The identification and analysis of the metabolic products of mephentermine.

Phentermine (Ib), N-hydroxymephentermine (Ic) and N-hydroxyphentermine (Id) were identified as metabolic products after in vitro incubation of mephentermine (Ia) with rabbit liver microsomal fractions. Compounds Ia, Ib and Ic were also identified as excretion products in the urine of a human subject given a single dose of mephentermine (Ia) sulphate. Derivatization with acetic anhydride, trifluoroacetic anhydride and the trimethylsilyl donor reagent N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) or hexamethyldisilazane (HMDS) were used for qualitative identification of the metabolic products Ib-Id by g.l.c.-mass spectrometry and for quantitative determination of Ia-Id after extraction from rabbit hepatic homogenates. The synthesis of N-hydroxymephentermine (Ic) and the properties of the metabolic products are reported.     

In vitro metabolism of amiodarone by rabbit and rat liver and small intestine

The present studies were designed to investigate whether amiodarone (Am) is metabolized in the major organs and tissues of the rat and rabbit. Incubations using Am and tissue homogenates (600 g supernatant) of rabbit and rat lung, liver, kidney, and gut revealed formation of desethylamiodarone (DEA) by the liver and gut. Subsequent experiments using the post-mitochondrial, cytosolic, and microsomal fractions of these tissues indicated that metabolism of Am was greatest in the microsomal fractions. In both species, greater DEA formation was detected for microsomes of hepatic origin. The hepatic microsomal mediated production of DEA was altered by protein concentration in both the rabbit and rat preparations with protein concentrations of 5 mg providing the greatest DEA production. DEA formation by gut microsomes was greatest at 3 mg of protein for the rabbit but exhibited no significant change from 1 mg to 10 mg of protein for the rat. In vitro metabolism of Am by rabbit and rat hepatic microsomal preparations was significantly reduced by 1 mM piperonyl butoxide, SKF 525-A, n-octylamine, and carbon monoxide. Effects of these inhibitors on rabbit and rat gut microsomal incubations were inconclusive. HPLC analysis of incubation samples revealed a species difference in the metabolism of Am as demonstrated by the detection of three metabolites in addition to DEA. The unidentified metabolites (I, II, III) were detected in rabbit hepatic microsomal incubations. Metabolite II was also detected in incubations using rabbit duodenal tissue microsomes. No metabolites other than DEA were found in incubations using rat tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of promethazine in vitro. Identification of N-oxidized products

1. Incubation of promethazine (Ia) and desmethylpromethazine (Ib) with 9000g supernatant fractions of rabbit liver homogenate resulted in formation of N-dealkylated, N-oxygenated and ring-hydroxylated products.

2. The N-oxidation products identified by t.l.c. and mass spectra using synthetic reference products are promethazine-N-oxide (IX) and the nitrone (VIII), which is believed to be formed chemically and metabolically from the metabolite N-hydroxydesmethylpromethazine (VII).     

Metabolic products of N-ethyl-beta-methoxy-beta-(3'-trifluoromethylphenyl)ethylamine (SKF 40652A)

The metabolsim of N-ethyl-beta-methoxy-beta-(3'-trifluoromethylphenyl)ethylamine (SKF 40652A, I) in vitro with 9000 g supernatants and washed microsomes of rabbit liver gave N-dealkylation, and N-oxidation of the parent secondary amine and the derived primary amine, anti and syn beta-methoxy-beta-(3'-trifluoromethylphenyl)acetaldoximes (V), and the beta-methoxy-beta-(3'-trifluoromethylphenyl(ethyl alcohol (VI). The identification and the physicochemical properties of these compounds are reported.     

Biological N-oxidation of piperidine in vitro

The biological N-oxidation of piperidine, a pharmacologically active biogenic amine of mammals and human beings, was studied in vitro. After incubation of piperidine-HCl in a fortified rat liver microsomal preparation (9000 x g supernatant) at 37 degrees C for 30 min, 2 metabolites were detected. They were identified as N-hydroxy piperidine and 2, 3, 4, 5-tetrahydro-pyridine-1-oxide as evidenced by TLC, GLC, HPLC, GC-MS and MS.     

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32- hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. K_m and V_max     

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.     

The biotransformation of allyl alcohol and acrolein in rat liver and lung preparations

The biotransformation of allyl alcohol and acrolein by rat lung and liver preparations was investigated by measuring acrolein, acrylic acid, glycidol, and glycidaldehyde. Acrolein was detected by high-pressure liquid chromatography from incubation mixtures containing allyl alcohol, NAD+, and liver 9000g supernatant fraction or cytosol. Acrolein was not formed when lung fractions were treated similarly. Addition of pyrazole in the incubation mixture inhibited the reaction. The metabolism of acrolein to acrylic acid by liver 9000g supernatant fraction, cytosol, and microsomes has been demonstrated; acrylic acid formation was greater with NAD+ than with NADP+ in all three fractions. Acrylic acid was also formed from allyl alcohol. Disulfiram inhibited the NAD+- and NADP+-dependent reactions. Acrylic acid was not formed when lung preparations were used. Lung and liver microsomal epoxidation products of allyl alcohol and acrolein have been identified. Conversion of glycidol to glycerol and glycidaldehyde to glyceraldehyde by liver epoxide hydrase has been demonstrated. Epoxides, glycidol, and glycidaldehyde were also found to be substrates for lung and liver cytosolic glutathione S-transferase.     

In vitro metabolic products of RWJ-34130, an antiarrythmic agent, in rat liver preparations

The in vitro metabolism of RWJ-34130, an antiarrhythmic agent, was conducted using rat hepatic 9000 x g supernatant (S9) and microsomes in an NADPH-generating system, and the rat liver perfusion. The 100 and 20 microg ml(-1) concentrations of RWJ-34130 aqueous solution were used for microsomal incubation and liver perfusion, respectively. Unchanged RWJ-34130 (approximately 77-78% of the sample in both S9 and microsomes) plus a major metabolite, RWJ-34130 sulfoxide (20% of the sample in both S9 and microsomes) were profiled, isolated and identified from both hepatic S9 and microsomal incubates (60 min) using HPLC and mass spectrometry (MS), and by comparison to a synthetic RWJ-34130 sulfoxide, which was synthesized by reacting RWJ-34130 with MCPBA (meta-chloroperoxy benzoic acid). No unchanged RWJ-34130 was detected in the 3 h liver perfusate, however, 1-phenyl-2-oxo-pyrrolidine was profiled, isolated and identified as a major hydrolyzed metabolite of liver perfusate. RWJ-34130 is not extensively metabolized in vitro in rat hepatic S9 and microsomes. All HPLC metabolic profiles of hepatic S9 and microsomal samples (30 min, 60 min) were qualitatively and nearly quantitatively identical.     

Acetaminophen nephrotoxicity in the rat. Renal metabolic activation in vitro

High doses of acetaminophen (APAP) result in hepatic centrilobular and renal cortical necrosis in man and the F344 rat. Hepatic necrosis is considered to be due to the generation of an arylating intermediate via a microsomal cytochrome P-450 dependent system. Renal microsomes also metabolize APAP to an arylating intermediate via a P-450 dependent mechanism. Thus, at least part of the renal damage from APAP may be due to a biochemical mechanism similar to that in liver. Additionally, APAP is deacetylated to p-aminophenol (PAP) in renal and hepatic cytosol and microsomes. Previous results demonstrated that PAP may be activated in renal microsomes via an NADPH-independent mechanism. Therefore, significant metabolic activation of APAP in the kidney may occur subsequent to deacetylation. Covalent binding of [ring-14C]APAP to renal subcellular fractions was used to substantiate this hypothesis. Under appropriate incubation conditions, enzymatic NADPH-independent covalent binding of [ring-14C]APAP could be demonstrated in renal microsomes but not in 100,000g supernatant fractions. Combination of these subcellular fractions resulted in greater covalent binding of [ring-14C]APAP than in the individual subcellular fractions alone. Addition of glutathione, bis(p-nitrophenyl)phosphate (a deacetylase inhibitor), or PAP inhibited this covalent binding. In contrast, NADPH-independent covalent binding of [ring-14C]APAP could not be demonstrated in any combination of hepatic subcellular fractions. Experiments comparing [ring-14C]APAP and [acetyl-14C]APAP covalent binding to renal 10,000g supernatant fractions indicate that the compound which binds to renal macromolecules is derived from PAP. Thus, these results are consistent with the hypothesis that APAP can be metabolically activated in the kidney after deacetylation to PAP.     

Differences in the effect of phenobarbital treatment on the in vitro metabolism of aflatoxin and aniline by duck and rat livers

Groups of ducks and rats were treated with phenobarbital sodium for 14 days (1 mg/ml in the drinking water) and the effects of this treatment on in vitro rates of microsomal aniline and aflatoxin metabolism were observed.

Aniline hydroxylase activity was enhanced in microsomes of both species. Normal levels of activity were comparable in duck and rat livers.

Aflatoxin metabolism was stimulated in crude microsomal preparations (9000 g supernatant fraction) from rat but not from duck liver. Normal mean rates of total aflatoxin metabolism were 0.31 nmole/g/min in rat liver and 47·0 nmoles/g/min in duck liver.

Phenobarbital treatment had the effect of stimulating total aflatoxin metabolism, hydroxylation and demethylation in isolated rat liver microsomes.

The failure of this treatment to stimulate aflatoxin metabolism in the duck liver suggested that microsomal metabolism was not rate limiting. Aflatoxin was actually metabolised 90 times more rapidly by soluble enzymes (105,000g supernatant) from duck liver than from rat liver.     

The metabolism of dibenzylamine: identification of NN-dibenzylhydroxylamine as the major in vitro metabolic product from rabbit fortified hepatic homogenates.

A study of the in vitro metabolism of dibenzylamine with fortified rabbit liver 9000 g supernatant fractions has shown that N-oxidation is the major metabolic process. Of the total amount of dibenzylamine metabolized, 90% is converted to NN-dibenzylhydroxylamine. The primary amine, benzylamine, is formed to the extent of only 6% of total substrate metabolized. The identity of the major metabolite, NN-dibenzylhydroxylamine has been established by thin-layer chromatography, gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry.     

The metabolism of N-ethyl-N-methylaniline by rabbit liver microsomes: the measurement of metabolites by gas-liquid chromatography.

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 formaldehyde during in vitro drug demethylation: Effectiveness of protection by semicarbazide

Formaldehyde formation is usually determined as a measure of the metabolism of drug substrates in vitro. We have studied the fate of formaldehyde added to incubates of rat liver microsomes or 9000 g supernatant fraction as well as the effectiveness of semicarbazide in protecting formaldehyde from metabolic degradation. Formaldehyde is known to be oxidized by a cytosolic NAD- and GSH-dependent (form)aldehyde dehydrogenase. We found that, in addition, some other NAD-independent reactions take place in the cytosol. We observed, moreover, that formaldehyde is also metabolized by the 9000 g supernatant fraction fortified with cofactors for hepatic monooxygenase in the absence of NAD. This finding could be attributed to a Hitherto unknown, cytosolic NADP-dependent, GSH-requiring dehydrogenase. The microsomal fraction metabolized formaldehyde only to a small extent. Therefore, in order to use formaldehyde formation as a parameter of drug metabolism, semicarbazide is necessary to protect formaldehyde from further metabolism in the 9000 g supernatant fraction and microsomes. By determining amounts of both formaldehyde and p-chlor-aniline duringp-clor-N-methylanilinedemethylation, it was shown that semicarbazide (4 mM) only partially protected for-maldehyde from further metabolism in the 9000 g supernatant, although semicarbazone formation from the added formaldehyde and semicarbazone progressed more rapidly than formaldehyde metabolism. As higher semicarbazide concentrations inhibit microsomal demethylations, it is concluded that determination of formaldehyde is not a suitable method for determining drug demethylation by the 9000 g liver supernatant. In microsomal incubates, only a low semicarbazide concentration (1.0 mM) was necessary to protect formaldehyde from further metabolism.     

Metabolism of strychnine in vitro

The in vitro metabolism of strychnine was studied in the 9000g supernatant fractions from rat and rabbit livers. The metabolism was markedly inhibited by cytochrome P-450 inhibitors, SKF-525A and n-octylamine, but only slightly by a microsomal FAD-containing monooxygenase inhibitor, methimazole. Five metabolites formed in vitro with rabbit liver were isolated and purified by Sep-Pak C18 cartridge chromatography and preparative TLC. Three of them were identified as 2-hydroxystrychnine, strychnine N-oxide, and 21 alpha, 22 alpha-dihydroxy-22-hydrostrychnine by comparison with their authentic samples by means of UV, NMR, and mass spectrometries. An additional two metabolites were tentatively identified as strychnine 21,22-epoxide and 11,12-dehydrostrychnine by spectral measurements. Four of these metabolites, with the exception of 2-hydroxystrychnine, were novel metabolites of strychnine. The in vitro formation of these metabolites by rabbit liver was determined by HPLC after partial purification. The major identified metabolite was strychnine N-oxide, which accounted for approximately 15% of the metabolized strychnine. All the other metabolites accounted for less than 1%. The presence of a larger quantity of other metabolites which have been neither isolated nor identified was also suggested.     

Identification of novel phencyclidine metabolites formed in vitro by rabbit microsomal metabolism.

1. Phencyclidine (PCP) was incubated with rabbit liver and brain microsomal fractions, and the structures of metabolites formed by oxidation determined by g.l.c.-mass spectrometry. 2. The formation of several known mono- and di-hydroxylated metabolites, as well as two new metabolites, was seen in the liver preparations. 3. Hydroxylated PCP metabolites were also formed after incubation of PCP with brain microsomes, indicating that PCP biotransformation may occur in the brain itself.     

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.     

The effect of solvents on drug metabolism in vitro

Nine water-miscible organic solvents, methanol, ethanol, acetone, ethoxyethanol, tetrahydrofuran, dioxane, dimethylformamide, acetonitrile, and dimethyl sulfoxide were each used with five commonly employed substrates of in vitro microsomal mixed-function oxidase assays containing liver 9,000g supernatant fractions from rats treated with phenobarbital or Aroclor 1254. When the metabolism of aminopyrine, aniline, 7-ethoxycoumarin, p-nitroanisole, and benzo[a]pyrene was determined in the presence of these solvents, varying degrees of stimulation and inhibition were observed. These effects were dependent on the substrate studied, the particular solvent incorporated into the assay, and the rat liver 9000g supernatant fraction used. These differential effects were also observed when 2-aminoanthracene was metabolically activated in the Ames Salmonella/mammalian-microsome mutagenicity test, but were not as dramatic when benzo[a]pyrene was tested.     

Identification of novel phencyclidine metabolites formed in vitro by rabbit microsomal metabolism

1. Phencyclidine (PCP) was incubated with rabbit liver and brain microsomal fractions, and the structures of metabolites formed by oxidation determined by g.l.c.-mass spectrometry.

2. The formation of several known mono- and di-hydroxylated metabolites, as well as two new metabolites, was seen in the liver preparations.

3. Hydroxylated PCP metabolites were also formed after incubation of PCP with brain microsomes, indicating that PCP biotransformation may occur in the brain itself.     

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax of the rifalazil-25-deacetylation in microsomes were 6.5 microM and 11.9 pmol/min/mg with NADPH, and 2.6 microM and 6.0 pmol/min/mg without NADPH, indicating that, although rifalazil-25-deacetylation did not require NADPH, NADPH activated it. Rifalazil-32-hydroxylation was NADPH dependent, and its Km and Vmax were 3.3 microM and 11.0 pmol/min/mg respectively. 3. Rifalazil-25-deacetylation in microsomes was completely inhibited by diisopropyl fluorophosphate, diethyl p-nitrophenyl phosphate and eserine, but not by p-chloromercuribenzoate or 5,5'-dithio-bis(2-nitrobenzoic acid), indicating that the enzyme responsible for the rifalazil-25-deacetylation is a B-esterase. 4. Rifalazil-32-hydroxylation in microsomes was completely inhibited by CYP3A4-specific inhibitors (fluconazole, ketoconazole, miconazole, troleandomycin) and drugs metabolized by CYP3A4 such as cyclosporin A and clarithromycin, indicating that the enzyme responsible for the rifalazil-32-hydroxylation is CYP3A4.