Microsomal Oxidation Research Articles

Isotopic sensitivity in the microsomal oxidation of the dihydropyridine calcium entry blocker nifedipine

The primary deuterium isotope effect on Vm for the microsomal oxidation of the dihydropyridine calcium entry blocker nifedipine [4-(2-nitrophenyl)-2,6-dimethyl-3,5- bis(methoxycarbonyl)-1,4-dihydropyridine] has been measured. The magnitude of the kinetic isotope effect, 6.7, suggests that the rate-limiting step in the mechanism of microsomal oxidation of nifedipine involves the loss of a hydrogen atom rather than nitrogen oxidation. Thus the microsomal oxidation of nifedipine is mechanistically different from that of other 1,4-dihydropyridines.

Inhibitors of the microsomal oxidation of psychotropic drugs: selectivity and clinical significance.

The use of human liver microsomal preparations as a tool in drug metabolism research has potentials in a number of areas, not least in pharmacogenetics.The testing of different compounds for their ability competitively to inhibit the oxidation of model compounds such as sparteine/debrisoquine or mephenytoin is a particularly useful primary screening procedure (Otton et al. 1983,1984; Boobis et al. 1983; von Bahr et al. 1985; Inaba et al. 1984,1985; see Table 1). Substances not acting as inhibitors of the sparteine/debrisoquine oxygenase can safely be excluded as substrates of this P450 isozyme and will, accordingly, not exhibit the corresponding monogenetic polymorphism in metabolism in vivo. There appear to be no false negatives contradicting this assumption. However, if a compound is extensively bound to tissue components in the in vitro preparation, apparent high K i values may lead to the erroneous conclusion that the compound is a weak in vitro inhibitor. Among the substances that act as competitive inhibitors of this oxygenase (Table 1), several different tricyclic antidepressants and β-blockers are notable substrates of this oxygenase and show in vivo the corresponding oxidation 1982).Several neuroleptics, both phenothiazines, thioxanthenes, and butyrophenones appear to be potent inhibitors of the metabolism of typical tricyclic antidepressants, such as imipramine, desipramine, amitriptyline and nortriptyline. These neuroleptics include chlorpromazine, perphenazine, thioridazine, levomepromazine, fluphenazine, chlorprothixene and haloperidol. Studies on the effect of perphenazine on formation and excretion of metabolites of imipramine and desipramine suggested that the major effect was an inhibition of the 2-hydroxylation of imipramine and, in particular, desipramine (Fig. 2; Gram 1975). This has been confirmed in studies on imipramine metabolite levels in plasma (Brosen et al. 1986)

Application of chemical P450 model systems to studies on drug metabolism. I. Phencyclidine: a multi-functional model substrate.

Cytochrome P450 model and liver microsomal oxidations of drugs were compared using phencyclidine. In general, the chemical reaction systems produced many oxidation products. Besides the formation of the cyclohexane-4-hydroxyl compound (2), hydroxylation of the aromatic ring was favored in the Fenton reaction system (Fe2+ + H2O2). Formation of an m-hydroxylated product (m-5) was the main aromatic oxidation pathway in the Udenfriend reaction (Fe2+-ascorbic acid-O2), and 2, the piperidine-3-hydroxyl compound (3), and the piperidine-4-hydroxyl compound (4) were also formed. In the system using meso-tetraphenylporphinatoiron chloride (Fe(III)TPPC1) with an oxidant, the main product was the piperidine-3-oxo compound (8). In the liver microsomes system, 2, 4, 8, and m-5, which were all generated by the chemical oxidation reactions, were detected as metabolites of phencyclidine. They were formed by cytochrome P450-dependent reactions. Chemical oxidation systems can be used to study drug metabolism; they can reveal some tendencies of the real metabolic reactions, are easy to operate, and yield sufficient amounts of product.

Teflubenzuron Resistance in Diamondback Moth (Lepidoptera: Plutellidae)

Laboratory selection of susceptible and field strains of diamondback moth, Plutella xylostella (L.), larvae for 20 generations or more resulted in only 8-12-fold resistance to teflubenzuron, a benzoylphenylurea (BPU) that interferes with chitin synthesis. Cross-resistance with another BPU, chlorfluazuron, or a number of conventional insecticides was not apparent. Selection of larvae with teflubenzuron caused the diamondback moth to develop considerable resistance to the ovicidal effect of this compound. Piperonyl butoxide, an inhibitor of microsomal oxidases, restored the effectiveness of teflubenzuron against larvae and eggs of the selected strains, indicating that microsomal oxidation was the major resistance mechanism. Larvae selected with teflubenzuron exhibited much higher aldrin epoxidase and aryl hydrocarbon hydroxylase activities than those of the susceptible and field strains. Lack of cross-resistance between conventional insecticides and teflubenzuron-chlorfluazuron, and between teflubenzuron and chlorfluazuron suggested that different microsomal oxidases in diamondback moth might be involved.

Microsomal S-demethylase activity in four lepidopterous insects

Microsomal S-demethylation in fall armyworm ( Spodoptera frugiperda) larvae was examined using 6-methylthiopurine as a model substrate. The S-demethylase system required NADPH for activity and was inhibited by CO and by piperonyl butoxide. The allelochemical indole 3-carbinol significantly induced the enzyme activity. The system metabolized numerous insecticides including carbamates such as methomyl, oxamyl, thiodicarb, and methiocarb and organophosphates such as sulprofos and fenthion and an allelochemical, methanethiol. The enzyme was also active in velvetbean catepillars, cabbage loopers, and corn earworms. On the basis of its sensitivity to inducers, inhibitor, and species variation, it is concluded that although the S-demethylation is a typical microsomal oxidation reaction, this system is quite different from other microsomal reactions including epoxidation, hydroxylation, N-demethylation, O-demethylation, desulfuration, and sulfoxidation.

Hepatic and extrahepatic microsomal oxidation of phorate by the cytochrome P-450 and FAD-containing monooxygenase systems in the mouse

The relative contribution of the cytochrome P-450-dependent monooxygenase system ( P-450) and the FAD-containing monooxygenase (FMO) in the oxidation of phorate ( O,O-diethyl S-((ethylthio)methyl)phosphorodithioate) was investigated in liver, lung, and kidney microsomes isolated from male and female Dub:ICR mice. Selective inhibition was used to define the role of each enzyme system independent of the other system. Liver microsomes exhibited the greatest sulfoxidase activity, with P-450 responsible for greater than 75% of the activity. Although overall rates of sulfoxidation were much lower in lung and kidney microsomes, the FMO activity, relative to P-450, was greater. In all three organs, FMO activity appeared higher in microsomes isolated from female mice than microsomes from male mice. Phorate sulfoxide was the only metabolite detected during the linear range of the time course incubations. Extended time incubations yielded metabolites in addition to phorate sulfoxide in liver microsomes (phorate sulfone and phorate oxon sulfoxide) and lung microsomes (phorate oxon and phorate oxon sulfoxide), but not in kidney microsomes.

Effect of oxygen concentration on microsomal oxidation of ethanol and generation of oxygen radicals.

The iron-catalysed production of hydroxyl radicals, by rat liver microsomes (microsomal fractions), assessed by the oxidation of substrate scavengers and ethanol, displayed a biphasic response to the concentration of O2 (varied from 3 to 70%), reaching a maximal value with 20% O2. The decreased rates of hydroxyl-radical generation at lower O2 concentrations correlates with lower rates of production of H2O2, the precursor of hydroxyl radical, whereas the decreased rates at elevated O2 concentrations correlate with lower rates (relative to 20% O2) of activity of NADPH-cytochrome P-450 reductase, which reduces iron and is responsible for redox cycling of iron by the microsomes. The oxidation of aniline or aminopyrine and the cytochrome P-450/oxygen-radical-independent oxidation of ethanol also displayed a biphasic response to the concentration of O2, reaching a maximum at 20% O2, which correlates with the dithionite-reducible CO-binding spectra of cytochrome P-450. Microsomal lipid peroxidation increased as the concentration of O2 was raised from 3 to 7 to 20% O2, and then began to level off. This different pattern of malondialdehyde generation compared with hydroxyl-radical production probably reflects the lack of a role for hydroxyl radical in microsomal lipid peroxidation. These results point to the complex role for O2 in microsomal generation of oxygen radicals, which is due in part to the critical necessity for maintaining the redox state of autoxidizable components of the reaction system.

Redox cycling and sulphydryl arylation; Their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes

Quinones are believed to be toxic by a mechanism involving redox cycling and oxidative stress. In this study, we have used 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ), which redox cycles to the same degree as menadione, but does not react with free thiol groups, to distinguish between the importance of redox cycling and arylation of free thiol groups in the causation of toxicity to isolated hepatocytes. Menadione was significantly more toxic to isolated hepatocytes than 2,3-diOMe-1,4-NQ. Both menadione and 2,3-diOMe-1,4-NQ caused an extensive GSH depletion accompanied by GSSG formation, preceeding loss of viability. Both compounds stimulated a similar increase in oxygen uptake in isolated hepatocytes and NADPH oxidation in microsomes suggesting they both redox cycle to similar extents. Further evidence for the redox cycling in intact hepatocytes was the detection of the semiquinone anion radicals with electron spin resonance spectroscopy. In addition we have, using the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide), demonstrated for the first time the formation of superoxide anion radicals by intact hepatocytes. These radicals result from oxidation of the semiquinone by oxygen and further prove that both these quinones redox cycle in intact hepatocytes. We conclude that while oxidative processes may cause toxicity, the arylation of intracellular thiols or nucleophiles also contributes significantly to the cytotoxicity of compounds such as menadione.

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.

Cytochrome P-450-mediated oxidation of substrates by electron-transfer; Role of oxygen radicals and of 1- and 2-electron oxidation of paracetamol

The mechanism by which the hepatic cytochrome P-450 (Cyt. P-450) containing mixed-function oxidase system oxidizes the analgesic drug paracetamol (PAR) to a hepatotoxic metabolite was studied. Since previous studies excluded the possibility of oxygenation of PAR, three other mechanisms, namely direct 1-electron oxidation by a Cyt. P-450-ferrousdioxygen complex under concomitant formation of H 2O 2 to N-acetyl- p-semiquinone imine (NAPSQI), direct 2-electron oxidation by a Cyt. P-450-ferric-oxene complex to N-acetyl- p-benzoquinone imine (NAPQI) and indirect oxidation by active oxygen species released from Cyt. P-450, were considered. Indirect oxidation by active oxygen species was not involved, as active oxygen scavengers such as superoxide dismutase, catalase and DMSO did not affect the oxidation of PAR in hepatic microsomes. No reaction products characteristic for a direct 1-electron oxidation of PAR by Cyt. P-450 were observed: neither NAPSQI radical formation was detectable by ESR, nor PAR-dimer formation, nor stimulation of the microsomal H 2O 2 production was found to occur. In fact, PAR inhibited the spontaneous microsomal H 2O 2 formation. Studies on the reactions of NAPSQI with glutathione (GSH) revealed that NAPSQI hardly conjugated with GSH to a 3-glutathionyl-paracetamol conjugate (PAR-GSH) conjugate. The reactions of the elusive reactive metabolite formed during microsomal oxidation of PAR in the presence of GSH closely resembled those of synthetic NAPQI: both PAR-GSH and oxidized glutathione (GSSG) formation occurred. Furthermore, in agreement with a 2-electron oxidation hypothesis, iodosobenzene-dependent oxidation of PAR by cyt. P-450 in the presence of GSH resulted in the formation of the PAR-GSH conjugate. It is concluded that bioactivation of PAR by the Cyt. P-450 containing mixed-function oxidase system consists of a direct 2-electron oxidation to NAPQI.

An unstable epoxy intermediate formed by the microsomal oxidation of 4-nitrophenyl vinyl ether.

An unstable epoxy intermediate formed by the microsomal oxidation of 4-nitrophenyl vinyl ether.

Calcium alginate-Immobilized hepatic microsomes: Effect of NADPH cofactor on oxidation rates

An important function of the liver is detoxification of drugs, toxins and foreign compounds. Within the liver cell, the endoplasmic reticulum, isolated as the microsomal fraction, is especially active. Microsomal oxidation is the major oxidation pathway for many compounds, and the requirement for NADPH, an expensive cofactor, is an important consideration in bioreactor design. This paper presents design information for NADPH- and substrate-dependent oxidation rates for free and immobilized microsomes. The primary goal of this paper is determining NADPH requirements for oxidation. The effect of various initial levels of nicotinamide adenine dinucleotide phosphate (NADPH) on chlorpromazine oxidation rate has been studied for a crude hepatic microsomal fraction immobilized in calcium alginate gel. At an initial NADPH concentration of 600 nmoles/ml, immobilized microsomes accelerate to a maximal velocity of ≈ 240 nmoles min −1 ml −1 of oxygen consumption. In comparison, free microsomes reach a maximal velocity of approximately 150 nmoles min −1 ml −1 at an initial NADPH concentration of 220 nmoles/ml. By fitting the “initial” rate as a function of NADPH concentration to Michaelis-Menten kinetics, the apparent half-saturation coefficients ( K m) app are 3.5 nmole/ml for free microsomes and 134.4 nmole/ml for immobilized microsomes, however the maximum reaction velocity, V max, for immobilized microsomes is calculated to be 322 nmoles min −1 ml −1 compared with 145 nmols min −1 ml −1 for free microsomes. Preliminary studies indicate that is is possible to obtain significant reaction rates using calcium alginate immobilized microsomes and that this system may offer advantages due to its simplicity and lower cost.

Flavin catabolites: identification and quantitation in human urine

Riboflavin is the primary flavin excreted in human urine but significant amounts of 7 alpha-hydroxyriboflavin and lesser amounts of 8 alpha-hydroxyriboflavin are present and reflect tissue microsomal oxidations. A newly found flavin catabolite of an 8 alpha-sulfonyl type may reflect intake and/or turnover of such thioether-linked flavin as occurs in monoamine oxidase. Additionally, lesser amounts of 10-hydroxyethylflavin (indicative of intestinal microbial action on the vitamin) and traces of lumiflavin (arising from photodecomposition) constitute part of the remaining flavin, which acutely reflects level of intake.

Characterization of the cytochrome P-450 monooxygenase system of hamster liver microsomes: Effects of prior treatment with ethanol and other xenobiotics

The cytochrome P-450 monooxygenase system of hamster liver microsomes and its response to prior treatment with ethanol and other xenobiotics have been examined. Male Syrian golden hamsters were administered ethanol (ETOH), phenobarbital (PB), 5,6-benzoflavone (BF) or isoniazid (INH). Each treatment resulted in a moderate increase (20–60%) in the specific content of liver microsomal cytochrome P-450 along with a unique hemeprotein ferrous carbonyl Soret maximum. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of liver microsomes revealed distinctive changes in protein banding patterns in the cytochrome P-450 (45–60 kDa) region with each treatment. NADPH : cytochrome c reductase activity was increased by both PB and INH, whereas cytochrome b 5 content was increased by INH only. Microsomal oxidation of ETOH and aniline p-hydroxylation (expressed per nmol cytochrome P-450) were enhanced dramatically by ETOH and INH, whereas PB and BF had no effect on these enzymatic activities. Both ETOH and INH also increased zoxazolamine 6-hydroxylation but, in contrast to other rodent species, this drug-metabolizing activity was decreased in hamster liver microsomes after treatment with either PB or BF. Microsomal benzphetamine N-demethylation was decreased by ETOH, INH and BF administration and was only modestly enhanced after treatment with PB. ETOH and INH had no effect on the O-dethylation of 7-ethoxycoumarin, and enzymatic activity increased by BF but decreased by PB. These results demonstrate that the cytochrome P-450-dependent monooxygenase system of hamster liver microsomes responds to treatment with ETOH and other xenobiotics in a manner that is quantitatively and, in certain respects, qualitatively different from that reported for the rat, rabbit, and mouse.

Increased sensitivity of the microsomal oxidation of ethanol to inhibition by pyrazole and 4-methylpyrazole after chronic ethanol treatment

Pyrazole and 4-methylpyrazole, inhibitors of the oxidation of ethanol by alcohol dehydrogenase, also inhibit microsomal metabolism of ethanol. The inhibitory effectiveness of these agents was increased in microsomes isolated from rats treated chronically with ethanol as compared to microsomes from pair-fed controls or from rats treated with other cytochrome P-450 inducers such as phenobarbital or 3-methylcholanthrene. Pyrazole and 4-methylpyrazole produced type II binding spectra with all the microsomal preparations. However, there was an increased affinity (lower K s value) for these agents by the microsomes from the ethanol-fed rats. A correlation between K s values and inhibitory effectiveness against ethanol oxidation by the various microsomal preparations could be observed. This suggests that an increase in affinity, which may reflect the induction of an alcohol-preferring isozyme of cytochrome P-450, is responsible for the increased inhibitory effectiveness of pyrazole and 4-methylpyrazole towards ethanol oxidation by microsomes after chronic ethanol treatment. One difference between pyrazole and 4-methylpyrazole was the increased affinity and inhibitory effectiveness of the latter but not the former with microsomes from rats treated with 3-methylcholanthrene. This could be due to the ability of 4-methylpyrazole, compared to pyrazole, to interact with and induce several isozymes of cytochrome P-450. Pyrazole and 4-methylpyrazole are often utilized to evaluate ethanol metabolism by alcoholdehydrogenase-dependent and -independent pathways. However, the sensitivity of microsomal ethanol oxidation to inhibition by these agents, especially after chronic ethanol treatment, would suggest that their use in this regard is complex and could tend to underestimate the contribution of the microsomal pathway towards the metabolic tolerance found after ethanol treatment.

Age and the Metabolism of Drugs

Many factors contribute to the altered responsiveness of the elderly to drug therapy [1]. Of these, particular attention has been given to investigating the effect of age upon the rate of drug elimination since, unless appropriate reductions in dosage are made, significant age-related reductions in clearance will tend to lead to higher blood levels, and hence a greater risk of toxicity [2]. The effects of age on renal drug clearance are now well-documented [3-5], and parallel the decline in renal function which occurs in the elderly. Physicians are thus accustomed to matching the dosages of drugs which undergo renal excretion to elderly patients' renal function. In many instances, serum creatinine levels alone provide sufficient guide to appropriate doses although, for drugs with low therapeutic ratio, it may be necessary to measure plasma drug levels to avoid toxicity. Age-related changes in the rates of drug metabolism have also been reported for many common therapeutic agents. Unfortunately, no clear pattern has emerged, and those changes that do occur are superimposed on .the wide range of genetic and environmental factors known to influence rates of drug metabolism. Two general trends have emerged: first, and with some notable exceptions, drugs undergoing hepatic microsomal oxidation are likely to be metabolised more slowly in the elderly, whilst those that are conjugated are usually uninfluenced by age; second, drugs with high hepatic clearances and extraction ratios (such as chlormethiazole and labetalol), and which undergo extensive 'first-pass', metabolism during oral absorption, may show substantially increased bioavailability in the elderly [2].

Vanadate-stimulated NADH oxidation in microsomes.

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD+ and H2O2. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations. The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn2+, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (Viv) gives the second electron to superoxide to reduce it H2O2. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.

Quantitative evaluation of the metabolic interactions between trichloroethylene and 1,1-dichloroethylene in vivo using gas uptake methods

Gas uptake simulation methods were used to determine kinetic constants for trichloroethylene (TCE) and 1,1-dichloroethylene (1,1-DCE) metabolism in vivo in male Fischer 344 rats. Both are metabolized by single, saturable, oxidative pathways with high-affinity substrate binding. The allometrically scaled maximum velocities ( V maxc) for TCE and 1,1-DCE were, respectively, 11 and 7.5 mg/hr (i.e., 84 and 77 μmol/hr). Gas uptake studies were also conducted with three mixed atmosphere exposures with the following initial concentrations in parts per million: 500 (1,1-DCE):2000 (TCE); 500 (1,1-DCE):500 (TCE); and 2000 (1,1-DCE):500 (TCE). Mixture uptake curves were described by a system of equations in which a full physiologically based pharmacokinetic (PB-PK) model was provided for each chemical and each was regarded as an inhibitor of the other's metabolism. A generic model was developed to accommodate multiple mechanisms of inhibitory interactions, i.e., competitive, noncompetitive, or uncompetitive. An excellent correspondence was obtained between predicted and observed behavior when the inhibition was assumed to be purely competitive with binding constants for TCE and 1,1-DCE set to 0.25 and 0.10 mg/liter, respectively; i.e., in vivo 1,1-DCE is a slightly better substrate for microsomal oxidation than is TCE. The PB-PK model which was successful in describing the mixture data was used to predict conditions under which 1,1-DCE hepatotoxicity would be expected in coexposure to constant concentration atmospheres of these two chloroethylenes. These predictions were compared with data on the increases in plasma liver enzymes resulting from exposures to either 1,1-DCE alone or to 1,1-DCE in combination with TCE.

Bromazepam pharmacokinetics: influence of age, gender, oral contraceptives, cimetidine, and propranolol.

Pharmacokinetics of the benzodiazepine bromazepam were evaluated in volunteer subjects who received single 6 mg oral doses followed by blood sampling during the next 48 hours. Age and gender effects were studied in 32 subjects, divided into young (aged 21 to 29 years) and elderly (aged 60 to 81 years) groups. Compared with young subjects, the elderly had significantly higher peak serum bromazepam concentrations (132 vs. 82 ng/ml), smaller volume of distribution (0.88 vs. 1.44 L/kg), lower oral clearance (0.41 vs. 0.76 ml/min/kg), and increased serum free fraction (34.8% vs. 28.8% unbound). However, gender had no significant influence on bromazepam kinetics. In 11 young female users of oral contraceptive steroids, compared with seven age- and weight-matched control women not using oral contraceptives, no differences in bromazepam kinetics were observed. Coadministration of cimetidine (1.2 gm daily) significantly reduced bromazepam clearance (0.41 vs. 0.82 ml/min/kg) and prolonged elimination half-life (29 vs. 23 hours). Propranolol (160 mg daily) significantly prolonged bromazepam half-life (28 vs. 23 hours), but the reduction in clearance associated with propranolol (0.65 vs. 0.82 ml/min/kg) did not reach significance. Bromazepam has the pharmacokinetic characteristics of benzodiazepines with half-life values between 20 and 30 hours. Consistent with its biotransformation pathway by hepatic microsomal oxidation, bromazepam clearance is significantly impaired in elderly individuals, by coadministration of cimetidine and possibly propranolol.

The microsomal ethanol oxidizing system and its interaction with other drugs, carcinogens, and vitamins.

The interaction of ethanol with the oxidative drug-metabolizing enzymes present in liver microsomes results in a number of clinically significant side effects in the alcoholic. Following chronic ethanol consumption, the activity of the microsomal ethanol oxidizing system (MEOS) increases. This enhancement of MEOS activity is primarily due to the induction of a unique microsomal cytochrome P-450 isozyme, which has a high capacity for ethanol oxidation, as shown in reconstituted systems. Normally present in liver microsomes at low levels, this form of cytochrome P-450 increases dramatically after chronic ethanol intake in many species, including baboons. The in-vivo role of cytochrome P-450 in hepatic ethanol oxidation, especially following chronic ethanol consumption, has been conclusively demonstrated in deer-mice lacking liver ADH. Induction of microsomal cytochrome P-450 by ethanol is associated with the enhanced oxidation of other drugs as well, resulting in metabolic tolerance to these agents. There is also increased cytochrome P-450-dependent activation of known hepatotoxins such as carbon tetrachloride and acetaminophen, which may explain the greater susceptibility of alcoholics to the toxicity of industrial solvents and commonplace analgesics. In addition, the ethanol-inducible form of cytochrome P-450 has the highest capacity of all known P-450 isozymes for the activation of dimethylnitrosamine, a potent (and ubiquitous) carcinogen. Moreover, cytochrome P-450-catalyzed oxidation of retinol is accelerated in liver microsomes, which may contribute to the hepatic vitamin A depletion seen in alcoholics. In contrast to chronic ethanol consumption, acute ethanol intake inhibits the metabolism of other drugs via competition for shared microsomal oxidation pathways. Thus, the interplay between ethanol and liver microsomes has a profound impact on the way heavy drinkers respond to drugs, solvents, vitamins, and carcinogens.