Biotransformation of Aryl Alkylamines by Cunninghamella Bainieri

1. Incubations of phenylethylamine and 11 of its analogues with Cunninghamella bainieri have been performed.

2. Products of all major routes of mammalian metabolism including ring and aliphatic hydroxylation, oxidative deamination, N-oxidation, N-dealkylation, and conjugation were found in extracts of the fungal incubation broths.

3. Metabolism was influenced by the nature and degree of substitution.     

Studies on N-demethylation of methamphetamine by liver microsomes of guinea-pigs and rats: The role of flavin-containing mono-oxygenase and cytochrome P-450 systems

1. Relative participation of flavin-containing mono-oxygenase and cytochrome P-4S0 systems in N-hydroxylation of and formaldehyde release from methamphetamine were studied in vitro using liver microsomes of guinea-pigs and rats. In guinea pigs, only methimazole, an inhibitor of flavin-containing mono-oxygenase, significantly suppressed the above reactions.

2. Formaldehyde release from methamphetamine was significantly inhibited not only by methimazole but also by inhibitors of the cytochrome P-450 system in liver microsomes from rats, but not guinea-pigs.

3. Pretreatment of guinea-pigs with phenobarbital and 3-methylcholanthrene did not enhance the metabolism of methamphetamine.

4. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene increased slightly the N-demethylation of methamphetamine by liver microsomes.

5. The results indicate that a marked species difference exists in the enzymes concerned with N-demethylation of methamphetamine. N-Oxidation predominates in guinea-pigs, whereas in rats, N-oxidation and C-oxidation of the methyl group participate equally as the initial reaction of the N-demethylation pathway.     

The Metabolism of N-Ethyl-N-methylaniline by Rabbit Liver Microsomes: The Measurement of Metabolites by Gas-liquid Chromatography

Abstract

1. A method for the determination of N-ethyl-N-methylaniline and its metabolites by g.l.c. is described.

2. Following incubation in N-ethyl-N-methylaniline with rabbit liver microsomes for 60 min, over 95% of the substrate was accounted for as unchanged compound or metabolites.

3. N-Ethyl-N-methylaniline is metabolized in vitro by rabbit tissues mainly by N-oxidation and N-demethylation and to a lesser extent by N-deethylation and di-dealkylation.

4. Both major routes of metabolism were observed in homogenates prepared from rabbit liver and lung; in addition N-oxidation occurred in kidney and bladder tissue homogenates.     

N-Hydroxylation

Abstract

1. Many drugs and foreign compounds contain nitrogen. Biological N-oxidation occurs with many types of N-containing compounds.

2. In mammals N-oxidation is catalyzed by enzyme systems in the endoplasmic reticulum of the liver and other tissues. Different mechanisms are involved in the N-hydroxylation of primary arylamines and the N-oxidation of secondary and tertiary amines.

3. Experimental data on the effects of stimulation and inhibition on microsomal N-oxidation of different types of amines are presented.

4. Kinetic parameters and pitfalls in experimental techniques of biological N-oxidation reactions are discussed.     

Dimethylaniline N-Oxidase and Aminopyrine N-Demethylase Activities of Human Liver Tissue

Abstract

1. N-Oxidation of N,N-dimethylaniline and oxidative demethylation of aminopyrine were studied in human liver hornogenates. At their respective pH optima (8·4 for N-oxidase and 7·4 for demethylase) N-oxidase activity was higher than that of demethylase in the majority of biopsies assayed.

2. Neither activity was preferentially influenced by a specific disease process, but both decreased upon severe tissue damage. There was no apparent relationship of either activity with age or sex.

3. Demethylase activities were highest in patients exposed to barbiturates, and increased N-oxidase activity was observed in patients exposed to amphetamines and some tranquilizers.     

Interaction of Primary Amines with a Mixed-Function Amine Oxidase Isolated from Pig Liver Microsomes

Abstract

1. This work describes the interaction of a variety of primary amines with a purified microsomal mixed-function oxidase that catalyzes the N-oxidation of sec.- and tert.-amines.

2. Most of the primary amines tested are not catalytically oxidized by this isolated microsomal oxidase. Only 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), 1-naphthylamine, and 2-naphthylamine appear to be oxidized at measurable rates.

3. 2-Naphthylamine is enzymically oxidized at a considerably faster rate than 1 -naphthylamine.

4. Primary alkylamines, while not enzymically oxidized by this enzyme, interact with a regulatory site on the oxidase and enhance the rate of N-oxidation of sec.- and tert.-amines catalyzed by this NADPH- and oxygen-dependent amine oxidase.

5. With microsomal-catalyzed oxidations, primary amines that enhance the rate of N-oxidation also inhibit the rate of oxidative N-demethylation.     

The Role of Nitrogen Oxidation in the Excretion of Drugs and Foreign Compounds

Abstract

1. Interest in arylhydroxylamines, because of their implication in ferrihaemoglobin formation and bladder carcinoma induction, has led to numerous investigations on the role of these compounds as a route of metabolism of aromatic amines. The literature on this subject has been reviewed.

2. The metabolic pathway of N-oxidation was first shown to be of importance on the fate of certain endogenous compounds in man and animals. A brief examination of the function of these N-oxides of endogenous compounds is given.

3. Recently N-oxidation as a route of drug metabolism has gained increasing interest although the detection of N-oxides so far has been fortuitous. Published data have been correlated with recent studies on the N-oxidation of nicotine, some antihistamines and amphetamine derivatives to elucidate the factors affecting the elimination of these N-oxides.     

In vivo and in vitro Inhibition of the Metabolism of N-Alkyl-substituted Amphetamines in Rat by Ferrocenylisopropylamine

Abstract

1. Ferrocenylisopropylamine (FIPA) inhibits the elimination of amphetamines in rat. The half-life of isopropylamphetamine was increased from approx. 30 to 85–100 min after administration of FIPA.

2. With isolated, perfused, rat liver, the half-lives of isopropylamphetamine, biamphetamine and benzylamphetamine were increased from 5–20 min to about 200 min by equimolar amounts of FIPA, indicating that the prolonging effect of FIPA is due to interference at the metabolic level.

3. Experiments with hepatic microsomal suspensions demonstrated that FIPA competitively inhibits the oxidative N-dealkylation of isopropylamphetamine; the K_i of FIPA is 4·1 × 10^?6 M.

4. Binding of isopropylamphetamine and FIPA to cytochrome P-450 was studied using hepatic microsomes of phenobarbital-treated rats. Isopropylamphetamine caused a type I, and FIPA a type II difference spectrum; FIPA showed a much higher binding affinity (K_s = 1·24 × 10^?2 M) than isopropylarnphetamine (K_s = 0·96 × 10^?3 M). FIPA acts as a modifier of the spectral changes induced by isopropylamphetamine.

5. Results suggest that the competitive inhibition of the N-dealkylation of N-alkylamphetamines, and thus the prolonging of their action, by FIPA is related to competition for binding to cytochrome P-450.     

Metabolic Oxidation of Aliphatic Basic Nitrogen Atoms and their α-Carbon Atoms

Abstract

1. Evidence is presented to show that in primary, secondary and tertiary aralkylamines, N-oxidation and α-C-oxidation involve separate metabolic routes; steric and stereochemical features, enzyme inhibition, induction, development and alteration are used in the studies.

2. Metabolic oxidation of tertiary nitrogen atoms gives N-oxides, while oxidation of secondary and primary aliphatic amines gives hydroxylamines.

3. The chemical breakdown of hydroxylamines to oximes, ketones and aldehydes under different conditions is discussed.

4. The importance of distinguishing between metabolites and their chemical breakdown products, ‘metabonates’, is stressed.     

Metabolic Interconversions and Binding of Imipramine,Imipramine-N-Oxide,and Desmethylimipramine in Rat Liver Slices

Abstract

1. Rat liver slices and microsomes were used to study metabolism, cellular transfer and binding of imipramine and imipramine-N-oxide (IPNO).

2. Imipramine permeates rapidly into slices and is avidly and strongly bound to microsomes. Concentrations in liver slice to incubation medium reach ratios of ten. The desmethylimipramine (DMI) formed shows even stronger binding than imipramine and little release into the cytosol occurs. IPNO is also formed from imipramine although part is reconverted to imipramine by extra-microsomal reductive enzymes. Both the capacity and strength of microsomal binding of IPNO are low, and the release of this metabolite into the medium is complete.

3. IPNO added to the medium also permeates into the liver cells, but more slowly than imipramine. This intracellular fraction is completely metabolized to imipramine and DMI. More DMI seems to be formed from IPNO by secondary microsomal metabolism of imipramine than by direct N-oxide demethylation. Again, imipramine and DMI are highly bound to the microsomes and released with difficulty. The characteristics of microsomal binding and release of IPNO are the same whether added exogenously or formed metabolically.

4. Time courses of the formation and disappearance of metabolites were measured in slices and homogenate as a function of temperature and NADPH-generating system added. Demethylation and N-oxidation of imipramine are higher in liver preparations from male than from female rats whereas the opposite holds for N-oxide reduction. Phenobarbital pretreatment increases demethylation and decreases N-oxidation of imipramine.     

Metabolism of trifluoperazine, fluphenazine, prochlorperazine and perphenazine in rats: in vitro and urinary metabolites

In vitro and in vivo metabolites of trifluoperazine, fluphenazine, prochlorperazine and perphenazine were isolated by solvent extraction and thin layer chromatography and quantified by u.v. spectroscopy. In liver microsomes from male rats all four drugs underwent N-dealkylation. N-oxidation, sulfoxidation and aromatic hydroxylation. The relative rates of these reactions depended on the substrate concentration, N-oxidation being favoured at higher concentrations. N-Demethylation of trifluoperazine proceeded faster than removal of the hydroxyethyl group from fluphenazine which led to the same metabolite N[γ-(2-trifluoromethyl-phenothiazinyl-10)-propyl] piperazine. The same applied to the dealkylation of prochlorperazine and perphenazine. Following oral administration of 10 mg/kg of the drugs, male rats excreted 1.8?4 per cent of the dose within the first 12 hr in urine in the form of the sulfoxide and the N-dealkylated sulfoxide. In vivo, too, the N-hydroxyethyl group was removed to a smaller extent than the N-methyl group. N-Oxides were not detected in urine at this dose level, but when 25 or 50 mg/kg prochlorperazine were administered, rats excreted small amounts of the N-oxide.     

Metabolism of chlorpromazine by pulmonary microsomal enzymes in the rat and rabbit

Pulmonary metabolism of chlorpromazine (CPZ) was compared using isolated microsomes of rat and rabbit lungs. CPZ-metabolizing activity of the rat lung was found to be 10-fold higher than that of the rabbit lung. The principal metabolic pathways were N-oxidation in the rat lung and N-demethylation in the rabbit lung. Kinetic analyses revealed that, although the values for apparent K_m were roughly similar for both pathways, V_max for N-oxidation by the rat lung was approximately ten times greater than that for N-demethylation by the rabbit lung. N-Oxidation by the rat lung had a broad range of pH optimum of 7–8, whereas N-demethylation by the rabbit lung had a pH optimum 8–9. SKF525-A, piperonyl butoxide, n-octylamine and CO did not inhibit N-oxidation by the rat lung, but inhibited N-demethylation by the rabbit lung. SKF525-A and n-octylamine stimulated the CPZ-N-oxidation by the rat lung. Hg²⁺ and Mg²⁺ inhibited N-oxidation by the rat lung. These results indicate that pulmonary metabolism of CPZ in the rat is catalyzed by a microsomal flavoprotein monooxygenase, while pulmonary metabolism in the rabbit is catalyzed by a cytochrome P-450 monooxygenase system, and that a marked species variation exists with respect to pulmonary metabolism of CPZ.     

2-Mercapto-1-methyl-5-methylmercapto-imidazole: a new metabolite of thiamazole

1. 2-Mercapto-1-methyl-5-methylmercapto-imidazole (13) was found in urine samples of man and rat after intake of thiamazole (1).

2. It is assumed that the metabolite is produced via a N-oxidation intermediate enabling a nucleophilic attack at carbon-5 in the thiazole ring.     

The Fate of an Experimental MAO Inhibitor,N-Cyclopropyl-2-Chlorophenoxyethylamine (Lilly 51641) in the Rat and in Man

Abstract

1. In vivo metabolism studies have shown N-cyclopropyl-2-chlorophenoxy-ethylamine (Lilly 51641) to be extensively metabolized in the rat. Those metabolites identified include 2-chlorophenoxyacetic acid, N-cyclopropyl-4-hydroxy-2-chlorophenoxyethylamine and 4-hydroxy-2-chlorophenoxyethanol, together with a number of minor products. The major route of elimination of these metabolites is through the kidney.

2. The major in vitro metabolites of Lilly 51641 have been identified as 2-chlorophenoxyethylamine, 2-chlorophenoxyacetaldehyde, 2-chlorophenoxyacetic acid and 2-chlorophenoxyethanol. It thus appears that this drug is metabolized by the following sequence: N-dealkylation, deamination and aldehyde oxidation or reduction.

3. Aromatic hydroxylation was found to be an independent but important mechanism involved in the metabolism of Lilly 51641.

4. Human urinary metabolites of Lilly 51641 were separated and identified. The nature of these metabolites indicated that the same mechanisms of detoxication were involved in the biotransformation in the human as were involved in the rat.

5. The major human urinary metabolite was 4-hydroxy-2-chlorophenoxy-acetic acid.     

Species Variation in the Metabolism of R-(+)- and S-(-)-Nicotine by α-C- and N-Oxidation in vitro

Abstract

1. Marked species, but not sex differences, were observed in the metabolism of R-(+)- and S-(-)-nicotine to cotinine and the diastereoisomeric nicotine-1′-N-oxides by liver 10 000 × g supernatant preparations from rats, rabbits, mice, guinea-pigs and hamsters.

2. S-(-)-nicotine formed predominantly R,S-cis-nicotine-1′-N-oxide, whereas R-(+)-nicotine gave predominantly S,R-trans-nicotine-1′-N-oxide; stereoselective metabolism to cotinine also occurred. A stereo-selective site for the N-oxidation of nicotine is proposed to explain these results.     

Species differences in N-dealkylation of oxamniquine

1. The major metabolite of the antischistosomal compounds desoxymansil (I) and oxamniquine (II) in various species is the 6-carboxylic acid (III). Mouse, rabbit, dog and monkey, but not rat, yield a second carboxylic acid metabolite (V) formed by oxidation of the isopropylaminomethyl side-chain.

2. Desoxymansil (I) incubated with microsomal preparations of rat, mouse or rabbit liver was metabolized to oxamniquine (II) by hydroxylation of the 6-methyl substituent; N-dealkylation of the isopropyl group did not occur. Oxamniquine (II) incubated with liver microsomal preparations did not undergo N-dealkylation, but the amine analogue (IV) of desoxymansil was deaminated by the liver microsomal preparations.

3. Oral administration of the amine analogue (IV) of desoxymansil to rat led to the excretion of the acid (V) in the urine, indicating that the rat can deaminate IV. Deamination of IV was shown to be catalysed by the mitochondrial fraction of liver, and mouse and rat showed similar levels of enzyme activity.

4. Intraperitoneal administration of desoxymansil to rat resulted in the biliary excretion of a conjugate of the alcohol (VI) but the acid (V) was not detected.

5. The anomalous metabolism of desoxymansil and oxamniquine in the rat is accounted for by two alternative metabolic pathways, namely, (i) N-dealkylation of the side-chain followed by oxidative deamination in the mitochondria and oxidation of the resultant aldehyde to the corresponding carboxylic acid, or (ii) oxidation of the α-carbon to give the carbinolamine which spontaneously decomposes in the cytosol to be subsequently reduced to the corresponding alcohol. The first pathway predominates in the mouse, rabbit, dog and monkey; the second pathway predominates in rat.     

Microsomal N-Hydroxylation of Dibenzylamine

1. The properties of the rabbit liver microsomal enzyme system(s) catalysing the formation of N,N-dibenzylhydroxylamine as the major metabolite of dibenzylamine have been investigated.

2. The system consists of NADPH- and NADH-dependent components which are differentiated by their different pH optima and sensitivity towards cyanide.

3. The effect of various metabolic inhibitors on the N-oxidation process in vitro are investigated.

4. The N-oxidation of the parent amine was inhibited by CO, SKF 525-A, and inhibitors known to interact with microsomal cytochrome P-450. Phenobarbitone pre-treatment stimulates further metabolism of the hydroxylamine.     

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor

Abstract

1.?Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent.

2.?Structure elucidation via high resolution, accurate mass LC-MSⁿ revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction.

3.?The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes.

4.?Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.     

Comparative Metabolic Studies of Phenacetin and Structurally-related Compounds in the Rat

1. A comparative study of the metabolism of [acetyl-¹⁴C]phenacetin, [acetyl-¹⁴C]methacetin, [acetyl-¹⁴C]paracetamol and [acetyl-¹⁴C]acetanilide in the rat is reported.

2. The extent of N-deacetylation, evidenced by the measurement of respired ¹⁴CO₂ varied, being greatest with acetanilide (25—31%) and least with paracetamol (6%).

3. The major urinary metabolites in each case were N-acetyl-p-aminophenyl sulphate and N-acetyl-p-aminophenyl glucuronide; the relative proportions varied with the sex of the animals and as a result of extended dosage.

4. The metabolism of [ethyl-¹⁴C]phenacetin and [ethyl-¹⁴C]phenetidine was investigated and the extent of O-dealkylation determined by measurement of respired ¹⁴CO₂

5. The metabolic pathways of some related glycolanilides and oxanilic acids included N-deacylation, and in the glycolanilides, oxidation of the glycollic group.     

The Metabolism of Chlorpromazine and Promethazine to give New ‘Pink Spots’

Abstract

1. The ‘pink spots’ observed in the in vivo and in vitro metabolism of chlorpromazine and promethazine were identified as N-oxidation products of 2-chlorophenothiazine and phenothiazine, respectively.

2. Incubations of the latter two compounds with fortified hepatic fractions from rabbit and guinea-pig gave the corresponding hydroxylamines, the nitroxides which were purplish-pink and gave characteristic e.s.r. signals, and the N-hydroperoxides which were the major pink compounds and gave no e.s.r. signals. Each hydroxylamine was readily oxidized to an N-hydroperoxide in air and the latter readily reduced back in solution to the corresponding hydroxylamine by ascorbic acid.

3. The synthesis, chemical properties and the i.r., u.v., n.m.r., e.s.r., and mass spectra of the above compounds, their sulphoxides and phenothiazine-N-peroxides are reported.