Erythromycin N-demethylation Research Articles

Identification of CYP3A4 as the principal enzyme catalyzing mifepristone (RU 486) oxidation in human liver microsomes

Various complementary approaches were used to elucidate the major cytochrome P450 (CYP) enzyme responsible for mifepristone (RU 486) demethylation and hydroxylation in human liver microsomes: chemical and immunoinhibition of specific CYPs; correlation analyses between initial rates of mifepristone metabolism and relative immunodetectable CYP levels and rates of CYP marker substrate metabolism; and evaluation of metabolism by cDNA-expressed CYP3A4. Human liver microsomes catalyzed the demethylation of mifepristone with mean (± SD) apparent K m and V max values of 10.6 ± 3.8 μM and 4920 ± 1340 pmol/min/mg protein, respectively; the corresponding values for hydroxylation of the compound were 9.9 ± 3.5 μM and 610 ± 260 pmol/min/mg protein. Progesterone and midazolam (CYP3A4 substrates) inhibited metabolite formation by up to 77%. The CYP3A inhibitors gestodene, triacetyloleandomycin, and 17α-ethynylestradiol inhibited mifepristone demethylation and hydroxylation by 70–80%; antibodies to CYP3A4 inhibited these reactions by approximately 82 and 65%, respectively. In a bank of human liver microsomes from 14 donors, rates of mifepristone metabolism correlated significantly with relative immunodetectable CYP3A levels, rates of midazolam 1′- and 4-hydroxylation and rates of erythromycin N-demethylation, marker CYP3A catalytic activities (all r 2 ⩾ 0.85 and P < 0.001). No significant correlations were observed for analyses with relative immunoreactive levels or marker catalytic activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. Recombinant CYP3A4 catalyzed mifepristone demethylation and hydroxylation with apparent K m values 7.4 and 4.1 μM, respectively. Collectively, these data clearly support CYP3A4 as the enzyme primarily responsible for mifepristone demethylation and hydroxylation in human liver microsomes.

Effects of food deprivation and adrenalectomy on CYP3A induction by RU486 in female rats.

We have studied the effects of food deprivation and adrenalectomy on the induction by RU486 of female rat liver microsomal CYP3A apoprotein, erythromycin N-demethylase and diazepam C3-hydroxylase activities. RU486 was a potent inducer of CYP3A apoprotein in intact animals and food deprivation enhanced this response. Food deprivation alone caused only weak CYP3A apoprotein induction suggesting a synergistic interaction in the regulation of protein expression. These results were reflected in the measurements of diazepam C3-hydroxylase activity. This confirms diazepam C3-hydroxylase as a useful and easily measured index of CYP3A monooxygenase content in female rat liver microsomes. Erythromycin N-demethylase did not show concordance with this pattern; this monooxygenase was much more strongly induced by food deprivation alone than by RU486 administration and, in addition, adrenalectomy abolished the induction response to food deprivation. The lack of correspondence between the apoprotein and erythromycin N-demethylase results suggests that non-CYP3A or novel, hitherto uncharacterized CYP3A isoforms may contribute to erythromycin N-demethylation in female rats. The close agreement between the results for CYP3A apoprotein and diazepam C3-hydroxylase indicates that although RU486 possesses a terminal acetylenic moeity it does not, at the dosages used here, cause mechanism-based inactivation of the CYP3A monooxygenase protein it induces. Current studies are directed to characterizing the particular CYP3A isoform(s) whose production is stimulated by RU486.

Effects of roxithromycin, erythromycin and troleandomycin on their N-demethylation by rat and human cytochrome P450 enzymes.

1. The effects of treatment of rat with roxithromycin, erythromycin and troleandomycin as well as other chemicals including typical cytochrome P450 inducers were examined in rat and human liver microsomes. 2. Erythromycin and troleandomycin but not roxithromycin caused slight increases in CYP3A1 levels and the N-demethylation of roxithromycin, erythromycin and troleandomycin and oxidation of nifedipine in rat, but none of these chemicals induced significantly CYP2B1 levels or benzphetamine N-demethylation activities. 3. Dexamethasone and pregnenolone 16 alpha-carbonitrile induced CYP3A1 levels and N-demethylation of roxithromycin, erythromycin and troleandomycin but not of benzphetamine, in rat liver microsomes. Treatment of rat with phenobarbital caused increases in both CYP2B1 and 3A1 levels and all of the N-demethylation activities examined. Phenytoin and metyrapone produced increases in contents of 2B1 and activities of benzphetamine N-demethylation as well as of roxithromycin, erythromycin and troleandomycin, although these two inducers did not induce 3A1 protein significantly. 4. In man, a liver sample that was high in CYP3A4 and nifedipine oxidation activity was found to be the most active in N-demethylation activities towards these substrates examined. In addition, recombinant 3A4 catalysed very efficiently the N-demethylation of roxithromycin, erythromycin and troleandomycin in reconstituted monooxygenase systems. 5. These data suggest that erythromycin and troleandomycin, but not roxithromycin, were able to induce CYP3A1 in rat liver microsomes, and that N-demethylation of roxithromycin, erythromycin and troleandomycin were catalysed mainly by 3A1 (and partly by 2B1) in rat and by 3A4 in man.

Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For comparison, another group received 300 mg of triacetyloleandomycin (TAO) per kg, which is equivalent to the 226-mg/kg tiamulin group. Subsequently, microsomal P-450 contents, P-450 enzyme activities, metabolic intermediate complex spectra, and P-450 apoprotein concentrations were assessed. In addition, effects on individual microsomal P-450 activities were studied in control microsomes at different tiamulin and substrate concentrations. In the rats treated with tiamulin, a dose-dependent complex formation as evidenced by its absorption spectrum and an increase in cytochrome P-4503A1/2 contents as assessed by Western blotting (immunoblotting) were found. The effects were comparable to those of TAO. Tiamulin induced microsomal P-450 content, testosterone 6 beta-hydroxylation rate, erythromycin N-demethylation rate, and the ethoxyresorufin O-deethylation activity. Other activities were not affected or decreased. When tiamulin was added to microsomes of control rats, the testosterone 6 beta-hydroxylation rate and the erythromycin N-demethylation were strongly inhibited. It is concluded that tiamulin is a potent and selective inducer-inhibitor of cytochrome P-450. Though not belonging to the macrolides, the compound produces an effect on P-450 similar to those of TAO and related compounds.

Involvement of cytochrome P450 3A4 enzyme in the N-demethylation of methadone in human liver microsomes.

Methadone has become one of the most widely used drugs for opiate dependency treatment. This drug is extensively metabolized by the cytochrome P450 hepatic enzyme family in man, yielding an N-demethylated metabolite that cyclizes spontaneously into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. The specific forms of cytochrome P450 involved in this oxidative N-demethylation were examined in a panel of 20 human liver microsomal preparations previously characterized with respect to their P450 enzyme contents. Methadone was demethylated with an apparent Km of 545 +/- 258 microM (n = 3). The metabolic rates were 745 +/- 574 pmol/(min.mg of protein). This metabolic pathway was strongly correlated with estradiol 2-hydroxylation, testosterone 6 beta-hydroxylation, nifedipine oxidation, erythromycin N-demethylation, and toremifene N-demethylation, all of these monooxygenase activities being supported by P450 3A4. Furthermore, the total P450 3A content of liver microsomal samples, determined by immuno-quantification using a monoclonal anti-human P450 3A4 antibody, was correlated with methadone demethylation (r = 0.72; p < 0.003). Methadone metabolism was 60-72% inhibited either by three mechanism-based inhibitors of P450 3A4 (gestodene, TAO, and erythralosamine) or by four reversible inhibitors of P450 3A (ketoconazole, dihydroergotamine, quercetin, and diazepam with an apparent Ki of 50 microM) and by two nonspecific inhibitors (metyrapone and SKF-525A). Conversely, quinidine (inhibitor of P450 2D6), 7,8-benzoflavone (inhibitor of P450 1A), or sulfaphenazole (inhibitor of P450 2C) did not significantly inhibit, and may even have activated, methadone metabolism. Four heterologously expressed P450 proteins were able to catalyze the N-demethylation of methadone, namely, P450 2C8, P450 2C18, P450 2D6, and P450 3A4. However, referring to their relative liver content, it can be asserted that P450 3A4 is the major enzyme involved in the N-demethylation of methadone on average. Accordingly, caution should be advised in the clinical use of methadone when other drugs are also administered that induce or inhibit P450 3A4, such as rifampicin or diazepam, respectively.

Expression of Cytochrome-P450-3A5 in Escherichia Coli: Effects of 5′ Modification, Purification, Spectral Characterization, Reconstitution Conditions, and Catalytic Activities

Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5′-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Rim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)−1]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of ∼40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6β-hydroxylation, aflatoxin 3α-hydroxylation and 8,9-epoxidation, N-ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6β-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b5, divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of N-ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems.

Involvement of cytochrome P450 3A enzyme family in the major metabolic pathways of toremifene in human liver microsomes

The anti-estrogen toremifen-Fc-1157a or 4-chloro-1,2-diphenyl-1-[4-[2( N, N-dimethylamino) ethoxy]-phenyl]-1-butene is now used for the treatment of breast cancer. This drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-demethyl-(DMTOR), 4-hydroxy-(4OH-TOR) and deamino-hydroxy- (TOR III) toremifene metabolites. The specific forms of cytochrome P450 involved in these oxidation reactions were examined in 32 human liver microsomal preparations previously characterized with respect to their contents of several known P450 enzymes. Toremifene was demethylated with an apparent K m of 124 μM while it was hydroxylated with an apparent K m of 139 μm. The metabolic rates were 71 ± 56, 13 ± 9 and 15 ± 4 pmol/min/mg microsomal protein, respectively, for DMTOR, 4-OH-TOR and TOR III. The N-demethylation activity was strongly correlated with estradiol 2-hydroxylation ( r = 0.75), nifedipine oxidation ( r = 0.86), tamoxifen N-demethylation ( r = 0.73), testosterone 6β-hydroxylation ( r = 0.78) and erythromycin N-demethylation ( r = 0.84), all these monooxygenase activities known to be supported by CYP3A4 isoform. Furthermore, the CYP3A content of liver microsomal samples, measured by western blot analysis using a monoclonal anti-human CYP3A4 antibody, was strongly correlated with DMTOR formation ( r = 0.80). Compounds such as cyclosporin, triacetyl-oleandomycin and testosterone inhibited the N-demethylation of toremifene metabolism at 80, 89 and 56% vs control, respectively, while the formation of TOR III was inhibited at 78, 82 and 73% vs control and the 4-hydroxylation pathway was inhibited no more than about 50% vs control. Prior incubation of microsomes with 100 μM gestodene, known to be a selective mechanism-based inhibitor of CYP3A4 in the presence of NADPH, led to 76 ± 6 and 76 ± 5% (N = 5 samples) reductions in the N-demethylation and formation of TOR III, respectively. Polyclonal antibody directed against human CYP3A enzymes inhibited formation of DMTOR and TOR III by 60 and 46%, respectively. The metabolism of toremifene was not activated by α-naphthoflavone. Finally, the use of yeasts genetically engineered for expression of human P4501A1, 1A2, 2C9 and 3A4 allowed us to demonstrate that DMTOR and TOR III formations are mediated by P4501A and 3A4 enzymes and by contrast these enzymes are not involved in the 4-hydroxylation pathway. All these results taken together suggest that the major metabolic pathways of toremifene, namely N-demethylation and TOR III formation, are mediated mainly by cytochrome CYP3A4 enzyme in the human liver microsomes owing to the relative hepatic contents in P4501A and 3A.

An evaluation of cytochrome P450 isoform activities in the female dark agouti (DA) rat: Relevance to its use as a model of the CYP2d6 poor metaboliser phenotype

The female dark agouti (DA) rat lacks CYP2D1, the equivalent enzyme in the rat to human CYP2D6 (debrisoquine hydroxylase), and shows impaired metabolism of a number of CYP2D6 substrates. However, from the data available in the literature it is not entirely clear whether the enzyme deficiency in the DA rat is restricted to CYP2D1, and whether factors such as age and substrate concentration are important determinants of interstrain differences in the activity of this enzyme. Given that the female DA rat is used as a model of the human CYP2D6 poor metaboliser phenotype, there is a need for a systematic evaluation of the P450 activities in the DA rat, and of its suitability as a model of the PM phenotype. In the present study metoprolol was used as a probe substrate to investigate CYP2D1 activity since both the α-hydroxylation and O-demethylation of this drug are catalysed by CYP2D6 in man. Formation of α-hydroxymetoprolol (AHM) and O-demethylmetoprolol (ODM) was 10- and 2.5-fold lower in liver microsomes from female DA rats compared with microsomes from agematched female Wistar rats, the latter representing the extensive metaboliser strain. Kinetic analysis suggested that in both strains of rat both the α-hydroxylation and O-demethylation of metoprolol were catalysed by more than one enzyme. By using quinine as a specific inhibitor of the enzyme, CYP2D1 was identified as an intermediate affinity site in the Wistar strain and was shown to have impaired activity in the DA strain. The activities of lower and higher affinity sites were similar in the two strains. Thus, the only difference between the two strains with respect to both routes of metoprolol metabolism appeared to be in the activity of CYP2D1. Interstrain differences were found to be highly dependent on the choice of substrate concentration, being more marked at lower concentrations. We have also investigated the metabolism of a number of probe compounds for some of the other P450 isoforms commonly involved in drug metabolism to determine the selectivity of the deficiency in the DA strain. p-Nitrophenol hydroxylation and erythromycin N-demethylation were catalysed at higher rates by DA than by Wistar liver microsomes, indicating higher levels of activity of CYP2E1 and CYP3A in the former strain. Felodipine oxidation, tolbutamide hydroxylation and both the hydroxylation and N-demethylation of S-mephenytoin were catalysed at similar rates by microsomes from the two strains, indicating similar activities of enzymes in the CYP2C and CYP3A families. However, both the hydroxylation and N-demethylation of R-mephenytoin were impaired in the DA strain. This indicates that at least one other isoform of P450, thought also to be a member of the CYP2C or CYP3A families, in addition to CYP2D1 is deficient in the DA strain. Our findings indicate that whilst the female DA rat could be used as a preliminary screen to identify CYP2D6 substrates, because of interspecies differences in metabolism it could not be used to provide quantitative information regarding the contribution of CYP2D6 to an oxidation in man. In addition, a small number of false positives would be identified owing to other enzyme deficiencies; no false negatives would be expected. Comparisons between strains should be performed using female, age-matched animals and low substrate concentrations.

Selective induction of rat hepatic CYP1 and CYP4 proteins and of peroxisomal proliferation by green tea.

Rats were exposed to freshly prepared aqueous extracts of green tea (2.5% w/v) as the sole source of drinking water for 4 weeks. Hepatic cytochrome P450 activity was determined using chemical probes, showing selectivity for particular isoforms, and by immunoblot analysis employing polyclonal antibodies. Exposure to green tea gave rise to increases in the O-demethylation of methoxyresorufin and, to a lesser extent, in the dealkylations of ethoxyresorufin and pentoxyresorufin. An increase was also seen in lauric acid hydroxylation but, in contrast, the N-demethylation of erythromycin was inhibited. p-Nitrophenol oxidase activity was unaffected by the same treatment. Immunoblot analysis revealed increases in the apoprotein levels of CYP1A2 and CYP4A1 following treatment with green tea. A significant increase was also noted in the CN(-)-insensitive palmitoyl CoA oxidation and this was paralleled by an increase in the levels of the peroxisomal trifunctional protein determined immunologically. Hepatic S9 and microsomal preparations from tea-treated animals were more effective than controls in activating 2-amino-3-methylimidazol[4,5-f]quinoline and 2-aminoanthracene to mutagens in the Ames test. When N-nitrosopyrrolidine served as the promutagen, tea did not influence its mutagenicity when isolated microsomes comprised the activation system but a significant inhibition was observed when hepatic S9 was used. The above findings are discussed within the context of the established anticarcinogenic and anti-mutagenic properties of green tea.

Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.

Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids; such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5 for the 6 beta-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.

Identification of rifampin-inducible P450IIIA4 (CYP3A4) in human small bowel enterocytes.

Enzymes within the P450IIIA (CYP3A) subfamily appear to account for significant "first pass" metabolism of some drugs in the intestine. To identify which of the known P450IIIA genes are expressed in intestine, enterocyte RNA was hybridized on Northern blots with synthetic oligonucleotides complementary to hypervariable regions of hepatic P450IIIA4, P450IIIA5, and P450IIIA7 cDNAs. Hybridization was detected only with the P450IIIA4-specific oligonucleotide. The identity of the hybridizing mRNA was confirmed to be P450IIIA4 by direct sequencing of a DNA fragment amplified from enterocyte cDNA by the polymerase chain reaction. To determine if enterocyte P450IIIA4 is inducible, biopsies of small bowel mucosa were obtained from five volunteers before and after they received 7d of treatment with rifampin, a known inducer of P450IIIA4 in liver. Rifampin treatment resulted in a five- or eightfold mean increase (P < 0.05) in the biopsy concentration of P450IIIA4 mRNA when normalized for content of sucrase isomaltase or intestinal fatty acid binding protein mRNAs, respectively. Rifampin also induced P450IIIA immunoreactive protein in enterocytes in each of the subjects, as judged by immunohistochemistry, and resulted in a 10-fold increase in P450IIIA4-specific catalytic activity (erythromycin N-demethylation) in the one patient studied. Our identification of inducible P450IIIA4 in enterocytes may in part account for drug interactions characteristic of P450IIIA4 substrates and suggests a strategy for controlling entry into the body of a major class of xenobiotics.

Nature of cytochromes P450 involved in the 2-/4-hydroxylations of estradiol in human liver microsomes

Kinetics of the 2- and 4-hydroxylations of estradiol (E2) by human liver microsomal samples were studied to determine the major P450 isoform involved in these endogenous reactions. Thirty human liver microsomal samples were analysed. Metabolism of 25 μM [ 14C]E2 produced 2-hydroxy and 4-hydroxy derivatives with a ratio of 3.2 ± 1.5 and a great inter-individual variation. Kinetic analysis of the 2- and 4-hydroxylations of E2 exhibited a curvilinear double reciprocal plot with an apparent K m of 15 μM. Further experiments demonstrated that α-naphthoflavone, testosterone and progesterone increased the 2-hydroxylation activity, suggesting the involvement of a substrate activation mechanism. These two hydroxylations of E2 were shown to be catalysed by cytochrome P450 with an apparent dissociation constant K s of 0.8 μM. These 2- and 4-hydroxylations inter-correlated significantly ( r = 0.93; N = 30). The 2-hydroxylation of E2 correlated with four monooxygenase activities known to be supported by P450 3A4/3A5, namely nifedipine oxidation ( r = 0.78; N = 29); erythromycin N-demethylation ( r = 0.69; N = 27), testosterone 6β-hydroxylation ( r = 0.66; N = 25) and tamoxifen N-demethylation ( r = 0.64; N = 29). On the other hand, E2-hydroxylations did not correlate with activities supported by P450 1A2 and P450 2E1. Furthermore, drugs as cyclosporin, diltiazem. triacetyl-oleandomycin and 17α-ethynylestradiol inhibited more than 90% of the E2-hydroxylations at concentrations < 250 μM, while weak inhibition was shown with 500 μM cimetidine and no significant inhibition with caffeine, phenacetin and omeprazole. Finally, 2- and 4-hydroxylations of E2 correlated significantly with the content of P450 3A4/3A5 immunodetected by a monoclonal antibody anti-human P450-nifedipine ( r = 0.84; N = 28). E2-hydroxylation activities were inhibited by more than 80% with polyclonal anti-human anti-P450-nifedipine. Preincubation of human liver microsomes with 100 μM gestodene (a suicide substrate of P450 3A4) inactivated this P450 isoform and accordingly allowed evaluation of the contribution of other P450 isoforms to the E2 metabolism to about 21% (± 17%. N = 29). All these results taken together suggest that P450 3A4 3A5 are the major forms involved in the formation of catecholestrogens in the human liver microsomes.

Effect of age and gender on the activity of human hepatic CYP3A

Many pharmacokinetic investigations in the elderly population reveal decreased clearance of lipophilic drugs metabolized by the cytochrome P450 enzymes; however, few studies have evaluated aging-dependent or gender-related changes in specific cytochrome P450 enzymes. The clearance of quinidine, midazolam, triazolam, erythromycin, and lidocaine declines with age; these drugs are metabolized by the isoform, CYP3A. To determine whether these metabolic effects are due to changes in CYP3A, the effects of age and gender on CYP3A activity were examined. The activity of the human hepatic cytochrome P450, CYP3A, was quantified in vitro as erythromycin N-demethylation in microsomes prepared from forty-three resected human liver specimens obtained from patients, age 27 to 83, with normal liver function. Erythromycin N-demethylation varied 5-fold in human liver microsomes. CYP3A activity was 24% higher in females than males (P = 0.027). CYP3A activity did not correlate with age, smoking status, ethanol consumption or percent ideal body weight. Large interindividual differences and a small female-specific increase in CYP3A activity were obtained. However, CYP3A activity was unaffected by age over the range of 27–83 years, suggesting that the aging-related alteration in the clearance of CYP3A substrates is secondary to changes in liver blood flow, size, or drug binding and distribution with aging.

Hepatic cytochrome P-4503A (CYP3A) activity in the elderly

Elderly patients exhibit decreased clearance of multiple drugs biotransformed by the hepatic cytochromes P-450. The cytochromes P-450 are a superfamily of enzymes, which comprise a central component of phase I drug metabolism. Distinct isoforms metabolize specific drugs. In human liver microsomes, the glucocorticoid-inducible cytochrome P-450 IIIA, CYP3A, catalyzes the N-demethylation of erythromycin. To examine the activity of hepatic CYP3A in elderly males and females, erythromycin N-demethylation was examined, as reflected by the recently described [ 14C]erythromycin breath test in 24 healthy volunteers, age 70–88. The [ 14C]erythromycin breath test was measured in normal elderly males and females to: (a) determine persistence of the gender-related dimorphism (evident in younger subjects) of CYP3A activity in the elderly population, (b) examine the effect of % ideal body weight, age, diet, and medication use on the activity of human hepatic CYP3A, and (c) compare breath test results obtained in normal geriatric volunteers with published results obtained in younger subjects, to determine aging-related alteractions in CYP3A enzyme activity. Erythromycin N-demethylation varied fivefold among these patients. Similar to earlier studies examining erythromycin N-demethylation in younger subjects, CYP3A activity was found to vary with gender in the geriatric cohort. [ 14C]Erythromycin N-demethylation at 60 min was 3.14% ± 0.75 ( n = 13) in females and 2.15% ± 0.77 ( n = 11) in males ( P = 0.005). In evaluating the role of % ideal body weight and % dietary fat using multivariable linear regression analyses. [ 14C]erythromycin N-demethylation, was found to decline significantly as % ideal body weight increased ( P = 0.001). This was not confounded by gender. [ 14C]Erythromycin N-demethylation was not related to dietary fat intake ( P < 0.13). [ 14C]Erythromycin N-demethylation in the elderly volunteers was similar to values reported for subjects aged 20–60. Performance of a new non-invasive test of the human hepatic glucocorticoid-inducible CYP3A in a geriatric cohort suggests that: (a) the gender-related heterogeneity in function of the glucocorticoid inducible human CYP3A persists during normal aging, (b) that the activity of CYP3A may decrease in obesity, and (c) that the activity of CYP3A is stable throughout normal aging.

BENZODIAZEPINE METABOLISM IN ETHANOL-TREATED MALE RATS: USE OF PAIR-FED AND AGE-MATCHED CONTROLS

The effects of chronic moderate (15%) ethanol consumption and ageing on rat hepatic cytochrome P450 monooxygenase activities were examined using diazepam, nordazepam, d-benzphetamine, erythromycin, ethylmorphine and nitrosodimethylamine (NDMA) as substrates. In addition, the effects of moderate ethanol alone on the oxidation of metoprolol, morphine and temazepam were examined. Cytochrome P450 specific content increased significantly only in the 6-week ethanol-treated rats, and no changes in percentage liver to body weights were apparent in any of the ethanol-treated animals compared with pair-fed controls. Only cytochrome P450IIIA enzyme activities displayed age-related decreases, these being identified in the pair-fed animals. C3-hydroxylation of diazepam and nordazepam (36% of controls) and N-demethylation of erythromycin and ethylmorphine (58% and 64% of controls) were decreased in 6-week ethanol-treated animals, these effects being less pronounced in the 12, 24 and 48-week ethanol-treated groups. The decrease seen for diazepam and d-benzphetamine N-demethylation caused by ethanol consumption was approximately 80% of control groups for the duration of the treatment. NDMA and morphine N-demethylations were increased to 120% of control activities and metoprolol alpha-hydroxylase was increased to 140% of control activities at 6 weeks, whilst metoprolol O-demethylase activity remained unaltered. NDMA N-demethylase activity showed a two-fold induction at 24 and 48 weeks of ethanol treatment, compared with corresponding pair-fed control groups. These results support previous findings from this laboratory showing that the same or similar P450IIIA family isozymes are involved in the C3-hydroxylation of diazepam and nordazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

The erythromycin breath test selectively measures P450IIIA in patients with severe liver disease.

There are significant interpatient differences in the activity of a major drug metabolizing enzyme termed P450IIIA. Because P450IIIA uniquely catalyzes the N-demethylation of erythromycin, we have proposed that the P450IIIA activity of a patient may be determined from the rate of 14CO2 production in the breath after an intravenous infusion of a test dose of [14C-N-methyl]erythromycin. However, direct evidence that this erythromycin breath test selectively measures P450IIIA and not other major human liver P450s in patients has been lacking. We therefore administered the erythromycin breath test to nine patients with severe liver disease who were awaiting liver transplantation. Microsomes were prepared from liver samples obtained during surgery and the concentration of P450IA2, P450IIC8, P450IIC9, P450IIE1, and P450IIIA were determined immunochemically. We found a significant correlation between patients' erythromycin breath test results and their liver P450IIIA levels (r2 = 0.56, p = 0.02). In contrast, there was no correlation at all between the erythromycin breath test result and the microsomal levels of any of the other four P450s assayed. The correlation of the erythromycin breath test and P450IIIA did not appear related to the extent of liver disease because neither correlated with prothrombin time or albumin or bilirubin levels. These data provide the best evidence to date that the erythromycin breath test is a specific assay of in vivo P450IIIA activity in patients.

Heterogeneity of CYP3A isoforms metabolizing erythromycin and cortisol

The N-demethylation of erythromycin and 6 beta-hydroxylation of cortisol are both functions of the glucocorticoid-inducible CYP3A in human liver microsomes. To determine whether 6 beta-hydroxylation and erythromycin N-demethylation are catalyzed by similar or distinct CYP3A isoforms, erythromycin N-demethylase activity, as reflected by the recently described 14[C]-erythromycin breath test, was compared with urinary 6 beta-hydroxycortisol/cortisol ratios, a measure of cortisol 6 beta-hydroxylase activity, in nine patients. Erythromycin N-demethylation varied fourfold and 6 beta-hydroxycortisol/cortisol ratios varied sevenfold among the subjects; no correlation was found between these activities (r2 = 0.065). New noninvasive tests of CYP3A strongly suggest cortisol 6 beta-hydroxylation and erythromycin N-demethylation are performed by distinct CYP3A isoforms.

Effects of ethinylestradiol and testosterone implants on hepatic microsomal cytochrome P450 monooxygenases of birth gonadectomized male and female Dark Agouti rats

Monooxygenases in the cytochrome P450 IIIA subfamily are induced by a number of their xenobiotic substrates and by testosterone, an endobiotic substrate of importance in their regulation. 17α-Ethinylestradiol (EE) is also metabolized by these enzymes and in this study Dark Agouti rats were used to examine the effects of subcutaneous implantation of controlled release silastic capsules containing EE to determine if this steroid also induces these enzymes. Data were compared with results obtained from equivalent groups of animals implanted with capsules containing testosterone propionate (TP). Liver microsomes prepared from male and female rats were used to identify intrinsic gender differences in the monooxygenases studied and gender differences in the responses to the implanted steroids were also determined. Effects due to imprinting of growth hormone secretion patterns were controlled by using male and female birth gonadectomized animals. Results obtained from groups with blank implants showed there were no effects due to the silastic implant material itself on the monooxygenases studied. The specific activities of erythromycin N-demethylation in liver microsomes of both EE and TP implanted male and female birth gonadectomized animals were enhanced relative to corresponding blank implanted controls consistent with both steroids having an effect to induce activity attributable to cytochrome P450 IIIA isoforms. Immunoinhibition studies using microsomes from EE treated female rats with erythromycin as substrate provided further evidence for this steroid having this induction effect. The specific activity of ethylmorphine N-demethylation was however not increased in microsomes prepared from the EE implanted female animals and was decreased in the corresponding male preparations. These findings distinguished the response to this steroid from that to TP and suggested induction by this estrogen of an isoform(s) having a more limited range of substrates than has characteristically been found in this subfamily. EE treatment also caused an increase in diazepam C 3 hydroxylase consistent with an effect to induce P450 IIIA activity but this was found only in microsomes from birth gonadectomized female animals. This was in contrast to the effect of TP treatment which produced increases in this monooxygenase in both male and female animals. Another gender specific effect of EE was a striking decrease in morphine N-demethylase activity seen only in birth gonadectomized male rats. This again contrasted with the effect of TP which caused a marked increase in this activity in liver microsomes of both male and female birth gonadectomized animals consistent with the proposal that testosterone is important in the regulation of this activity. EE also caused decreases in diazepam N-demethylase specific activity in both male and female rats and spectral evidence indicated that mechanisms other than heme alkylation may be responsible for the decreased activities of these monooxygenases.

Inducing effect of oxfendazole on cytochrome P450IA2 in rabbit liver: Consequences on cytochrome P450 dependent monooxygenases

Male New Zealand rabbits were dosed with either 0.9, 4.5 or 22.5 mg/kg/day of oxfendazole by gastric intubation for 10 days. Oxfendazole administered at the therapeutic dose (4.5 mg/kg) and at the highest dose (22.5 mg/kg) increased 1.54- and 2.36-fold the total liver microsomal cytochrome P450 and more particularly the isoenzyme P450IA2 (95 and 184% increases) as demonstrated by western blotting. Increases in ethoxyresorufin O-deethylation and hydroxylations of benzopyrene and acetanilide occurred in livers of the same animals without any change in N-demethylation of aminopyrine, benzphetamine or erythromycin. Because of the unchanged level of mRNA specific to cytochrome P450IA2, as shown by northern blot analysis of poly mRNA, an enzyme stabilization rather than a transcriptional activation of IA2 genes should be involved in the P450IA2 regulation mechanisms. Oxfendazole bound strongly to cytochrome P450, giving rise to a type II spectrum, and inhibited noncompetitively the ethoxyresorufin O-deethylase and acetanilide hydroxylase activities, this confirmed that oxfendazole interacts only with the P450IA family. On the basis of a comparison of the enzymatic activities induced by various imidazole drugs, it was concluded that oxfendazole, like omeprazole and albendazole, behaved as a 3-methylcholanthrene-type inducer. These three benzimidazoles did not all belong to the same category of cytochrome P450 inducers as the antifungal drugs miconazole, clotrimazole and ketoconazole.

Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme

Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage lambda gt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83, 8064-8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8-2 [Cullin, C. & Pompon, D. (1988) Gene 65, 203-217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous-CO spectrum typical of cytochrome P-450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4-oxidation, quinidine 3-hydroxylation and N-oxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH-cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH-cytochrome P-450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.