera litura)

Biotransformation of sinesetin (1) by larvae of Spodoptera litura was investigated. Compound 1 was converted to a new flavonoid, (+)-5,7,3',4'-tetramethoxyflavone-6-O-beta-D-glucoside (4), and two known flavones, 4'-hydroxy-5,6,7,3'-tetramethoxyflavone (2) and 6-hydroxy-5,7,3',4'-tetramethoxyflavone (3). These structures were established by IR, HR-EI-MS, HR-FAB-MS, 1D NMR, and 2D NMR spectral studies. The results indicate that the metabolic reaction of compound 1 by larvae of S. litura proceeded along two pathways; the main pathway is demethylation at the C-6 position followed by glucosylation, and the minor pathway is demethylation at the C-4' position.