Compound mixtures in Caco-2 cell permeability screens as a means to increase screening capacity.

The purpose of this investigation was a study of simultaneous permeability measurement using compound mixtures (cassette dosing) as an alternative to single compound evaluation in order to increase the capacity of screens for intestinal drug permeability. Drug transport across Caco-2 monolayers was studied, both in the apical to basolateral and the basolateral to apical direction. The apparent permeability coefficients for ten compounds displaying different intestinal transport mechanisms were determined, first as single compounds and then as components of a mixture. Seven beta-adrenoceptor antagonists and baclofen were analysed simultaneously using reversed phase HPLC with UV detection, D-glucose and mannitol were measured by scintillation counting. The results indicated that the Papp from the mixture as donor phase correlated well with that of the single compounds and merely small changes in the Papp of each compound were observed between the single compound and mixture experiments. This minor variation resulted in a change in rank-order of the poorly permeable compounds in the mixture, however, without affecting their association with the permeability class according to the biopharmaceutics classification system (BCS). It can be concluded that the use of compound mixtures is a suitable method for improving the capacity in permeability screens. Further improvement of the throughput may be expected upon automatisation of permeability measurements using robotics combined with increased selectivity using LC-MS analysis.

An assessment of human liver-derived in vitro systems to predict the in vivo metabolism and clearance of almokalant.

The ability of various human derived in vitro systems to predict various aspects of the in vivo metabolism and kinetics of almokalant have been investigated in a multicenter collaborative study. Although almokalant has been withdrawn from further clinical development, its metabolic and pharmacokinetic properties have been well characterized. Studies with precision-cut liver slices, primary hepatocyte cultures, and hepatic microsomal fractions fortified with UDP-glucuronic acid all suggested that almokalant is mainly glucuronidated to the stereoisomers M18a and M18b, which is in good agreement with the results in vivo. Both in vivo and in vitro studies indicate that the formation of M18b dominates over that of M18a, although the difference is more pronounced with the in vitro systems. Molecular modeling, cDNA-expressed enzyme analysis, correlation analysis, and inhibition studies did not clearly indicate which P450 enzymes catalyze the oxidative pathways, which may indicate a problem in identifying responsible enzymes for minor metabolic routes by in vitro methods. All of the in vitro systems underpredicted the metabolic clearance of almokalant, which has previously been reported to be a general problem for drugs that are cleared by P450-dependent metabolism. Although few studies on in vivo prediction of primarily glucuronidated drugs have appeared, in vitro models may consistently underpredict in vivo metabolic clearance. We conclude that in vitro systems, which monitor phase II metabolism, would be beneficial for prediction of the in vivo metabolism, although all of the candidate liver-derived systems studied here, within their intrinsic limitations, provided useful information for predicting metabolic routes and rates.

Presystemic elimination of the beta-blocker pafenolol in the rat after oral and intraperitoneal administration and identification of a main metabolite in both rats and humans.

Pafenolol is a beta 1-adrenoreceptor antagonist exhibiting some interesting oral absorption properties in both rat and humans. The blood concentration-time profile exhibits two peaks, and the bioavailability is low and dose-dependent due to an incomplete and nonlinear intestinal uptake. The origin of the presystemic metabolism was studied in rats after oral and intraperitoneal administration of tritium-labeled pafenolol with reference to the intravenous route by means of urinary excretion data of pafenolol and metabolites specifically assayed by HPLC and radioisotope detection. The oral-bioavailability increased from 15.8 +/- 4.1% (1.0 mumol/kg) to 33.3 +/- 5.8% (25 mumol/kg, p < 0.001). This was primarily due to a change in the fraction of the absorbed dose (fa) from 21.9 +/- 4.6 to 39.5 +/- 7.9% (p < 0.01). The bioavailability following an intraperitoneal dose was almost complete indicating that the presystemic metabolism was due to gut wall metabolism. Saturation of the presystemic metabolism contributed only by approximately 15-20% to the 2-fold increase of bioavailability. This clearly indicates that the underlying mechanism for the low and dose-dependent bioavailability was an incomplete and nonlinear intestinal uptake. The metabolic pattern showed that at least eight metabolites are formed in the rat. One of these is an alpha-OH pafenolol, identified as the main metabolite in human urine by mass spectrometry.

Determination of a renin inhibitor in plasma by solid-phase extraction using acetone as protein binding displacer followed by on-line high-performance liquid chromatography.

H 218/54 is a potent inhibitor of human renin activity (pIC50 = 8.3 at pH 6) and is therefore a potential agent for blood pressure reduction. This lipophilic compound is highly bound to plasma proteins, e.g. 99.7% in rats and 99.6% in humans. For pharmacokinetic studies, a quantitative assay for 3H-H 218/54 in plasma has been developed. On top of an AASP phenyl solid-phase cartridge 70 microliters of rat plasma or 1 ml of cynomolgus plasma was mixed with 200 microliters of water containing 20% acetone. The acetone displaced the substance from plasma proteins without precipitation of the sample and clogging of the extraction column. The mixture was passed through the cartridge, which adsorbed 3H-H 218/54. The cartridge was placed in an AASP autosampler connected to a reversed-phase LC system, with a Vydac C-18 column and CH3CN-H2O-TFA (60:40:0.1, v/v/v) as mobile phase. The effluent from the separation column was collected in fractions for radioactivity counting. Recovery, as measured after adding various amounts of tritium-labelled H 218/54 to blank plasma followed by repeated analysis of the samples, was close to 100% with relative standard deviations between 1.4 and 3.0%. At the lowest level tested, 200 dpm per sample, the recovery was 120% with a relative standard deviation of only 10%. The sensitivity of the method will depend on the specific radioactivity of the dose given.

Direct coupled column separation and determination of the diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine.

A reversed-phase coupled column separation (CCS) system for the analysis of two diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine is described. After direct injection of urine samples (50 microliters) the glucuronides were isolated by complex formation on a terbium(III) loaded strong cation exchanger at alkaline pH. The solutes were eluted from the precolumn by an acidic mobile phase, enriched and separated on Hypercarb (porous graphitic carbon) as analytical column with 0.1 M acetic acid pH 2.8 and 30% acetonitrile as mobile phase. The calibration graph was linear (r2 = 0.9999) and the detection limits were in the low picomole (UV) or femtomole (fluorescence) range. Optimization of the analytical column revealed that elution order and selectivity for the glucuronides were dependent on the buffer agent and temperature used. By appropriate choice of mobile phase conditions all four diastereomers could be separated.

Determination of four carboxylic acid metabolites of felodipine in plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method with ultraviolet detection at 220 nm was developed to determine four carboxylic acid metabolites in plasma following therapeutic doses of the calcium antagonist felodipine. After the addition of an internal standard the analytes were isolated by liquid-liquid and solid-phase extraction. The metabolites were applied to a C2 cartridge in their free acid form, but they were transformed and retained as ion pairs with tetrabutylammonium during a wash with phosphate buffer (pH 7), prior to automated elution and injection by the Varian AASP system onto the analytical C18 column. Using a sample volume of 1 ml of plasma, the lower limit of determination for the metabolites was about 20 nmol/l. The influence of the pH of the mobile phase on the retention time of the metabolites and the structural requirements for the internal standard were studied. The method was applied to plasma samples from four dogs collected after an oral dose of felodipine. The plasma concentration-time profiles of the metabolites gave useful information about the mechanisms by which they were formed and eliminated.

Pharmacokinetics of the enantiomers of felodipine in the dog after oral and intravenous administration of a pseudoracemic mixture.

1. A pseudoracemic mixture of deuterated (S)-felodipine and unlabelled (R)-felodipine was administered as single i.v. or oral doses to four dogs. Plasma concentrations of the enantiomers and their corresponding pyridine metabolites were determined by g.l.c.-mass spectrometry. 2. No isotope effects were observed after oral administration of equimolar amounts of deuterated and unlabelled (S)-felodipine. 3. The pharmacokinetic parameters of the enantiomers were similar after i.v. administration, indicating that the disposition of felodipine was not stereoselective. 4. After oral administration the bioavailability of (R)-felodipine was slightly higher than that of (S)-felodipine in two of the dogs, presumably due to a lower first-pass extraction of the (R)-enantiomer, while no difference was observed in the other two dogs. 5. No substantial differences in Cmax or AUC were observed between the deuterated and unlabelled pyridine metabolites, indicating that the oxidative clearances of the felodipine enantiomers were similar.

Mechanistic studies on the metabolic activation of acetaminophen in vivo.

Three analogues of acetaminophen (APAP), labeled at specific positions with either oxygen-18 or deuterium, were administered by ip injection to male BALB/c mice at the moderately hepatotoxic dose of 200 mg kg-1 in order to probe the mechanism by which APAP undergoes metabolic activation in vivo. The thioether conjugates of APAP present in bile, urine, and feces, which are believed to derive from the electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI), were isolated following aqueous-phase derivatization, separated by HPLC, and converted to a common volatile derivative for analysis by GC-MS. The observed labeling patterns of these conjugates indicated that APAP undergoes metabolism to NAPQI by a process that does not involve the generation of a free oxygenated intermediate, but which more likely entails the sequential removal of two electrons from the substrate. On the basis of these findings, an integrated metabolic scheme is proposed which invokes initial cytochrome P-450 mediated generation of a caged oxygen-centered APAP radical species. Subsequent reactions of this intermediate may account for the formation of all known oxidative metabolites of APAP.

Analysis and stereoselective metabolism after separate oral doses of tocainide enantiomers to healthy volunteers.

The potential of stereoselective metabolism of tocainide was studied in six healthy volunteers after separate oral administration of the pure enantiomers in solution. A method was developed to convert the N-carbamoylglucuronide of tocainide in plasma and urine by base treatment to a hydantoin derivative which after extraction and silation was analysed by selected ion monitoring using a deuterated internal standard. Analytical problems concerning side-reactions during derivatization of the conjugate are discussed. The peak plasma levels of the enantiomers, observed at less than or equal to 2 h after dosing, were similar but plasma clearances and terminal half-lives were different after oral administration of (R)-tocainide (195.5 +/- 20.1 ml min-1 and 9.7 +/- 0.8 h) and (S)-tocainide (110.2 +/- 10.5 ml min-1 and 14.5 +/- 1.7 h). Over 0-96 h the averaged urinary recovery of (R)-tocainide was 36 per cent and of (S)-tocainide 50 per cent. Stereoselective metabolism was a likely mechanism for the observed differences as the urinary recovery of the conjugate formed from (R)-tocainide differed substantially from that of (S)-tocainide (45 vs 1.2 per cent of given dose). Plasma t1/2 of the (R)- and (S)-conjugate were 9.9 and 18.7 h, respectively, indicating formation rate limited kinetics of the metabolite. The renal clearances of the conjugates were not significantly different (131 vs 97 ml min-1).

Identification of two main urinary metabolites of [14C]omeprazole in humans.

The excretion and metabolism of [14C]omeprazole given orally as a suspension was studied in 10 healthy male subjects. An average of 79% of the dose was recovered in the urine in 96 hr, with most of the radioactivity (76% of dose) being eliminated in the first 24 hr. Pooled urine (0-2 hr) from five subjects, containing about 47% of the dose, was analyzed by reverse phase gradient elution LC with radioisotope detection. Omeprazole was completely metabolized to at least six metabolites. The two major metabolites were extensively purified by LC and their structures were determined by MS with derivatization and use of stable isotopes, 1H NMR, and comparison with synthetic references. They were formed by hydroxylation of a methyl group in the pyridine ring, followed by further oxidation of the alcohol to the corresponding carboxylic acid. Both metabolites retained the sulfoxide group of omeprazole, rendering them as unstable as the parent compound at pH less than 7. They accounted for approximately 28% (hydroxyomeprazole) and 23% (omeprazole acid) of the amount excreted in the 0-2-hr collection interval. Based on in vitro studies with the synthetic metabolites in isolated gastric glands, it is unlikely that M1 and M2 will contribute to the pharmacological effect of omeprazole in humans.

The use of alkoxycarbonyl derivatives for the mass spectral analysis of drug-thioether metabolites. Studies with the cysteine, mercapturic acid and glutathione conjugates of acetaminophen.

Alkoxycarbonyl derivatives of the cysteine-, N-acetylcysteine- and glutathione conjugates of acetaminophen have been prepared in aqueous buffer solutions and their chromatographic and mass spectrometric properties examined. Structurally informative fragmentation patterns of the cysteine- and N-acetylcysteine derivatives were obtained when their methyl esters were subjected to analysis by direct insertion chemical ionization (CH4) mass spectrometry, although field desorption and liquid secondary ion mass spectrometric techniques were required in order to obtain satisfactory spectral data for derivatives of the glutathione adduct. Treatment of ethoxycarbonyl derivatives of the three acetaminophen metabolites with N-methyltrifluoroacetamide-based silylating reagents led to the formation of a common volatile product which was ideally suited to analysis by gas chromatography/electron impact mass spectrometry. A mechanism is proposed for the formation of this novel derivative, which appears to possess a benzo-1,3-thioxalane structure, and its mass spectral characteristics are reported. Finally, the utility of alkoxycarbonyl derivatives for the analysis of drug-thioether conjugates in biological fluids is discussed in terms of their advantages for aqueous phase derivatization, purification by high-performance liquid chromatography and characterization by mass spectrometry.

Covalent binding of acetaminophen to mouse hemoglobin. Identification of major and minor adducts formed in vivo and implications for the nature of the arylating metabolites.

When hepatotoxic doses of [ring-U-14C]acetaminophen ([ring-U-14C]APAP) were administered to mice, radioactivity became bound irreversibly to hemoglobin as well as to proteins in the liver and kidney. The covalent binding to hemoglobin was dose-dependent, and in phenobarbital-pretreated mice occurred to the extent of approximately 8% of the corresponding binding to liver proteins. Degradation of the modified globin by acid hydrolysis yielded 3-cystein-S-yl-4-hydroxyacetanilide as the major radioactive product, accounting for approximately 70% of protein-bound drug residues. This finding is consistent with the view that the majority of covalent binding of APAP to proteins is mediated by N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite which preferentially arylates cysteinyl thiol residues. However, after administration of [acetyl-3H]APAP to mice, it was found that approximately 20% of the drug bound to hemoglobin had lost the N-acetyl side-chain, indicating the existence of a second type of APAP-protein adduct. One minor component of the globin hydrolysate was identified as S-(2,5-dihydroxyphenyl)-cysteine, which most likely arises from binding to hemoglobin of p-benzoquinone, a hydrolysis product of NAPQI. The two adducts reported represent the first identified examples of arylating drugs binding to hemoglobin. Experiments on the influence of different cytochrome P-450 inducing agents on the ratio of drug bound to hemoglobin versus hepatic proteins suggested that the reactive metabolites of APAP are formed in the liver and migrate to the erythrocyte, rather than being produced by hemoglobin-catalyzed oxidation of APAP. These findings imply that the reactive metabolites of APAP escape from hepatocytes in some latent forms, which then participate in the arylation of protein thiols in red blood cells and, possibly, at other remote sites.

Analysis of alpha-hydroxy metabolites of metoprolol in human urine after phosgene/trimethylsilyl derivatization.

Three metoprolol metabolites containing an alpha-hydroxy group were identified in human urine by capillary column gas chromatography/mass spectrometry. After aqueous phase cyclization with phosgene the neutral or acidic derivatives formed were isolated by solvent extraction at pH 10 or 3, respectively. Following silylation the electron impact mass spectra of the metabolites exhibited a characteristic ion at m/z 336 of high abundance which originated from cleavage of the bond adjacent to the alpha-OTMS group. Most probably the identified compounds were formed by further biotransformations of alpha-hydroxy metoprolol, which is a primary metabolite. The analytical method is applicable to detect the metoprolol metabolites reported so far. A quantitative assay for one of the metabolites (H 119/72) with nitrogen selective detection is described. The total amount of this metabolite excreted by one subject within 24 h after dosing was about 0.25% of the given dose.

Metabolism of [14C] felodipine, a new vasodilating drug, in healthy volunteers.

After oral administration of [14C] felodipine (27.5mg) to 4 healthy volunteers, 6 main urinary metabolites were identified by gas chromatography-mass spectrometry. The compounds were isolated by solvent extraction at pH 2.2 and silylated prior to analysis. They were formed by dehydrogenation of felodipine followed by ester hydrolysis, hydroxylation of the alkyl groups and conjugation. These metabolites were excreted both as free acids and as conjugates accounting on average for 37% of the excreted amount (23% of the dose). A specific liquid chromatographic assay with radioactive detection was developed to determine the acidic metabolites in all collected samples. The urinary excretion rate declined biphasically for the mono-acids III and IV, whereas the excretion rates of metabolites VI, VII and VIII, formed via aliphatic hydroxylation, were better fitted to equations of first-order processes.

Biotransformation of felodipine in liver microsomes from rat, dog, and man.

The biotransformation of the calcium antagonist felodipine was investigated in liver microsomes from rat, dog, and man. The metabolites were quantified and identified by gradient elution reverse phase liquid chromatography, liquid scintillation analysis, and GC/MS. Ten metabolites were identified, including the pyridine analogue of felodipine, two carboxylic mono acids, two ester lactones, as well as the corresponding open hydroxy acid forms, and a lactonic compound with a carboxylic acid group. The presence of two decarboxylated products was also verified. Metabolites with an intact dihydropyridine nucleus were not detected. The total pool of metabolites formed in vitro was more lipophilic than that excreted in urine from the same species. The metabolic pathways were similar in the three species studied, although quantitative differences were observed. Comparison between incubations with liver microsomes from male and female rats indicated that the females metabolized felodipine more slowly than the males. From a more detailed quantitative analysis of eight metabolites, in relation to incubation time, it was apparent that the hydroxylation of the 2- and 6-methyl groups occurred at a faster rate (0.027 min-1) than did the ester hydrolysis (0.016 min-1). These hydroxy metabolites rearranged spontaneously to lactones. The results from this study indicate that the open hydroxy acid metabolites were formed enzymatically from the corresponding lactones. A metabolic scheme for the overall metabolism of felodipine is given and discussed with reference to the in vivo situation.

Comparative metabolic disposition of oral doses of omeprazole in the dog, rat, and mouse.

The metabolic disposition of [14C]omeprazole was studied in dogs, rats, and mice after the administration of pharmacologically active, single oral doses of drug in buffer solutions (pH 9). Averages of 38% (dogs), 43% (rats), and 55% (mice) of the radiolabeled doses were excreted in the urine in 72 hr. Most of the remaining dose was recovered in the feces. Omeprazole was extensively metabolized in all species studied and the metabolites were eliminated rapidly. No unchanged drug could be detected in the urine samples (less than 0.1% of dose). In each species at least 10 metabolites were detected in urine (pH 9) by gradient elution reverse phase HPLC. Based on liquid chromatographic retention data, the metabolic patterns were very complex and exhibited some quantitative differences between species. Bile was collected from rats and from chronic bile-fistulated dogs. Biliary excretion was a major route of elimination of omeprazole metabolites, and four polar metabolites were detected in the rat bile. The stability of omeprazole metabolites at varying pH values is discussed with reference to reductive metabolism of the parent compound.

Identification of the main urinary metabolites of omeprazole after an oral dose to rats and dogs.

The structures of seven urinary metabolites of omeprazole following high oral doses to rats and dogs were determined unambiguously by combining different analytical and spectroscopic techniques including derivatization and stable isotopes. Omeprazole was metabolized by aromatic hydroxylation at position 6 in the benzimidazole ring followed by glucuronidation. There was also oxidative O-dealkylation of both methoxy groups, and aliphatic hydroxylation of a pyridine methyl group followed by oxidation to the corresponding carboxylic acid. Due to the experimental design, implying no pH control of collected samples, all metabolites were isolated as sulfides. They were formed in both species with quantitative variations in the metabolic pattern. As far as identified metabolites are concerned, aromatic hydroxylation and subsequent glucuronide formation were the major biotransformation routes in the dog. In the rat, aliphatic hydroxylation and the formation of the carboxylic acid represented the major metabolic pathways. The identified metabolites corresponded approximately to 50% (rat) and 70% (dog) of the amount excreted in the 0-24-hr urine (about 12% of the given dose in both species).

Identification of felodipine metabolites in rat urine.

After intragastric administration of 100 mumol kg-1 [14C]felodipine to rats eight urinary metabolites were isolated. Batch extraction at pH 2.2 and semipreparative reversed-phase liquid chromatography were used for trace enrichment of the metabolites. Trimethylsilylation followed by transesterification with diazomethane blocked the carboxylic acid and alcohol groups selectively before gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. Deuterated derivatives of the metabolites and chemical ionization measurements added complementary structural information. All metabolites reported in this study were formed from oxidized felodipine by ester hydrolysis. Hydroxylation of the pyridine methyl group represented an important metabolic pathway and metabolites oxidized to the corresponding carboxylic acids were detected as well. Lactone formation from hydroxy acid metabolites in urine as a possible analytical artefact is discussed.

Identification of the major covalent adduct formed in vitro and in vivo between acetaminophen and mouse liver proteins.

Improved analytical methodology has been developed for the structural characterization of covalently bound drug-protein adducts and has been applied to an investigation of the conjugates formed in vivo and in vitro between [14C]acetaminophen and mouse liver proteins. The major adduct released by acid hydrolysis of hepatic protein samples, which accounted for approximately 70% of the bound radioactivity in vivo and in vitro, was identified as 3-cystein-S-yl-4-hydroxyaniline, a derivative whose structure reflects the predominance of acetaminophen thioether adducts in drug-modified proteins. It is concluded that the reactive, electrophilic metabolite of acetaminophen, which most likely is N-acetyl-p-benzoquinoneimine, binds with a high degree of selectivity to cysteinyl thiol groups on protein, formally in a Michael-type addition reaction. Cysteine residues thus represent primary target sites for arylation by the reactive metabolite of acetaminophen, and proteins rich in free thiols may be especially vulnerable to damage by this toxic intermediate.