Jürgen Schmider

Midazolam Hydroxylation by Human Liver Microsomes In Vitro: Inhibition by Fluoxetine, Norfluoxetine, and by Azole Antifungal Agents

Biotransformation of the imidazobenzodiazepine midazolam to its α‐hydroxy and 4‐hydroxy metabolites was studied in vitro using human liver microsomal preparations. Formation of α‐hydroxy‐midazolam was a high‐affinity (Km = 3.3 μmol/L) Michaelis‐Menten process coupled with substrate inhibition at high concentrations of midazolam. Formation of 4‐hydroxy‐midazolam had much lower apparent affinity (57 μmol/L), with minimal evidence of substrate inhibition. Based on comparison of Vmax/Km ratios for the two pathways, α‐hydroxy‐midazolam formation was estimated to account for 95% of net intrinsic clearance. Three azole antifungal agents were inhibitors of midazolam metabolism in vitro, with inhibition being largely consistent with a competitive mechanism. Mean competitive inhibition constants (Ki) versus α‐hydroxy‐midazolam formation were 0.0037 μmol/L for ketoconazole, 0.27 μmol/L for itraconazole, and 1.27 μmol/L for fluconazole. An in vitro‐in vivo scaling model predicted inhibition of oral midazolam clearance due to coadministration of ketoconazole or itraconazole; the predicted inhibition was consistent with observed interactions in clinical pharmacokinetic studies. The selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine and its principal metabolite, norfluoxetine, also were inhibitors of both pathways of midazolam biotransformation, with norfluoxetine being a much more potent inhibitor than was fluoxetine itself. This finding is consistent with results of other in vitro studies and of clinical studies, indicating that fluoxetine, largely via its metabolite norfluoxetine, may impair clearance of P450‐3A substrates.

Five Distinct Human Cytochromes Mediate Amitriptyline N-Demethylation In Vitro: Dominance of CYP 2C19 and 3A4

The human cytochromes P450 (CYPs) mediating amitriptyline N‐demethylation have been identified using a combination of enzyme kinetic and chemical inhibition studies. Amitriptyline was N‐demethylated to nortriptyline by microsomes from cDNA transfected human lymphoblastoid cells expressing human CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. CYP 2E1 showed no detectable activity. While CYP 2C19 and CYP 2D6 showed high affinity, CYP 3A4 showed low affinity; CYP 2C9 and 1A2 showed intermediate affinities. Based on these kinetic parameters and estimated relative abundance of the different CYPs in human liver, CYP 2C19 was identified as the major amitriptyline N‐demethylase at low (therapeutically relevant) amitriptyline concentrations, whereas CYP 3A4 may be more important at higher amitriptyline concentrations. Chemical inhibition studies with ketoconazole and omeprazole indicate that CYP 3A4 is the major amitriptyline N‐demethylase at 100 μmol/L amitriptyline, while CYP 2C19 is equally important at a substrate concentration of 5 μmol/L. The CYP 1A2 inhibitor α‐naphthoflavone and the CYP 2C9 inhibitor sulfaphenazole produced much less inhibition of amitriptyline N‐demethylation at both substrate concentrations. Quinidine produced no detectable inhibition. The kinetics of amitriptyline N‐demethylation by human liver microsomes were consistent with a two enzyme model, with the high affinity component exhibiting Michaelis Menten kinetics and the low affinity component exhibiting Hill enzyme kinetics. No difference was apparent in the kinetics of amitriptyline N‐demethylation in two liver samples with low levels of CYP 2C19 activity compared with two other samples with relatively normal 2C19 activity. This may reflect the importance of higher substrate concentration values in estimation of kinetic parameters in vitro.

O- and N-demethylation of venlafaxine in vitro by human liver microsomes and by microsomes from cDNA-transfected cells: effect of metabolic inhibitors and SSRI antidepressants.

The biotransformation of venlafaxine (VF) into its two major metabolites, O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) was studied in vitro with human liver microsomes and with microsomes containing individual human cytochromes from cDNA-transfected human lymphoblastoid cells. VF was coincubated with selective cytochrome P450 (CYP) inhibitors and several selective serotonin reuptake inhibitors (SSRIs) to assess their inhibitory effect on VF metabolism. Formation rates for ODV incubated with human microsomes were consistent with Michaelis-Menten kinetics for a single-enzyme mediated reaction with substrate inhibition. Mean parameters determined by non-linear regression were: Vmax = 0.36 nmol/min/mg protein, Km = 41 μM, and Ks 22901 μM (Ks represents a constant which reflects the degree of substrate inhibition). Quinidine (QUI) was a potent inhibitor of ODV formation with a Ki of 0.04 μM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 μM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, −2C19, and −2D6. CYP2D6 was dominant with the lowest Km, 23.2 μM, and highest intrinsic clearance (Vmax/Km ratio). No unique model was applicable to the formation of NDV for all four livers tested. Parameters determined by applying a single-enzyme model were Vmax = 2.14 nmol/min/mg protein, and Km = 2504 μM. Ketoconazole was a potent inhibitor of NDV production, although its inhibitory activity was not as great as observed with pure 3A substrates. NDV formation was also reduced by 42% by a polyclonal rabbit antibody against rat liver CYP3A1. Studies using expressed cytochromes showed that NDV was formed by CYP2C9, −2C19, and −3A4. The highest intrinsic clearance was attributable to CYP2C19 and the lowest to CYP3A4. However the high in vivo abundance of 3A isoforms will magnify the importance of this cytochrome. Fluvoxamine (FX), at a concentration of 20 μM, decreased NDV production by 46% consistent with the capacity of FX to inhibit CYP3A, 2C9, and 2C19. These results are consistent with previous studies that show CYP2D6 and −3A4 play important roles in the formation of ODV and NDV, respectively. In addition we have shown that several other CYPs have important roles in the biotransformation of VF.

Metabolism of Drugs by Cytochrome P450 3A Isoforms

SummaryMembers of the P450 3A subfamily are the most abundant of the human hepatic cytochromes. CYP3A isoforms mediate the biotransformation of many drugs, including a number of psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents. Activity of CYP3A in humans is variable among individuals, but there is no evidence of genetic polymorphism. Significant amounts of CYP3A are present in the gastrointestinal tract, and may contribute to presystemic extraction of drugs such as cyclosporin. The azole antifungal agents ketoconazole and itraconazole are potent inhibitors of human CYP3A isoforms. Selective serotonin reuptake inhibitor (SSRI) antidepressants are also CYP3A inhibitors, but much less potent than ketoconazole or itraconazole. In vitro models can provide important information on the qualitative and quantitative activity of potential inhibitors of human cytochromes. However, in vitro inhibition constant (Ki) values alone do not predict the magnitude of an in vivo interaction, nor whether an interaction will be of clinical importance. For example, SSRIs are predicted to impair clearance of the antihistamine terfenadine in humans. However, the magnitude of this effect is much less than would be associated with a pharmacokinetic interaction of clinical importance.

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

Abstract 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-Km (mean Km1 = 68 μM) and high-Km (mean Km2 = 7691 μM) components. The low-Km 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-Km enzyme (Ki1 = 0.013 μM); furafylline also was a moderately active inhibitor (Ki1 = 4.4 μM), but its inhibiting potency was increased by preincubation with microsomes. Ketoconazole was a relatively weak inhibitor (Ki1 = 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 Ki1 = 0.24 μM). The other SSRIs were more than tenfold less potent. Mean Ki1 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.

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro : role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3‐methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA‐transfected human lymphoblastoid cells.

Metabolism of dextromethorphan in vitro: involvement of cytochromes P450 2D6 and 3A3/4, with a possible role of 2E1.

Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O‐demethylation to dextrorphan (DOP), and N‐demethylation to 3‐methoxy‐morphinan (MEM), in humans. 3‐hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O‐demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N‐demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4. DMO O‐demethylation displayed a biphasic pattern with a high‐affinity site reflecting CYP2D6 activity (mean Ki for quinidine, 0·1 ± 0·13 μM). Kinetic parameters for the two O‐demethylation mediating enzymes predict an average relative intrinsic clearance (Vmax/Km ratio) of 96% of total O‐demethylation mediated via the high‐affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O‐demethylation as an index reaction to monitor CYP2D6 activity. The Eadie–Hofstee plot of DMO N‐demethylation was consistent with single‐enzyme Michaelis–Menten kinetics (Vmax varying from 3·3 to 6·8 nmol mg−1 min−1, Km from 231 to 322 μM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N‐demethylation only by 60% and had a mean Ki an order of magnitude higher (0·37 μM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC50 varying from 1 to 2·6 μM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 μM between 33 and 43%. Chemical inhibitors of CYP2D6 (quinidine), CYP1A1/2 (α‐naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N‐demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo.

Nefazodone, meta-chlorophenylpiperazine, and their metabolites in vitro: cytochromes mediating transformation, and P450-3A4 inhibitory actions

Rationale: Understanding of the mechanisms of biotransformation of antidepressant drugs, and of their capacity to interact with other medications, is of direct relevance to rational clinical psychopharmacology. Objectives: To determine the human cytochromes P450 mediating the metabolism of nefazodone, and the inhibitory activity of nefazodone and metabolites versus human P450–3A. Methods: Biotransformation of nefazodone to its metabolic products, and of meta-chlorophenylpiperazine (mCPP) to para-hydroxy-mCPP, was studied in vitro using human liver microsomes and heterologously expressed human cytochromes. Nefazodone and metabolites were also tested as inhibitors of alprazolam hydroxylation, reflecting activity of cytochrome P450–3A isoforms. Results: mCPP and two hydroxylated derivatives were the principal metabolites formed from nefazodone by liver microsomes. Metabolite production was strongly inhibited by ketoconazole or troleandomycin (relatively specific P450–3A inhibitors), and by an anti-P450-3A antibody. Only heterologously expressed human P450-3A4 mediated formation of nefazodone metabolites from the parent compound. Nefazodone, hydroxy-nefazodone, and para-hydroxy-nefazodone were strong 3A inhibitors, being more potent than norfluoxetine and fluvoxamine, but less potent than ketoconazole. The triazoledione metabolite and mCPP had weak or negligible 3A-inhibiting activity. Formation of para-hydroxy-mCPP from mCPP was mediated by heterologously expressed P450-2D6; in liver microsomes, the reaction was strongly inhibitable by quinidine, a relatively specific 2D6 inhibitor. Conclusion: The complex parallel biotransformation pathways of nefazodone are mediated mainly by human cytochrome P450-3A, whereas clearance of mCPP is mediated by P450-2D6. Nefazodone and two of its hydroxylated metabolites are potent 3A inhibitors, accounting for pharmacokinetic drug interactions of nefazodone with 3A substrate drugs such as triazolam and alprazolam.

Human cytochromes mediating N-demethylation of fluoxetine in vitro

Abstract Biotransformation of the selective serotonin reuptake inhibitor antidepressant, fluoxetine, to its principal metabolite, norfluoxetine, was evaluated in human liver microsomes and in microsomes from transfected cell lines expressing pure human cytochromes. In human liver microsomes, formation of norfluoxetine from R,S-fluoxetine was consistent with Michaelis-Menten kinetics (mean Km = 33 μM), with evidence of substrate inhibition at high substrate concentrations in a number of cases. The reaction was minimally inhibited by coincubation with chemical probes inhibitory for P450-2D6 (quinidine), -1A2 (furafylline, α-naphthoflavone), and -2E1 (diethyldithiocarbamate). Substantial inhibition was produced by coincubation with sulfaphenazole (Ki = 2.8 μM), an inhibitory probe for P450-2C9, and by ketoconazole (Ki = 2.5 μM) and fluvoxamine (Ki = 5.2 μM). However, ketoconazole, relatively specific for P450-3A isoforms only at low concentrations, reduced norfluoxetine formation by only 20% at 1 μM, and triacetyloleandomycin (≥ 5 μM) reduced the velocity by only 20–25%. Microsomes from cDNA-transfected human lymphoblastoid cells containing human P450-2C9 produced substantial quantities of norfluoxetine when incubated with 100 μM fluoxetine. Smaller amounts of product were produced by P450-2C19 and -2D6, but no product was produced by P450-1A2, -2E1, or 3A4. Cytochrome P450-2C9 appears to be the principal human cytochrome mediating fluoxetine N-demethylation. P450-2C19 and -3A may make a further small contribution, but P450-2D6 is unlikely to make an important contribution.

Relative Contribution of CYP3A to Amitriptyline Clearance in Humans: In Vitro and In Vivo Studies

The relative contribution of cytochrome P450 3A (CYP3A) to the oral clearance of amitriptyline in humans has been assessed using a combination of in vitro approaches together with a clinical pharmacokinetic interaction study using the CYP3A‐selective inhibitor ketoconazole. Lymphoblast‐expressed CYPs were used to study amitriptyline N‐demethylation and E‐10 hydroxylation in vitro. The relative activity factor (RAF) approach was used to predict the relative contribution of each CYP isoform to the net hepatic intrinsic clearance (sum of N‐demethylation and E‐10 hydroxylation). Assuming no extrahepatic metabolism, the model‐predicted contribution of CYP3A to net intrinsic clearance should equal the fractional decrement in apparent oral clearance of amitriptyline upon complete inhibition of the enzyme. This hypothesis was tested in a clinical study of amitriptyline (50 mg, PO) with ketoconazole (three 200 mg doses spaced 12 hours apart) in 8 healthy volunteers. The RAF approach predicted CYP2C19 to be the dominant contributor (34%), with a mean 21% contribution of CYP3A (range: 8%‐42% in a panel of 12 human livers). The mean apparent oral clearance of amitriptyline in 8 human volunteers was decreased from 2791 ml/min in the control condition to 2069 ml/min with ketoconazole. The average 21% decrement (range: 2%‐40%) was identical to the mean value predicted in vitro using the RAF approach. The central nervous system (CNS) sedative effects of amitriptyline were slightly greater when ketoconazole was coadministered, but the differences were not statistically significant. In conclusion, CYP3A plays a relatively minor role in amitriptyline clearance in vivo, which is consistent with in vitro predictions using the RAF approach.