Metabolic fate of delmopinol in man after mouth rinsing and after oral administration.

1. The metabolism of delmopinol, an inhibitor of plaque formation and gingivitis. has been studied after mouth rinsing or an oral dose of 14C-delmopinol to healthy male volunteers. The metabolite pattern was studied in urine and plasma samples (unhydrolysed or hydrolysed with conjugate cleaving enzymes) by liquid chromatography (LC) with radio detection. Metabolite identifications were carried out by gas chromatography-electron-impact mass spectrometry (GC-MS) and by liquid chromatography-thermospray mass spectrometry (LC-MS). 2. The metabolic clearance of delmopinol was high, and < 0.2% of the dose was excreted as intact delmopinol in the urine. The main metabolites were, for both administration routes, glucuronide conjugates of delmopinol and of (omega-1-hydroxy) delmopinol. These metabolites were predominant and accounted for nearly the entire urinary radioactivity and most of the plasma radioactivity. After mouth rinsing, parent delmopinol was also one of the main compounds in plasma. 3. Several other products of oxidation of the aliphatic side-chain were present in minor amounts in urine. These metabolites also appeared to be excreted as glucuronic acid conjugates. 4. Glucuronidated (omega-1-hydroxy) delmopinol separated into three peaks by the LC system used. This could be due to different chromatographic properties of conjugate isomers, positional or optical.

Mass balance and metabolism of [(3)H]Formoterol in healthy men after combined i.v. and oral administration-mimicking inhalation.

Mass balance and metabolism of formoterol were investigated in six healthy men in an open study. Mean age was 49.7 years (range: 40-63). Simultaneous oral (mean dose 88.6 nmol, 49.3 MBq) and i.v. (mean dose 38.2 nmol, 21.4 MBq) doses of tritium-labeled formoterol were administered. The combination of these two administrations was aimed at simulating the fate of inhaled formoterol. Total radioactivity was monitored for 24 h in blood plasma and for at least 4 days in urine and feces. Formoterol and metabolites were determined using liquid chromatography plus radiodetection, directly after centrifugation in urine and after sample workup in blood plasma and feces. Metabolites were identified in urine, sampled from two subjects, using liquid chromatography-electrospray ionization mass spectrometry. Mean total recovery was 86% of the administered formoterol dose, 62% in urine and 24% in feces. Tritiated water was generated and because its in vivo turnover is slow, the terminal decline of total radioactivity was slow and dose recovery was incomplete during the sampling period. Formoterol was conjugated to inactive glucuronides and a previously unidentified sulfate. The phenol glucuronide of formoterol was the main metabolite in urine. Formoterol was also O-demethylated and deformylated. Plasma exposure to these pharmacologically active metabolites was low. O-demethylated formoterol was seen mainly as inactive glucuronide conjugates and deformylated formoterol only as an inactive sulfate conjugate. Intact formoterol and O-demethylated formoterol dominated recovery in feces. Mean recovery of unidentified metabolites was 7. 0% in urine and 2.0% in feces.

Pharmacokinetics of 14C-delmopinol in the healthy male volunteer.

1. The absorption and excretion of delmopinol, an inhibitor of plaque formation and gingivitis, were studied in healthy male volunteers. Each subject received a single dose of a 10-ml aqueous solution of 14C-delmopinol at 2 mg/ml as a 1-min mouth rinse (the intended route of administration; n=6) or as an oral dose (n=6). 2. After mouth rinsing, 72% of the dose was expectorated. The retained portion of the dose was rapidly absorbed and eliminated. Of the dose, 25% was recovered in the urine. Renal excretion was predominant also after oral administration with 92% of the dose excreted in the urine. The total recovery of 14C-activity after 6 days was 99% (rinsing) and 95% (oral) with the bulk, 20% (rinsing) and 80% (oral), of the dose excreted already within 8 h. 3. Peak plasma levels of 14C-labelled compounds were attained at 1.5 h after rinsing and 0.5 h after oral administration; total concentrations were approximately 200 and 3000nmol/l, respectively. For parent delmopinol, peak plasma levels of 49 nmol/l (rinsing) and 8 nmol/l (oral) occurred after 1.5 and 1.3 h, respectively. After rinsing, the concentration of delmopinol declined with a half-life of 2.4 h. The plasma level of 14C-labelled compounds declined multiphasically. In the terminal elimination phase (after 72 h) remaining radioactivity, < 2% of dose (rinse) and < 5% of dose (oral), decreased with a half-life of approximately 130 and 160 h, respectively. 4. Results indicate a very low bioavailability (1-3%) due to extensive first-pass metabolism of delmopinol after oral administration. A high plasma clearance (> 60 l/h) and a large volume of distribution (> 200 l) were also indicated. 5. The results of rapid and almost complete recovery in the urine support that delmopinol can safely be used in the treatment of patients with plaque or gingivitis.

Reversible formation of fatty acid esters of budesonide, an antiasthma glucocorticoid, in human lung and liver microsomes.

Microsomes from human lung and liver catalyze the formation of fatty acid esters of budesonide, a glucocorticoid used for inhalation treatment of asthma. The conjugation was dependent on coenzyme A and ATP. Addition of free fatty acids to the incubations affected the pattern of metabolites, but ester formation was observed also without such addition. Budesonide oleate, palmitate, linoleate, palmitoleate, and arachidonate were identified as metabolites. The fatty acid conjugates of budesonide were shown to be substrates for lipase in vitro, thus budesonide is regainable from the conjugates. The data suggest that an equilibrium between budesonide and these pharmacologically inactive lipoidal conjugates will be established in tissues at repeated exposure to budesonide. Since the fatty acid conjugates most likely will be retained intracellularly for a longer time than unchanged budesonide, the duration of tissue exposure to budesonide will depend partly on the rate of lipase-catalyzed hydrolysis of the conjugates. The findings in this study provide a possible explanation for the efficacy of budesonide in mild asthmatics also when inhaled once daily.

Effects of alpha-carbon substituents on the N-demethylation of N-methyl-2-phenethylamines by rat liver microsomes.

The effects of methyl, ethyl, isopropyl, isobutyl, and benzyl substituents at the alpha-carbon of N-methyl-2-phenethylamine on the kinetics of its N-demethylation in liver microsomes from both control and phenobarbital pretreated rats were studied. In control microsomes, the kinetic studies indicated that more than one enzyme was active for N-demethylation of N-methyl-alpha-methylphenethylamine (methamphetamine) while the other N-methyl-2-phenethylamines appeared to be demethylated by a single enzyme. In microsomes from phenobarbital pretreated animals, there appeared to be more than one enzyme system which was active for N-demethylation of all compounds except N-methyl-alpha-benzylphenethylamine. One of these had a much higher affinity for alpha-ethyl, isopropyl, and isobutyl N-methylphenethylamines while another exhibited affinities for substrates similar to the constitutive enzyme in control microsomes. A correlation was observed between the octanol-buffer or heptane-buffer distribution ratios of the compounds and the negative logarithm of the Michaelis constant (pKm) for the enzyme in control microsomes and for each of the enzyme systems in microsomes from phenobarbital-pretreated animals. Therefore, it is indicated that the concentration of a substrate at the active site of these microsomal enzymes is a function of its lipid solubility.

Phencyclidine metabolism in vitro. The formation of a carbinolamine and its metabolites by rabbit liver preparations.

Four products of the in vitro oxidative metabolism of the piperidine ring of phencyclidine, 5-(1-phenylcyclohexylamino)valeraldehyde, V; N-(1-phenylcyclohexyl)-1,2,3,4-tetrahydropyridine, VIII; 5-(1-phenylcyclohexylamino)valeric acid, VII; and 1-phenylcyclohexylamine, IX, have been identified following derivatization, by GC/MS with stable isotope labeling and/or synthesis. The enamine, VIII, may be a work-up elimination product of a carbinolamine, alpha-hydroxy-N-(1-phenylcyclohexyl)piperidine, IV. The formation of all metabolites requires microsomal enzymes, but VII and the previously described N-(5-hydroxypentyl)-1-phenylcyclohexylamine, VI, also require soluble enzymes. Quantitative or semiquantitative data show that VI and VIII appear and disappear with time, whereas VII seems to be a terminal metabolite.

Cytochrome P-455-nm complex formation in the metabolism of phenylalkylamines. VI. Structure--activity relationships in metabolic intermediary complex formation with a series of alpha-substituted 2-phenylethylamines and corresponding N-hydroxylamines.

The formation of cytochrome P-450 metabolic intermediary (MI) complexes from a homologous series of alpha-substituted 2-phenylethylamines and corresponding N-hydroxylamines was investigated during NADPH-dependent metabolism in liver microsomes from phenobarbital-pretreated rats. The alpha-alkyl substituent consisted of branched and unbranched alkyl chains ranging from 0-4 carbons and the benzyl group. All the compounds but 2-phenylethylamine generated the complex and double-reciprocal plots of the highest observed rate of complex formation vs. substrate concentration gave linear relations over a defined substrate range. The Vmax(obs) values for complex formation by the amines increased markedly with increasing size of the alkyl group and a good correlation was obtained between log Vmax(obs) and the logarithm of the octanol/buffer partition coefficient of the substrates. With the N-hydroxy compounds, complex formation was a much less selective phenomenon and without exception the rates were quite high with Vmax(obs) values as much as 100 times greater than those of the amines. The disappearance of substrate amines was independent of structure and at an initial substrate concentration of 100 microM about a 50% decrease was noted during a 20-min period in all cases. The results substantiate the previous notion that N-oxidation is a prerequisite for MI-complex formation from primary amines. The results also suggest that C- and N-oxidation have different rate-limiting steps and the microsomal enzymes catalyzing the N-oxidation seem to be deeply submerged in the lipid matrix, as amines with a low distribution are inactive or poor substrates for generating the cytochrome P-450 ligand. Also, it is evident that the MI complex formation does not impair the overall metabolism of the amines.

Metabolism of Oxybutynin: establishment of desethyloxybutynin and oxybutynin N-oxide formation in rat liver preparation using deuterium substitution and gas chromatographic mass spectrometric analysis.

Oxybutynin is rapidly metabolized in rat liver microsomes. Two major primary oxidation products were identified as N-desethyl oxybutynin and oxybutynin N-oxide. Deuterium substituted substrate was used to aid the identification. N-Desethyl oxybutynin was characterized by gas chromatography electron impact mass spectrometry as its trifluoroacetamide derivative and oxybutynin N-oxide was indicated by the presence of a decomposition product, 2-oxo-3-butenyl-2 cyclohexyl-2-phenylglycolate, as elucidated from the gas chromatographic mas spectrometric analysis. The formation of this product from synthetic oxybutynin N-oxide was verified and occurs by two consecutive rearrangements upon thermolysis of the unstable N-oxide. Attempted titanous chloride reduction of oxybutynin N-oxide resulted in the formation of the hydrolytic products 2-cyclohexyl-2-phenylglycolic acid and 4-diethylamino-2-butynol.

Metabolic N-oxidation of alpha-acetylenic amines-a gas chromatographic mass spectrometric investigation of the chemical decomposition of N-(5-pyrrolidinopent-3-ynyl)succinimide N'-oxide using deuterium substitution.

On gas chromatographic analysis, N-(5-pyrrolidinopent-3-ynyl)succinimide N'-oxide gave rise to three main decomposition products, which were assigned the structures N-(3-oxopent-4-enyl)succinimide, N-(penta-3, 4-dienyl)succinimide and N-3(3-pyrrolidino-3-oxopropyl)succinimide. These three products were identified by electron impact mass spectrometry, using deuterium substituted N-oxides, and mechanisms for the formation of the former two are proposed. By gas chromatographic mass spectrometric analysis, extracts from metabolic incubation of N-(5-pyrrolidinopent-3-ynyl)succinimide were shown to contain the N-oxide, as previously indicated by indirect methods.

Metabolism of N-(5-pyrrolidinopent-3-ynyl)-succinimide (BL 14) in rat liver preparations. Characterization of four oxidative reactions.

1. Four non-acidic primary metabolites of N-(5-pyrrolidinopent-3-ynyl)succinimide (BL 14) were identified and quantified using g.l.c. and mass spectrometry. The metabolites are alpha-hydroxy-N-(5-pyrrolidinopent-3-ynyl)succinimide (A), N-(5-(2-oxopyrrolidino)-pent-3-ynyl)succinimide (B), N-(2-hydroxy-5-pyrrolidinopent-3-ynyl)succinimide (C) and N-(5-pyrrolidinopent-3-ynyl)succinimide N-oxide (E), the latter analysed after reduction to the parent amine. 2. In rat liver preparations, all metabolites are formed by microsomal, NADPH-dependent enzyme systems, but with different characteristics. The response to inhibitors such as CO and SKF 525A indicates participation of cytochrome P-450 enzymes in the formation of all metabolites. Phenobarbital pretreatment markedly enhances propynylic hydroxylation (C) but has little or no effect on the other metabolic pathways. Succinimide hydroxylation (A) exhibits a pH optimum at 7.0, while the formation of metabolism B and C increases at pH values between 6.4 and 7.7. 3. Kinetic studies on the formation of metabolites A-C revealed differences in the Michaelis constant, while the Vmax values were similar. Succinimide hydroxylation (A) is most efficient with a Km of 3.7 X 10(-5) M, compared with a Km of 1.7 X 10(-3) M for propynylic hydroxylation (C). 4. The formation of metabolites B and E conforms to the corresponding mechanisms for lactam and N-oxide formation for other xenobiotics. The formation of metabolites A and C represents two extremities, reflected in their different responses to phenobarbital pretreatment, pH changes and in their different Km values. Although little can be discerned about the mechanisms from the literature, the enzymes catalysing both reactions appear to be cytochromes.

Microsomal formation and chemical decomposition of pargyline N-oxide.

Pargyline undergoes metabolic N-oxidation in rat and rabbit liver microsomal preparations. The reaction requires oxygen and is NADPH dependent. N-oxidation and N-demethylation are equal in both control and induced rat liver microsomes, while N-oxidation is more dominant in rabbit tissue. Experiments investigating the CO-sensitivity and the effects of metyrapone suggest that cytochrome P-450 systems are involved in both reactions in the rat while an additional enzyme is responsible for the N-oxidation in the rabbit. Pargyline N-oxide is characterized by chemical instability and undergoes two consecutive rearrangements to yield propenal and Schiff bases, the latter undergoing hydrolysis to aldehydes and primary amines. Accordingly, due to the inherent instability of the N-oxide, metabolic N-oxidation of pargyline is, in addition to alpha-carbon oxidation, indicated as a metabolic route to benzaldehyde. Similarly the ease with which pargyline N-oxide generates propenal implicates N-oxidation as a metabolic route to be considered when evaluating the toxicity of pargyline.

Enzymic oxidation alpha to the acetylenic group in the metabolism of N-(5-pyrrolidinopent-3-ynyl)-succinimide (BL 14) in vitro.

1. The product of alpha-acetylenic oxidation of N-(5-pyrrolidinopent-3-ynyl)-succinimide (BL 14) by rat liver preparations was identified as N-(5-pyrrolidino-2-hydroxypent-3-ynyl)succinimide, by mass spectral analysis of metabolites of deuterium-labelled and non-labelled substrate. 2. The synthesis and physicochemical characteristics of the metabolite are reported. 3. Substantial amounts of the metabolite were obtained in preparations from phenobarbital-treated rats, while only minute amounts were formed by non-induced preparations. 4. Evidence for the involvement of an inducible cytochrome P-450 system in effecting this alpha-acetylenic oxidation is presented.