Fibrin-stabilizing factor inhibitors. 12. 5-Dibenzylaminopentylamine and related compounds, a new type of FSF inhibitors.

A series of omegadibenzylaminoalkylamines and related compounds have been prepared and tested as inhibitors of fibrin cross-linking. This structural type was chosen in an attempt to develop noncompetitive inhibitors of fibrinoligase. By the combination of the dibenzylamino moiety at one end and the primary amino group at the other end of a polymethylene chain, the same compound could function both as a pseudo donor substrate and as a noncompetitive alkylating inhibitor. Some of the compounds, notably 74-79, are among the most active fibrinoligase inhibitors described. However, the data indicate that the compounds probably function only as pseudo donor inhibitors.

The effect of impaired renal function on the plasma concentration and urinary excretion of metoprolol metabolites.

Plasma concentration and urinary excretion of total and 2 active metabolites of metoprolol have been studied in patients with varying degrees of renal impairment and in healthy subjects after intravenous and oral administration of 20 and 50 mg of 3H-metoprolol tartrate respectively. Renal clearance of total metabolites correlated directly with 51Cr-EDTA clearance (r = 0.95, p less than 0.001). A reduction of glomerular filtration rate (GFR) by 70 to 80% increased the elimination half-life of total metabolites and of the active metabolite alpha-hydroxymetoprolol about 3-fold. Significant accumulation was, however, only observed in the patients with a GFR of about 5 ml/min. Even in these patients, the contribution of alpha-hydroxymetoprolol to the beta-adrenoceptor blocking effect of metoprolol will be negligible. The second active metabolite studied is eliminated via biotransformation, and the urinary excretion as well as the plasma concentration of this metabolite were extremely low in comparison with those of the parent drug.

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.

Absorption, distribution and elimination of felodipine in man.

The objectives of these investigations were to study the absorption and disposition characteristics of felodipine in young healthy male volunteers following acute administration of different intravenous and oral doses, and to study urinary metabolites of [14C]felodipine following oral administration. Felodipine is rapidly and extensively absorbed from the gastrointestinal tract but owing to presystemic elimination, probably primarily in the liver, only 15% on average is systemically available. The systemic availability is independent of the oral dose in the 5 to 40 mg dose interval. The major fraction of the felodipine dose is localised extravascularly with a volume of distribution of about 10 L/kg. Less than 1% is confined to the blood. Felodipine is extensively bound to plasma proteins (greater than 99%). The mean elimination half-life of felodipine is greater than 10 hours. The urinary metabolic pattern of felodipine, using high pressure liquid chromatography, reveals 3 major metabolites (carboxylic acids of oxidised felodipine) in human urine.

Structural characterization of the major covalent adduct formed in vitro between acetaminophen and bovine serum albumin.

The structure of the covalent adduct formed in vitro between [14C]-acetaminophen ([14C]APAP) and bovine serum albumin (BSA) has been investigated with the aid of new analytical methodology. The APAP-BSA adduct, isolated from mouse liver microsomal incubations to which the radiolabeled drug and BSA had been added, was cleaved using a combination of specific (cyanogen bromide) and non-specific (acid hydrolysis) procedures, following which the mixture of amino acids obtained was derivatized, in aqueous solution, with ethyl chloroformate. The resulting ethoxycarbonyl derivatives were recovered by extraction into ethylacetate, methylated and subjected to profile analysis using both reverse-phase and normal-phase HPLC techniques. In each HPLC step, one major radioactive amino acid adduct was detected and was identified by mass spectrometry as the derivative of 3-cystein-S-yl-4-hydroxyaniline. Based on this finding, and with a knowledge of the behavior under acidic hydrolysis conditions of the 3-cysteinyl conjugate of APAP, it could be concluded that the major APAP-BSA adduct is one in which the drug is bound, via a thioether linkage at the C-3 position, to a sulfhydryl group on the protein. Furthermore, it could be established that this -SH function almost certainly is that associated with the cys-34 residue of BSA.

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.

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.

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.

Stereoselective disposition of RS-tocainide in man.

The disposition of RS-tocainide in three healthy volunteers has been studied after oral administration of a pseudoracemic mixture containing S(+) [3H] tocainide as a radioactive tracer together with a therapeutic dose of the racemate. Analytical methods based on HPLC have been developed to measure S(+) and R(-) tocainide in urine samples. Selected ion detection has been used for quantification of a tocainide conjugate. The radioactive dose was efficiently absorbed and mainly cleared via the kidneys. The elimination half-life of RS-tocainide was found to be 14.3 hours. The elimination half-lives of the two stereoisomers of tocainide differed significantly, i.e. R(-) tocainide 10 hours, and S(+) tocainide 16.7 hours. The observed t1/2 for the tocainide conjugate of 10.3 hours was close to that of R(-) tocainide, indicating that the metabolite was preferably formed from the R(-) stereoisomer of tocainide. Of the given dose, between 45 and 70% can be accounted for.

Species differences in the metabolism of pamatolol, a cardioselective beta--adrenoceptor antagonist.

The metabolism of pamatolol was studied in man, dogs, rats and mice after oral administration of a single dose. The drug was well absorbed in the gastro-intestinal tract and excreted in the urine, mainly in unchanged form, within 24 hrs. Four urinary metabolites were identified by gas chromatographic-mass spectrometric techniques. The metabolic data, in man, dog and mouse was found to be similar, both qualitatively and quantitatively. One metabolism route, involving aliphatic hydroxylation and subsequent oxidation, was found, to a significant extent only in the rat. The species variation between the mouse and the rat with regard to long-term toxicity of pamatolol is discussed. Artefact formation during trace analysis was observed.

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).

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.

Simultaneous determination of metoprolol and metabolites in urine by capillary column gas chromatography as oxazolidineone and trimethylsilyl derivatives.

A method for the determination of metoprolol and its main metabolites in urine is presented. The method comprises derivatization of the aminopropanol side-chain with phosgene at alkaline pH and isolation in an organic phase at acidic pH. After trimethylsilylation, separation and quantification are performed by capillary column gas chromatography with flame ionization detection. The reaction is performed at pH 12 with 60 microliters of 2 M phosgene in toluene added in three portions. Diethyl ether--dichloromethane is used as extraction medium and bis(trimethylsilyl) acetamide as silylating agent. With spiked samples linear standard curves were obtained for metoprolol and three of its main metabolites with a detection limit varying between 4 and 20 mumol/l of urine. The method was applied to urine samples from a normal individual who had taken 292 mumol of metoprolol as tartrate.

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.

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.

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.

Urinary metabolites of felodipine, a new vasodilator drug, in man, dog, rat and mouse.

The urinary excretion of total 14C after oral administration of 25 mg (approximately 1 mumol/kg) 14C-felodipine to man, and intragastric administration (5 mumol/kg) to dog, rat and mouse, was 70, 39, 44 and 53% dose, respectively, in 72 h. Metabolites of felodipine were separated and quantified by h.p.l.c. Unchanged felodipine and its oxidized analogue were not excreted by any of the species studies. Three metabolites, present in all species studied, were isolated from urine and identified as products of the oxidation of felodipine to its pyridine analogue followed by hydrolysis of one or both of the pyridine carboxylic acid esters.

Biotransformation of alprenolol in dog, guinea-pig and rat liver microsomes.

1. Metabolites of alprenolol were isolated and identified in dog, guinea-pig and rat liver microsomes by means of g.l.c.-mass spectrometry and comparison with synthetic reference compounds. 2. The compounds were chromatographed as n-butylboronate derivatives, giving a series of diagnostic ions in the mass spectral fragmentation, which was elucidated by using stable isotopes. 3. Alprenolol was metabolized by aromatic ring hydroxylation, oxidation of the allylic function, and degradation of the isopropylaminopropanol side-chain. Alprenolol and four metabolites were quantified by h.p.l.c. and batch extraction techniques based on radioactivity measurements. 4. Five metabolites were detected in rat and guinea-pig liver microsomes and four in the dog. A species variation in the biotransformation of the allyl function in alprenolol was observed. The metabolite formed by oxidation of the allyl double bond was detected in significant amounts in the guinea-pig, and was also formed in the rat but could not be detected in dog liver microsomes.