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PubMed1997

Myoglobin toxicity in proximal human kidney cells: roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport.

The purpose of this study was to gain direct insights into mechanisms by which myoglobin induces proximal tubular cell death. To avoid confounding systemic and hemodynamic influences, an in vitro model of myoglobin cytotoxicity was employed. Human proximal tubular (HK-2) cells were incubated with 10 mg/ml myoglobin, and after 24 hours the lethal cell injury was assessed (vital dye uptake; LDH release). The roles played by heme oxygenase (HO), cytochrome p450, free iron, intracellular Ca2+, nitric oxide, H2O2, hydroxyl radical (-OH), and mitochondrial electron transport were assessed. HO inhibition (Sn protoporphyrin) conferred almost complete protection against myoglobin cytotoxicity (92% vs. 22% cell viability). This benefit was fully reproduced by iron chelation therapy (deferoxamine). Conversely, divergent cytochrome p450 inhibitors (cimetidine, aminobenzotriazole, troleandomycin) were without effect Catalase induced dose dependent cytoprotection, virtually complete, at a 5000 U/ml dose. Conversely, -OH scavengers (benzoate, DMTU, mannitol), xanthine oxidase inhibition (oxypurinol), superoxide dismutase, and manipulators of nitric oxide expression (L-NAME, L-arginine) were without effect. Intracellular (but not extracellular) calcium chelation (BAPTA-AM) caused approximately 50% reductions in myoglobin-induced cell death. The ability of Ca2+ (plus iron) to drive H2O2 production (phenol red assay) suggests one potential mechanism. Blockade of site 2 (antimycin) and site 3 (azide), but not site 1 (rotenone), mitochondrial electron transport significantly reduced myoglobin cytotoxicity. Inhibition of Na, K-ATPase driven respiration (ouabain) produced a similar protective effect. We conclude that: (1) HO-generated iron release initiates myoglobin toxicity in HK-2 cells; (2) myoglobin, rather than cytochrome p450, appears to be the more likely source of toxic iron release; (3) H2O2 generation, perhaps facilitated by intracellular Ca2+/iron, appears to play a critical role; and (4) cellular respiration/terminal mitochondrial electron transport ultimately helps mediate myoglobin's cytotoxic effect. Formation of poorly characterized toxic iron/H2O2-based reactive intermediates at this site seems likely to be involved.

Antioxidants↗
PubMed1997

Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery.

1. In rat mesenteric artery, acetylcholine (ACh) causes endothelium-dependent hyperpolarization by releasing endothelium-derived hyperpolarizing factor (EDHF). Recent evidence suggests that EDHF may be a cytochrome P450-derived arachidonic acid metabolite. The aim of the present study was to investigate whether such a metabolite is indeed contributing to ACh-induced hyperpolarization observed in rat mesenteric artery. 2. The phospholipase A2 inhibitor quinacrine (30 microM) nearly completely eliminated ACh-induced hyperpolarization. However, the hyperpolarizing effect of pinacidil was also abolished in the presence of quinacrine. 3. The imidazole antimycotic agents ketoconazole (50 microM), clotrimazole (30 microM) and miconazole (10 microM), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 microM), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 microM), an inhibitor of all arachidonic acid metabolic pathways, altered ACh-induced hyperpolarization. Furthermore, the hyperpolarization was unaffected by the preferential inhibitors of specific cytochrome P450 isozymes, alpha-naphtoflavone (1 microM), diedthyldithiocarbamate (50 microM), metyrapone (20 microM) and troleandomycin (10 microM). 4. Pretreatment of rats with lipopolysaccharide (2 mg kg-1) and exposure to nitroprusside (10 microM), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without effect on ACh-induced hyperpolarization. Pretreatment of rats for 3 days with pentobarbitone (80 mg kg-1 day-1), a cytochrome P450 inducer, also did not affect the hyperpolarizing response to ACh. 5. Arachidonic acid in concentrations up to 100 microM had no detectable effect on smooth muscle membrane potential. 11, 12-Epoxyeicosatrienoic acid (EET, 10 microM), one of cytochrome P450-derived epoxygenase metabolites of arachidonic acid, elicited a small endothelium-independent membrane hyperpolarization. The hyperpolarizing response to EET was blocked by glibenclamide (30 microM), in contrast to the response to ACh. 6. These results suggest that the contribution of a cytochrome P450-derived metabolite of arachidonic acid to ACh-induced hyperpolarization via EDHF release is minimal or absent in rat mesenteric artery.

Acetylcholine↗
PubMed2000

Identification of the cytochrome P450 enzymes involved in the metabolism of cisapride: in vitro studies of potential co-medication interactions.

Cisapride is a prokinetic drug that is widely used to facilitate gastrointestinal tract motility. Structurally, cisapride is a substituted piperidinyl benzamide that interacts with 5-hydroxytryptamine-4 receptors and which is largely without central depressant or antidopaminergic side-effects. The aims of this study were to investigate the metabolism of cisapride in human liver microsomes and to determine which cytochrome P-450 (CYP) isoenzyme(s) are involved in cisapride biotransformation. Additionally, the effects of various drugs on the metabolism of cisapride were investigated. The major in vitro metabolite of cisapride was formed by oxidative N-dealkylation at the piperidine nitrogen, leading to the production of norcisapride. By using competitive inhibition data, correlation studies and heterologous expression systems, it was demonstrated that CYP3A4 was the major CYP involved. CYP2A6 also contributed to the metabolism of cisapride, albeit to a much lesser extent. The mean apparent K(m) against cisapride was 8.6+/-3.5 microM (n = 3). The peak plasma levels of cisapride under normal clinical practice are approximately 0.17 microM; therefore it is unlikely that cisapride would inhibit the metabolism of co-administered drugs. In this in vitro study the inhibitory effects of 44 drugs were tested for any effect on cisapride biotransformation. In conclusion, 34 of the drugs are unlikely to have a clinically relevant interaction; however, the antidepressant nefazodone, the macrolide antibiotic troleandomycin, the HIV-1 protease inhibitors ritonavir and indinavir and the calcium channel blocker mibefradil inhibited the metabolism of cisapride and these interactions are likely to be of clinical relevance. Furthermore, the antimycotics ketoconazole, miconazole, hydroxy-itraconazole, itraconazole and fluconazole, when administered orally or intravenously, would inhibit cisapride metabolism.

Anti-Ulcer Agents↗
PubMed2003

Upregulation of cytochromes P450 2B in rat liver by orphenadrine.

1 The alkylamine drug orphenadrine (ORPH) is an inducer and inhibitor of the microsomal cytochrome P450 (CYP) system in mammals. This study evaluated the selectivity of CYP induction by ORPH in rat liver. 2 Immunoblot analysis indicated that ORPH was a selective inducer of the phenobarbitone (PB)-inducible CYP2B in rat liver. CYP2B protein was increased to approximately 14-fold of levels in untreated rat liver. By comparison PB increased CYP2B expression 40-fold. Corresponding increases in the activity of CYP2B-dependent androstenedione 16beta-hydroxylation were measured in microsomes from ORPH and PB-induced rats. 3 Northern analysis indicated that CYP2B1/2 mRNA was increased in ORPH-induced rat liver. Consistent with this finding, ORPH was found to activate a PB-responsive enhancer module in constitutive androstane receptor (CAR)-transfected Hep G2 cells. 4 Other alkylamines like troleandomycin impair CYP turnover. We tested whether ORPH induction of CYP2B may include a post-translational component. In PB-pretreated animals ORPH administration delayed the loss of CYP2B after PB withdrawal, but no evidence for altered turnover was found. 5 These studies establish ORPH as a selective inducer of CYP2B in rat liver. Induction appears to be mediated pretranslationally by CAR activation of CYP2B gene transcription. Post-translational stabilisation by an ORPH metabolite does not elicit induction. Induction of CYP2B may influence pharmacokinetic interactions involving ORPH.

Animals↗
PubMed1999

Review article: cardiac adverse effects of gastrointestinal prokinetics.

Gastrointestinal prokinetics, such as metoclopramide, cisapride and levosulpiride, are widely used for the management of functional gut disorders. Recently, several studies have shown that cisapride (a partial 5-HT4 receptor agonist) can induce dose-dependent cardiac adverse effects, including lengthening of the electrocardiographic QT interval, syncopal episodes and ventricular dysrhythmias. Until recently, it was not clear whether these effects were dependent on 5-HT4 receptor activation or related to peculiar characteristics in the molecular structure of single agents within the benzamide class. Experimental evidence now favours the second hypothesis: cisapride possesses Class III antiarrhythmic properties and prolongs the action potential duration through blockade of distinct voltage-dependent K+ channels, thus delaying cardiac repolarization and prolonging the QT interval. Patients at risk of cardiac adverse effects are children, subjects with idiopathic, congenital or acquired long QT syndrome and, in particular, those receiving concomitant medication with Class III antiarrhythmic agents, some H1-receptor antagonists (e.g. terfenadine), or drugs such as azole antifungals (e.g. ketoconazole, itraconazole, miconazole and fluconazole) and macrolide antibacterials (e.g. erythromycin, clarithrod-mycin and troleandomycin), which can inhibit cisapride metabolism by interfering with the CYP3A4 isoenzyme.

Benzamides↗
PubMed1997

Evidence for involvement of human CYP3A in the 3-hydroxylation of quinine.

AIMS: Our previous studies using in vitro hepatic microsomal preparations suggested that the hepatic metabolism of quinine to form the major metabolite 3-hydroxyquinine is most likely catalysed by human P450 3A (CYP3A). The present study was carried out to investigate the kinetics and to identify and further characterise the human liver CYP isoforms involved in the metabolism of quinine. METHODS: In vitro human microsomal techniques were employed. RESULTS: The mean apparent Km value for 3-hydroxyquinine formation was 83 +/- 19 (s.d.) microM, ranging from 57 microM to 123 microM in microsomes from ten human livers. There was a 6.7-fold variation in Vmax values (mean 547 +/- 416 pmol min-1 mg-1). Quinine 3-hydroxylation was inhibited by the specific CYP3A inhibitors, troleandomycin, midazolam and erythromycin. Inhibitors selective for CYP1A1/2, CYP2D6, CYP2E1, CYP2C9/10 or CYP2C19 had little or no effect on quinine 3-hydroxylation. Using microsomes from a panel of livers, significant correlations were found only between 3-hydroxyquinine activity and other CYP3A activities (caffeine 8-oxidation, omeprazole sulphoxidation, midazolam 1'-hydroxylation and midazolam 4-hydroxylation) and immunoreactive CYP3A content. There were no statistically significant correlation with activities selective for CYP1A2, CYP2C9 and CYP2E1. Competitive inhibition of quinine 3-hydroxylation was observed with a substrate known to be specifically metabolized by human CYP3A, i.e. midazolam, with an apparent Ki value of 11.0 microM. CONCLUSIONS: The present results strongly indicate that the conversion of quinine to 3-hydroxyquinine is the major metabolic pathway in human liver in vitro and that the reaction is catalysed by CYP3A isoforms.

Antimalarials↗
PubMed1997

Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine.

AIMS: Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated. METHODS: The kinetics of formation of N- and O-demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied. RESULTS: Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N-demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N-demethylation in both extensive and poor metabolizers. The CYP3A inducer, alpha-naphthoflavone, increased N-demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar Km values. The kinetic constants for O-demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O-demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer. CONCLUSIONS: CYP2D6 is the major enzyme mediating O-demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A.

Analgesics, Opioid↗
PubMed1999

Methadone N-demethylation in human liver microsomes: lack of stereoselectivity and involvement of CYP3A4.

AIMS: To investigate the kinetics of CYP-mediated N-demethylation of methadone in human liver microsomes, and examine the role of stereoselectivity and CYP isoforms involved. METHODS: The kinetics of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) formation via N-demethylation of rac-, (R)- and (S)-methadone in human liver microsomes prepared from six liver samples were determined by h.p.l.c., and inhibition of metabolic function was studied using isoform-specific chemical inhibitors and monoclonal antibodies. Microsomes containing expressed CYP3A4, CYP2D6 and CYP2C19 were also used to examine the formation of EDDP. RESULTS: The V max, Km, and CLint values for the formation of EDDP from rac-, (R)- and (S)-methadone were in the ranges of 20-77 nmol mg-1 protein h-1, 125-252 microm, and 91-494 ml h-1 g-1 protein. Km and CLint values for (R)- and (S)-methadone were not statistically significantly different (P >0.05), while V max values for (S)-methadone were 15% (P=0.045) lower than for (R)-methadone. Expressed CYP3A4 and CYP2C19 showed similar reaction rates for both (R)- and (S)-methadone, while CYP2D6 did not catalyse this reaction. Selective chemical inhibitors of CYP3A (troleandomycin, ketoconazole) and monoclonal human CYP3A4 antibodies significantly inhibited (P<0.05) the formation of EDDP in a concentration dependent manner by up to 80%. Sulphaphenazole (CYP2C9) also significantly inhibited (P<0.05) EDDP formation (range 14-25%). There were no statistically significant differences in the inhibition observed between the three substrates. Selective inhibitors of CYP1A2 (furafylline), CYP2A6 (coumarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (quinidine) and CYP2E1 (diethyldithiocarbamic acid sodium salt and monoclonal human CYP2E1 antibodies) had no significant (P >0.05) effect. CONCLUSIONS: The N-demethylation of methadone in human liver microsomes is not markedly stereoselective, and is mediated mainly by CYP3A4 with the possible involvement of CYP2C9 and CYP2C19. Thus, the large interindividual variation reported for methadone pharmacokinetics may be due to variability in the expression of these CYP isoforms, and the reported stereoselectivity in the systemic clearance of methadone in vivo is not due to stereoselectivity in N-demethylation.

Adult↗
PubMed2003

The human hepatic metabolism of simvastatin hydroxy acid is mediated primarily by CYP3A, and not CYP2D6.

AIMS: To identify the cytochrome P450 (CYP) isoforms responsible for the metabolism of simvastatin hydroxy acid (SVA), the most potent metabolite of simvastatin (SV). METHODS: The metabolism of SVA was characterized in vitro using human liver microsomes and recombinant CYPs. The effects of selective chemical inhibitors and CYP antibodies on SVA metabolism were assessed in human liver microsomes. RESULTS: In human liver microsomes, SVA underwent oxidative metabolism to three major oxidative products, with values for Km and Vmax ranging from about 50 to 80 microM and 0.6 to 1.9 nmol x min(-1) x mg(-1) protein, respectively. Recombinant CYP3A4, CYP3A5 and CYP2C8 all catalysed the formation of the three SVA metabolites, but CYP3A4 was the most active. CYP2D6 as well as CYP2C19, CYP2C9, CYP2A6, CYP1A2 did not metabolize SVA. Whereas inhibitors that are selective for CYP2D6, CYP2C9 or CYP1A2 did not significantly inhibit the oxidative metabolism of SVA, the CYP3A4/5 inhibitor troleandomycin markedly (about 90%) inhibited SVA metabolism. Quercetin, a known inhibitor of CYP2C8, inhibited the microsomal formation of SVA metabolites by about 25-30%. Immunoinhibition studies revealed 80-95% inhibition by anti-CYP3A antibody, less than 20% inhibition by anti-CYP2C19 antibody, which cross-reacted with CYP2C8 and CYP2C9, and no inhibition by anti-CYP2D6 antibody. CONCLUSIONS: The metabolism of SVA in human liver microsomes is catalysed primarily (> or = 80%) by CYP3A4/5, with a minor contribution (< or = 20%) from CYP2C8. CYP2D6 and other major CYP isoforms are not involved in the hepatic metabolism of SVA.

Aryl Hydrocarbon Hydroxylases↗
PubMed2004

Large interindividual variability in the in vitro formation of tamoxifen metabolites related to the development of genotoxicity.

AIMS: To characterize the interindividual variability and the individual CYP involved in the formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen from tamoxifen. METHODS: Microsomes from 50 human livers were used to characterize the interindividual variability in the alpha-hydroxylation, N-desmethylation and N-didesmethylation of tamoxifen. Selective inhibitors and recombinant enzymes were used to identify the forms of CYP catalysing these reactions. RESULTS: The rates of formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen were highly variable, and correlated with each other (P < 0.0001). The respective ranges were 0.7-11.4, 25.7-411, and below the limit of quantification--4.4 pmol mg(-1) protein min(-1). Formation of all metabolites was observed with expressed recombinant CYP3A4, inhibited by troleandomycin (65, 77 and 35%, respectively, P < 0.05) and associated with CYP3A4 expression (rs = 0.612, rs = 0.585 and rs = 0.430, P < 0.01, respectively). CONCLUSIONS: Formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen in vitro is highly variable and mediated predominantly by CYP3A4.

Adolescent↗
PubMed2004

CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes.

AIM: To determine the Michaelis-Menten kinetics of hydrocodone metabolism to its O- and N-demethylated products, hydromorphone and norhydrocodone, to determine the individual cytochrome p450 enzymes involved, and to predict the in vivo hepatic intrinsic clearance of hydrocodone via these pathways. METHODS: Liver microsomes from six CYP2D6 extensive metabolizers (EM) and one CYP2D6 poor metabolizer (PM) were used to determine the kinetics of hydromorphone and norhydrocodone formation. Chemical and antibody inhibitors were used to identify the cytochrome p450 isoforms catalyzing these pathways. Expressed recombinant cytochrome p450 enzymes were used to characterize further the metabolism of hydrocodone. RESULTS: Hydromorphone formation in liver microsomes from CYP2D6 EMs was dependent on a high affinity enzyme (Km = 26 microm) contributing 95%, and to a lesser degree a low affinity enzyme (Km = 3.4 mm). In contrast, only a low affinity enzyme (Km = 8.5 mm) formed this metabolite in the liver from the CYP2D6 PM, with significantly decreased hydromorphone formation compared with the livers from the EMs. Norhydrocodone was formed by a single low affinity enzyme (Km = 5.1 mm) in livers from both CYP2D6 EM and PM. Recombinant CYP2D6 and CYP3A4 formed only hydromorphone and only norhydrocodone, respectively. Hydromorphone formation was inhibited by quinidine (a selective inhibitor of CYP2D6 activity), and monoclonal antibodies specific to CYP2D6. Troleandomycin, ketoconazole (both CYP3A4 inhibitors) and monoclonal antibodies specific for CYP3A4 inhibited norhydrocodone formation. Extrapolation of in vitro to in vivo data resulted in a predicted total hepatic clearance of 227 ml x h-1 x kg-1 and 124 ml x h-1 x kg-1 for CYP2D6 EM and PM, respectively. CONCLUSIONS: The O-demethylation of hydrocodone is predominantly catalyzed by CYP2D6 and to a lesser extent by an unknown low affinity cytochrome p450 enzyme. Norhydrocodone formation was attributed to CYP3A4. Comparison of recalculated published clearance data for hydrocodone, with those predicted in the present work, indicate that about 40% of the clearance of hydrocodone is via non-CYP pathways. Our data also suggest that the genetic polymorphisms of CYP2D6 may influence hydrocodone metabolism and its therapeutic efficacy.

Analgesics, Opioid↗
PubMed2000

Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes.

Although cytochrome P-450 (CYP)-generated reactive metabolites can cause hepatocyte apoptosis, the mechanism of this effect is incompletely understood. In the present study, we assessed the hepatotoxicity of skullcap, a diterpenoid-containing herbal remedy. Male rat hepatocytes were incubated for 2 hours with skullcap diterpenoids (100 microg/mL). This treatment decreased cell glutathione and protein thiols and increased cell [Ca(2+)]. This activated Ca(2+)-dependent tissue transglutaminase, forming a cross-linked protein scaffold, and also opened the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, increased cytosolic cytochrome c, activation of procaspase 3, internucleosomal DNA fragmentation, and ultrastructural features of apoptosis. Cell death was increased by a CYP3A inducer (dexamethasone) or a sulfur amino acid-deficient diet increasing glutathione depletion. In contrast, cell death was prevented by decreasing CYP3A activity (with troleandomycin), preventing glutathione depletion (with cysteine or cystine), blocking Ca(2+)-modulated events (with calmidazolium), preventing mitochondrial permeability transition (with cyclosporin A), or inhibiting caspase 3 (with acetyl-Asp-G u-Va-Asp-a dehyde). Both calmidazolium and cyclosporin A also prevented the increase in cytosolic cytochrome c and procaspase 3 activation. In conclusion, CYP3A activates skullcap diterpenoids into reactive metabolites that deplete cellular thiols and increase cell [Ca(2+)]. This activates Ca(2+)-dependent transglutaminase and also opens the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, cytochrome c release, and caspase activation. Preventing mitochondrial permeability transition pore opening and/or caspase activity blocks apoptosis, showing the fundamental role of these final events in metabolite-mediated hepatotoxicity.

Animals↗
PubMed1999

Macrolide antibiotics, drug interactions and microsomal enzymes: implications for veterinary medicine.

The macrolide group of antibiotics includes natural members, pro-drugs and semi-synthetic derivatives, thus named because they are composed of a large aglycone ring (from 14 to 16 carbon atoms), to which are attached several sugars. Some of them are amino-sugars, containing a diethylamino, tertiary amine function. A number of antibiotics, including erythromycin, oleandomycin, triacetyl-oleandomycin (troleandomycin), carbomycin, spiramycin, tylosin, rosamicin, azithromycin, clarithromycin, dirithromycin and others, are members of this group. On a comparative basis, erythromycin and oleandomycin are similar, with the same basic 14-carbon lactone ring and side chain sugars. The remaining compounds contain a basic 15- or 16-carbon lactone ring and one or two side-chain sugars. Most of the macrolides are produced by Streptomyces spp bacteria. An exception is rosamicin, which is produced by Micromonospora. Clarithromycin and azithromycin are new semi-synthetic derivatives of erythromycin.

Animals↗
PubMed2003

Macrolides as biological response modifiers in cystic fibrosis and bronchiectasis.

For 50 years, macrolide antibiotics have been used to treat community acquired pneumonia and atypical infections such as Chlamydia pneumonia and Mycoplasma. In the late 1960s it was noted that when the 14-member ring macrolide antibiotic troleandomycin was given to asthma patients who required large doses of systemic corticosteroids, they could often reduce their steroid dose or even stop steroids completely without exacerbation of their asthma. Because of this experience, Prof. S. Kodoh and colleagues first used erythromycin as an immunomodulatory agent to treat diffuse panbronchiolitis (DPB). DPB is a cystic fibrosis (CF)-like condition seen predominantly in young, nonsmoking adults in Japan and Korea. The introduction of erythromycin profoundly improved survival, and in many of these very ill patients the illness disappeared. Since then, research has focused attention on many non-antibacterial, disease modifying effects of this class of compounds. These include downregulation of proinflammatory cytokines via an effect on nuclear transcription factors, reduction in adhesion molecule expression, suppression of inducible nitric oxide synthase (iNOS), reduced neutrophil chemotaxis and degranulation, inhibition of neutrophil elastase, cytoprotection against bioactive phospholipids, improvement in the rheological properties of mucus, reduction in bronchial hyperreactivity, and, perhaps, modulation of neutrophil death by apoptosis pathways, and in the end, airway remodeling. Additionally, they have unconventional effects on microorganisms, including inhibiting Pseudomonas aeruginosa twitching motility and thus biofilm formation. There are small case series and three large randomized controlled trials that have established unequivocal evidence of benefit in CF. There is less evidence for an immunomodulatory effect in bronchiectasis. Future work is likely to focus on the development of macrolides with disease-specific modes of action.

Journal Article↗
PubMed2001

A pilot evaluation of alfentanil-induced miosis as a noninvasive probe for hepatic cytochrome P450 3A4 (CYP3A4) activity in humans.

OBJECTIVE: The opioid alfentanil is a CYP3A4 substrate whose plasma clearance depends exclusively on hepatic CYP3A4 activity. Alfentanil clearance is an excellent in vivo probe for hepatic CYP3A4 activity and drug interactions in humans. However, such pharmacokinetic studies are invasive and time-consuming, and they require extensive analytical effort. This investigation tested the hypothesis that alfentanil-induced miosis (drug effect) can be used as a surrogate measure for alfentanil plasma concentrations and that alfentanil effect clearance will reflect plasma clearance; thus alfentanil can serve as a noninvasive probe for hepatic CYP3A4. METHODS: Six healthy volunteers were studied in a 3-way randomized crossover design. Each volunteer received 1 mg intravenous midazolam, followed 1 hour later by 15 microg/kg intravenous alfentanil, after CYP3A4 induction (rifampin [INN, rifampicin]), CYP3A4 inhibition (troleandomycin), and control. Dark-adapted pupil diameter and dynamic light response were measured coincident with venous blood sampling for up to 8 hours. Midazolam and alfentanil were quantified by gas chromatography-mass spectrometry. Plasma concentrations of alfentanil and midazolam (an additional CYP3A4 probe) and pupil diameter versus time data were analyzed by use of noncompartmental modeling. Pupil diameter change was analyzed analogously to determine the area under the alfentanil effect (miosis)-time curve (AUEC), effect clearance (CL(miosis)), and effect half-time. RESULTS: Compared with control, CYP3A4 induction and inhibition significantly altered the clearances of alfentanil (2.8 +/- 1.4, 5.3 +/- 1.0, and 0.42 +/- 0.1 ml/kg/min, respectively; P <.05 versus control) and midazolam. Dark-adapted resting diameter (in millimeters) was the best measure of alfentanil pupil effects. Alfentanil-dependent miosis was significantly altered by CYP3A4 modulation, and log(diameter(0) - diameter(t)) versus time curves resembled alfentanil plasma disposition. AUEC(infinity) values after control, CYP3A4 induction, and inhibition were 280 +/- 150, 120 +/- 22, and 1030 +/- 240 mm x min, respectively (P <.05 versus control). Effect clearances (CL(miosis)) were 4.2 +/- 1.3, 8.8 +/- 2.4, and 1.2 +/- 0.8 microg/mm x min, respectively, and effect half-times were 62 +/- 23, 34 +/- 27, and 211 +/- 35 minutes, respectively (P <.05 versus control). CL(miosis) was significantly correlated with plasma clearances of alfentanil (r = 0.77, P <.001) and midazolam (r = 0.80; P <.001). CONCLUSIONS: Alfentanil effect (miosis) may be a sensitive and reliable surrogate for plasma alfentanil concentrations. Alfentanil effect kinetics may be used as a noninvasive surrogate for conventional pharmacokinetics. CL(miosis) appears to be a suitable noninvasive in vivo probe for hepatic CYP3A4 activity, and it merits further investigation.

Adult↗
PubMed2000

Interspecies comparison and role of human cytochrome P450 and flavin-containing monooxygenase in hepatic metabolism of L-775,606, a potent 5-HT(1D) receptor agonist.

1. Quantitative species differences and human liver enzymes involved in the metabolism of L-775,606, a potent and selective 5-HT1D receptor agonist developed for the acute treatment of migraine headache, have been investigated in vitro. 2. In human, monkey, dog and rat liver microsomes, formation of the hydroxylated M1 and the N-dealkylated M2 was mediated by enzyme(s) of high-affinity (apparent Km approximately 1-6 microM), and that of the two N-oxide isomers (M3) was catalysed by those of low affinity (apparent Km approximately 50-110 microM). In dog, M3 constituted a major pathway (approximately 40%), whereas in all other species it was a minor metabolite (< 5%). 3. In human liver microsomes, a marked inhibition (> or =80%) of M1 and M2 formation was observed by SKF525-A, troleandomycin, ketoconazole and anti-CYP3A antibodies, whereas the inhibition was modest (approximately 20-40%) with quercetin. Of seven cDNA-expressed human P450 tested, only CYP3A4 and CYP2C8 were capable of oxidizing L-775,606, resulting primarily in M1 and M2. However, CYP3A4 possessed much higher affinity (> or = 20-fold) and much higher intrinsic activity (> 100-fold) than CYP2C8. 4. In contrast, N-oxidation was not inhibited by any inhibitors of P450 tested, but rather was reduced significantly by heat treatment and methimazole, and was increased substantially with an incubation pH>7.4. Human flavin-containing monooxygenase form 3 (FMO3) catalysed exclusively the N-oxidation to M3, with apparent Km and optimum pH comparable with those observed in human liver microsomes. 5. These results demonstrated quantitative interspecies differences in the metabolism of L-775,606. In human, metabolism of L-775,606 to the principal metabolites, M1 and M2, was mediated primarily by CYP3A4 with minimal contribution from CYP2C8, whereas the minor N-oxidative pathway was catalysed mainly by FMO3.

Animals↗
PubMed2000

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes.

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax of the rifalazil-25-deacetylation in microsomes were 6.5 microM and 11.9 pmol/min/mg with NADPH, and 2.6 microM and 6.0 pmol/min/mg without NADPH, indicating that, although rifalazil-25-deacetylation did not require NADPH, NADPH activated it. Rifalazil-32-hydroxylation was NADPH dependent, and its Km and Vmax were 3.3 microM and 11.0 pmol/min/mg respectively. 3. Rifalazil-25-deacetylation in microsomes was completely inhibited by diisopropyl fluorophosphate, diethyl p-nitrophenyl phosphate and eserine, but not by p-chloromercuribenzoate or 5,5'-dithio-bis(2-nitrobenzoic acid), indicating that the enzyme responsible for the rifalazil-25-deacetylation is a B-esterase. 4. Rifalazil-32-hydroxylation in microsomes was completely inhibited by CYP3A4-specific inhibitors (fluconazole, ketoconazole, miconazole, troleandomycin) and drugs metabolized by CYP3A4 such as cyclosporin A and clarithromycin, indicating that the enzyme responsible for the rifalazil-32-hydroxylation is CYP3A4.

Acetylation↗
PubMed2000

Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (+/- SEM) Km and Vmax of 8.3 +/- 1.3 microM and 454 +/- 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 microM (i.e. about two and six times Km respectively). With 20 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 microM sulphaphenazole, 50-500 microm S-mephenytoin and 2-50 microM quinidine had little effect. 6. The metabolism of 20 microM BFC to HFC in human liver microsomes was also inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8/9 and CYP2D6 had no effect. 7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms. 8. The results also demonstrate the importance of the selection of an appropriate substrate concentration when conducting reaction phenotyping studies with human hepatic CYP isoforms.

Antibodies↗