Values For Microsomes Research Articles

In Vitro–In Vivo Inaccuracy: The CYP3A4 Anomaly

When predicting hepatic clearance using in vitro to in vivo extrapolation (IVIVE), microsomes or hepatocytes are commonly used. Here, we examine intrinsic clearance values and IVIVE results in human hepatocytes and microsomes for compounds metabolized by a variety of enzymes. The great majority of CYP3A4 substrates examined had higher intrinsic clearance values in microsomes compared with hepatocytes, whereas the values were more similar between the two incubations for substrates of other enzymes. We hypothesize that this may be due to interplay between CYP3A4 and the efflux transporter P-glycoprotein, as they have been shown to exhibit coordinated regulation. When examining the prediction accuracy for substrates of other enzymes between microsomes and hepatocytes, average fold errors as well as overall error were similar, demonstrating once again that IVIVE methods are not adequately defined and understood. SIGNIFICANCE STATEMENT: For CYP3A4 substrates, microsomes give markedly higher predictive in vitro to in vivo extrapolation than for other metabolic enzymes, which is not found for hepatocytes. We hypothesize that this could be a result of CYP3A4-P-glycoprotein interplay or coordinated regulation in hepatocytes that would not be observed in microsomes.

A Primer of Deuterium in Drug Design

A Primer of Deuterium in Drug Design

Orally available pyridinylpyrimidine derivatives as novel RANKL-induced osteoclastogenesis inhibitors

An HTS campaign led to the identification of 4-pyrroldino-2-(pyridin-2-yl)pyrimidine compound 1 as an RANKL-induced osteoclastogenesis inhibitor. The compound 1 showed high clearance values in microsomes, however. Modification of the pyrrolidino group to a benzylamino group improved human microsomal stability with a slight loss of in vitro activity. Substitution at the ortho position of the benzyl group ameliorated in vitro activity, and further fluorination of the benzyl group improved microsomal stability in rodents. Representative members of this series, compounds 20 and 23, exhibited efficacy in RANKL-induced osteopenic mice when administered orally at 0.3mg/kg.

Chlorzoxazone: a probe drug whose metabolism can be used to monitor toluene exposure in rats.

In this study we investigated cytochrome P450 (CYP) 2E1 expression using a probe drug, chlorzoxazone (CZX), whose metabolism can be used to monitor toluene exposure in rats. The animals received an i.p. injection of toluene (0.25, 0.5 and 1 ml/kg) once a day for 3 days. The total CYP and CYP2E1 content and the aniline and CZX hydroxylase activity (Vmax and CL(int)) increased depending on the dose of toluene administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from toluene-treated rats was reduced by about 80%. The IC50 values in microsomes from toluene-treated rats were between 3 and 5 microM. The production of HCZX and the activity of aniline hydroxylase in toluene-treated rats were correlated with the amount of rat CYP2E1 protein (r = 0.88 and r = 0.88, respectively). The elimination of CZX by toluene-treated rats was increased and the HCXZ production in the toluene-treated group was greater than that in the olive oil control group. The correlations between intrinsic clearance (CL(int): Vmax/Km) in vitro and total body clearance (CL(tot)) of CZX hydroxylation and the elimination half-life (t1/2) of CZX in vivo in toluene-treated rats were high (r = 0.784, P < 0.001; r = -0.678, P < 0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and 2 h after CZX administration in vivo there was also a high correlation with CL(int) (Vmax/Km) in vitro (r = -0.729, P < 0.001). In conclusion, these results demonstrate that CZX is a very good probe for monitoring induction in toluene-treated rats.

In-vitro metabolism of retinoic acid by different tissues from male rats.

Significant differences between the metabolism of retinoic acid by different tissues might be an important determinant of the effectiveness of a systemically administered inhibitor at a particular tissue site. Here the metabolism of retinoic acid has been studied in microsomal fractions from different tissues (liver, kidney, intestinal mucosa, lung, skin, brain) of the male rat to determine their relative metabolic activity. Kinetic analysis revealed major differences between the activity of different tissue microsomes. This is shown by the Vmax values for the metabolism of retinoic acid-liver (102+/-39.0 pmol (mg protein)(-1) min(-1)) was 100 times more active than the lung (1+/-0.03 pmol (mg protein)(-1) min(-1)), which was the least active. The range of Km values for microsomes from the different tissues was narrow (0.48-1.40 microM). Taking into account the mass of the tissue, the gross activity ranking for metabolism of retinoic acid was liver >> skin = kidney > brain > intestinal mucosa >> lung. It is concluded that metabolism of administered retinoic acid occurs mainly in the liver but that cellular retinoic acid levels in some other tissues (skin, kidney, brain) could be reduced (metabolized) to such an extent that higher levels might be observed after the use of inhibitors of retinoic acid metabolism.

The regulation of CoA-independent transacylation reactions in neuronal nuclei by lysophospholipid, free fatty acid, and lysophospholipase: the control of nuclear lyso platelet-activating factor metabolism.

CoA-independent transacylase activities generating alkylacylglycerophosphocholine (AAGPC) from alkylglycerophosphocholine (1-alkyl GPC) were considerably enriched in neuronal nuclei isolated from rabbit cerebral cortex. Specific nuclear transacylation activities were 13 times the corresponding microsomal values. Several lysophospholipids, notably 1-acyl glycerophosphocholine (1-acyl GPC), 1-alkenyl GPC and 1-alkenyl GPE (1-alkenyl glycerophosphoethanolamine) inhibited the transacylation of 1-alkyl GPC. The inhibitory effects of 1-acyl GPC were seen in the presence of MAFP (methyl arachidonoylfluorophosphonate) or free oleate, compounds that inhibit neuronal nuclear lysophospholipase. When neuronal nuclei were preincubated with 1-alkyl GPC, the radioactive AAGPC product served as donor in transacylation reactions, to generate 1-alkyl GPC. In these nuclear reactions, 1-palmitoyl GPE and 1-palmitoyl GPC appeared to be poor acceptor substrates, when compared with corresponding 1-alkyl and 1-alkenyl analogues. The presence of free oleate or MAFP in the reactions containing 1-acyl GPC boosted the release of 1-alkyl GPC from AAGPC. These observations are of particular relevance to brain ischemia in which lysophospholipid, free fatty acid, and platelet-activating factor (PAF) levels rise dramatically. PAF can be made by the nuclear acetylation of 1-alkyl GPC, which is formed by nuclear transacylation mechanisms. Yet transacylase also removes 1-alkyl GPC, and thus this enzyme activity can regulate 1-alkyl GPC availability. Our observations indicate that lysophospholipids promote the formation of 1-alkyl GPC from nuclear AAGPC via transacylation, while free fatty acid likely prolongs the lifetime of 1-acyl lysophospholipids substrates by lysophospholipase inhibition. Similarly, once 1-alkyl GPC is formed, other lysophospholipids effectively compete with this 1-alkyl analogue and reduce its conversion back to AAGPC by transacylation. Free oleate, in this case, sustains 1-acyl lysophospholipid inhibitors of 1-alkyl GPC transacylation. Thus the cycle of transacylation may favour 1-alkyl GPC formation during ischemia, increasing levels of 1-alkyl GPC for nuclear acetylation reactions and PAF formation. The nuclear generation of PAF is of considerable importance as PAF can play regulatory roles in transcription events associated with inflammation.

Identification of Cytochrome P450 3A1/2 as the Major P450 Isoform Responsible for the Metabolism of Fentanyl by Rat Liver Microsomes

The metabolism of fentanyl was investigated using rat liver microsomes to determine whether fentanyl is metabolized by rat liver microsomal cytochrome P450 and, if so, which isoform is responsible for the metabolism. Microsomes isolated from rats pretreated with phenobarbital were more active in metabolizing fentanyl than were microsomes from saline controls. The major metabolic pathway of fentanyl was an oxidative N-dealkylation to norfentanyl, which was detected by a gas chromatograph-mass selective detector (GC-MSD) method. The apparent Vm values for microsomes isolated from saline- and phenobarbital-treated rats were 2 and 9 nmol norfentanyl.min-1.mg-1 microsomal protein, and the apparent Km values were 32 and 47 microM, respectively. Fentanyl metabolism was inhibited by antibodies specific for CYP3A1/2, as well as by chemical inhibitors specific for CYP3A. These results indicate that CYP3A1/2 plays a major role in the oxidation of fentanyl to norfentanyl by rat liver microsomes.

Comparison of one-electron reduction activity against the bipyridylium herbicides, paraquat and diquat, in microsomal and mitochondrial fractions of liver, lung and kidney ( in vitro)

The first one-electron reduction steps of paraquat and diquat were compared using microsomal and mitochondrial fractions of rat liver, lung and kidney. Both fractions reduced each herbicide effectively, with the order of the V max values in microsomes and mitochondria being liver > lung > kidney and kidney > liver > lung, respectively. Although similar V max values were obtained from the liver and lung with the two subcellular fractions, the affinity of mitochondrial enzymes was lower, suggesting that the reduction of both herbicides in a microsomal site would be dominant in these two organs. The V max values for radical formation of paraquat were higher than those of diquat in all the endogenous one-electron reducing systems. The apparent K m values for diquat, however, were lower than those for paraquat in both subcellular fractions from the ttree tissues, indicating the superiority of the reduction for diquat to that for paraquat at low concentrations. This difference in the K m values supported the finding that the reduction velocity for diquat was significantly higher than that for paraquat at 1 mM concentration. Thus, at low concentrations, diquat would be reduced more easily than paraquat. In addition, tissue enzymatic specificity for paraquat was not obtained. From these data, it seems reasonable to conclude that the tissue-selective accumulation of paraquat previossly proposed determines its toxicity.

Enzymic characteristics of benzylamine oxidase and monoamine oxidase in rat lung

Many studies on various tissues indicate the presence of benzylamine oxidase (BZO), and there are two forms of BZO in rat heart and aorta. Here, we compared enzymic properties of rat lung BZO and MAO and studied the possible existence of two forms of BZO. Subcellular distribution studies revealed that MAO activity was mainly localized in mitochondria, but for BZO, high recoveries of activity were observed in both mitochondria and microsomes. Mitochondrial MAO activity towards tyramine was more highly sensitive to deprenyl than to clorgyline, with a single-sigmoidal curve, indicating MAO-B predominance in the lung. In kinetic analyses, Km values of BZO and MAO for benzylamine in the two fractions were markedly different (about 3-6μM for BZO and 80uM for MAO, respectively). Vmax values of MAO in mitochondria were remarkably higher compared to those in microsomes, but in contrast, BZO values in microsomes were significantly higher. Substrate specificities of these two amine oxidases were extremely different, but those of each enzyme in the two fractions were similar. MAO activities towards various substrates under 20 and 5% of O2 concentrations similarly decreased in proportion to O2 concentration, but those for BZO increased to a similar extent, except towards benzylamine. These results indicate cross-contamination of the two fractions and that BZO might play a role in deaminations of exogenous amines when MAO activity is lowered.

Comparison of nuclear and microsomal epoxide hydrase from rat liver

The specific activities of hydration of nine arene and alkene oxides by purified nuclei prepared from the livers of 3-methylcholanthrene-pretreated rats were found to fall within the range of 2.2 to 9.1% of the corresponding microsomal values. Pretreatment with phenobarbital enhanced both the nuclear and microsomal hydration of phenanthrene-9,10-oxide, benzo( a)pyrene-11,12-oxide, and octene-1,2-oxide. 3-Methylcholanthrene pretreatment enhanced the nuclear hydration of these three substrates by 30–60% but had no significant effect on microsomal hydration. An epoxide hydrase modifier, metyrapone, stimulated the hydration of octene-1,2-oxide by the two organelles to quantitatively similar extents, but affected the nuclear and microsomal hydration of benzo( a)pyrene-4,5-oxide differentially. Cyclohexene oxide also exerted differential effects on nuclear and microsomal epoxide hydrase which were dependent both on the substrate and on the organelle. The inhibition by this agent of nuclear and microsomal epoxide hydrase was quantitatively similar only for a single substrate, benzo( a)anthracene-5,6-oxide. When purified by immunoaffinity chromatography, nuclear and microsomal epoxide hydrases from 3-methylcholanthrene-pretreated rats were shown to have identical minimum molecular weights (≅ 49,000) on polyacrylamide gels in the presence of sodium dodecyl sulfate. These findings support the assertion that microsomal metabolism can no longer be considered an exclusive index of the cellular activation of polycyclic aromatic hydrocarbons.

Environmental Effects on Glutathione-Insulin Transhydrogenase in Rat Liver

The dietary and hormonal regulation of the level of glutathione-insulin tranhydrogenase in rat liver was investigated in these studies. In order to make valid comparisons, the assay of glutathione-insulin transhydrogenase was performed at near zero-order kinetics wherein enzyme rate was proportional to enzyme amount. Changing the protein content of the diet or administration of glucagon or cortisone did not significantly affect the specific activity of glutathione-insulin transhydrogenase in microsomes from rat liver. However, the Vmax and Km of this enzyme in the livers of adrenalectomized rats were increased three-and fourfold over these values in microsomes from normal liver. Administration of cortisone resulted in a return to the normal kinetic constants of microsomal GIT within 4 hr.

Stimulation of drug metabolism by ascorbic acid in weanling guinea-pigs

Drug metabolism activities such as O-demethylase and N-demethylase and liver microsomal electron transport components including cytochrome P-450 and NADPH-cytochrome P-450 reductase were determined in weanling guinea-pigs under vitamin C deficiency as well as with normal and high intake of the vitamin. Animals depleted of the vitamin for only 8 days, with no signs of scurvy or weight loss, had decreased drug enzyme activities. Further depletion of the vitamin resulted in marked reduction of the microsomal drug-metabolizing system. Furthermore, administration of high amounts of ascorbic acid (2–200 mg/day) led to significant increases in liver drug oxidation activities and electron transport components. Studies of K m values in microsomes from vitamin C-deficient, normal and animals given high supplements of vitamin C showed no difference in the apparent affinity or V max of a typical drug oxidation reaction, N-demethylation. Specificity studies indicate that ascorbyl palmitate or d-isoascorbic acid can replace the vitamin, in part, in maintaining drug metabolism.

The Purification and Biochemical Characterization of Bovine Liver Nuclear Membranes

Abstract Bovine liver nuclear membranes were purified by means of a high salt (0.5 m MgCl2) discontinuous sucrose gradient. The purified nuclear membranes were isolated at a density of ρ = 1.05 to 1.23 and contained 62% of the protein recovered from the gradient. The presence of nuclear pore complexes identified the fraction as nuclear membrane. Twenty-six per cent of the protein was recovered at a density of ρ = 1.23 to 1.30. Electron microscopy demonstrated both nuclear membranes and nonmembranous nucleolar components with a predominance of the latter. A fraction sedimenting through the most dense high salt sucrose solution (ρ ≥ 1.30) made up 12% of the total recovered protein. Ultrastructural analysis revealed nucleolar constituents in both a granular-fibrillar network and in the more condensed form characteristic of nucleoli in intact nuclei. In the absence of high salt, the nuclear membranes and nucleolar material aggregated and sedimented at a density intermediate between the lighter membranes and the heavier nonmembranous components. Purified nuclear membranes consisted of 70.4% protein, 5.8% RNA, 1.1% DNA, and 22.6% phospholipid. The comparatively nonmembranous nature of the heavier fractions was indicated by the much lower phospholipid contents of 9.7% and 9.8%, respectively. Since nucleoli have a characteristically high RNA content, the much higher RNA levels of the heavier fractions (8.0% and 11.4%), as compared to the lighter nuclear membranes, were consistent with the ultrastructural demonstration of nucleolar components in these fractions. Eighty to 90% of the endoplasmic reticulum-type enzymes NADH-cytochrome c reductase, glucose 6-phosphatase, and Mg++-stimulated ATPase, and the characteristic inner mitochondrial membrane activity, cytochrome c oxidase, were not due to microsomal and mitochondrial contamination. The low succinate dehydrogenase activity in purified nuclear membranes is correlated with the absence of acid nonextractable, trypsin-releasable flavin, which is associated with mitochondrial succinate dehydrogenase flavoprotein. In addition, coenzyme Q, a characteristic component of inner mitochondrial membranes, was not detected. The above enzyme activities were increased 1.2- to 1.9-fold in purified nuclear membranes compared to the unpurified membranes. In contrast, the high salt sucrose gradient fractions of higher density demonstrated progressive decreases in specific activities from 0.32 to 0.61 in the intermediate density fraction to 0.15 to 0.41 in the heaviest fraction. This was paralleled by the progressive decreases in nuclear membranes in these two fractions. Moreover, 84 to 90% of the total enzyme activities were found in the purified nuclear membranes, whereas only 7 to 15% and 1 to 4% were present in the intermediate and heaviest fractions, respectively. The membranes contained 0.322 nmole of cytochrome b5 per mg of protein and 0.045 nmole of cytochrome a, a3 per mg of protein, which were 33% and 30% of values in microsomes and mitochondria. The cytochrome P-450 content of nuclear membranes was only 4.4% of microsomes and can be attributed to microsomal contamination. Direct enzymatic comparison of nuclear and microsomal membranes demonstrated a strong endoplasmic reticulumtype character in nuclear membranes. Nuclear membranes differed from endoplasmic reticulum, however, by the presence of cytochrome c oxidase activity and cytochrome a, a3 and the only trace amounts of NADPH-cytochrome c reductase and cytochrome P-450.

Benzpyrene pretreatment changes the kinetics and pH optimum for aniline hydroxylation in vitro, but not those for benzphetamine demethylation in vitro by rat liver microsomes

The effects of benzpyrene pretreatment on aniline para-hydroxylation, benzphetamine demethylation and benzpyrene hydroxylation in rat liver microsomes were studied and the following results were obtained: The pH optimum for the metabolism in vitro of aniline is shifted from pH 7.0 to pH 8.1 by benzpyrene pretreatment. When the spectral dissociation constants ( K s ) with aniline are compared at pH 7.0 and pH 8.1 using microsomes from benzpyrene-pretreated rats, a pH dependency of K s , is seen. The K s , values for microsomes from control rats are the same at both pH's, but they are different from both the K s , value at pH 7.0 for microsomes from benzpyrene-pretreated rats and the K s , value at pH 8.1 for microsomes from benzpyrene-pretreated rats. K m values for aniline hydroxylase are altered by changes in pH with hepatic microsomes from either control or benzpyrene-pretreated rats. Neither the K m nor the pH optimum for benzphetamine demethylase is changed by benzpyrene pretreatment. The K s for benzphetamine is also unchanged by pretreatment with benzpyrene. The K m value for benzpyrene hydroxylase is changed by pretreatment with benzpyrene, but the pH optimum for the metabolism in vitro of benzpyrene does not seem to be altered by benzpyrene pretreatment.