Björn Lindeke

The p-Hydroxylation of Amphetamine and Phentermine by Rat Liver Microsomes

1. The products of p-hydroxylation of amphetamine and phentermine by two different preparations of rat liver microsomes were identified and quantitatively determined. At low concentrations (muM) significant proportions of the substrates were metabolized to the p-hydroxy derivatives by an NADPH-dependent system. The enzyme system was inhibited by higher substrate concentrations (mM) and was not induced by either phenobarbital or 3-methylcholanthrene. 2. The properties of this in vitro system are consistent with reports on in vivo studies of this reaction.

Cytochrome P-455 complex formation in the metabolism of phenylalkylamines--IV. Spectral evidences for metabolic conversion of methamphetamine to N-hydroxyamphetamine.

Abstract Incubation of liver microsomes from phenobarbital-treated rats with N -methylamphetamine (1) and its N -oxidized congeners N -hydroxy- N -methylamphetamine (2), N -methylene-1-phenyl-2-pro-pylamine N -oxide (3a) and N -(1-phenyl-2-propylidene)methylamine N-oxide (3b) gave rise to the formation of cytochrome P-450 product complexes characterized by maximum absorbances in the 453–457 nm region. Compounds 1, 2 and 3a showed maximum absorbances at 456 nm and for 2 and 3a both the rate and extent of complex formation was increased several fold over those of 1, with the complexing activity being about 90 per cent of that of N -hydroxyamphetamine (4a). Contrary to 1, 2, 3a and 4a, nitrone 3b showed its maximum absorbance at 453 nm and the spectral perturbations were identical to those seen with N -hydroxymethylamine (4b). Demethylation of 1 and 2, as monitored by formaldehyde production, showed good correlation with the complex formation. From the results it seems safe to conclude that 3a, formed after metabolic N -oxidation of N -methylamphetamine (1), undergoes further conversion to N -hydroxyamphetamine (4a), the latter being the ultimate precursor to the ligand forming the cytochrome P-455 complex. The results substantiate the notion that there is a preference for the formation of nitrones related to 3a rather than 3b during the metabolism of N -alkylamphetamines. Thus, in addition to α-carbon oxidation, N -oxidation is indicated as a route instrumental to the metabolic demethylation of N -methylamphetamine.

Peroxidative N-oxidation and N-dealkylation reactions of pargyline

The hydrogen peroxide-supported oxidation of pargyline in rat-liver microsomes was investigated and compared to that promoted by cytochrome P-450 in the presence of an NADPH-generating system. The metabolic conversions promoted by hydrogen peroxide and cytochrome P-450 comprised N-demethylation, N-depropargylation, N-debenzylation and N-oxidation. For the hydrogen peroxide-cytochrome P-450-promoted oxidation, cyanide, but not carbon monoxide, was an effective inhibitor of all the reactions. Similarly, 2,4-dichloro-6-phenyl phenoxyethylamine (DPEA) inhibited all reactions, particularly N-demethylation and N-oxidation more extensively than the NADPH-dependent microsomal oxidation. Using microsomes from rats pretreated with phenobarbital caused no increase in the metabolites above the levels seen with microsomes from untreated animals. Various other peroxidase systems which were investigated were essentially unable to promote oxidation of pargyline.

Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The nature of the reactive metabolites formed during HRP/H2O2 catalyzed oxidation of p-phenetidine was investigated. Interaction with DNA measured as the induction of DNA single strand breaks and DNA binding resulted in a time-dependent decrease in the interaction and could be related to the primary oxidation of p-phenetidine. Oxygen uptake observed during p-phenetidine metabolism in the presence of GSH also exhibited such a correlation. GSH-conjugate formation and protein binding on the other hand exhibited an initial increase and did not appear to be directly related to primary p-phenetidine oxidation since maximal interaction was obtained when p-phenetidine had been completely metabolized. The GSH-conjugate and protein binding ratio of ring labelled to ethyl labelled p-phenetidine of approx. 2:1 indicated that these reactive metabolites(s) may be dimer(s) whose formation presumably involved loss of one ethoxy group of p-phenetidine. Accordingly formation of ethanol, indicative of ethoxy group elimination, could be observed during p-phenetidine metabolism. Only one metabolite generated from p-phenetidine oxidation exhibited a concentration dependent binding to protein. This metabolite also reacted with GSH to form water-soluble conjugates. Prior reduction of the metabolite by ascorbic acid prevented this conjugate formation. The mass spectral fragmentation pattern of the reactive protein- and GSH-binding metabolite was compatible with the structure N(4-ethoxyphenyl)-p-benzoquinoneimine.

Cytochrome P450 product complexes and glutathione consumption produced in isolated hepatocytes by norbenzphetamine and its N-oxidized congeners

Abstract Incubation of liver microsomes from phenobarbital-treated rats with norbenzphetamine (I) and its two N -oxidized metabolites, N -hydroxynorbenzphetamine (II) and the corresponding nitrone (III), in the presence of NADPH and molecular oxygen, gave rise to the formation of cytochrome P450 product complexes characterized by maximal absorbance at 455 nm. The complex forming activity increased in the order I, II and III, with the nitrome (III) exhibiting an activity of about 60 per cent of that of N -hydroxyamphetamine (IV). The same relative order of complex forming activity was seen also when incubations were performed with hepatocytes isolated from phenobarbital-treated rats, but the complex formation was less rapid as well as less extensive. Liver tissues from untreated rats exhibited the same complex forming pattern, but with considerably lower activity. Incubations of hepatocytes with I, II and III caused a decrease in the cellular level of reduced glutathion (GSH) and II and III caused the most significant drops in the GSH level. The decrease in GSH was enhanced in hepatocytes from phenobarbital-treated rats. N -hydroxyamphetamine had no effect on the cellular GSH level and the amount of oxidized glutathione (GSSG) was unaffected by addition of I-IV. It is suggested that N -hydroxyamphetamine (IV), formed by hydrolysis of the nitrone (III), is the ultimate substrate for the reaction leading to complex formation. The results indicate that the nitrone (III) is a common intermediate in the reactions leading to the interaction with GSH and cytochrome P450 complex formation and by reacting with III, GSH decreases the concentration of cytochrome P450-binding norbenzphetamine metabolites in the hepatocyte.

Decomposition of prop-2-ynylic N-oxides to acrolein and schiff bases via O-allenyl hydroxylamines

Abstract Prop-2-ynylic N-oxides rearrange thermally via O-allenyl hydroxylamines to acrolein and an imine. The second rearrangement step, falling in the class of thermal cis -eliminations, is shown with variously 2 H-substituted pargyline N-oxides.

Biotransformation of terodiline. III. Opposed stereoselectivity in the benzylic and aromatic hydroxylations in rat liver microsomes

1. Terodiline (N-tert-butyl-4,4-diphenyl-2-butylamine) is a racemic drug with anticholinergic and/or calcium antagonistic activity, which is subject to renewed interest as a potential remedy for urinary incontinence. As part of the current investigations on terodiline, the metabolism of its enantiomers is being investigated. 2. The metabolism of the enantiomers of terodiline in rat liver microsomes is slow, as for the racemate, though the S-enantiomer is metabolized more rapidly than its optical antipode. Phenobarbitone pretreatment of the rats enhances the metabolism with a marked increase in the conversion of the S-enantiomer. 3. While aromatic p-hydroxylation greatly exceeds benzylic oxidation in the metabolism of R-terodiline, this situation is reversed in the metabolism of S-terodiline. Moreover, the rate of aromatic p-hydroxylation of racemic terodiline follows that of R-terodiline, while the rate of benzylic hydroxylation of racemic terodiline follows that of S-terodiline. Phenobarbital pretreatment of the rats had little or no effect on aromatic p-hydroxylation but markedly increased benzylic oxidation. 4. Separation of the mixture of p-hydroxylated metabolites into diastereomeric pairs showed that their composition is highly dependent on which form of terodiline is used as substrate. 5. The results from the study are compatible with the participation of multiple forms of cytochrome P-450 enzymes.

Autoxidation of N‐hydroxyphenylalkylamines: the inhibitory effect of some anions on copper catalysed autoxidation of N‐hydroxyphentermine*

The inhibitory effect of certain electrolytes and buffers on the copper catalysed autoxidation of N‐hydroxyphentermine (2‐hydroxylamino‐2‐methyl‐1‐phenylpropane) has been investigated. The presence of ions such as SO42−, Cl− or Br− markedly reduced the rate of oxidation. Phosphate and carbonate buffers had a similar effect with halides and phosphate buffers being the most inhibitory. The occurrence of 2‐methyl‐2‐nitro‐1‐phenylpropane and 2‐methyl‐1‐phenylpropene‐(1) as secondary oxidation products was also established.

Biotransformation of terodiline. v. stereoselectivity in hydroxylation by human liver microsomes

The stereoselective hydroxylation of N-tert-butyl-4,4-diphenyl-2-butylamine (Terodiline) was studied in human liver microsomes. Formation of the two main metabolites, N-tert-butyl-4(4-hydroxyphenyl)-4-phenyl-2-butylamine (II) and N-(2-hydroxymethyl-2-propyl)-4,4-diphenyl-2-butylamine (VI), was found to be stereoselective. R-Terodiline was preferentially transformed by phenolic hydroxylation to the 2R,4S-II and 2R,4R-II forms with a pronounced selectivity for the former. The formation rate ratio 2R,4S-II/2R,4R-II was about 6, obtained from two liver preparations. S-Terodiline was mainly hydroxylated to the alcohol 2S-VI although phenolic hydroxylation to the 2S,4S-II and 2S,4R-II also occurred, yielding about equal amounts of the two phenols.

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.