Interaction of miconazole, ketoconazole and itraconazole with rat-liver microsomes

The interaction of the antimycotics miconazole, ketoconazole and itraconazole with liver microsomes from untreated rats or from rats pretreated with phenobarbital or 3-methylcholanthrene, gave rise to type II difference spectra. The interactions of the antimycotics with control, phenobarbital-induced or 3-methylcholanthrene-induced microsomes were biphasic, except for the monophasic binding of ketoconazole to phenobarbital-induced microsomes. The N-demethylation of N,N-dimethylaniline, the O-demethylation of p-nitroanisole and the hydroxylation of aniline in microsomes from untreated and inducer-treated rats were lowered by miconazole and ketoconazole, the former being the more potent inhibitor. Control microsomes were less sensitive than induced microsomes. Itraconazole was almost devoid of inhibitory properties. The three antimycotics were non-competitive (mixed) inhibitors of enzyme activities in phenobarbital-induced microsomes. The Ki values were of the same order of magnitude as the Ks values, except for itraconazole. For the latter drug, Ki values were much greater than could be expected from the spectral studies. It is concluded that the antimycotics affect microsomal enzyme activities via a direct interaction of an azole-nitrogen with the haem group of cytochrome P-450. The interaction with mammalian cytochrome P-450 decreases from miconazole greater than ketoconazole much greater than itraconazole and is much weaker than the interaction of the antimycotics with yeast cytochrome P-450.     

Comparative effects of the antimycotic drugs ketoconazole, fluconazole, itraconazole and terbinafine on the metabolism of cyclosporin by human liver microsomes.

Four antimycotic drugs, the azoles ketoconazole, itraconazole and fluconazole, and the allylamine terbinafine have been studied for their effect on the metabolism of cyclosporin by human liver microsomes (n = 3) in vitro. Ketoconazole caused marked inhibition of cyclosporin hydroxylase (to metabolites M17 and M1) with IC50 and Ki values of 0.24 +/- 0.01 and 0.022 +/- 0.004 microM, respectively. Based on IC50 values, itraconazole was ten times less potent (IC50 value of 2.2 +/- 0.2 microM) and both fluconazole and terbinafine had values above 100 microM. Ki values for itraconazole and fluconazole were 0.7 +/- 0.2 and 40 +/- 5.6 microM, respectively. No kinetic parameters were calculated for terbinafine because of the lack of inhibitory effects. Based on these data, ketoconazole is confirmed as being a potent inhibitor of cyclosporin metabolism and this has clinical relevance. Although inhibition by fluconazole was much less than that by itraconazole at equimolar concentrations, it should be noted that in patients plasma concentrations of fluconazole are much greater than those of itraconazole. Clinical interactions of cyclosporin with both fluconazole and itraconazole have been reported. In contrast to the azoles, terbinafine does not have the same potential for interaction.     

Terfenadine-antidepressant interactions: an in vitro inhibition study using human liver microsomes

AIMS: Inhibition of the metabolism of terfenadine has been associated with torsades de pointes ventricular arrhythmias. The aim of this study was to assess in vitro the potency of the antidepressants nefazodone, sertraline and fluoxetine in inhibiting terfenadine biotransformation. METHODS: Human liver microsomes were incubated with terfenadine and the antidepressants at various concentrations. Formation of the two major metabolites of terfenadine was determined by h.p.l.c. RESULTS: The apparent Km for microsomes from four human livers was 11+/-5 and 18+/-3 microM (mean +/-s.e.mean) for the N-dealkylation and C-hydroxylation pathways, respectively. Nefazodone, sertraline and fluoxetine inhibited terfenadine N-dealkylation with K(i) values of 10+/-4, 10+/-3 and 68+/-15 microM respectively. Inhibition of the C-hydroxylation pathway yielded noncompetitive K(i) values of 41+/-4, 67+/-13 and 310+/-40 microM respectively. CONCLUSIONS: Nefazodone and sertraline were moderately weak in vitro inhibitors of terfenadine metabolism while fluoxetine was a very weak inhibitor. Clinically significant interaction of terfenadine is more likely with nefazodone than sertraline or fluoxetine since therapeutic plasma levels of nefazodone are comparatively higher.     

Phenacetin O-deethylation by human liver microsomes in vitro: inhibition by chemical probes,SSRI antidepressants,nefazodone and venlafaxine

Biotransformation of phenacetin via O-deethylation to acetaminophen, an index reaction reflecting activity of Cytochrome P450-1A2, was studied in microsomal preparations from a series of human livers. Acetaminophen formation was consistent with a double Michaelis-Menten system, with low-K_m (mean K_m1 = 68 μM) and high-K_m (mean K_m2 = 7691 μM) components. The low-K_m enzyme accounted for an average of 96% of estimated intrinsic clearance, and was predicted to contribute more than 50% of net reaction velocity at phenacetin concentrations less than 2000 μM. Among index inhibitor probes, α-naphthoflavone was a highly potent inhibitor of the low-K_m enzyme (K_i1 = 0.013 μM); furafylline also was a moderately active inhibitor (K_i1 = 4.4 μM), but its inhibiting potency was increased by preincubation with microsomes. Ketoconazole was a relatively weak inhibitor (K_i1 = 32 μM); quinidine and cimetidine showed minimal inhibiting activity. Among six selective serotonin reuptake inhibitor (SSRI) antidepressants, fluvoxamine was a potent inhibitor of 1A2 (mean K_i1 = 0.24 μM). The other SSRIs were more than tenfold less potent. Mean K_i1 values were: fluoxetine, 4.4 μM; norfluoxetine, 15.9 μM; sertraline, 8.8 μM; desmethylsertraline, 9.5μM; paroxetine, 5.5 μM. The antidepressant nefazodone and four of its metabolites (meta-chloro-phenylpiperazine, two hydroxylated derivatives, and a triazoledione) were very weak inhibitors of P450-1A2. Venlafaxine and its O- and N-desmethyl metabolites showed minimal inhibitory activity. Received: 18 March 1996/Final version: 10 July 1996     

Calcium channel antagonists and cyclosporine metabolism: in vitro studies with human liver microsomes.

The effects of four Ca2+ channel antagonists on the metabolism of cyclosporine (CsA) by human liver microsomes (n = 4) in vitro have been examined. Nicardipine produced marked inhibition of both M17 and M21 (IC50 = 7.0 microM) formation. In contrast nifedipine produced less than 20% inhibition of M17 and M21 even at the highest concentration examined (50 microM). Diltiazem data were comparable to those for nifedipine. Verapamil (50 microM) produced 30 and 28% inhibition of M17 and M21 formation, respectively. These findings give a basis to the increase in CsA blood concentrations seen in transplant patients who are also given nicardipine.     

Diazepam–omeprazole inhibition interaction: an in vitro investigation using human liver microsomes

1 The metabolism of diazepam to its primary metabolites 3-hydroxydiazepam (3HDZ) and nordiazepam (NDZ) was evaluated in human liver microsomes. The 3HDZ pathway was the major route of metabolism representing 90% of total metabolism with a V _max /K _m ratio of 0.50–7.26  μl  min⁻¹  mg ⁻¹ protein.
2 Inhibition of the two metabolic pathways of diazepam by omeprazole was investigated. The NDZ pathway was not affected by omeprazole whilst a K _i of 201±89  μm was obtained for the 3HDZ pathway ( K _m /K _i ratio of 3.0±0.9).
3 Inhibitory effects of omeprazole sulphone on the 3HDZ and NDZ pathways were also investigated. Omeprazole sulphone inhibited both pathways with similar K_is of 121±45 and 188±73  μm respectively ( K _m /K _i ratios of 5.2±2.3 and 3.3±1.5 respectively).
4 These in vitro data provide direct evidence for cytochrome P450 inhibition as the mechanism for the well documented diazepam-omeprazole clinical interaction and indicate that omeprazole sulphone, as well as the parent drug, contribute to the inhibition effect.     

体外人肝微粒体中阿米替林N-去甲基代谢的酶促动力学分析和抑制

采用6例人的肝微粒体应用酶促动力学分析和抑制研究阐明参与阿米替林（AT）N-去甲基代谢的CYP450的种类及性质. 去甲替林（NT）生成的酶促动力学数据符合一两酶模型，其中高亲和力酶具有Michaelis-Menten 动力学特征，而低亲和力酶则具有底物别构激活的特性. 当AT为2 μmol·L^-1时S-美芬妥英和呋喃茶碱均可使NT生成被抑制达50%左右；较高浓度的酮康唑也可使NT生成明显受到抑制，但醋竹桃霉素几乎对此没有影响; 而当AT为100 μmol·L^-1 时，酮康唑和醋竹桃霉素均是NT生成的强抑制剂. 结果提示：当底物的浓度较低时，CYP1A2和CYP2C19是催化AT体外人肝微粒体中N-去甲基代谢的主要CYP450酶类；当底物浓度较高时，由于底物别构激活的特性，CYP3A4逐渐占据主导地位.     

Catalepsy induced by calcium channel blockers in mice

It is known that calcium channel blockers induce Parkinsonism. In this study, amlodipine-, diltiazem-, and verapamil-induced catalepsy was investigated in mice. All of these three calcium channel blockers induced catalepsy. Dopamine D₁, D₂, and mACh receptor occupancies were estimated under the same conditions, and the affinities of these drugs for each receptor were also estimated in vitro. Intensity of catalepsy was predicted by dopamine D₁, D₂, and mACh receptor occupancies with the dynamic model which had already been constructed and was compared with the observed values. The predicted and the observed values were comparable (r = 0.98, p <0.001). In conclusion, the dynamic model considering D₁, and D₂, and mACh receptor occupancy may be useful for quantitative prediction of drug-induced catalepsy. © 1998 John Wiley & Sons, Ltd.     

Metabolism of lovastatin by rat and human liver microsomes in vitro

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry. One fraction consisted of a 2:1 mixture of 6-hydroxy-lovastatin and the rearrangement product delta 4,5-3-hydroxy lovastatin. Addition of a trace of acid to this mixture resulted in the formation of a single aromatized product, the desacyl-delta 4a,6,8-dehydro analog of lovastatin. Another microsomal metabolite was determined to be the delta 4,8a,1-3-hydroxy-lovastatin derivative. The chromatographic pattern of metabolites produced from lovastatin by human liver microsomes was similar to that obtained with rat liver microsomes. Metabolism of lovastatin by rat liver microsomes was both time and concentration dependent; optimal microsomal metabolism occurred with 0.1 mM lovastatin, whereas higher lovastatin concentrations inhibited the reaction. The open acid form of lovastatin was poorly metabolized by both the rat and the human liver microsomes.     

Enzyme kinetic analysis and inhibition of amitriptyline N-demethylation in human liver microsomes in vitro

采用６例人的肝微粒体应用酶促动力学分析和抑制研究阐明参与阿米替林（ＡＴ）Ｎ－去甲基代谢的ＣＹＰ４５０的种类及性质．去甲替林（ＮＴ）生成的酶促动力学数据符合一两酶模型，其中高亲和力酶具有Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ动力学特征，而低亲和力酶则具有底物别构激活的特性．当ＡＴ为２μｍｏｌ·Ｌ－１时Ｓ－美芬妥英和呋喃茶碱均可使ＮＴ生成被抑制达５０％左右；较高浓度的酮康唑也可使ＮＴ生成明显受到抑制，但醋竹桃霉素几乎对此没有影响；而当ＡＴ为１００μｍｏｌ·Ｌ－１时，酮康唑和醋竹桃霉素均是ＮＴ生成的强抑制剂．结果提示：当底物的浓度较低时，ＣＹＰ１Ａ２和ＣＹＰ２Ｃ１９是催化ＡＴ体外人肝微粒体中Ｎ－去甲基代谢的主要ＣＹＰ４５０酶类；当底物浓度较高时，由于底物别构激活的特性，ＣＹＰ３Ａ４逐渐占据主导地位．     

In vitro inhibition studies of tolbutamide hydroxylase activity of human liver microsomes by azoles, sulphonamides and quinolines.

1. A number of compounds have been examined for their ability to inhibit tolbutamide hydroxylase activity in human liver microsomes (control value at a substrate concentration of 150 microM being 0.27 +/- 0.12 nmol min-1 mg-1 protein; mean +/- s.d.; n = 7). 2. IC50 (concentration of inhibitor producing 50% inhibition) values were determined for a range of sulphonamides, imidazoles and aminoquinoline compounds. The most potent inhibition was evident with the 1-substituted imidazole antimycotic drugs ketoconazole, clotrimazole and miconazole and the sulphonamide sulphaphenazole (IC50 values of 16.5, 2.5, 0.85 and 0.5 microM respectively). A number of compounds showed little or no inhibition of tolbutamide hydroxylase as judged by an IC50 of greater than or equal to 500 microM. 3. The Km value for tolbutamide hydroxylase was 125 microM and Vmax, 0.44 nmol min-1 mg-1 protein. All the substituted imidazoles examined in kinetic studies 1v vs 1s, Line-weaver-Burk plots) produced either non-competitive or mixed inhibition. The sulphonamides exhibited competitive inhibition, the Ki for sulphaphenazole being 0.22 microM. Primaquine showed mixed inhibition. Dixon plots confirmed the type of inhibition produced. 4. Although the competitive inhibition between some sulphonamides and tolbutamide is consistent with metabolism by the same isozyme of cytochrome P-450 it does not prove it and further studies with purified enzymes will be necessary to confirm this.     

Cyclosporin metabolism in human liver microsomes and its inhibition by other drugs

The metabolism of cyclosporin was studied in human liver microsomes. There was no metabolism in the presence of cytochrome C or carbon monoxide or in the absence of cofactors, suggesting metabolism by cytochrome P-450 enzymes. The metabolism was inhibited by ketoconazole and erythromycin, by the steroids methylprednisolone and oestradiol, and by the calcium antagonists diltiazem, nifedipine, prenylamine and verapamil. These in vitro findings correlate well with previously published clinical reports suggesting that these drugs may inhibit the metabolism of cyclosporin in vivo. Our observations suggest that metabolic interactions between cyclosporin and other drugs in vivo may be predicted in vitro under proper experimental conditions.     

Effect of calcium channel blockers and trifluoperazine on rat liver regeneration

The activity of hepatic thymidylate synthetase and thymidine kinase at 24 h after 70% partial hepatectomy of rats was suppressed significantly compared with that in the control group by the administration of calcium channel blockers (verapamil, diltiazem and nifedipine) 8 h after partial hepatectomy. The decrease of thymidylate synthetase and thymidine kinase activities was accompanied by a reduction of DNA content in 24 h regenerating liver. Trifluoperazine showed an effect similar to that of the calcium channel blockers on DNA synthesis during liver regeneration. These results suggest that calcium entry into the hepatic cell is an essential event in liver regeneration.     

Comparative effects of two antimycotic agents, ketoconazole and terbinafine on the metabolism of tolbutamide, ethinyloestradiol, cyclosporin and ethoxycoumarin by human liver microsomes in vitro.

Two antimycotic agents, the azole ketoconazole and the allylamine terbinafine, have been examined for their effects on the metabolism of tolbutamide, ethinyloestradiol, cyclosporin and ethoxycoumarin by human liver microsomes (n = 4) in vitro. Ketoconazole caused marked inhibition of all enzyme activities with mean IC50 values (concentration producing 50% inhibition) of 17.9 microM (tolbutamide hydroxylase), 1.9 microM (ethinyloestradiol 2-hydroxylase), 2.0 microM (cyclosporin N-demethylase), 2.1 microM (cyclosporin hydroxylase) and 25 microM (ethoxycoumarin O-deethylase). At 50 microM terbinafine concentration, inhibition was less than 5% for tolbutamide and ethoxycoumarin, approximately 12% for both cyclosporin pathways and 35% for ethinyloestradiol. Terbinafine does not have the same inhibitory potential for cytochrome P-450 isozymes as ketoconazole.     

Release of secretoneurin and noradrenaline from hypothalamic slices and its differential inhibition by calcium channel blockers

Secretoneurin is a newly discovered peptide found in high concentrations in brain. We have studied the release of secretoneurin and noradrenaline from superfused hypothalamic slices from rat brain. Both electrical stimulation and potassium induced depolarisation released secretoneurin and noradrenaline from these slices in a calcium-dependent manner. Electrical stimulation caused a preferential release of noradrenaline when compared to the secretion elicited by high potassium. The time course of secretoneurin release was more protracted than that of noradrenaline. The calcium channel blocker -conotoxin inhibited only the electrically induced release of noradrenaline, whereas nifedipine inhibited only that of secretoneurin. These results establish that secretoneurin is secreted from neurons. Inhibition of this release by nifedipine is consistent with the concept that secretion from large dense core vesicles occurs at sites different from that of small vesicles and depends on calcium influx via L-type calcium channels. Correspondence to: H. Winkler at the above address     

西尼地平在人肝微粒体内代谢及代谢抑制

目的：在体外研究西尼地平在人肝微粒体内的代谢及选择性细胞色素P-450（CYP450）酶抑制剂对其代谢的影响。方法：在体外用人肝微粒体研究西尼地平的代谢,并用CYP450酶的选择性抑制剂探讨其对西尼地平代谢的影响及人肝微粒体中参与西尼地平二氢吡啶环脱氢代谢的CYP450酶。结果：西尼地平在人肝微粒体内被迅速代谢物M1,二氢吡啶环侧链脱甲基代谢物M2,二氢吡嘧环脱氢及其侧链脱甲基代谢物M3,酮康唑竞争性地抑制西尼地平二氢吡啶环的脱氢代谢,同时降低西尼地平的代谢速率,而其它抑制剂,奎尼丁,α-Naphthoflavone,diethyldithiocarbamate,sulfaphenazole和tra-nylcypromine对西尼地平二氢吡啶环的脱氢代谢没有明显的影响。结论：西尼地平在人肝微粒体内被迅速代谢,其二氢吡啶环的脱氢代谢是其代谢的关键性的步骤,CYP3A作为主要的CYP酶参与了西尼地平二氢吡啶环的脱氢代谢,CYP3A的抑制剂可能会与西尼地平发生代谢相互作用。     

Metabolic inhibition and kinetics of raloxifene by pharmaceutical excipients in human liver microsomes

This study was originally undertaken to establish the in vitro metabolic conditions and then evaluate the effect of pharmaceutical excipients (PEs) on drug metabolism in uridine diphosphoglucuronic acid-supplemented human liver microsomes. Poorly bioavailable raloxifene was chosen as a model drug. Intact drug and its two glucuronide metabolites were successfully isolated using gradient HPLC analysis and LC/MS analysis. Formation of raloxifene metabolites was affected by buffer compositions, incubation time and initial raloxifene concentrations. Under optimized metabolic conditions, 41.0% of raloxifene was converted to its metabolites after 2h incubation. This metabolic inhibition of raloxifene by the PEs occurred in a dose-dependent manner and accordingly formed two glucuronide metabolites. In the metabolic kinetics using Lineweaver-Burk analyses, Cremophor EL competitively inhibited formation of metabolites while sodium lauryl sulfate (SLS), polyvinylpyrrolidone K30 (PVP) and Tween 80 significantly inhibited in a mixed competition. Although some PEs showed inhibition on glucuronidation of raloxifene in vitro, their effects on in vivo bioavailability of raloxifene need to be confirmed directly due to the dilution factors and other complicated situations influencing the bioavailability.     

Quantitative prediction of metabolic inhibition of midazolam by itraconazole and ketoconazole in rats: implication of concentrative uptake of inhibitors into liver.

To evaluate the extent of drug-drug interaction concerning metabolic inhibition in the liver quantitatively, we tried to predict the plasma concentration increasing ratio of midazolam (MDZ) by itraconazole (ITZ) or ketoconazole (KTZ) in rats. MDZ was administered at a dose of 10 mg/kg through the portal vein at 60 min after bolus administration of 20 mg/kg ITZ or during 0.33 mg/h/body of KTZ infusion. The ratio values in the area under the plasma concentration curve of MDZ in the presence of ITZ and KTZ was 2.14 and 1.67, respectively. The liver-unbound concentration to plasma-unbound concentration ratios of ITZ and KTZ were 11 approximately 14 and 1.3, respectively, suggesting a concentrative uptake of both drugs into the liver. ITZ and KTZ competitively inhibited the oxidative metabolism of MDZ in rat liver microsomes, and Ki values of ITZ and KTZ were 0.23 microM and 0.16 microM, respectively. We predicted the ratio values of MDZ in the presence of ITZ and KTZ, using Ki values and unbound concentrations of both drugs in the plasma or liver. The predicted ratio values in the presence of ITZ or KTZ calculated by using unbound concentration in the plasma were 1.03 approximately 1.05 and 1.39, whereas those calculated using unbound concentration in the liver were 1.73 approximately 1.97 and 1.51, respectively, which were very close to the observed ratio values. These findings indicated the necessity to consider the concentrative uptake of inhibitors into the liver for the quantitative prediction of the drug-drug interactions concerning metabolic inhibition in the liver.