The role of cytochrome P-450 in the dual pathways of N-demethylation of N,N-dimethylaniline by hepatic microsomes

The role of cytochrome P-450 in the demethylation of N,N'-dimethylaniline (DMA) has been investigated by studying carbon monoxide (CO) inhibition and its reversal by light. Two distinct pathways of N-demethylation are present in hepatic microsomes from phenobarbital-(PB) and 3-methylcholanthrene-(3-MC) induced rats. Pathway A, the demethylation of DMA, is catalysed by a microsomal cytochrome P-450 system similar to the one we have reported for the dealkylation of benzphetamine and 3-ethylmorphine, in that there is a maximal, although weak, light reversal of CO inhibition by wavelengths of light around 445 nm. The second pathway consists of two enzyme steps (pathways B + C). Pathway B is the step in which the N-oxide of DMA, N,N'-dimethylaniline N-oxide (DMAO) is formed and this step is catalysed by the flavin amine mixed-function oxidase described by Ziegler and his co-workers. Pathway C, the demethylation of DMAO, requires a cytochrome P-450 with rather unusual properties. This reaction is inhibited by SKF 525A and CO, but is not significantly affected by the removal of oxygen even though CO strongly inhibits in this anaerobic atmosphere. Higher CO/O2 ratios (2:1 to 4:1) are required for inhibition equal to that of pathway A and light reversal of this inhibition is almost complete and maximally produced by wavelengths of light around 455 nm.     

Oxidative N-demethylation of N,N-dimethylaniline by purified isozymes of cytochrome P-450

The metabolism of N,N-dimethylaniline (DMA) by rabbit liver microsomes results in the formation of N-methylaniline (NMA) and formaldehyde. The N-oxide of DMA (DMA N-oxide) has been suggested as an intermediate in the cytochrome P-450-catalyzed demethylation reaction. The role of DMA N-oxide as an intermediate in demethylation has been investigated in a reconstituted system consisting of NADPH-cytochrome P-450 reductase, phospholipid, and several different purified isozymes of cytochrome P-450. The abilities of several cytochrome P-450 isozymes from rabbit liver (P-450 form 2 and P-450 form 4) and rat liver (P-450b and P-450c) to catalyze N-oxide formation and their abilities to catalyze demethylation of the N-oxide were determined and compared with their abilities to catalyze the demethylation of DMA. The metabolism of DMA by the purified isozymes of cytochrome P-450 in the reconstituted system did not result in the formation of measurable amounts of the N-oxide. The turnover numbers for the metabolism of DMA and DMA N-oxide to formaldehyde by the reconstituted system containing cytochrome P-450 form 2 were 25.6 and 3.4 nmol/min/nmol cytochrome P-450, respectively. The three other isozymes (P-450 form 4, P-450b, and P-450c) also exhibited significantly greater rates for the demethylation of DMA than for the N-oxide. If the N-oxide were an intermediate in the demethylation reaction, it should be metabolized at a rate greater than or at least equal to DMA. Therefore, these data, along with the inability to detect N-oxide formation during the cytochrome P-450-catalyzed demethylation of DMA, suggest that the N-oxide of DMA is not an intermediate in demethylation of DMA by these forms of cytochrome P-450 and that DMA N-oxidase activity is not associated with these isozymes.     

N-Oxide Formation and Related Reactions in Drug Metabolism

Abstract

1. Tertiary amine drugs are converted into dealkylated and N-oxide metabolites by liver microsomal enzymes. The two reactions are catalyzed by NADPH-dependent microsomal electron transfer chains; the first involving NADPH-cytochrome c reductase and cytochrome P-450, the latter a different flavo-protein and no cytochrome P-450. The individual rates are highly dependent on the animal species and experimental conditions.

2. N-oxides can be further metabolized by dealkylation and/or by reduction. However, N-oxides are not obligatory intermediates in the dealkylation of tertiary amines. Rather, two alternative pathways are open to these compounds: C-oxygenation and N-oxygenation.

3. With imipramine and imipramine-N-oxide as substrates it could be shown that tertiary amine dealkylation and N-oxidation are catalyzed by microsomal enzymes only, whereas N-oxide dealkylation and reduction occur only in extra-microsomal compartments. With isotope trapping experiments the simultaneous occurrence of all four reactions could be demonstrated in NADPH-fortified liver homogenate, and the individual reaction rates could be determined. Experiments with liver slices, perfused livers and whole animals suggest a similar kinetic situation whereby N-oxide formation may well be a major pathway.     

Microsomal N-Oxygenation of Adenine to Adenine 1-N-Oxide

During investigations on the N-oxygenation of adenine ( 1 ) the enzymatic formation of adenine 1-N-oxide 3 was demonstrated for the first time. The identity of this metabolite was confirmed by its chromatographic behaviour and UV-spectrum recorded after HPLC separation. Adenine 1-N-oxide ( 3 ) and similar oxygenated derivatives of adenine were synthesized as reference substances. The enzymatic formation of 3 exhibits the typical characteristics of a reaction catalysed by microsomal mono-oxygenases. In induction experiments, an increase in the rate of formation of 3 after pretreatment with phenobarbital was observed. A participation of those isoenzymes of the cytochrome P-450 enzyme system which can be induced by phenobarbital is assumed.     

Non-enzymatic reduction of aliphatic tertiary amine N-oxides mediated by the haem moiety of cytochrome P450

1. The mechanism of reduction of aliphatic tertiary amine N-oxides to tertiary amines in liver microsomes was examined and a novel type of reduction by cytochrome P450 was found. 2. Rat liver microsomes exhibited a significant N-oxide reductase activity toward brucine N-oxide and imipramine N-oxide in the presence of both NAD(P)H and FAD under anaerobic conditions. These N-oxide reductase activities were inhibited by carbon monoxide or air. However, the activities were not abolished by boiling the microsomes; indeed, in the case of brucine N-oxide, the activity was enhanced. 3. The activity toward brucine N-oxide was also observed after the conversion of cytochrome P450 to cytochrome P420. Cytochrome P4502B1 alone exhibited the reductase activity in the presence of both NAD(P)H and FAD. After the removal of haem from cytochrome P4502B1, the activity was observed in the haem moiety, but not in the cytochrome P450 apoprotein. 4. Photochemically reduced FAD was effective in the reduction in place of NAD(P)H and FAD. 5. The N-oxide reduction appears to proceed non-enzymatically, catalysed by the haem group of cytochrome P450 in the presence of a reduced flavin.     

Induction of liver microsomal cytochrome P-450 isozymes by 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide

1. 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (RP 52028), an antagonist of peripheral type benzodiazepine binding sites, a potential anticonvulsant, has been shown to have an inducing effect on drug-metabolizing enzymes.

2. RP 52028 was administered orally at 20–800?mg/kg for 1 week, and enzymic activities were determined using a panel of substrates. Western blot analyses were performed using several specific polyclonal and monoclonal antibodies directed against cytochrome P-450 isozymes.

3. RP 52028 appears to be an inducer of cytochrome P-450 II B1 (P-450b) and related enzymic activities; i.e. benzphetamine, ethylmorphine and aminopyrine demethylation.     

“Detoxication,a most Stimulating Concept of Toxicology”

1. Large, independent variations occur among New Zealand White rabbits in the 21-and 6β-hydroxylation of progesterone as catalysed by liver microsomes.

2. These reactions are catalysed respectively by two electrophoretically distinct types of rabbit-liver microsomal cytochrome P-450, 1 and 3b, as judged by their catalytic efficiency and the capacity of specific monoclonal antibodies to extensively inhibit the respective microsomal hydroxylases.

3. The relatively large variations in progesterone 6β-hydroxylase activity do not appear to be associated with differences in microsomal content of cytochrome P-450 3b, whereas differences in the microsomal concentration of cytochrome P-450 1 may underlie variations in 21-hydroxylase activity.

4. Preparations of cytochrome P-450 3b contain at least two catalytically distinct subforms, one of which catalyses both 6β- and 16α-hydroxylation of progesterone with a low K_m while the other subform catalyses predominantly 16α-hydroxylation with a significantly greater K_m.

5. The two catalytic subforms of cytochrome P-450 3b can be independently modulated in vitro by positive and negative effectors that can arise in vivo from the metabolism of progesterone.

6. The 6β-hydroxylase subform of cytochrome P-450 3b is not expressed in a genetically defined strain of rabbits, IIIVO/J, indicating a heritable basis for the differential expression of the two subforms of cytochrome P-450 3b.

7. These results indicate that the extent of cytochrome P-450 multiplicity may be greater than is evident from the isolation of electrophoretically distinct forms of cytochrome P-450, and that small differences in structure may underlie large differences in catalytic properties.

8. It is not known whether the differences among outbred New Zealand White rabbits in the expression of either cytochrome P-450 1 or the subforms of cytochrome P-450 3b reflect regulatory phenomena or genetic polymorphism.     

Effects of inducers and/or inhibitors on metabolism of lysergic acid diethylamide in rat liver microsomes

1. When lysergic acid diethylamide (LSD) was incubated with liver microsomes obtained from untreated rats, SKF 525-A inhibited most potently the hydroxylation at the 13-position, moderately inhibited N-demethylation at the 6-position, and least affected the metabolism of the side-chain at the 8-position. Furthermore, an atmosphere of 80% CO and 20%O₂ (v/v) caused max. inhibition in N-demethylation, moderate inhibition in 13-hydroxylation, and the minimum in metabolism of the side-chain at the 8-position. These data suggested that the metabolism of LSD is catalysed by three separate enzyme systems.

2. The formation of 13-hydroxy-lysergic acid diethylamide (13-hydroxy-LSD) in liver microsomes obtained from 3-methylcholanthrene-treated rats was not inhibited by CO, although the hydroxylation required NADPH and oxygen.

3. The results of experiments using various inhibitors suggest that the 13-hydroxylation in liver microsomes from 3-methylcholanthrene-treated rats is catalysed by an enzyme system involving an unusual type of cytochrome P-448.     

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.     

Cytochrome P-450 and oxidative metabolism in molluscs

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs.

2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc.

3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)).

4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, α-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra.

5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes.

6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH.

7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones.

8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.     

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24?h after the last treatment.

2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene.

3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes.

4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes.

5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30?min after treatment with the drug.     

Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B₂ PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B₂ BNF IG (against the β-naphtoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, /gb-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine, N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, β-naphtoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.     

Reduced nicotinamide adenine dinucleotide-dependent reduction of tertiary amine N-oxide by liver microsomal cytochrome P-450.

NADH-supported reduction of tertiary amine N-oxides in rat liver microsomes was investigated with imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide as substrate in the presence of NADH. The reductase activity is sensitive to carbon monoxide. NADH-cytochrome b₅ reductase or cytochrome b₅, solubilized by trypsin or subtilisin, showed no N-oxide reductase activity. The NADH-dependent reduction of tertiary amine N-oxides was markedly inhibited by antibody to NADH-eytochrome b₅ reductase and antibody to cytochrome b₅. These results confirmed that NADH-dependent N-oxide reduction was catalyzed by cytochrome P-450 and the reducing equivalent for the N-oxide reduction was transferred from NADH to cytochrome P-450 mainly via NADH-cyto-chrome b₅ reductase and cytochrome b₅ in the microsomal membranes. NADH-dependent N-oxide reductase is also sensitive to oxygen and 4 μM oxygen gave 50 per cent inhibition with imipramine N-oxide. Kinetic study shows that K_m values for the reduction of imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide were 0.05 mM, 0.14 mM and 0.16 mM, respectively. NADH-dependent N-oxides reductase activity is affected by azo and nitroso compounds and hydrazide, although the degree of inhibition was rather weak compared with those of NADPH-dependent activity. Furthermore, NADH-dependent reduction of tertiary amine N-oxide was only slightly affected by n-octylamine, 2,4-diehloro-6-phenylphenoxyethylamine and aniline. NADH-dependent N-oxide reductase activity in liver microsomes was less sensitive to phenobarbital or 3-methyleholanthrene pretreatment than NADPH-dependent activity. Some characteristics of NADH-dependent N-oxide reductase activity were discussed and compared with those of NADPH-dependent activity.     

An investigation of the antigenic determinants on chloroperoxidase and purified rat liver microsomal cytochrome P-450b

Chloroperoxidase (CPO) exhibits many physicochemical and catalytic properties similar to those of the bacterial and microsomal cytochromes P-450. Therefore, the possible similarities between the antigenic determinants of CPO and rat liver microsomal cytochrome P-450b were investigated. Polyclonal antibodies against CPO and rat liver cytochrome P-450b were raised in rabbits and used to investigate the antigenic cross-reactivity between CPO and P-450b. Although anti-CPO antibodies were capable of inhibiting the ethyl hydroperoxide-supported N,N-dimethylaniline (DMA) demethylation activity of CPO by more than 80%, they were unable to inhibit the NADPH-supported demethylation of DMA by cytochrome P-450b in the reconstituted system. The ethyl hydroperoxide-supported demethylation of DMA by CPO was not affected by the addition of anti-P-450b antibodies which inhibited cytochrome P-450 activity greater than 90%. In order to probe for the possible existence of common antigenic determinants which were not involved in catalytic activity, the cross-reactivities were investigated using enzyme-linked immunosorbent assays. There was no cross-reactivity between anti-CPO and cytochrome P-450b, or anti-P-450b and CPO using enzyme-linked immunosorbent assays. When control, phenobarbital-, isosafrole-, and beta-naphthoflavone-induced rat and rabbit liver microsomes and CPO were analyzed by Western blotting and developed with anti-P-450 antibodies, only the phenobarbital- and isosafrole-induced microsomes showed a positive reaction in the P-450 region. When anti-CPO antibodies were used on Western blots of the same series of proteins, a positive reaction was observed only with CPO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective Destruction of Cytochrome P-450d and Associated Monooxygenase Activity by Carbon Tetrachloride in the Rat

1. The effects of acute treatment of 3-methylcholanthrene (MC)-induced rats with carbon tetrachloride (CCI₄) on the content and activity of the polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 (P-450c and P-450d) in liver and kidney have been determined.

2. Post-treatment of MC-induced rats with CCl₄ in vivo decreased the specific content of total, spectrally determined, P-450 in both hepatic and renal microsomes, by 60% and 40%, respectively. CCl₄ treatment destroyed almost all of the hepatic P-450d (specific content after 6h, <2% of control), but had no effect on P-450c, which increased slightly over the 6h, to 30% above control values.

3. Immunocytochemical measurements demonstrated greater loss of P-450d from the centrilobular and midzonal than from periportal regions of the liver.

4. Hepatic phenacetin O-deethylase, an activity catalysed specifically by P-450d in this tissue, was dramatically decreased following administration of CCl₄ to MC-induced rats. Loss of monooxygenase activity was highly correlated with the decrease in P-450d content (r=0.947, P<0.001). Aryl hydrocarbon hydroxylase activity of liver, catalysed almost entirely by P-450c, was unchanged and neither activity was affected in kidney.

5. Treatment of MC-induced rats with CCl₄ causes a selective loss of hepatic P-450d and associated monooxygenase activities. Phenacetin O-deethylation is catalysed specifically by P-450d in liver, but not in kidney. The mechanism for this destruction of P-450d may be suicide activation of CCl₄, but the rate of such activation appears to be much lower than with P-450b. Alternatively, P-450d may be particularly sensitive, and P-450c particularly resistant, to the active metabolite of CCl₄ diffusing from a distant site of formation.     

The interactions of hydrazine derivatives with rat-hepatic cytochrome P-450

1. The ability of different classes of hydrazine derivatives to modify cytochrome P-450 function during turnover as judged by loss of absorbance at 416 nm, loss of CO-reactive cytochrome P-450, or destruction of haem has been studied.

2. Addition of monosubstituted hydrazines to rat-liver microsomes caused considerable loss of CO-reactive cytochrome P-450 and haem destruction; monosubstituted hydrazides caused mainly loss of CO-reactive cytochrome P-450, most likely due to abortive complex formation. Metabolism of 1,1-disubstituted hydrazines by microsomal cytochrome P-450 resulted in loss of CO-reactive cytochrome P-450 only, with no haem destruction. The 1,2-disubstituted hydrazines and hydrazides, procarbazine and iproniazid, acted similarly to the monosubstituted hydrazines, while 1,2-dimethyl-hydrazine elicited no response, cither in observable spectral changes or loss of CO-reactive cytochrome P-450.

3. Synthetic diazene intermediates of phenylhydrazine and N-aminopiperidine reacted rapidly with microsomal cytochrome P-450 to form a spectral intermediate resembling the putative iron porphyrin-diazenyl complex. The decomposition of certain iron porphyrin-diazenyl derivatives apparently leads to destruction of the porphyrin prosthetic group, most likely due to haem alkylation.     

Inhibition of antipyrine metabolite formation in rats in vivo

1. The effect of four inhibitors of cytochrome P-450-mediated drug oxidations (SKF 52SA, cimetidine, metyrapone and α-naphthoflavone) on the urinary metabolite pattern and ¹⁴CO₂ exhalation rate (CER)-time profile following [N-methyl-¹⁴C]antipyrine administration has been investigated.

2. The CER-time profiles indicated that inhibition of antipyrine metabolism was in the rank order SKF 525A >cimetidine > metyrapone >ANF.

3. The urinary metabolite patterns showed selectivity in action towards particular pathways, 3-hydroxylation being primarily decreased by SKF 525A and cimetidine, and N-demethylation by ANF. The results provide further evidence for involvement of multiple forms of cytochrome P-450 in antipyrine metabolism.     

Spectral interaction of orphenadrine and its metabolites with oxidized and reduced hepatic microsomal cytochrome P-450 in the rat

Product inhibition has been suggested to be a determinant in orphenadrine pharmacokinetics. Two possibilities for the mechanism of product inhibition in orphenadrine metabolism are explored in this study. Orphenadrine and its metabolites may compete for cytochrome P-450 catalytic binding sites. Therefore the interaction of orphenadrine and some of its metabolites with hepatic microsomal ferricytochrome P-450 of the rat was investigated. The spectral dissociation constant for the type I (substrate) interaction of orphenadrine and its metabolites displayed no relationship with the lipophilicity of the compounds. Orphenadrine is only partially displaced from its cytochrome P-450 binding sites by its respective metabolites. For this mechanism to be significant in vivo. the metabolites need to reach concentrations near cytochrome P-450 similar to that of orphenadrine. This is not known yet. The significance of this mechanism for the product inhibition phenomenon is therefore uncertain. In this study it is also established that during both in vitro as well as in vivo metabolism of orphenadrine, a metabolic intermediate is formed, which binds irreversibly to ferrous-cytochrome P-450 (MI complex). In vitro, both the rate and extent of the MI complex formation with orphenadrine and metabolites as precursor, decreased in the order N-hydroxytofenacine >; tofenacine > orphenadrine > bisnororphenadrine. The metabolite orphenadrine-N-oxide did not produce an MI complex, in vitro. Furthermore. in vitro, it was shown that the N-demethylation of tofenacine paralleled the concomitant MI complex formation. Together, the data suggest that the first N-demethylation step of orphenadrine occurs via α-carbon oxidation, whereas the second N-demethylation step mainly comes about via N-oxidation. Both metabolic pathways eventually lead to the MI complex forming species. These two parallel pathways also account for the complicated substrate dependency and concentration dependency in MI complex formation. Finally, the formation of the nitroxide radical (the ultimate ligand for MI complexation) has been shown to be susceptible to inhibition by its precursors.The occurrence of MI complex formation resulting in metabolic inactive cytochrome P-450 is probably the main mechanism for the product inhibition phenomenon in orphenadrine metabolism.     

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from β-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation.

2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex.

3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c.

4. Both ethoxyresorufin and benzo[α]pyrene inhibited the development of the type 1 optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[α]pyrene was noncompetitive in nature.

5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type 1 optical difference spectrum non-competitively.

6. The results indicate that ethoxyresorufin and benzo[α]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.     

Human cytochromes P-450

1. Studies in vivo have provided evidence for a multiplicity of cytochromes P-450 in man, some of which are under independent monogenic control.

2. Although the activity of cytochromes P-450 in man are generally lower than those of rat, this is by no means always the case. There are several important exceptions including the N-hydroxylation of 2-acetamidofluorene.

3. Studies in vitro by a number of different techniques have confirmed the evidence from studies in vivo that there are multiple forms of human cytochrome P-450. In addition to differences in V_{m ax}' the different forms of cytochrome P-450 may also exhibit marked differences in their apparent K_m values. The implications that this may have for pharmacokinetics and toxicology are discussed.

4. The polymorphism in the 4-hydroxylation of debrisoquine observed in vivo has been shown to be due to a defect in a specific form of cytochrome P-450 which appears to be under monogenic regulation. Cross-inhibition studies have enabled the specificity of this isozyme to be characterized. Such studies have also enabled the contribution of this isozyme of cytochrome P-450 to the oxidation of other substrates to be determined. Compounds investigated include bufuralol and phenytoin.

5. Evidence from studies both in vivo and in vitro suggest that selective induction of different forms of cytochrome P-450 can occur in man. However, the number of different classes of inducer in man is not yet known.

6. Human cytochromes P-450 have been purified to near homogeneity in several laboratories. Different forms of cytochrome P-450 purified from the same liver sample vary in molecular weight, chromatographic characteristics and substrate specificities.