Identification with liquid chromatography-ionspray mass spectrometry of the metabolites of the enantiomers N-methyl dextrorphan and N-methyl levorphanol after rat liver perfusion.

To gather more information on stereochemical factors in the hepatic disposition of organic cations, mass spectrometry coupled to liquid chromatography was used to determine the identity of the metabolites excreted in bile after isolated rat liver perfusions with the quaternary ammonium derivatives of the enantiomeric drugs dextrorphan and levorphanol. Ionspray mass spectrometry was chosen for its soft ionization and absence of thermal degradation of labile compounds. The drugs were labelled with a stable (2H) isotope and mixed with unlabelled drugs to create an artificial isotope pattern in the mass spectrum and facilitate the recognition of unknown metabolites. In mass spectra that were recorded under normal conditions, fragmentation was absent and metabolites of N-methyl dextrorphan and N-methyl levorphanol were visible as parent-ion 'doublets'. Collision-induced fragmentation studies were performed to support the identification of the metabolites. For N-methyl dextrorphan the glucuronide, the glutathione conjugate and the glucuronide of the N-demethylated metabolite were found in bile. For N-methyl levorphanol the glucuronide, the glutathione conjugate, the sulphate conjugate and the glucuronide of a hydroxylated N-methyl levorphanol were excreted in bile. Thus a remarkable stereoselectivity occurs in the metabolism of these quaternary ammonium compounds in the rat liver.

Impact of structural differences on the in vitro glucuronidation kinetics of potentially dopaminergic hydroxy-2-aminotetralins and naphthoxazines using rat and human liver microsomes.

The in vitro glucuronidation of seven monohydroxy-2-aminotetralins and two naphthoxazines has been determined using human and rat liver microsomes. All these compounds stimulate the D2 dopamine receptor. The influence of the position of the phenolic hydroxyl group was studied with rat microsomes in monohydroxy-2-(N,N-dipropylamino)-tretralins. The highest activity and intrinsic clearance was found for 7-OH-DPAT, but the latter values for 5-OH-DPAT and 6-OH-DPAT were much lower by a factor of 9 and 30, respectively. The 8-OH-isomer was not glucuronidated at all. Substitution of a propyl side chain by a thienylethyl-, or phenylethyl side chain, in 5-hydroxy-DPAT, or in (+)-4-propyl-9-hydroxyhexahydronaphthoxazine (PHNO, N-0500), showed a large increase of the UDPGT affinity and intrinsic clearance especially for N-0437. It also resulted for N-0437 in a much higher affinity towards the dopaminergic D2 receptor. Although the glucuronidation activity of human microsomes was found to be considerably lower than that of rat microsomes, the latter phenomenon was clearly visible with human microsomes as well. These findings may have serious implications for the ability of these drugs to adequately reach the brain.

Metabolism and disposition of the dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in conscious monkeys after subsequent i.v. oral, and ocular administration.

The metabolism of 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437) was investigated in conscious monkeys after subsequent i.v., oral, and ocular administration. The administration of the drug caused some physiological effects, such as bradycardia and sedation of the monkeys. During a collection period of 120 hr, on average 83% was recovered after iv administration and 90% after p.o. dosing. After i.v. administration, 44% was excreted in the bile, as compared to 38% in the urine and about 1% in the feces. After oral administration, bile is the major excretion route, accounting for about 60% of the dose, as compared to 25% in the urine and about 5% in the feces. After ocular administration, on average 62% was recovered after 7 hr, excreted in bile and urine in about equal amounts. All percentages given above reflect the total amount of radioactivity recovered, thus comprising the unchanged drug plus various metabolites. After all three dosing routes, N-0437 was metabolized almost completely prior to elimination. Direct glucuronidation of the phenolic group proved to be the major metabolic pathway of N-0437, comprising about 44% of the dose after i.v. and ocular administration and 72% after oral dosing. Hydroxylation of N-0437 at the position ortho to the phenolic group present yielded a catechol intermediate, which was excreted as a glucuronide and accounted for about 10% of the dose. In the monkey, a clear regioselective preference towards glucuronidation at the 6-position was observed. Besides the glucuronide, the sulfoconjugate of N-0437 was a major metabolite after i.v. and ocular administration, accounting for about 15% of the dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Ocular and systemic disposition of the dopamine agonist N-0437 in monkeys after ocular administration.

The ocular and systemic disposition of the new dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437) was investigated in conscious monkeys after ocular administration of 0.56 mg N-0437. HCl (corresponding to 0.50 mg free base), containing 200 microCi [3H]N-0437, into the right eye. After killing the animal, the eyes were removed and several eye tissues were dissected and assessed for their content of radioactivity. In the treated eye, the iris contained by far the highest concentration of radioactivity, while in the untreated eye the lower conjunctiva, the iris the ciliary body and the choroid possessed the highest levels of radioactivity. In all dissected eye tissues, a substantially higher concentration of radioactivity was established for the treated eye compared to the untreated eye, except for the vitreous in which for both eyes about an equal concentration was measured. This result suggests the presence of a systemic transport, which was confirmed by the occurrence of bradycardia, starting immediately after ocular application of the drug and lasting for about 1.5 hr. As the total amount of radioactivity in both eyes 7 hr after ocular dosing is very low (0.3-0.5% of the dose), one can conclude that N-0437 is almost completely taken up into the general circulation. The radioactive measurements of bile and urine samples collected up to 7 hr after administration revealed that the elimination of N-0437 and its metabolites is very fast, with the urinary excretion (35% of the dose) slightly higher than to the biliary one (31% of the dose).

Metabolic conversion of cyoctol during skin passage in humans.

Upon application of 14C-labeled cyoctol to the forearm of healthy volunteers, no parent cyoctol was detectable in ipsilateral blood plasma. The 14C activity was largely accounted for by a component with higher lipophilicity than the parent compound, as justified from their HPLC retention. Thus, this study suggests that human skin is capable of nearly complete cutaneous first-pass metabolism, resulting in negligible systemic availability of cyoctol. In a comparable experiment, rabbits were also able to convert cyoctol during skin absorption to a more lipophilic metabolite, which was identified as the palmitoleic acid ester of O-demethylated cyoctol by GC/MS. However, chromatographic evidence indicates that the human ipsilateral metabolite differs from the rabbit cyoctol metabolite.

Disposition and metabolic profiling of the penetration enhancer Azone. I. In vitro studies: urinary profiles of hamster, rat, monkey, and man.

Chain-labeled 14C-Azone was intravenously administered to hamster, monkey, and rat, to compare its metabolic profile with that obtained previously in humans after dermal application. Azone-derived radioactivity was excreted predominantly in the urine for both hamster and monkey, which is similar to the disposition in humans. Metabolic profiling in urine revealed extensive systemic metabolism to occur in all species studied. The main fraction of the metabolites was most polar in man, followed by rat, monkey, and hamster. Traces of the parent compound were detectable only in hamster urine. Although some of the polar major human metabolites were also present in rat urine, the animals were unsuitable for collecting metabolites of Azone observed in humans. In rats, complete cleavage of the dodecyl side chain was ruled out by administering Azone that had been labeled at two distinct positions of the molecule. Additionally, oral administration of Azone to rats resulted in the same metabolic profile as intravenous administration, indicating that gastrointestinal metabolism does not occur or is similar to systemic metabolism.

The metabolic fate of the dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in rats after intravenous and oral administration. I. Disposition and metabolic profiling.

1. The disposition and metabolic profiling of 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin(I), a dopamine agonist, were studied in anaesthetized rats after i.v. administration and in non-anaesthetized rats after i.v. and oral dosing. No major differences due to narcosis were observed. 2. Independent of dosing route or anaesthetic, clearance of I was rapid. Bile was the main route of excretion, accounting for 88% dose, compared with 9% in urine. 3. Drug metabolic profiling revealed that I is almost completely metabolized before elimination; less than 0.5% total radioactivity in bile and urine was due to parent compound. 4. The biliary metabolic profiles after i.v. and oral administration were similar. One major metabolite was detected, accounting for 50% (i.v.) or 65% (oral) dose. The major biliary metabolite was identified as the glucuronide of I. 5. Urinary metabolic profiles were quantitatively different from those of bile. After i.v. administration one major metabolite was detected in urine, but this was not the major biliary metabolite. After oral administration, the major urine metabolite was the same as the major biliary metabolite. These differences can be explained by first-pass gastro-intestinal metabolism.

The metabolic fate of the dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in rats after intravenous and oral administration. II. Isolation and identification of metabolites.

1. The in vivo metabolic pathways of 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (I) in rats have been established, using in vitro metabolism as a complementary technique. 2. All identified metabolites were conjugates. Glucuronidation at the phenolic group yields the major metabolite, accounting for 50% (i.v.) or 65% (oral) of the dose. The corresponding sulphate conjugate of I is virtually absent (less than 0.2% dose). 3. Hydroxylation of I, at the ortho position to the phenolic hydroxy group, yields 6-hydroxy-I (II), accounting for about 13% (i.v.) or 9% (oral) dose. This catechol is excreted, as a glucuronide, almost exclusively into the bile. Both the 5- and the 6-glucuronide of II were detected in about equal amounts. 4. Metabolism of I in vitro showed that under oxidative conditions, depropylation of I occurred. Conjugation of 3H-I in the presence of UDPGA or PAPS, was successful in yielding the glucuronide and sulphate conjugates.

Percutaneous absorption, metabolic profiling, and excretion of the penetration enhancer azone after multiple dosing of an azone-containing triamcinolone acetonide cream in humans.

Radioactive Azone (1.6%; 1-dodecylazacycloheptan-2-one) was incorporated in a therapeutic formulation containing triamcinolone acetonide at a concentration of 0.05%. This cream (TAZ) was applied for four consecutive days to human volunteers on the same 24-cm2 application area on the forearm for 12 h under occlusion. The percutaneous absorption of Azone as measured in the excreta appeared to be only 3.47 +/- 0.33% during the whole study period. Azone-derived radioactivity was predominantly excreted by the kidneys (97.8 +/- 0.4%). From the urinary excretion plot, it could be deduced that the flux of Azone through human skin increased during the study period, reaching a plateau within 2-3 d. Accumulation of Azone in the stratum corneum did not occur. Only unchanged Azone could be detected in the stratum corneum. Excretion was mainly in the form of very polar metabolites. Compared with pure Azone, the therapeutic formulation did not influence the metabolism, excretion route, or urinary elimination rate of the penetration enhancer.

Synthesis of d-[11C]oxyphenonium iodide, a potential radioligand for in vivo visualization of human cholinergic muscarinic receptor-sites by positron emission tomography.

Carbon-11 labeled d-oxyphenonium iodide, a cholinergic antagonist is synthesized for in vivo visualization of muscarinic receptor-sites on airway tissue by positron emission tomography (PET). Methylation with [11C]CH3I of d-demethyloxyphenonium, followed by HPLC purification affords the desired radiopharmaceutical with a radiochemical yield of 66% (based on [11C]CH3I, and corrected for decay) and with a specific activity of 110-300 Ci/mmol. The biologically active labeled d-enantiomer is prepared within 40 min after EOB. Optical and chemical purity proved to be better than 99.9%. Radiochemical purity was determined to be higher than 99%.

Unequal disposition of enantiomers of the organic cation oxyphenonium in the rat isolated perfused liver.

This paper describes the results of pharmacokinetic experiments in the rat isolated perfused liver with enantiomers of oxyphenonium. The study was performed with the [14C]methyl labelled compounds. In this preparation both metabolism and biliary excretion were significantly different for the (+)- and the (-)-isomer. Hepatic uptake rate was similar, but total biliary excretion (including metabolites) of the (-)-isomer was only 55% compared with the excretion of the (+)-isomer. In line with these data, after 2 h only 30% of the dose of the (+)-isomer and over 50% of the dose of the (-)-isomer was still found in the liver, predominantly in the form of metabolites. The metabolic profile was investigated using ion pair TLC. At least two metabolites were detected in bile for both enantiomers. However, unchanged (-)-oxyphenonium persisted for longer in bile, indicating either a more rapid canalicular transport of the (+)-isomer and/or a more rapid metabolism of (+)-oxyphenonium to cholephilic metabolites.

Determination of enantiomeric purity of the new D-2 dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437) by reversed-phase high-performance liquid chromatography after pre-column derivatization with D(+)-glucuronic acid.

This paper describes an enzymic derivatization procedure that allows accurate determination of very small amounts of enantiomeric impurities in the D-2 dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437). After pre-column glucuronidation of the individual enantiomers, two diastereoisomers were formed which were separated by reversed-phase high-performance liquid chromatography. An enantiomeric purity of 99.84% was calculated for the (-)-enantiomer, against 99.89% for the (+)-enantiomer. The assay was validated by spiking 1% of the (-)-enantiomer in the (+)-enantiomer. A high accuracy (error 4.5%) and precision (coefficient of variation 2.9%, n = 5) of the method were established.

Isotopic separations of the drug N-0437 and its diastereoisomeric glucuronides by high-performance liquid chromatography.

During the investigations of the metabolic pathways of the new dopaminergic drug N-0437 we encountered a substantial difference in HPLC-retention times between the metabolites, detected by a uv spectrophotometer, and their tritium-labeled markers, measured off-line by a scintillation counter. These distinct retention times can be ascribed to a phenomenon known as isotopic fractionation. In this article we quantified the isotopic separation by reversed-phase HPLC of the unlabeled N-0437, its deuterated and tritiated analogs, and their corresponding glucuronides, synthesized in vitro by rat liver microsomes. In the separation of the glucuronides we demonstrated that this isotope effect is dependent largely on the eluent pH.

Chromatographic separation of enantiomers.

In this paper a review is presented on the chromatographic analysis of enantiomers with special attention to high pressure liquid chromatography. Also, some examples of resolution of racemates by thin layer chromatography and gas chromatography are given. The various procedures in the surveyed literature have been divided into three main classes: procedures with formation of diastereomeric compounds prior to the chromatographic separation, procedures in which a chiral mobile phase is used, and procedures with the use of a chiral stationary phase. These methods are subdivided and some examples of their application to drugs and related compounds are presented.

Percutaneous absorption and elimination of the penetration enhancer Azone in humans.

The percutaneous absorption and elimination of Azone, a new penetration enhancer, were investigated in humans. The distribution and accumulation of Azone in the skin were studied by means of tape stripping. These studies reveal that pure Azone is poorly absorbed. Furthermore, what little Azone is absorbed appears to be rapidly cleared from the circulation by the kidneys. In order to explain the urinary excretion profile, the formation of at least one metabolite is suggested. No accumulation of Azone in the skin was observed.

Determination of cyclodextrins in biological fluids by high-performance liquid chromatography with negative colorimetric detection using post-column complexation with phenolphthalein.

A rapid and sensitive high-performance liquid chromatographic method for the analysis of beta- and gamma-cyclodextrin in aqueous biological fluids such as plasma, urine, or tissue homogenate is described. The chromatographic system consists of a microBondapak Phenyl column as stationary phase and a mobile phase of water with 10% methanol. After post-column addition of an alkaline solution of phenolphthalein, negative colorimetric detection is used. The elution solvent and post-column reagent were mixed in a capillary tubing of 1.5 m (1.0 mm I.D.). Two methods of sample treatment are given, one for large (1.0 ml) and one for small (0.1 ml) sample volumes. Both methods were shown to be linear and reproducible. The detection limit for beta-cyclodextrin was 1.0 microgram/ml (0.77 nmol/ml). The method was used in the determination of some pharmacokinetic parameters of beta-cyclodextrin in rats after intravenous injection.

On-line diode array UV-visible spectrometry in screening for drugs and drug metabolites by high-performance liquid chromatography.

The direct coupling of a multi-channel diode array UV-visible spectrophotometer to a powerful reversed-phase HPLC separation system is considered, especially for use in qualitative analysis, e.g., screening/identification of drugs and drug metabolites. The approach is illustrated by the screening for metabolites of butoprozine and ticlopidine directly in human and rat bile.

Screening for impurities in butoprozine.

The purity analysis of butoprozine is described. Both gas chromatography-mass spectrometry (GC-MS) and high pressure liquid chromatography (HPLC) with UV-vis detection (conventional and multichannel) were used. In the butoprozine example disadvantages for both techniques became apparent: incorrect conclusions with regard to the purity of the drug would have been drawn if only one of these chromatographic techniques had been used. GC-MS allowed the identification of an impurity not found by HPLC.