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.     

Uptake and metabolism of chlorpromazine by rat and rabbit lungs

Isolated perfused rat lung and in vitro rat and rabbit lung preparations were used to study the pulmonary uptake and metabolism of chlorpromazine (CPZ). Rat lungs were artificially ventilated and perfused via pulmonary artery with recirculating perfusate containing 1.7--180 microM 14C-CPZ. In the perfusate, 14C-CPZ-derived radioactivity declined rapidly, representing maximal pulmonary uptake at 5 min. Thereafter, the total radioactivity in the perfusate increased steadily. During the latter phase, the appearance of CPZ metabolites in the perfusate more than offset the continued decline in CPZ, suggesting metabolism of previously sequestered CPZ and a net, steady efflux of metabolites from the lung. Increasing concentration of metabolites paralleled the increase in total 14C in the perfusate. The principal metabolite was identified as CPZ-N-oxide. Biotransformation of CPZ by the perfused rat lung was temperature-sensitive and dependent on substrate concentration. Substrate saturation was apparent at 120 microM CPZ in the perfusate. In vitro incubation of 14C-CPZ with 9000g supernatant fractions of rat and rabbit lungs revealed that CPZ-metabolizing activity of rat lungs is far greater than that of rabbit lungs. In accordance with the perfusion studies, the principal pathway was N-oxidation of CPZ in the rat lung incubations. Although quantitatively much less significant, CPZ was metabolized in rabbit lung incubations, via demethylation. SKF 525-A did not inhibit CPZ metabolism by perfused or in vitro rat lung preparations, but selectively enhanced N-oxidation of CPZ. Piperonyl butoxide was without any effect. Metabolism of CPZ by rabbit lung incubations was inhibited by SKF 525-A and piperonyl butoxide. These results suggest that CPZ is metabolized by the rat lungs principally via N-oxidation by a pathway not involving cytochrome P-450; the resultant metabolite, CPZ-NO, is released into the circulation, indicating significantly lower affinity for the lung tissue than the parent compounds.     

Conversion of N-hydroxyamphetamine to phenylacetone oxime by rat liver microsomes

These in vitro studies indicate that N-oxidation of N-hydroxyamphetamine (NOHA) by rat liver homogenates yields phenylacetone oxime (PAOx) as the major metabolite. This oxidation was NADPH and oxygen dependent but was not appreciably increased in microsomes from phenobarbital-pretreated animals. The addition to microsomal incubations of Superoxide dismutase (SOD), catalase (CAT), azide or mannitol did not alter the rate of oxidation, suggesting that O₂su?. H₂O₂, or OH^. are not involved in this process. The reaction was minimally inhibited by a 2:1 ratio of CO/O₂, and there was no significant reduction in the formation of product by the presence of diethylaminoethyl diphenylvalerate (SKF-525A) or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) in micromolar concentrations. Thus, although this NADPH-dependent N-oxidation pathway was catalyzed by rat hepatic microsomes, the data suggest that it was not a cytochrome P-450 mediated monooxygenase reaction.     

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.     

Metabolism of 15N-labelled N-nitrosodimethylamine and N-nitroso-N-methylaniline by isolated rat hepatocytes

The N-demethylation of ¹⁵N-labeled N-nitrosodimethylamine (DMN) and N-nitroso-N-methylaniline (NMA) by isolated rat hepatic cells has been investigated. The values obtained in this system for molecular nitrogen formed during metabolism, compared with substrate consumed, were DMN 47%, NMA 23%, and N-nitroso-N-methylurea (NMU) 105%. The results for DMN are roughly halfway between those previously determined with rat liver S-9 fraction in vitro (33%) and in vivo (67%). For NMA, the hepatocyte data are closer to those obtained from S-9 in vitro (19%), rather than the in vivo (52%). No mixed nitrogen (¹⁵N¹⁴N) or labeled nitrogen oxides were found.     

Species variations in the N-oxidation of chlorphentermine

The metabolism and excretion of orally administered or injected [¹⁴C]chlorphentermine has been studied in man, rhesus monkey, marmoset, rabbit, guinea-pig and rat. These species excreted 55–95% of the administered radioactivity in the urine over 5 days. Two metabolites were characterised by thin-layer and paper chromatography, gas-liquid chromatography and g.c.-m.s. and these were N-hydroxychlorphentermine and 1-(4'-chlorophenyl)-2-methyl-2-nitropropane. There are marked species differences in the excretion of N-oxidation products which were found in the urine of human volunteers. rhesus monkeys, rabbits and guinea-pigs, but not in the urine of marmosets or rats. The rat, rabbit and marmoset also excreted an unidentified unstable acid-labile precursor of chlorphentermine. The results are discussed in relation to the toxicity of the drug and to the metabolism of amphetamines in general.     

The influence of methyl substitution on the N-demethylation and N-oxidation of normethadone in animal species

N-Monodemethylation and N-oxidation were shown to be the major routes of metabolism of normethadone, (—)-methadone and (—)-isomethadone in vitro by hepatic microsomal preparations from rat, rabbit, guinea-pig, mouse and hamster. The rate of N-oxidation was decreased and the rate of N-demethylation was increased by the introduction of the methyl substituent into normethadone; the configuration of the methyl substituent influenced these processes. K_m and V_max values were determined for liver microsomal N-demethylation of normethadone, (—)-methadone and (—)-isomethadone by rat and guinea-pig and for the N-oxidation of normethadone by guinea-pig. The use of selective inhibitors showed that N-demethylation was not preceeded by N-oxidation in these compounds.     

Inhibition of brain epinephrine synthesis by 3,4-dichlorophenylethanolamine,a competitive substrate for norepinephrine N-methyltransferase

3,4-Dichlorophenylethanolamine (DCPE) is a substrate for norepinephrine N-methyltransferase (NMT) from rat brain stem or from rabbit adrenal glands and competitively inhibits the methylation of (?)norepinephrine by NMT in vitro. The K_i value for inhibition of rat brain NMT by DCPE was 6 × 10^?5 M. When DCPE was injected i.p. into rats at a dose of 50 mg/kg, epinephrine concentration in hypothalamus was reduced, although NMT activity measured in hypothalamic homogenates in vitro was not inhibited. The findings contrast in several ways to those with 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (LY134046), a competitive inhibitor of rat brain NMT with an in vitroK_i of 2.4 × 10^?8 M. Although the potency of DCPE was 1/2500th that of LY134046 in vityro, it lowered hypothalamic epinephrine concentrations at similar doses in vivo. The NMT activity measured in vivo in homogenates of hypothalamic tissue from LY134046-treated rats was reduced by a greater percentage than was epinephrine concentration. The reduction of epinephrine concentration in vivo by DCPE may relate to its ability to be a substrate for NMT, which would result in conversion of S-adenosylmethionine (SAMe) to S-adenosylhomocysteine (SAH) in NMT-containing neurons. The resulting increase in the SAH/SAMe ratio may inhibit norepinephrine N-methylation, since SAH is a competitive inhibitor of NMT when SAMe is the variable substrate. This mechanism is similar to that suggested for the decrease in epinephrine concentration in rat brain following l-dopa injection. l-Dopa injection increases SAH and decreases SAMe concentration measured in hypothalamus, due presumably to extensive O-methylation of dopa and its decarboxylated metabolites. In contrast, SAH and SAMe concentrations in hypothalamus were not measurably altered after DCPE injection, a not-unexpected finding since SAH/SAMe changes would occur only in NMT-containing neurons, which make up a negligibly small percentage by weight of hypothalamic tissue. DCPE and other NMT substrates may be able to inhibit epinephrine synthesis in vivo as effectively as non-substrate, competitive inhibitors having much higher affinity for NMT because their N-methylation elevates SAH/SAMe ratios specifically within NMT-containing neurons.     

N-demethylation as an example of drug metabolism in isolated rat hepatocytes

The capacity of isolated rat hepatocytes to carry out drug metabolism in vitro was studied. The aromatic hydroxylation of quinine sulfate was measured fluorometrically from two starting concentrations of the drug. The N-demethylation of dansylamide and antipyrine was measured by following the release of tritium into water after hydroxylation of ³H-labeled methyl groups. The N-demethylation of antipyrine and dansylamide was inhibited by SKF-525A and ethylmorphine. The metabolism was rapidly increased (approximately 10 per cent) by the addition in vitro of 0.1 mM phenobarbital. Isolated rat hepatocytes are well suited for drug metabolism studies. They are readily prepared, retain their viability for hours, and allow many, well-controlled experiments from the same preparation of cells. The formation of ³H₂O from suitably labeled precursors is a sensitive and promising technique to study certain aspects of drug metabolism.     

Genotoxicity of N-methyl-N-nitrosourea in the presence of amphiphilic membrane-active compounds

With V79 Chinese hamster lung cells the dose-response relationship of sister chromatid exchange (SCE) induction by NMU shows a sigmoidal-shaped curve. In vitro, the time schedule of the short-lived (<60 min) SCE inducer (NMU) and the sister chromatid marker 5-bromodeoxyuridine (BrdU) addition to the cells has a significant effect on the SCE response suggesting a direct chemical and/or indirect biochemical interaction between both compounds. Certain amphiphilic molecules of a definite length, consisting of a hydrophobic chain and a polar end, have been shown to alter functions of various membrane integrated proteins by modifying membrane lipid structures. In vitro, three chemically distinct membrane-active compounds inhibit induction of SCEs following N-methyl-N-nitrosourea (NMU) treatment to different extents in a concentration-dependent manner. These findings with Chinese hamster lung cells confirm the results of in vivo experiments with bone marrow cells from Chinese hamsters. The anti-SCEs inducer effects are interpreted in terms of a decrease of nuclear bioavailability of NMU. Experimental results are not in favor of a competitive inhibition of SCE induction as a consequence of eventual alkylations of the amphiphilic molecules at their polar end. It is hypothesized that cell membrane structure modifications enhance the probability of chemical reactions of the strongly reactive SN₁ alkylating compound NMU with nucleophilic membrane sites. Membrane-active compounds with anti-SCE inducer capacities should be useful probes for the study of possible relationships between SCE induction and the carcinogenic as well as antitumor efficiency of NMU.     

Bleomycin toxicity: alterations in oxidative metabolism in lung and liver microsomal fractions

Separate groups of male rats received low doses (5 units) or high doses (15 units) of bleomycin i.p. twice weekly for 1.5, 3 or 6 weeks. The susceptibility of tissue lipid to peroxidation and the activities of mixed function oxidations were examined in microsomal fractions prepared from lung and liver. ADP-Fe (III)-initiated lipid peroxidation was stimulated in lung microsomal fractions only in animals treated with high-dose bleomycin for 1.5 weeks, whereas a 2- to 4-fold enhancement was observed in liver preparations from all bleomycin-treated animals. Microsomal ADP-Fe (III)-initiated lipid peroxidation was inhibited, however, by the in vitro addition of bleomycin in both lung and liver preparations, but this inhibition was an artifact resulting from the chelation of Fe (III) by bleomycin. Soybean lipoxygenase I-initiated microsomal lipid peroxidation, which does not require added iron, was unaffected by bleomycin in lung preparations but was inhibited in liver. Following in vivo treatment, lung microsomal hydrogen peroxide generation was inhibited by 1.5 weeks of high-dose bleomycin treatments, whereas benzphetamine N-demethylation was unchanged. These cytochrome P-450-dependent reactions were both suppressed, however, in liver microsomal fractions. In vitro, both reactions were also inhibited by bleomycin in liver but not in lung microsomal fractions. The lack of effect of in vitro bleomycin treatments on Superoxide generation in lung or liver preparations suggests that the NADPH cytochrome P-450 reductase component of the mixed function oxidase system was not affected. Minimal alterations in lipid peroxidizability and mixed function oxidase activities in lung microsomal fractions of bleomycin-treated animals suggest that the insensitivity could be due to: (1) the site of toxicity not being at the level of the endoplasmic reticulum; or (2) the target of bleomycin toxicity being limited to a small population of pulmonary cell types. Even though the liver is not susceptible to bleomycin toxicity, the hepatic microsomal mixed function oxidase system is highly sensitive to this chemical.     

Halidohydrolytic conversion of N,N'-bis-(dichloroacetyl)-N,N'-diethyl-1,4-xylylenediamine in rat liver

N,N′-bis-(dichloroacetyl)-N,N′-diethyl-1,4-xylylenediamine (WIN 13099) was synthesized with tritium on the xylylene carbon which did not exchange with body water. This compound was metabolized by 140,000 g supernatant liquid and the mitochondrial fraction of rat liver. Deproteinized 140,000 g supernatant liquid was required for the mitochondrial activity. Although WIN 13099 effectively inhibits drug metabolism by rat liver microsomes in vitro and in vivo, it is not metabolized by rat liver microsomes in vitro. The major metabolite in vitro of WIN 13099, N-dichloroacetyl-N′-hydroxyacetyl-N,N′-diethyl-1,4-xylylenediamine, was isolated and characterized and found not as effective as the parent compound in inhibiting electron transport by sub-mitochondrial particles or drug metabolism by rat liver microsomes.     

Formation of α-methylnorepinephrine as a metabolite of metaraminol in guinea pigs

Metaraminol was metabolized to α-methylnorepinephrine by homogenates from guinea pig liver in vitro, and α-methylnorepinephrine was found in tissues of guinea pigs treated with metaraminol in vivo. The conversion of metaraminol to α-methylnorepinephrine with rat liver homogenates in vitro was low, and no α-methylnorepinephrine was found in metaraminol-treated rats in vivo. α-Methyl-norepinephrine concentrations were measured in liver, heart, spleen, kidney and lung of guinea pigs treated with metaraminol. The ratio of tissue concentrations of α-methylnorepinephrine/metaraminol varied from near 0 in the adrenal glands to greater than 1 in liver at early times, though α-methyl-norepinephrine disappeared from liver at a slightly faster rate than did metaraminol, possibly due to the ability of α-methylnorepinephrine to be metabolized by O-methylation. Iprindole pretreatment reduced the concentration of α-methylnorepinephrine in heart and liver of metaraminol-treated rats but did not alter the depletion of norepinephrine in these tissues. The possibility that α-methylnore-pinephrine is involved in some of the pharmacological effects of metaraminol in guinea pigs is raised.     

The in vitro metabolism of mephentermine

The metabolism of mephentermine (Ie) to phentermine (Ia), N-hydroxyphentermine (Ib) and N-hydroxymephentermine (If) was investigated using hepatic microsomal preparations from various species. Liver microsomes from rabbit were the best homogenate fractions to metabolise Ie. The effect of incubation times, various substrate concentrations, addition of selective inhibitors and activators and species differences suggested that the dealkylation of Ie to Ia involved a separate metabolic route than the N-oxidation of Ie to Ib and If and that both hydroxylamino compounds Ib and If resulted from metabolic oxidation at the nitrogen atom of Ie. A mechanism explaining the separate formation of Ib and If through a common intermediate resulting from N-oxidation of Ie is proposed. The synthesis and the properties of N-methyl-(α,α-dimethyl-β-phenethyl)nitrone (Ig) are reported.     

Inhibition of monoamine oxidase by N-phenacyl-cyclopropylamine

N-phenacyl-cyclopropylamine hydrobromide (54761) was evaluated in vitro and in vivo as a monoamine oxidase (MAO) inhibitor in rats. In contrast to 51641, which has an o-chlorophenoxy group in place of the phenacyl group and which is a highly selective inhibitor of type A MAO, 54761 showed a slight preference as a type B MAO inhibitor, since it inhibited phenylethylamine oxidation at slightly lower concentrations than were required to inhibit serotonin oxidation in vitro by rat liver MAO. Twelve analogs of 54761 with various substituents on the phenyl ring were also studied, but none was substantially more selective than 54761 as a type B inhibitor and most were preferential type A inhibitors. When 51641 and 54761 were injected into rats and MAO activity was assayed in tissue homogenates, the oxidation of serotonin in brain, heart and liver was inhibited more by 51641 than by 54761. In contrast, the oxidation of phenylethylamine was inhibited more by 54761 than by 51641 in brain and liver. In heart, however, 51641 was a more effective inhibitor of phenylethylamine oxidation than was 54761, supporting earlier evidence that phenylethylamine is destroyed in heart mainly by type A MAO. The oxidation of exogenous [¹⁴C]phenylethylamine was inhibited in vivo more effectively by 54761, whereas the oxidation of endogenous serotonin in brain was inhibited more by 51641. Although 54761 is not as selective an inhibitor of type B MAO as some other compounds such as deprenyl, it illustrates that a large range of selectivity in MAO inhibition can exist within the N-cyclopropylamine series. Further, selective type B inhibition could be achieved in vivo 24 hr after injection of 54761 by co-administration of harmaline. Harmaline selectively protected against the inactivation of type A MAO by 54761 but permitted the inactivation of type B MAO to occur.     

Kinetics and disposition of picumeterol in animals

1. The pharmacokinetics and disposition of picumeterol, a novel β₂ receptor agonist agent, have been studied in the rat and dog following administration by inhalation, intravenous and oral routes at various dose levels.

2. Picumeterol was found to be transferred across the lung of the rat and dog following inhalation dosage. After i.v. dosage picumeterol was eliminated from plasma with a half-life of about 1?h in the rat and about 2?h in the dog. Plasma clearance in the rat was about twice liver blood flow and the plasma levels of picumeterol were low after oral administration.

3. Following instillation of ¹⁴C-picumeterol to the trachea of isolated respiring rat lung preparations radioactivity was transferred from the airways to perfusion media as unchanged drug within 2?min. After 2?h perfusion, no metabolites were detected in the recirculation perfusate or lung.

4. Picumeterol was extensively metabolized in vivo in the rat (about 95%) and dog (about 90%) and in vitro in microsomal preparations of rat, dog and human liver. O-dealkylation and β-oxidation are important as routes of metabolism.

5. Radioactivity was largely excreted in the urine of the rat and dog (> 50% of dose), as metabolites, following i.v. administration. There was some excretion of radioactivity in dog bile. Extensive first-pass metabolism was found after oral administration in the rat.     

Flavin-containing monooxygenase 1-catalysed N,N-dimethylamphetamine N-oxidation

N,N-dimethylamphetamine (DMA) is a methamphetamine analogue known to be a weaker central nervous system stimulant than methamphetamine. Although a major metabolite of DMA is known to be DMA N-oxide (DMANO), which may be catalysed by flavin-containing monooxygenase (FMO), the specific enzyme(s) involved in this biotransformation has not been identified. In this study, the specific enzyme(s) involved with DMA N-oxidation was characterized by several assays. When DMA was incubated with different human recombinant drug-metabolizing enzymes, including FMOs and cytochrome P450s (CYPs), the formation of DMANO by FMO1 was the most predominant. The Michaelis–Menten kinetic constants for DMA N-oxidation by FMO1 were: K_m of 44.5 μM, V_max of 7.59 nmol min^?1 mg^?1 protein, and intrinsic clearance of 171 μl min^?1 mg^?1 protein, which was about twelve-fold higher than that by FMO3. Imipramine, an FMO1-specific inhibitor, selectively inhibited DMA N-oxidation. The resulting data showed that DMA N-oxidation is mainly mediated by FMO1.     

Studies on the hepatic microsomal metabolism of [14C]phenanthrene

[¹⁴C]Phenanthrene was metabolished in vitro by hepatic microsomes obtained from untreated, sodium phenobarbital (PB), and 3-methylcholanthrene (MC) pretreated Wistar rats, guinea pigs, SW and DBA/2J mouse strains. Metabolite profiles were obtained by comparison of thinlayer radiochromatographs of the products with R_fs of authentic reference compounds, trans-dihydrodi-hydroxyphenanthrenes (dihydrodiols) were the major metabolites in all preparations. In every case, except with microsomes from MC pretreated guinea pigs, trans — 9,10 — dihydro — 9,10 — dihydroxyphenanthrene predominated. With microsomes from MC pretreated guinea pigs, the 1,2-dihydrodiol was the major metabolite. Addition of cyclohexene oxide (5.0 mM) to the incubation mixture inhibited dihydrodiol formation in rat and mouse preparations by 90 per cent, and in guinea pig preparations by 70 per cent. Phenols and phenanthrene — 9,10 — oxide were produced instead.     

Mutagenicity and α-hydroxylation of N-nitrosopyrrolidine and N-nitrosopiperidine: A possible corelation

N-Nitrosopyrrolidine (NPyrr) and N-nitrosopiperidine (NPip)are carcinogenic and mutagenic cyclic nitrosamines. Their biotransformation by rat liver post-mitochondrial fraction into 1,4-butanediol and 1,5-pentanediol, respectively, is evaluated by determining these ultramate metabolites with a sensitive and suitable method. Their mutagenic activity towards the Salmonella typhimurtum strain TA 1530 was simultaneously observed. A relationship exists between their metabolism and their mutagenicity. α-Hydroxylation is probably the critical metabolic mechanism of cyclic nitrosamines.     

In vitro metabolism of lidocaine in subcellular post-mitochondrial fractions and precision cut slices from cattle liver

In vitro models are widely used to study the biotransformation of xenobiotics and to provide input parameters to physiologically based kinetic models required to predict the kinetic behavior in vivo. For farm animals this is not common practice yet. The use of slaughterhouse-derived tissue material may provide opportunities to study biotransformation reactions in farm animals. The goal of the present study was to explore the potential of slaughterhouse-derived bovine liver S9 (S9) and precision cut liver slices (PCLSs) to capture observed biotransformation reactions of lidocaine in cows. The in vitro data obtained with both S9 and PCLSs confirm in vivo findings that 2,6-dimethylaniline (DMA) is an important metabolite of lidocaine in cows, being for both PCLSs and S9 the end-product. In case of S9, also conversion of lidocaine to lidocaine-N-oxide and monoethylglycinexylidine (MEXG) was observed. MEGX is considered as intermediate for DMA formation, given that this metabolite was metabolized to DMA by both PLCSs and S9. In contrast to in vivo, no in vitro conversion of DMA to 4-OH-DMA was observed. Further work is needed to explain this lack of conversion and to further evaluate the use of slaughterhouse-derived tissue materials to predict the biotransformation of xenobiotics in farm animals.