Molecular orbital calculations and nicotine metabolism: a rationale for experimentally observed metabolite ratios.

The results of molecular orbital calculations and molecular modelling studies on nicotine are reported. It is shown that the product ratio of nicotine metabolism can be directly related to HOMO electron densities on the relevant hydrogen atoms associated with oxidation sites in S-nicotine. In addition, molecular modelling of nicotine within the putative active site of CYP2A6, the enzyme most closely associated with nicotine metabolism, indicates that the substrate is orientated for oxidation at the 5'-position via a combination of hydrogen bonding and pi-pi stacking interactions. Alternative routes of metabolism may require rotation of the pyrrolidine ring system and could, therefore, involve a degree of re-orientation of the nicotine molecule which is energetically less favourable than the modelled interaction indicating formation of cotinine via 5'-oxidation.

Studies on the in vitro hepatic microsomal formation of amides during the metabolism of certain secondary and tertiary benzylic amines.

Part of our interest during the last few years has been to investigate the possible intermediate(s) and mechanism(s) involved in the formation of amides from N-benzylic amines. A number of benzylic amines with different aryl and alkyl moieties introduced onto the constituent nitrogen were prepared, thus creating a wide variety of secondary, tertiary and heterocyclic benzylic amines with different logP and pKa characteristics (Tables I & II). In some experiments, the possible intermediates of this reaction, i.e. nitrones (Table III), imines (Table IV) and amides themselves (Table V), were used as substrates in our metabolic studies. Their in vitro hepatic microsomal metabolism was studied in order to obtain a structure/metabolic activity relationship for the formation of amides from benzylic amines. This communication reviews these studies and reports our conclusions as to the mechanism of formation of amides from N-benzylic amines.

Effects of cAMP-dependent protein kinase and ATP on N1-oxidation of 9-benzyladenine by animal hepatic microsomes.

N1-Oxidation is a major metabolic pathway for 9-benzyladenine (BA) catalyzed by the cytochrome P450 system in animal hepatic microsomes. After normal hamster hepatic microsomes or phenobarbital induced rabbit hepatic microsomes were preincubated in the presence of cyclic AMP-dependent protein kinase catalytic subunit (PKA), MgCl2 and ATP, BA-N1-oxidation was significantly decreased. However, further investigation indicated that the decrease of BA-N1-oxidation seemed to be a combination of the effects of PKA and ATP, as ATP alone showed a biphasic regulatory effect on BA-N1-oxidation when microsomes were preincubated in the presence of various concentrations of ATP. In the lower ATP concentration range (0.5-2.5mM), BA-N1-oxidation increased along with the increase of ATP concentration; whereas BA-N1-oxidation decreased when the ATP concentration was higher (>5mM). The biphasic regulatory effects of ATP on BA-N1-oxidation seem dependent on the incubation process, as preincubation markedly strengthened the effects. When microsomes were incubated at 37 degrees C for different time lengths in the absence or presence of ATP (2.5 or 20mM), the activity of BA-N1-oxidase decreased at similar rates in all groups, but the activity levels of BA-N1-oxidase were different among the groups. The cytochrome P450 content was not changed parallel to the variation of BA-N1-oxidation when microsomes were incubated in the presence of ATP, indicating that the effects of ATP on BA-N1-oxidation were not mediated by affecting CYP stability. In addition, the activity of NADPH-cytochrome P450 reductase was not markedly affected by ATP without incubation. The result implied that ATP did not inhibit the reductase directly. After microsomes were incubated in the presence of low ATP concentration (2.5mM), the reductase was slightly inhibited, whilst high ATP concentration (20mM) showed marked inhibition (83% of control). This may partially contribute to the down-regulatory effect of ATP on BA-N1-oxidation. Furthermore, it was found that the presence of magnesium ions during preincubation weakened the up-regulatory effect of ATP (2.5mM) on BA-N1-oxidation, but showed no effect on the down-regulatory effect of ATP (20mM). Since these observed phenomena are not readily explained, a possible mechanism, i.e. phosphorylation and dephosphorylation of cytochrome P450, is suggested.

In vitro hepatic microsomal metabolism of N-benzyl-N-methylaniline.

In the present study, the in vitro microsomal metabolism of a tertiary aniline, N-benzyl-N-methylaniline (NBNMA) was studied to determine whether this compound produces an amide derivative (benzoyl) together with N-dealkylation and C- and N-oxidation products as metabolites. The preparations of the corresponding potential metabolites were undertaken and were separated using TLC and HPLC. Incubations were performed using rat microsomal preparations fortified with NADPH. The substrate and its potential metabolites were extracted into dichloromethane in the presence of NaCl and examined by TLC and HPLC-UV. The results indicated that NBNMA did not produce the corresponding amide (benzoyl derivative) or N-oxide metabolite but was dealkylated to the corresponding secondary amine. Two p-hydroxylated phenolic metabolites were also observed. These findings support the concept that nitrones are essential intermediate metabolites for the formation of amides from secondary aromatic amines (chemical rearrangement to amide via an oxaziridine intermediate). The carbinolamine produced from NBNMA does not seem stable enough to allow further oxidation to the amide and therefore this intermediate is broken down to the dealkylation products. N-Dealkylations and p-hydroxylations are major metabolic reactions following in vitro hepatic microsomal metabolism of the benzylic tertiary aniline, NBNMA.

Metabolism, pharmacogenetics, and metabolic drug-drug interactions of antipsychotic drugs.

1. Antipsychotic drugs are extensively metabolised by cytochrome P450 (CYP) enzymes. 2. Dispositions of a number of antipsychotic drugs have been shown to cosegregate with polymorphism of CYP2D6. 3. Metabolic drug-drug interactions have frequently been observed when antipsychotics are coadministered with other drugs. 4. Many antipsychotic drugs are converted to active metabolites which can contribute to the therapeutic or side effects of the parent drug. 5. Information concerning the individual CYP isoenzymes involved in the metabolism of antipsychotic drugs is important for the safe clinical use of this group of drugs.

Effect of the nicotine metabolite 5'-hydroxycotinine on glucose transport and glycogen synthase activity in rat skeletal muscle.

Cigarette smoke contains many potentially harmful substances, including nicotine and nicotine metabolites, which are likely to contribute to altered glucose homeostasis. We determined the effects of nicotine and nicotine derivatives on glucose transport in skeletal muscle. Split rat soleus muscles were pre-incubated in the presence of nicotine (range 0.01-100 microg/ml) or nicotine metabolites including nicotine 1'-N-oxide, cotinine, trans-3'-hydroxycotinine, 5'-hydroxycotinine, gamma-3-pyridyly-oxo-butyric acid and nicotine iminium ion before measurement of 3-O-methylglucose transport rate and glycogen synthase activity. Nicotine (100 microg/ml) did not alter basal 3-O-methylglucose transport. Insulin-stimulated (0.6 nmol/l) glucose transport was unaltered following acute (50 min) exposure to nicotine (0.01-100 microg/ml). The nicotine metabolite 5'-hydroxycotinine increased basal glucose transport and glycogen synthase activity (up to 50%; P<0.05), with no effect on insulin-stimulated glucose transport and glycogen synthase activity. None of the other nicotine metabolites had any effect on basal or insulin-stimulated glucose transport. Acute exposure of skeletal muscle to the nicotine derivative 5'-hydroxycotinine appears to directly increase basal glucose transport and metabolism. Whether this leads to changes in whole-body glucose homeostasis in cigarette smokers requires further investigation.

Evidence for the biosynthesis of A glucuronide conjugate of (S)-(-)-nicotine, but not (S)-(-)-cotinine or (+/-)-trans-3'-hydroxycotinine by marmoset hepatic microsomes.

Recently, the detection of urinary glucuronide conjugates of nicotine and its two major metabolites, trans-3'-hydroxycotinine and cotinine, showed that glucuronidation is an important pathway of nicotine metabolism in humans. (S)-(-)-Nicotine-N(+)-1-beta-glucuronide (quaternary N-glucuronide with linkage through the pyridino-nitrogen of nicotine) was shown to be an important nicotine metabolite of humans in vivo. The present study was undertaken to develop an animal model for this process, in order to ascertain the factors influencing quaternary N-glucuronide formation. (S)-(-)-Nicotine-N(+)-1-beta-glucuronide was formed in vitro when [2'-14C]-nicotine was incubated with Triton X-100 activated marmoset hepatic microsomes in the presence of uridine diphosphoglucuronic acid; it was not formed when activated microsomal preparations of rabbit, guinea-pig, or rat were used as enzyme source. The glucuronide was characterised by comparison with authentic synthetic (S)-(-)-nicotine-N(+)-1-beta-glucuronide using HPLC. The rate of formation of the glucuronide was almost linear during up to four hours of incubation, but still only accounted for a maximum of 6.0% of the available substrate at the end of five hours incubation. The synthetic and biosynthetic (S)-(-)-nicotine-N(+)-1-beta-glucuronides were hydrolysed by beta-glucuronidase and alkali, but were resistant to acid hydrolysis. The results support the concept that the marmoset may be a good animal species to mimic man in studies of nicotine metabolism during exposure to tobacco smoke. In vitro studies using (+/-)-trans-3'-hydroxycotinine or (S)-(-)-cotinine (as potential substrate) and [14C]-uridine diphospho-glucuronic acid (as cofactor) failed to produce any new radiolabelled glucuronide when the above microsomal preparations were used.

In vitro metabolism of (S)-(-)-[2'-14C]nicotine, using various tissue preparations of marmoset.

The nicotine metabolite profile produced by marmoset liver, lung and kidney preparations was investigated after 30 minutes incubation of (S)-(-)-[2'-14C]nicotine. Cation-exchange high performance liquid radiochromatography was employed to separate and quantify nicotine and its metabolites. Cotinine-N-oxide (CNO, 0.7%), 3'-hydroxy-cotinine (3'-OH-C, 0.2%), norcotinine (NORC, 0.9%) and nornicotine (NORN, 0.4%) were formed in the incubates of marmoset lung homogenates; when marmoset kidney homogenates were used, CNO, 0.4%; 3'-OH-C, 0.2%; NORC, 0.7%; NORN, 0.7%; and cotinine (COT, 0.4%) were detected in the incubates. These nicotine metabolites constituted only approximately 2.2% and 2.4% of the original nicotine substrate used by lung and kidney homogenates respectively. When marmoset hepatic homogenates and microsomes were used, both COT and NORN were detected as the major nicotine metabolites. In addition, traces of CNO and 3'-OH-C were also detected in both incubates. The amounts of COT (6.4%) and NORN (1.8%) in the hepatic homogenates were approximately twice that of those formed by hepatic microsomes (3.8% and 0.9%, respectively). Nicotine-1'-N-oxide (NNO, 1.1%) was only detected in the latter preparation. Under the experimental conditions, these nicotine metabolites constituted only 8.2% and 5.8% of the substrate nicotine used in the respective incubates. The present results showed that both primary C-oxidation pathways, i.e. cotinine formation and N-demethylation of nicotine, occurred in the lung, kidney and liver of marmoset in vitro. However, N-oxidation of nicotine was only observed when a marmoset hepatic microsomal preparation was used.

The in vitro hepatic microsomal metabolism of N-benzyladamantanamine in rats.

The metabolism of N-benzyladamantanamine (NBAD) was studied in vitro using rat hepatic microsomal preparations. The substrate and proposed metabolites were synthesized and characterized using spectroscopic techniques and separated using a reverse phase HPLC system. NBAD was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl and evaporated under a stream of nitrogen. The results from HPLC studies showed that NBAD produced the corresponding nitrone and hydroxylamine. This experiment also revealed that dealkylation occurred. No metabolites were observed which corresponded to authentic amide or oxaziridine. The reactions required a microsomal enzyme source and NADPH as a cofactor. The results indicate that the nitrone observed as a metabolite of NBAD is not an intermediate leading to the formation of an oxaziridine and hence an amide, under careful experimental conditions excluding light.

Induction and inhibition of the in vitro N1-oxidation of 9-benzyladenine and isomeric 9-(nitrobenzyl)adenines.

The present study investigated some aspects of the enzymology of the in vitro N1-oxidation of 9-benzyladenine (BA) and isomeric 9-(nitrobenzyl)adenines (NBAs) using various potential inducers and inhibitors of cytochrome P-450 (CYP). When incubated with phenobarbital-induced rabbit hepatic microsomes, the N1-oxidation rates of BA and 9-(4-nitrobenzyl)adenine were about 6- and 2-fold higher than that of the control, respectively; while the N1-oxidation of 9-(2-nitrobenzyl)adenine and 9-(3-nitrobenzyl)adenine was not markedly affected. In contrast, beta-naphthoflavone and Arochlor 1254 showed no inductive effects towards the N1-oxidation of any of these substrates. Using 12 typical CYP inhibitors, it was found that nifedipine (CYP3A inhibitor) and haloperidol (CYP2D inhibitor) showed significant inhibition towards the N1-oxidation of BA and NBAs. Therefore, the N1-oxidation of BA and NBAs is probably catalysed by CYP3A and CYP2D subfamilies. Furthermore, when 9-(4-nitrobenzyl)adenine was incubated with compounds which possessed a certain chemical similarity to the adenine substrate, various degrees of inhibition of N1-oxidation of 9-(4-nitrobenzyl)adenine were observed. These observations allowed a preliminary indication as to the structure-metabolism relationship of 9-substituted adenine derivatives.

Comparison of metabolic rates of some 9-aralkyladenines obtained using hamster hepatic microsomes.

Previous investigations have revealed that N1-oxidation is a major metabolic pathway in vitro for some 9-aralkyladenines (AAs) such as 9-benzyladenine (BA). However, dealkylation and other metabolic pathways are also involved. In addition, various substituents on the benzyl moiety of BA seem to have a marked effect on the metabolic rate. In order to establish the potential structure-metabolism relationship of this class of compounds, the enzyme kinetics of the substrates, which possess 2'-nitro (2NBA), 3'-nitro (3NBA), 4'nitro (4NBA), 2'-chloro (2CBA), 2'-methyl (2MBA), or 2-methoxy (2MOBA) substitution of the benzyl moiety of BA, were compared using hamster hepatic microsomes. The results show that the formation rates of the N1-oxides are in the order 2NBA > 2CBA > BA > 3NBA and 4NBA > 2MBA and 2MOBA; the formation rates of the total metabolites except N1-oxides are in the order 2MOBA and 2MBA > 2CBA > BA > 4NBA > 3NBA > 2NBA; however, the total biotransformation rates of the substrates are in the order 2MBA and 2MOBA > 2CBA > BA and 2NBA > 4NBA > 3NBA. The results strongly imply that the electronic, steric and other physicochemical properties are potential controlling factors for AA metabolism.

Comutagenesis-I: the in vitro metabolism of 2-amino-3-methylpyridine.

The metabolism of the comutagen 2-amino-3-methylpyridine has been studied in vitro using rat and rabbit hepatic preparations. 2-Amino-3-methylpyridine-N-oxide, 2-amino-3-hydroxymethylpyridine and 2-amino-5-hydroxy-3-methylpyridine were formed by both rat and rabbit hepatic preparations. No evidence was obtained for the formation of the corresponding 2-hydroxylamine, 2-nitroso, 2-nitro-3-methyl-pyridine or their condensation products i.e. azo, azoxy or hydrazo. The results are discussed in relation to the possible mechanism of action of the substrate.

Comutagenesis-III. In vitro metabolism of 2-amino-3-methylpyridine: the effect of various potential inhibitors, activators and inducers.

1. The effects of various potential inhibitors, activators and inducers on the metabolism of the comutagen 2-amino-3-methylpyridine (2A3MP) by rabbit hepatic microsomes and S9 supernatants have been studied. 2. The 1-N-oxidation of 2A3MP to 2-amino-3-methylpyridine-1-N-oxide (2A3M-PNO) was inhibited by 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525-A) and n-octylamine. 3. The C-oxidation products of 2A3MP, i.e. 2-amino-3-hydroxymethylpyridine (2A3HMP) and 2-amino-3-methyl-5-hydroxypyridine (2A3M5HP), were also inhibited by these compounds. 4. Pretreatment of animals with phenobarbitone (PB) resulted in an increase in the production of 2A3MPNO and 2A3HMP, whereas beta-naphthoflavone (BNF) pretreatment had a greater effect on the formation of 2A3M5HP. 5. Pretreatment with pyridine or pyrazine also had an appreciable effect on the formation of 2A3HMP. 6. It is suggested that different cytochrome P450 isozymes are responsible for the metabolic profile of 2A3MP. CYP2B was involved in the N-oxidation; 2E and/or 2B in the formation of 2A3HMP, and 3A and/or 1A in the formation of 2A3M5HP.

Solid phase extraction and HPLC determination of 9-benzyladenine and isomeric 9-(nitrobenzyl)adenines and their metabolic N1-oxides present in microsomal incubates.

9-Benzyladenine, 9-(2-nitrobenzyl)adenine, 9-(3-nitrobenzyl)adenine and 9-(4-nitrobenzyl)adenine were metabolized to 9-benzyladenine-N1-oxide, 9-(2-nitrobenzyl)adenine-n1-oxide, 9-(3-nitrobenzyl)adenine-N1-oxide and 9-(4-nitrobenzyl)adenine-N1-oxide, respectively, by animal hepatic microsomes. For the quantitative determination of the substrates and metabolites present in microsomal incubates, an off-line solid phase extraction procedure, using columns paced with C18 silica bonded phase, was developed. The extraction recovery for these 9-alkyladenines and their N1-oxides was in the range of 92-101%. A reversed-phase HPLC method was established with an ODS column at a column temperature of 50 degrees C. The mobile phase consisted of H20-MeOH-diethylamine (65:35:0.5, v/v/v). pH 6.8. The above analytes were monitored at 233 nm and retention times of all analytes were within 6-14 min. The within-day coefficients of variation (CV) for the determinations were in an acceptable range. The biotransformation of BA and NBAs to N1-oxides by hamster microsomes was determined under the experimental conditions employed.

In vitro studies on the metabolic fate of mifentidine, a novel histamine H2-receptor antagonist.

The in vitro metabolism of mifentidine and several of its metabolites was studied using hepatic microsomes from seven animal species. The effects of potential enzyme inducers, inhibitors and activators were also studied. Mifentidine metabolites identified and characterised were: 4-imidazolylphenylamine (amine), 4-imidazolylphenyl-formamide (formamide), the urea derivative of mifentidine (urea) and the imidazole-hydroxylated derivative of the amine (i-OH-amine), along with three unidentified metabolites, M1, M2 and M3. Evidence for the presence of the amine, formamide, urea and i-OH-amine was obtained by comparison with authentic reference compounds: (i) HPLC retention times; (ii) UV spectra; and (iii) MS spectra of metabolites. The postulated intermediates are: carbinolimine (for formamide, amine, i-OH-amine and urea formation); formamide (for amine and i-OH-amine formation); amine (for i-OH-amine formation), and nitrone (for urea formation). One 'metabonate' of mifentidine was also identified, namely the nitro analogue of the amine. A possible prerequisite for the formation of this nitro is the corresponding hydroxylamine or nitroso compound. Cytochromes P450I and P450II were shown to be involved in the in vitro microsomal biotransformation of mifentidine, but the involvement of the flavin monooxygenase system was not proven.

Recognition of novel artifacts produced during the microsomal incubation of secondary alicyclic amines in the presence of cyanide.

1. During the in vitro microsomal metabolism of the secondary alicyclic amines, nornicotine, anabasine or 4-benzylpiperidine in the presence of cyanide, a new product was formed. 2. Comparison of these new products with authentic compounds by hplc and ms showed they were the corresponding 1'-N-cyanomethyl compounds. 3. The source of the methylene group was shown to be formaldehyde, arising during incubation, from either microsomal lipid and/or glycerol used to store microsomes for long periods at low temperature. 4. The formation of the 1'-N-cyanomethyl compounds was enhanced in the presence of substrates producing formaldehyde during their metabolism, in direct relationship to the amount of formaldehyde generated. 5. The results indicate that care should be taken in interpreting data obtained from incubates containing secondary amines and cyanide, especially in the presence of substrates capable of donating formaldehyde.

Microsomal metabolism of N-benzyl-N-ethylaniline and N-benzyl-N-ethyl-p-toluidine.

The in vitro hepatic microsomal metabolism of two tertiary anilines, N-benzyl-N-ethylaniline (NBNEA) and N-benzyl-N-ethyl-p-toluidine (NBNEPT), was examined in order to determine whether these compounds produce amide derivatives (benzoyl or acetyl) in addition to N-dealkylation and N-oxidation products as metabolites. The preparation of these tertiary anilines and their corresponding potential metabolites was undertaken. The amines and metabolites were separated using TLC and HPLC. Incubations were performed using hamster microsomal preparations fortified with NADPH. The substrates and their potential metabolites were extracted into dichloromethane and examined by TLC and HPLC. The metabolic process of particular interest was the formation of amides from NBNEA and NBNEPT. The results from these experiments indicated that neither tertiary aniline (NBNEA and NBNEPT) produced amide (acetyl or benzoyl) or N-oxide metabolites. These substrates were dealkylated to the corresponding secondary amines via debenzylation and de-ethylation. Uncharacterised metabolites observed with substrates are proposed to be phenolic (for NBNEA) and hydroxymethyl (for NBNEPT). These findings support the concept that: nitrones are essential intermediates for the formation of amides from secondary aromatic amines (chemical rearrangement to amide via an oxaziridine intermediate); carbinolamines produced by NBNEA and NBNEPT are not stable enough to allow further oxidation to amides and therefore these intermediates are broken down to dealkylated products. The results are discussed in relation to the mechanism of metabolic amide formation from amines.

Comutagenesis-II. Some factors influencing the in vitro metabolism of 2-amino-3-methylpyridine.

Factors affecting the metabolism of 2-amino-3-methylpyridine (2A3MP) in vitro have been studied and the conditions which allow maximal metabolism established. Ring nuclear and methyl hydroxylation, and 1-N-oxidation of 2A3MP were linear with respect to arochlor 1254 induced rat S9 supernatant (10,000 g fraction) up to 4.86 mg per ml. The results showed that 20 min incubation time was adequate to observe metabolites formed from 2A3MP. The rate of metabolite production increased with increase in substrate concentration up to 2 µmol per incubate. Using the data obtained the apparent K(m) and V(max) values were calculated using Hanes-Wolf and Lineweaver-Burk plot. No N-hydroxylation of the exo-amino group was observed.

The in vitro hepatic microsomal metabolism of benzoic acid benzylidenehydrazide.

The in vitro hepatic microsomal metabolism of benzoic acid benzylidenehydrazide was studied using rat microsomal preparations fortified with NADPH. The substrate and its potential metabolites were synthesised and their structures were elucidated by use of their UV, IR, ¹H-NMR and mass spectral characteristics. The results showed this compound produced hydrolytic metabolites, i.e. benzoic hydrazide and benzaldehyde, together with a phenolic metabolite, benzoic acid phydroxybenzylidene hydrazide.

Metabolic N-oxidation of 9-benzyladenine and isomeric 9-(nitrobenzyl)adenines by hamster hepatic microsomes.

The metabolism of 9-benzyladenine (BA) and isomeric 9-(nitrobenzyl) adenines (NBAs), i.e. 9-(2-nitrobenzyl)adenine (2NBA), 9-(3-nitrobenzyl)adenine (3NBA) and 9-(4-nitrobenzyl)adenine (4NBA), was investigated with normal hamster hepatic microsomes. Using HPLC, TLC, UV and MS techniques, it was established that metabolic N¹-oxidation occurred for all BA and NBAs. The N¹-oxidative rate was in the order 2NBA>BA>3NBA and 4NBA. The results suggest that the introduction of a nitro group may interfere with the binding between substrates and related N¹-oxidase(s). This may be the reason for the lower N¹-oxidative rates of 3NB A and 4NB A. It is also suggested that 2NBA may possess the most favourable conformation and binding characteristics for the enzymes among the substrates studied as it was metabolised even faster than BA.