Stereoselective distribution of hydroxychloroquine in the rabbit following single and multiple oral doses of the racemate and the separate enantiomers

Hydroxychloroquine (HCQ) stereoselective distribution was investigated in rabbits after 20 mg/kg po of racemic-HCQ (rac-HCQ) and 20 mg/kg po of each enantiomer, 97% pure (?)-(R)-HCQ and 99% pure (+)-(S)-HCQ. Concentrations were 4 to 6 times higher in whole blood than in plasma. Melanin did not affect plasma and whole blood levels since concentrations did not differ between pigmented and nonpigmented animals. After single and multiple doses of the separate enantiomers, only 5–10% of the antipode could be measured, in blood or plasma. Therefore, there was no significant interconversion from one enantiomer into the other. Following rac-HCQ, plasma (+)-(S)-levels always surpassed (?)-(R)-ones while in whole blood, (?)-(R)-HCQ concentrations were always the highest. When the enantiomers were administered separately, blood concentrations achieved after (?)-(R)-HCQ were higher, especially after multiple doses. These observations suggest that (?)-(R)-HCQ is preferentially concentrated by cellular components of blood. This enantioselective distribution of HCQ could be secondary to a stereoselective protein binding to plasma proteins, although a more specific binding of (?)-(R)-HCQ to blood cells cannot be ruled out. Since in whole blood (?)-(R)-HCQ is retained in cellular components, metabolism would favour the more available (+)-(S)-enantiomer. © 1994 Wiley-Liss, Inc.     

Enantioselective determination of hydroxychloroquine and its major metabolites in urine and the observation of a reversal in the (+)/(−)-hydroxychloroquine ratio

A sequential achiral-chiral high-performance liquid chromatographic system has been developed for the quantitation in urine of the enantiomers of hydroxychloroquine (HCQ), and of its 3 major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ). HCQ and its metabolites were separated and quantified on a cyano-bonded phase, and the enantiomeric ratios were determined using a Chiral-AGP chiral stationary phase. The assay validation and application of this method to a preliminary study in a human volunteer are presented. In this subject, the initial 0-4 h urine contained the 2 HCQ enantiomers in a ratio of (+)-HCQ:(?)-HCQ of 3:2; by the 2,064 h of the study, this ratio had reversed to (+)-HCQ:(?)-HCQ of 3:7. © 1993 Wiley-Liss, Inc.     

Stereoselective disposition of hydroxychloroquine and its metabolites in rats

Racemic hydroxychloroquine-sulfate (HCQ-sulfate) was administered to rats orally. Groups of 9 male and 9 female rats received doses of 0, 8, 16, or 24 mg/kg/day for 6 weeks, followed by a reduction of the higher doses to 8 mg/kg/day for the duration of the study. Whole blood samples were collected at 0, 3, 6, 8, and 10 weeks, and eleven tissues were harvested after the tenth week. The concentrations and enantiomer ratios of the parent drug and three metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ), were determined. The highest concentration of HCQ was found in the intestinal smooth muscle, and the lowest in the brain and adipose tissue. The highest concentrations of the metabolites were found in the liver, adrenals, and lung tissue. The metabolism of HCQ in the rats was found to be stereoselective with R/S > 1 for the drug and < 1 for the metabolites. Gender-specific differences in the proportions of the drug and its metabolites and their enantiomers in blood and tissue were found. Varying dosages appeared to have only a temporary influence on blood concentrations and not to effect the enantiomer ratios in blood. Only a limited number of tissues exhibited significant differences between dose groups. There were no observed differences in enantiomer ratios among the blood collection times. © 1995 Wiley-Liss, Inc.     

Disposition and absorption of hydroxychloroquine enantiomers following a single dose of the racemate

The disposition of hydroxychloroquine enantiomers has been investigated in nine patients with rheumatoid arthritis following administration of a single dose of the racemate. Blood concentrations of (?)-(R)-hydroxychloroquine exceed those of (+)-(S)-hydroxychloroquine following both an oral and intravenous dose of the racemate. Maximum blood concentrations of (?)-(R)-hydroxychloroquine were higher than (+)-(S) -hydroxychloroquine after oral dosing (121 ± 56 and 99 ± 42 ng/ml, respectively, P = 0.009). The time to maximum concentration and the absorption half-life, calculated using deconvolution techniques, were similar for both enantiomers. The fractions of the dose of each enantiomer absorbed were similar, 0.74 and 0.77 for (?)-(R)- and (+)-(S)-hydroxychloroquine, respectively (P = 0.77). The data suggest that absorption of hydroxychloroquine is not enantioselective. The stereoselective disposition of hydroxychloroquine appears to be due to enantioselective metabolism and renal clearance, rather than stereoselectivity in absorption and distribution. © 1994 Wiley-Liss, Inc.     

Determination of the enantiomers of citalopram,its demethylated and propionic acid metabolites in human plasma by chiral HPLC

A stereoselective HPLC assay has been developed to analyze the enantiomers of citalopram and of its three main metabolites in plasma after their separation on a Chiracel OD column. Using a fluorescence detector, the limit of quantification in plasma samples was 15, 4, 5, and 2 ng/ml for the enantiomers of citalopram (CIT), desmethylcitalopram (DCIT), didesmethylcitalopram (DDCIT), and for the citalopram propionic acid derivative (CIT-PROP), respectively. Except for CIT, all metabolites were derivatized with achiral reagents. Identification of the enantiomers was realized with an optical rotation detector which showed that the enantiomers invert their rotation depending on the polarity and nature of the solvent. Under varying conditions, a racemization study has shown that the pure enantiomers of CIT and its demethylated metabolites are configurationally stable. Preliminary results obtained with five patients treated with CIT show a mean S/R ratio of 0.7 for both CIT and its active metabolite DCIT and of 3.6 for CIT-PROP in plasma. This suggests that the pharmacologically relevant (+)-(S)-isomers of CIT and DCIT could be preferentially and steroselectively metabolized to CIT-PROP. © 1995 Wiley-Liss, Inc.     

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.     

Chiral assay methods for lifibrol and metabolites in plasma and the observation of unidirectional chiral inversion following administration of the enantiomers to dogs

Lifibrol, a new drug for the treatment of hypercholesterolemia, contains a stereogenic center bearing a secondary alcohol group. A normal-phase achiral–chiral HPLC separation of the enantiomers of lifibrol and two of its metabolites was developed and validated for quantitation in dog plasma. A silica and a Chiralcel OD-H column were operated in series and all six enantiomeric components and internal standard were directly separated. An initial solid-phase extraction (phenyl) clean-up step and a column-switching step to eliminate late-eluting compounds were also utilized. The solid-phase extraction step was automated using a robotic system. Assay development, validation, and application of the method to a bioavailability study of the racemate and enantiomers of lifibrol in dogs are described. The lower limit of quantitation was 0.0125 μg/ml for each enantiomer of lifibrol using 200 μl of dog plasma with UV detection (255 nm). In dog plasma following oral or intravenous administration of the racemate, the (R)/(S) ratio of the enantiomers of lifibrol was greater than one and increased with time. Following administration of the individual enantiomers, chiral inversion of the (S)-enantiomer but not the (R)-enantiomer was observed. © 1994 Wiley-Liss, Inc.     

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.     

Kinetics of kavain and its metabolites after oral application

Kavain metabolism in humans was the target of this current investigation. In the present study a high-performance liquid chromatographic (HPLC-DAD) assay method for the simultaneous determination of kavain and its main metabolites (p-hydroxykavain, p-hydroxy-5,6-dehydrokavain and p-hydroxy-7,8-dihydrokavain) in serum and urine was developed and validated. The metabolites were mainly excreted in the form of their conjugates. All kavain metabolites were detectable in serum and urine, except for p-hydroxy-7,8-dihydrokavain, which was found in urine only. Confirmation of the results and identification of the metabolites were performed by LC-MS or LC-MS-MS. Kinetics of kavain and its metabolites in serum were investigated after administration of a single oral dose (800 mg kavain). Within 1 and 4 h after uptake, the serum concentrations ranged between 40 and 10 ng/ml for kavain, 300 and 125 ng/ml for p-hydroxykavain, 90 and 40 ng/ml for o-desmethyl-hydroxy-5,6-dehydrokavain, and 50 and 30 ng/ml for 5,6-dehydrokavain.     

Steady-state pharmacokinetics of the enantiomers of citalopram and its metabolites in humans

The steady-state pharmacokinetics in serum and urine of the enantiomers of citalopram and its metabolites, demethylcitalopram (DCT) and didemethylcitalopram (DDCT), were investigated after multiple doses of rac-citalopram for 21 consecutive days (40 mg per day) to healthy human subjects who were extensive metabolisers of sparteine and mephenytoin. Comparable pharmacokinetic variability was noted for (+)-(S)-, (−)-(R)- and rac-citalopram. Enantiomeric (S/R) serum concentration ratios for citalopram were always less than unity and were constant during the steady-state dosing interval. A modest, but statistically significant, stereoselectivity in the disposition of citalopram and its two main metabolites was observed. Serum levels of the (+)-(S)-enantiomers of citalopram, DCT, and DDCT throughout the steady-state dosing interval investigated were 37 ± 6%, 42 ± 3% and 32 ± 3%, respectively, of their total racemic serum concentrations. The (+)-(S)-enantiomers of citalopram, DCT, and DDCT were eliminated faster than their antipodes. For (−)-(R)- and (+)-(S)-citalopram, respectively, the serum t½ averaged 47 ± 11 and 35 ± 4 h and AUC_ss averaged 4,193 ± 1,118 h · nmol/l and 2,562 ± 1,190 h · nmol/l. The observed enantiospecificities were apparently more related to clearance, rather than to distributional mechanisms. Chirality 9:686–692, 1997. © 1997 Wiley-Liss, Inc.     

Distribution of [14C]-trans-resveratrol,a cancer chemopreventive polyphenol,in mouse tissues after oral administration

Trans-resveratrol, a phenolic compound present in wine, has been reported to be a potential cancer chemopreventive agent. However, although it has numerous biological activities in vitro, there are few data about its bioavailability and tissue distribution in vivo. The objectives of this study were to investigate the absorption and tissue distribution of 14C-trans-resveratrol following oral administration to mice. Male Balb/c mice were given a single oral dose of 14C-trans-resveratrol and were sacrificed at 1.5, 3 or 6 h postdose. The distribution of radioactivity in tissues was evaluated using whole-body autoradiography, quantitative organ-level determination and microautoradiography. In addition, identification of radioactive compounds in kidney and liver was done with high-performance liquid chromatography. Autoradiographic survey of mice sections as well as radioactivity quantification in various organs revealed a preferential fixation of 14C-trans-resveratrol in the organs and biological liquids of absorption and elimination (stomach, liver, kidney, intestine, bile, urine). Moreover, we show that 14C-trans-resveratrol derived radioactivity is able to penetrate the tissues of liver and kidney, a finding supported by microautoradiography. The presence of intact 14C-trans-resveratrol together with glucurono- and/or sulfoconjugates in these tissues was also shown. This study demonstrates that trans-resveratrol is bioavailable following oral administration and remains mostly in intact form. The results also suggest a wide range of target organs for cancer chemoprevention by wine polyphenols in humans.     

Distribution of various nickel compounds in rat organs after oral administration

In this study, eight kinds of nickel (Ni) compounds were orally administered to Wistar male rats and the distribution of each compound was investigated 24 h after the administration. The Ni compounds used in this experiment were nickel metal [Ni−M], nickel oxide (green) [NiO(G)], nickel oxide (black) [NiO(B)], nickel subsulfide [Ni₃S₂], nickel sulfide [NiS], nickel sulfate [NiSO₄], nickel chloride [NiCl₂], and nickel nitrate [Ni(NO₃)₂]. The solubilities of the nickel compounds in saline solution were in the following order; [Ni(NO₃)₂>NiCl₂>NiSO₄]≫[NiS>Ni₃S₂]>[NiO(B)>Ni−M>NiO(G)]. The Ni level in the visceral organs was higher in the rats given soluble Ni compounds; Ni(NO₃)₂, NiCl₂, NiSO₄, than that in the rats receiving other compounds. In the rats to which soluble Ni compounds were administered, 80–90% of the recovered Ni amounts in the examined organs was detected in the kidneys. On the other hand, the Ni concentration in organs administered scarcely soluble Ni compounds; NiO(B), NiO(G), and Ni−M were very low. The estimated absorbed fraction of each Ni compounds was increased with the increase of the solubility. These results suggest that the kinetic behavior of Ni compounds administered orally is closely related with the solubility of Ni compounds, and that the solubility of Ni compounds is one of the important factors for determining the health effect of Ni compounds.     

Stereospecific pharmacokinetics and toxicodynamics of ketorolac after oral administration of the racemate and optically pure enantiomers to the rat

To determine the stereospecific pharmacokinetics and gastrointestinal permeability (GI) changes (surrogate measures of toxicity) in the rat following oral administration of S, R, and racemic ketorolac (KT), optically pure enantiomers (S and R 2.5 mg/kg), and racemic KT (5 mg/kg) were administered orally to male Sprague-Dawley rats and plasma samples were collected for 6 h post-dose for pharmacokinetic assessments. KT-induced changes in GI permeability were assessed using sucrose and 51Cr-EDTA as markers of gastroduodenal and distal intestinal permeability, respectively. After the racemate, R-KT was predominant in plasma (AUC S/R, 0.45). No significant differences in pharmacokinetic indices were evident following administration of the racemate as compared with individual enantiomers. In plasma, there was only negligible S-KT after administration of R-KT. After S-KT, on the other hand, AUC of R-KT was found to be 6.7% of that of S-KT. Both permeability markers showed considerable interanimal variability. Gastroduodenal permeability was significantly increased from baseline by the racemate but not by either of the two enantiomers administered alone. Permeability to 51Cr-EDTA was not significantly increased above baseline for any of the treatments. The plasma concentration of R-KT found after administration of S-KT may be from the < 2% chiral impurity which appears magnified due to its slower clearance as compared with its antipode. There is no evidence of a pharmacokinetic interaction between the enantiomers. Since 2.5 mg/kg S-KT is somewhat less toxic on the gastroduodenum than 5 mg/kg racemate, it may be a safer alternative to the latter, at least in the rat model.     

Plasma protein binding of ketoprofen enantiomers in man: method development and its application.

The protein binding of ketoprofen enantiomers was investigated in human plasma at physiological pH and temperature by ultrafiltration. 14C-labelled (RS)-ketoprofen was synthesized and purified by high-performance liquid chromatography and utilized as a means of quantifying the unbound species. In vitro studies were conducted with plasma obtained from six healthy volunteers. The plasma was spiked with (R)-ketoprofen alone, (S)-ketoprofen alone, and (RS)-ketoprofen in the enantiomeric concentration range of 1.0 to 19.0 micrograms/ml. The plasma protein binding of ketoprofen was nonenantioselective. At a racemic drug concentration of 2.0 micrograms/ml the mean (+/- SD) percentage unbound of (R)-ketoprofen was 0.80 (+/- 0.15)%. The corresponding value for (S)-ketoprofen, 0.78 (+/- 0.18)%, was not statistically different (P greater than 0.05). At this racemic drug concentration (2.0 micrograms/ml) the percentage unbound of each enantiomer was unaffected (P greater than 0.05) by the presence of the glucuronoconjugates of ketoprofen (10 micrograms/ml) in plasma. At clinically relevant concentrations, the plasma binding of ketoprofen did not exhibit enantioselectivity or concentration dependence nor was the binding of either enantiomer influenced by its optical antipode (P greater than 0.05).     

Pharmacokinetics of ketoprofen enantiomers in monkeys following single and multiple oral administration

Pharmacokinetic studies are reported after single oral administration of 3 mg/kg of stereochemically pure (S)-ketoprofen [(S)-KP] and (R)-ketoprofen [(R)-KP] to three male Cynomolgus monkeys and after repeated administration for 6 months of 3, 15 and 75 mg/kg/day of (S)-KP to both male and female monkeys. A high-performance liquid chromatographic (HPLC) analysis was performed without derivatization of the samples, using a chiral column. The pharmacokinetic parameters for (S)-KP after administration of (S)-KP and for (R)-KP after administration of (R)-KP were, respectively, elimination half-life 2.32 ± 0.36 and 1.64 ± 0.40 h; oral clearance 3.50 ± 0.66 and 7.50 ± 3.20 ml/min/kg; apparent volume of distribution 0.74 ± 0.24 and 1.16 ± 0.76 liter/kg; mean residence time 1.79 ± 0.77 and 1.41 ± 0.65 h; area under the concentration/time curve 14.16 ± 2.93 and 7.31 ± 2.98 μg·h/ml. Forty-nine percent unidirectional bioinversion of (R)-KP to (S)-KP was observed in this species and the pharmacokinetic parameters for the (S)-KP resulting from this inversion were also calculated. In the study of 6-month repeated administration of (S)-KP, linear pharmacokinetic behavior and no evidence of drug accumulation were observed at the three dose levels. © 1994 Wiley-Liss, Inc.     

Achiral and chiral high-performance liquid chromatographic determination of tramadol and its major metabolites in urine after oral administration of racemic tramadol

A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of tramadol and its major metabolites O-demethyltramadol and N-demethyltramadol in urine has been developed. The determination of the enantiomeric ratios of the three compounds was achieved using a Chiralpak AD column and a Chiralcel OD column, respectively. After oral administration of racemic tramadol to five healthy volunteers, inter-individual differences of the excreted amounts and the enantiomeric ratios of the compounds were observed.     

Peroxiredoxin I and II in Human Eyes: Cellular Distribution and Association with Pterygium and DNA Damage

Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2’-deoxyguanosine antibody with the nuclear dye 4’,6’-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition.     

Determination of the concentrations of 5-fluorouracil and its metabolites in rabbit plasma and tissues by high-performance liquid chromatography

The concentrations of 5-fluorouracil, 5-fluoro-5,6-dihydrouracil, 5-fluorouridine and 5-fluoro-2′-deoxyuridine in plasma, liver, kidney, lung and heart of rabbits were determined by high-performance liquid chromatography (HPLC) after drug administration by two different routes. HPLC was carried out by using a Spherisorb 5 ODS 2 column and 0.05 M phosphate buffer as the mobile phase with UV detection at 200 nm. The pH of the mobile phase, organic modifier content and column temperature were found to have a profound influence on the results, hence it was necessary to optimize a procedure for each matrix. A comparison of the efficiency of intravenous and peritoneal administration revealed that the latter provides higher drug concentrations in the liver and minimal contents in plasma and all other tissues studied.