Direct high pressure liquid chromatographic analysis and preliminary pharmacokinetics of enantiomers of oxazepam and temazepam with their corresponding glucuronide conjugates

Three high pressure liquid chromatographic systems for the separation of oxazepam, temazepam and their glucuronides (system A), the separation of theirR,S glucuronide diastereomers (system B) and the chiral separation of the parent drugs (system C) are described. Preliminary pharmacokinetics ofR,S-oxazepam andR,S-temazepam in a human volunteer reveal that the protein binding of the glucuronides is lower than that of the parent drugs, but that there is no difference in protein binding between theR-oxazepam/temazepam andS-oxazepam/temazepam and their corresponding glucuronides. TheS-glucuronide is the main metabolite formed and excreted by man. The plasma ratioR/S-glucuronide is 11 for both oxazepam and temazepam. The renal clearances ofR-temazepam, andS-temazepam are similar, and those ofR-oxazepam andS-oxazepam tend to be different.     

Stereoselective Taurine Conjugation of (<Emphasis Type="Italic">R</Emphasis>)-Benoxaprofen Enantiomer in Rats: <Emphasis Type="Italic">In Vivo</Emphasis> and <Emphasis Type="Italic">in Vitro</Emphasis> Studies Using Rat Hepatic Mitochondria and Microsomes

No HeadingPurpose. Identify (R)-BOP-T in rat bile after administration of (R)-BOT over a 12 h period.Methods. Each benoxaprofen (BOP) enantiomer was administered i.v. to bile duct-cannulated rats at a dose of 5 mg/kg. The optical isomers of BOP and its metabolites in plasma, urine, and bile were quantified using a chiral HPLC column. The amounts of BOP glucuronide (BOP-G), BOP taurine conjugate (BOP-T), and BOP enantiomers excreted into the bile over 12 h after administration of (R)-BOP were as follows: (R)-BOP-G and (S)-BOP-G, 2.1 ± 0.5 and 6.2 ± 1.4% of the dose; (R)-BOP-T and (S)-BOP-T, 5.6 ± 1.8 and 0.7 ± 0.3% of the dose; (R)-BOP and (S)-BOP, 0.7 ± 0.1 and 1.7 ± 0.2% of the dose, respectively, whereas after (S)-BOP administration, (S)-BOP-G and (S)-BOP were mainly excreted into the bile (14.3 ± 1.8 and 3.0 ± 0.4% of the dose, respectively). Only after (R)-BOP administration was the taurine conjugate of BOP found in the bile, and the configuration was R. BOP-T could not be found in the bile after (S)-BOP administration. To investigate the stereoselectivity of the conjugation enzymes responsible for BOP-T formation, in vitro studies were performed using rat hepatic organelles.Results. When (R)-BOP was used as a substrate, rat hepatic mitochondrial and microsomal fractions exhibited stereoselective BOP-T formation activity, with microsomal activity approximately 3.0 times greater than that of the mitochondria. That of (S)-BOP was approximately 2.1. Mean (R)/(S) ratios of BOP enantiomer for BOP-T formation in the mitochondrial and microsomal incubations were approximately 1.7 and 2.4, respectively.Conclusion. Although in the in vivo studies, only (R)-BOP-T originated from (R)-BOP was found in the bile, the configuration of BOP-T formed by the incubations of (R)-BOP or (S)-BOP with rat hepatic mitochondria or microsomes was S for both.     

Enantioselective disposition of (R,R)‐formoterol, (S,S)‐formoterol and their respective glucuronides in urine following single inhaled dosing and application to doping control

Formoterol is a long‐acting beta2‐adrenoceptor agonist (LABA) used for the treatment of asthma and exercise‐induced bronchoconstriction. Formoterol is usually administered as a racemic (rac‐) mixture of (R,R)‐ and (S,S)‐enantiomers. While formoterol is restricted by the World Anti‐Doping Agency (WADA), inhalation of formoterol is permitted to a predetermined dose (54 μg/24 hours) and a urine threshold of 40 ng/mL. However, chiral switch enantiopure (R,R)‐formoterol is available, effectively doubling the therapeutic advantage for the same threshold. The aim of this study was to investigate whether formoterol exhibits enantioselective urinary pharmacokinetics following inhalation. Six healthy volunteers were administered a 12 μg inhaled dose of rac‐formoterol. Urine was collected over 24 hours and analyzed by enantioselective ultra‐performance liquid chromatography?tandem mass spectrometry (UPLC?MS/MS) assay. Total (free drug plus conjugated metabolite) median (min‐max) rac‐formoterol urine levels following inhalation were 1.96 (1.05–13.4) ng/mL, 1.67 (0.16–9.67) ng/mL, 0.45 (0.16–1.51) ng/mL, 0.61 (0.33–0.78) ng/mL, and 0.17 (0.08–1.06) ng/mL at 2, 4, 8, 12, and 24 hours, respectively, well below the 2019 urine threshold. The proportion of conjugation differed between enantiomers with glucuronide conjugation much greater for (R,R)‐formoterol (around 30%–60% of total) compared to (S,S)‐formoterol (0%–30%). There was clear evidence of inter‐individual enantioselectivity observed in the ratios of (R,R):(S,S)‐formoterol, where (S,S)‐ was predominant in free formoterol, and (R,R)‐ predominant in the conjugated metabolite. In conclusion, rac‐formoterol delivered by inhalation exhibits enantioselective elimination in urine following single‐dose administration. Enantioselective assays should be employed in doping control to screen for banned beta2‐agonist chiral switch products such as (R,R)‐formoterol, and total hydrolyzed rac‐formoterol is warranted to account for inter‐individual differences in enantioselective glucuronidation.     

Correlation betweenin vitro andin vivo drug metabolism rate: Oxidation of ethoxybenzamide in rat

In vitroand in vivocorrelations of the microsomal oxidation of drugs were examined, using ethoxybenzamide as a model drug. Ethoxybenzamide disappearance time course from rat plasma in vivowas analyzed by a two-compartment model assuming a Michaelis-Menten type elimination process. Ethoxybenazmide oxidation in vitrowas measured by the appearance rate of salicylamide in rat liver microsomal suspension. Parameters obtained were V_max=3.46 and 3.77 moles/min/kg body weight and K_m=0.378 and 0.192mM, in vitroand in vivo,respectively.     

Chiral Amines Derived from 2-Arylpropionic Acids: Novel Reagents for the Liquid Chromatographic (LC) Fluorescence Assay of Optically Active Carboxylic Acid Xenobiotics

For the enantiospecific analysis of optically active carboxylic acids, the availability of readily detectable coupling components is desirable, but highly fluorescent chiral amines are rare. From activated enantiomers of fluorescent 2-arylpropionic acids fluorescent chiral amines were synthesized via Curtius degradation, i.e., under formation of the acyl azide, the isocyanate, and finally, the amine. The formation of isocyanates and of amine hydrochlorides led to an inversion of the direction of rotation of polarized light. Amines derived from R- and S-flunoxaprofen, R- and S-naproxen, and R/S-benoxaprofen were characterized. The amines were found to be applicable for the chiral separation of carboxylic acids (such as 2-arylpropionic acids) as diastereomeric derivatives via high-performance liquid-chromatographic (normal and reversed-phase) and thin-layer chromatographic techniques.     

In Vivo and in Vitro Assessment of Baseline Blood-Brain Barrier Parameters in the Presence of Novel Nanoparticles

Purpose. Nanoparticles have advantage as CNS drug delivery vehicles given they disguise drug permeation limiting characteristics. Conflicting toxicological data, however, is published with regard to blood-brain barrier integrity and gross mortality. Methods. To address this issue two novel nanoparticle types: emulsifying wax/Brij 78and Brij 72/Tween 80 nanoparticles were evaluated in vivo for effect on cerebral perfusion flow, barrier integrity, and permeability using the in situ brain perfusion technique. Additional evaluation was completed in vitro using bovine brain microvessel endothelial cells for effect on integrity, permeability, cationic transport interactions, and tight junction protein expression. Results. In the presence of either nanoparticle formulation, no overall significant differences were observed for cerebral perfusion flow in vivo. Furthermore, observed in vitro and in vivo data showed no statistical changes in barrier integrity, membrane permeability, or facilitated choline transport. Western blot analyses of occludin and claudin-1 confirmed no protein expression changes with incubation of either nanoparticle. Conclusions. The nanoparticle formulations appear to have no effect on primary BBB parameters in established in vitro and in vivo blood-brain barrier models.     

In vitro cleavage of diisocyanate-glutathione conjugates by human gamma-glutamyl transpeptidase-1

1. Isocyanates differ from many other xenobiotics in their ability to form S-linked conjugates with glutathione (GSH) through direct nucleophilic addition reactions (e.g. without enzymatic “preactivation” and/or transferase activity), potentially predisposing them to metabolism via the mercapturic acid pathway. In vivo, mono-isocyanates are metabolized via the mercapturic acid pathway and excreted as N-acetylated cysteine conjugates, however, the metabolism of di-isocyanates remains unclear.

2. We assessed the ability of purified human gamma-glutamyl transpeptidase-1 (GGT-1), a primary enzyme of the mercapturic acid pathway, to cleave S-linked GSH conjugates of 4,4′-methylene diphenyl diisocyanate (MDI) and 1,6-hexamethylene diisocyanate (HDI), two widely used industrial chemicals.

3. A combination of liquid chromatography (LC), tandem mass spectrometry (MS/MS) and hydrogen-deuterium exchange studies confirmed GGT-1 mediated formation of the 607.2 and 525.2 m/z (M?+?H)⁺?ions corresponding to bis(cys-gly)-MDI and bis(cys-gly)-HDI, respectively, the cleavage products expected from the corresponding bis(GSH)-diisocyanate conjugates. Additional intermediate metabolites and mono(cys-gly)-conjugates with partially hydrolyzed diisocyanate were also observed.

4. Consistent with GGT enzyme kinetics, metabolism proceeded more rapidly under conditions that favored transpeptidation versus hydrolytic mechanisms of cleavage. Together the data demonstrate the capacity of human GGT-1 to cleave GSH conjugates of both aromatic and aliphatic diisocyanates, suggesting a potential role in their metabolism.

     

The Measurement of Warfarin Enantiomers in Serum Using Coupled Achiral/Chiral,High-Performance Liquid Chromatography (HPLC)

An assay for the serum concentration of the enantiomers of warfarin, R-warfarin and S-warfarin, has been developed using a bovine serum albumin chiral stationary phase (BSA-CSP) coupled to a Pinkerton internal-surface reverse-phase (ISRP) achiral column. The ISRP column is used to separate R,S-warfarin from the serum components and warfarin metabolites and to quantitate the total R,S-warfarin concentration. The eluent containing R,S-warfarin is then selectively transferred to the BSA-CSP, where the enantiomers are stereochemically resolved ( = 1.19) and the enantiomeric composition is determined. This system is sensitive and accurate, does not require extensive precolumn manipulations, and can be automated for use in large-scale clinical studies.     

In Vivo Bioavailability and Metabolism of Topical Diclofenac Lotion in Human Volunteers

Purpose. The primary objective of this study was to determine the rate and extent of transdermal absorption for systemic delivery of diclofenac from Pennsaid (Dimethaid Research, Inc.) topical lotion into the systemic circulation after the lotion was applied to human volunteers, in an open treatment, non-blinded, non-vehicle controlled study. In addition, the in vivo metabolism of this topical diclofenac lotion has also been studied. Methods. Human volunteers were dosed with topical [¹⁴C]-diclofenac sodium 1.5% lotion on the knee for 24 h. Sequential time blood and urine samples were taken to determine pharmacokinetics, bioavailability and metabolism. Results. Topical absorption was 6.6% of applied dose. Peak plasma ¹⁴C occurred at 30 h after dosing, and peak urinary ¹⁴C excretion was at 24–48 h. The urinary ¹⁴C excretion pattern exhibits more elimination towards 24 h and beyond, as opposed to early urinary ¹⁴C excretion. This suggests a continuous delivery of [¹⁴C]-diclofenac sodium from the lotion into and through skin which only ceased when the dosing site was washed. Skin surface residue at 24 h was 26 ± 9.5% dose (remainder assumed lost to clothing and bedding). Extraction of metabolites from urine amounted to 7.4–22.7% in untreated urine, suggesting substantial diclofenac metabolism to more water soluble metabolites, probably conjugates, which could not be extracted by the method employed. Two Dimensional TLC analysis of untreated urine showed minimal or no diclofenac, again emphasizing the extensive in vivo metabolism of this drug. Treatment of the same urine samples with the enzymes sulfatase and (-glucuronidase showed a substantial increase in the extractable material. Three spots were consistently present in each sample run, namely diclofenac, 3hydroxy diclofenac and an intermediate polar metabolite (probably a hydroxylated metabolite). Therefore, there was significant sulfation and glucuronidation of both diclofenac and numerous hydroxy metabolites of diclofenac, but many of the metabolites/conjugates remain unidentified. Conclusions. There was a continuous delivery of diclofenac sodium from the lotion into and through the skin, which ceased after the dosing site was washed. The majority of the material excreted in the urine were conjugates of hydroxylated metabolites, and not the parent chemical, although further identification is required.     

Drug Distribution in Human Skin Using Two Different In Vitro Test Systems: Comparison with In Vivo Data

Purpose. Two in vitro test systems used to study drug penetration into human skin—the Franz diffusion cell (FD-C) and the Saarbruecken penetration model (SB-M)—were evaluated, and the results were compared with data gained under analogous in vivo conditions. Methods. Excised human skin was used in all in vitro experiments. Flufenamic acid dissolved in wool alcohols ointment, was chosen as a model drug, and the preparation was applied using infinite dose conditions. To acquire quantitative information about the drug penetration, the skin was segmented into surface parallel sections at the end of each experiment, first by tape stripping the stratum corneum (SC), and second by cutting the deeper skin layers with a cryomicrotome. The flufenamic acid was extracted from each sample and assayed by high performance liquid chromatography (HPLC). For in vivo experiments, only the tape stripping technique was used. Results. a) Drug penetration into the SC: In both in vitro test systems the total drug amounts detected in the SC were found to increase over the different incubation times. Similar conditions were obtained in vivo, but on a lower level. Using Michaelis-Menten kinetics, the m_max value was calculated for the skin of two donors. The relations of the m_max values for the FD-C and the SB-M closely correspond (1.26 [donor 1] and 1.29 [donor 2]). A direct linear correlation of the drug amount in the SC and the time data were found for in vivo with both in vitro test systems.%b) Drug penetration into the deeper skin layers: The detected drug amounts in the deeper skin layers continuously increased with the incubation time in the SB-M, while in the FD-C, only very small drug amounts were observed after incubation times of 30 and 60 minutes. It was also noticed, that the drug amounts rose steeply at time points 3 and 6 hours. Additional studies showed a remarkable penetration of water into the skin from the basolateral acceptor compartment in the FD-C. This could explain the different drug transport into the deeper skin layers between the two in vitro test systems. Conclusions. Both in vitro models showed comparable results for the drug penetration into the SC and a robust correlation with in vitro data. Different results were obtained for the deeper skin layers. Whether a correlation between in vitro and in vivo data is also possible here has to be investigated by further experiments.     

Enantioselective pharmacokinetics of sibutramine in rat

Racemic sibutramine is widely used to treat obesity owing to its inhibition of serotonin and noradrenaline reuptake in synapses. Although the enantioselective effects of sibutramine and its two active desmethyl-metabolites, monodesmethylsibutramine (MDS) and didesmethylsibutramine (DDS), on anorexia and energy expenditure have been elucidated, the enantioselective pharmacokinetics of sibutramine are still unclear. Therefore, we aimed to characterize the enantioselective pharmacokinetics of sibutramine and its metabolites in plasma and urine following an intravenous and a single oral administration of sibutramine in rats. The absolute bioavailability of sibutramine was only about 7%. The pharmacologically less effective S-isomer of DDS was predominant in the plasma: the C _max and the AUC _inf were 28 and 30 times higher than those of the R-isomer, respectively (p<0.001). In the urine, the concentrations of the R-isomers of hydroxylated DDS and hydroxylated and carbamoylglucuronized MDS and DDS appeared to be 11.3-, 5.1-, and 5.3-times the concentrations of the respective S-isomers. Thus, regardless of increased potency than the S-enantiomers, the R-enantiomers of the sibutramine metabolites MDS and DDS were present at lower concentrations, owing to their rapid biotransformation to hydroxylated and/or carbamoylglucuronized forms and their faster excretion in the urine. The present study is the first to elucidate the enantioselective pharmacokinetics of sibutramine in rats.     

An in vivo evaluation of the ontogeny of stereoselective fluoxetine metabolism and disposition in lambs from birth to one year of age

Abstract

1. The objective was to determine the ontogeny of stereoselective fluoxetine (FX) disposition in postnatal sheep from newborn to adulthood.

2. Catheters were implanted in a carotid artery and jugular vein. FX was administered intravenously, followed by serial arterial blood and cumulative urine collection. The concentrations of R,S-FX and R,S-norfluoxetine (R,S-NFX) in samples were measured using a validated enantioselective LC/MS/MS analytical method.

3. The metabolism of FX at 4.2?±?0.4?days was limited compared to adults, but had developed compared to the fetus. Total body clearance (Cl_TB) did not significantly increase up to 33.6?±?0.9?days, but significantly increased at 98.5?±?2.0?days, with no further changes up to 397.3?±?8.5?days. Up to 13.4?±?0.8?days, the disposition of FX included Phase I metabolism to NFX and trifluoromethylphenol (TFMP), and renal elimination. At 32.9?±?0.9?days, metabolism included Phase II conjugates of FX and NFX. Renal elimination of these compounds was low.

4. The elimination of FX increased in a non-linear manner during the first year in sheep. The metabolism and disposition of FX and NFX in plasma and urine were stereoselective and this appeared due to both stereoselective protein binding and metabolism.     

Application of a chiral high‐performance liquid chromatography‐tandem mass spectrometry method for the determination of 13 related amphetamine‐type stimulants to forensic samples: Interpretative hypotheses

Interpretation of amphetamine‐type stimulant (ATS) findings in urine samples can be challenging without chiral information. We present a sensitive enantioselective high‐performance liquid chromatography–tandem mass spectrometry method for the quantification of (R)‐amphetamine, (S)‐amphetamine, (R)‐methamphetamine, (S)‐methamphetamine, (1R,2R)‐pseudoephedrine, (1S,2S)‐pseudoephedrine, (1R,2S)‐ephedrine, (1S,2R)‐ephedrine, (1R,2S)‐norephedrine, (1S,2R)‐norephedrine, (R)‐cathinone, (S)‐cathinone, and (1S,2S)‐norpseudoephedrine (cathine) in urine. The method was successfully applied to more than 100 authentic urine samples from forensic casework. In addition, samples from a controlled self‐administration of (1S,2S)‐pseudoephedrine (Rinoral, 1200 mg within 6 days) were analyzed. The results strengthen the hypothesis that (1R,2S)‐norephedrine is a minor metabolite of amphetamine and methamphetamine. We suggest cathine and (1S,2R)‐norephedrine as minor metabolites of amphetamine racemate in humans. Small methamphetamine concentrations detected in samples with high concentrations of amphetamine could result from a metabolic formation by methylation of amphetamine although in samples with an (R)/(S) ratio for methamphetamine < 1 an additional (previous) (S)‐methamphetamine consumption seems likely. Our data suggest that even amphetamine concentrations exceeding methamphetamine concentrations in urine can be caused by the biotransformation of methamphetamine to amphetamine as long as no (R)‐amphetamine is detected. However, without chiral information, such findings might be (falsely) assumed as a co‐consumption of both substances. Cathinone enantiomers detected in urine samples with high amphetamine concentrations can be interpreted as metabolites of amphetamine. In addition, the results of the self‐administration study revealed that both cathinone enantiomers are minor metabolites of (1S,2S)‐pseudoephedrine, which is the active ingredient of various medicines used for cold. The enantioselective analysis is a powerful tool to avoid the misinterpretation of ATS findings in urine samples.     

Metoprolol does not reduce platelet aggregability during sympatho-adrenal stimulation

Summary The possibility that -adrenoceptor blockers, especially ₁-selective agents might inhibit platelet function is of considerable interest, as this might be of pathophysiological importance in cardiovascular diseases. Platelet function, however, is difficult to assess and in vivo related data are scarce.The effect of one week of treatment with metoprolol 200 mg/day on platelet aggregability during mental stress (colour word conflict test; CWT) and low and high dose adrenaline infusions has been evaluated in a double-blind, placebo-controlled, cross-over study in 10 healthy male volunteers. Platelet function in vivo was assessed using ex vivo filtragometry, and the urinary excretions of -thromboglobulin (HMW -TG) and 11-dehydro-TxB₂ (a thromboxane metabolite). Conventional in vitro aggregometry and the urinary levels of 2,3-dinor-6-keto-PGF₁ (a prostacyclin metabolite) were also studied.During the interventions there was increased platelet aggregability in vivo, as filtragometry readings were shortened by 41±11% during high dose adrenaline infusion, urinary HMW -TG levels increased and urinary 11-dehydro-TxB₂ tended to increase. In contrast, platelet sensitivity to ADP in vitro was reduced. The urinary 2,3-dinor-6-keto-PGF₁ levels were increased during the interventions.Despite the cardiovascular and biochemical signs of -adrenoceptor blockade at rest and during the interventions, metoprolol failed to influence platelet function in vivo, as measured by ex vivo filtragometry, or urinary HMW -TG or 11-dehydro-TxB₂ levels. It tended rather to enhance the stress response measured by ex vivo filtragometry. Platelet aggregability in vitro and urinary 2,3-dinor-6-keto-PGF₁ levels were not altered by metoprolol.Thus, metoprolol was not found to reduce platelet aggregability in healthy male volunteers either at rest or during sympatho-adrenal activation. The effect of treatment may still differ in patients; studies in patients with ischaemic heart disease are under way.     

The 5-HT1A receptor selective ligands, (R)-8-OH-DPAT and (S)-UH-301, differentially affect the activity of midbrain dopamine neurons

Summary The effects of the selective 5-HT_1A receptor agonist (R)-8-hydroxy-2(di-n-propylamino)tetralin [(R)-8-OH-DPAT] and the novel 5-HT_1A antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)-tetralin [(S)-UH-301] were studied with regard to the firing pattern of single mesencephalic dopamine (DA) neurons with extracellular recording techniques in chloral hydrate anesthetized male rats. Neuronal activity was studied with respect to firing rate, burst firing and regularity of firing. In the ventral tegmental area (VTA) low doses of (R)-8-OH-DPAT (2–32 g/kg i.v.) caused an increase in all three parameters. The effect on firing rate of DA neurons was more pronounced in the parabrachial pigmentosus nucleus than in the paranigral nucleus, the two major subdivisions of VTA. In the substantia nigra zona compacta (SN-ZC), (R)-8-OH-DPAT (2–256 g/kg i.v.) had no effect on firing rate and regularity of firing and only slightly increased burst firing. High doses of (R)-8-OH-DPAT (512–1024 g/kg i.v.) decreased the activity of DA cells in both areas, an effect that was prevented by pretreatment with the selective DA D₂ receptor antagonist raclopride. (S)-UH-301 (100–800 g/kg i.v.) decreased both firing rate and burst firing without affecting regularity of DA neurons in the VTA. In the SN-ZC, (S)-UH-301 decreased the firing rate but failed to affect burst firing and regularity of firing. These effects of (S)-UH-301 were blocked by raclopride pretreatment. Local application by pneumatic ejection of 8-OH-DPAT excited the DA cells in both the VTA and the SN-ZC, whereas (S)-UH-301 inhibited these cells when given locally. These results show that 5-HT_1A receptor related compounds differentially affect the electrophysiological activity of central DA neurons. The DA receptor agonistic properties of these compound appear to contribute to the inhibitory effects of high doses of (R)-8-OH-DPAT and (S)-UH-301 on DA neuronal activity. Given the potential use of 5-HT_1A receptor selective compounds in the treatment of anxiety and depression their effects on central DA systems involved in mood regulation and reward related processes are of considerable importance.Correspondence to T. H. Svensson at the above address     

Occlusive Properties of Monolayer Patches: In Vitro and in Vivo Evaluation

Purpose. Patches can cause a different grade of skin occlusion, depending on matrix composition and thickness, backing layer material. The aim of this work was to verify if in vitro water vapour permeability (WVP) values are predictive of transepidermal water loss (TEWL) and Fourier-transform infrared (FTIR) spectroscopy values measured in vivo after 24 h of methacrylic or acrylic monolayer patches application. The correlation between both in vivo methods has been evaluated. Methods. The WVP, TEWL and FTIR measurements were performed by using four patches made of a methacrylic or an acrylic polymeric system (250 and 500 m thickness on a polyurethane backing layer). A fifth patch was made of the methacrylic matrix on a polyvinyl chloride backing layer. Results. A good correlation was found between TEWL values and IR water/lipid absorbance ratios. The in vitro WVP values are in a good correlation with the results of both in vivo methods: TEWL = –0.01WVP + 21.31 (R² = 0.9312); FTIR water/lipid ratio = –0.01WVP + 27.15 (R² = 0.9447). Conclusions. The in vitro method proposed for measuring the WVP is predictive of the degree of occlusion resulting from the in vivo application of monolayer patches.     

Carvedilol Stereopharmacokinetics in Rats: Affinities to Blood Constituents and Tissues

Carvedilol, a lipophilic β-adrenoceptor antagonist with vasodilating activities, is characterized by a high as well as stereoselective metabolic clearance and distribution volume. Tissue distribution of carvedilol enantiomers and their conjugates were determined under steady-state conditions in rats (p.o., 10 mg/kg, repetitive dosage; n = 5) and after single i.v. administration in control rats and rats with surgical portacaval shunt (pcs) (10 mg/kg; n = 3 each group). In addition, in vitro plasma protein binding was evaluated. - The plasma protein binding of carvedilol in rats is > 98% for total plasma (tp) and > 96% for rat serum albumin (rsa) solution (4%), with enantioselectivity ratios of 1.53 (tp) and 1.27 (rsa). Significantly higher unbound fractions were observed in pcs rats, in part due to reduced protein concentrations. - In contrast to plasma, where a preponderance of the R-enantiomer with an S/R ratio of 0.6 was found, S-carvedilol was predominant in all tissues (heart, liver, kidneys, lung, spleen, muscle, and adipose tissue), with S/R ratios of 1.3-1.4 in most of these tissues and 2.3 in liver. This preferential tissue partitioning of S-carvedilol was in accordance with its higher unbound fraction in plasma. Carvedilol accumulated predominantly in the highly perfused and/or eliminating organs liver, kidneys, and lung (tissue/plasma ratios; lung: S 76, R 34; liver: S 21, R 5; kidney: S 8, R 3). A similarly enantioselective distribution into the heart of control as well as pcs rats was observed, where the S-enantiomer concentrations exceeded the plasma concentrations 7-fold. Probably because of the impaired liver function in pcs rats with increased importance of the renal route, kidney concentrations were higher in these rats. The kidney/plasma ratio was elevated approximately 2-fold for the parent compound (control: S 7, R 2; pcs: S 14, R 4) and 4-fold for the R-carvedilol conjugate (control: S 2, R 1; pcs: S 3, R 4). The conjugates of carvedilol were detectable in all organs, with significantly smaller concentrations than those of the aglycones and with varying stereoselectivities.     

Association with polymorphic marmoset cytochrome P450 2C19 of in vivo hepatic clearances of chirally separated R-omeprazole and S-warfarin using individual marmoset physiologically based pharmacokinetic models

1. Simulated clearances of R-warfarin and efavirenz were recently reported for individual cynomolgus monkeys genotyped for cytochrome P450 2C19 and 2C9, respectively. To expand and verify this modeling procedure, simulations of R/S-omeprazole and R/S-warfarin clearances after oral administrations in individual marmosets were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling consisting of gut, liver and central compartments.

2. Pharmacokinetics of R/S-omeprazole were chirally determined using the previously reported plasma microsamples in this study. The areas under the plasma concentration/time curves (AUC) of R-omeprazole and S-warfarin, but not S-omeprazole and R-warfarin, after oral administrations in the P450 2C19 homozygous mutant group were significantly higher than those in the wild-type group. These modeled hepatic intrinsic clearances were also significantly associated with the marmoset P450 2C19 genotypes. Other parameter values, e.g. absorption rate constants or systemic circulation volumes, were not likely determining factors.

3. The reported individual AUC values measured in 4–6 marmosets after oral R-omeprazole and S-warfarin administrations were significantly correlated with the AUC values predicted using the PBPK models after virtual administrations.

4. This study indicates that clearances of R-omeprazole, S-warfarin and related medicines associated with polymorphic P450 2C19 in individual marmosets can be simulated using simplified individual PBPK models.     

N-Glucuronidation of N-hydroxy aromatic amines: A mechanism for their transport and bladder-specific carcinogenicity

Glucuronide conjugates of carcinogenic N-hydroxy metabolites of the primary aromatic amines, 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 1-naphthylamine (1-NA) were isolated from the urine of dogs administered the respective primary amine and from the in vitro incubation of N-hydroxy metabolites with uridine-5′-disphosphoglucuronic acid-fortified dog liver microsomes. The urinary and microsomal conjugates were purified by several sequential chromatographic procedures, including Sephadex G-15, Amberlite XAD-2, and cellulose CF-11 chromatography for microsomal conjugates and Sephadex G-10, DEAE, and Amberlite XAD-2 chromatography for urinary conjugates. The infrared spectra of purified urinary and microsomal conjugates of these three N-hydroxy aromatic amines were identical to spectra of authentic N---C glucuronides prepared by two different synthetic procedures. The urinary and microsomal conjugates comigrated with synthetic N---C glucuronides in two solvent systems. These observations in conjunction with previous studies provide evidence that N---C glucuronidation represents a general metabolic reaction of carcinogenic N-hydroxy aromatic amines which provides the means of transport of these compounds to their site of action in the bladder.