L. L. von Moltke

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.

Alprazolam Metabolism in vitro: Studies of Human, Monkey, Mouse, and Rat Liver Microsomes

Biotransformation of the triazolobenzodiazepine alprazolam (ALP) was studied in vitro using hepatic microsomal preparations from human, monkey, mouse, and rat liver tissue. Two principal hydroxylated metabolites were identified: 4-hydroxy- and alpha-hydroxy-alprazolam (4-OH-ALP and alpha-OH-ALP). In all species, rates of 4-OH-ALP formation exceeded those of alpha-OH-ALP. In human liver microsomes, ratios of 4-OH-ALP/alpha-OH-ALP reaction velocities calculated at clinically relevant plasma concentrations of ALP ranged from 7 to 17, qualitatively consistent with, but numerically larger than, the ratio of the plasma levels of the two metabolites during clinical use of ALP in humans. Km values for both 4-OH-ALP (170-305 microM) and alpha-OH-ALP (63-441 microM) considerably exceeded the usual maximum plasma concentration observed in humans (200 ng/ml, 0.65 microM), consistent with the linear (dose-independent) pharmacokinetic characteristics of ALP observed in humans. Thus formation of 4-OH-ALP via hydroxylation is the major route of ALP metabolism. This pathway is probably mediated by the cytochrome P-450-3A subfamily. Factors that impair the activity of this cytochrome subtype are likely to impair clearance of ALP in vivo.

Potent mechanism-based inhibition of human CYP3A in vitro by amprenavir and ritonavir: comparison with ketoconazole

AbstractObjective: Biotransformation of triazolam to its α-hydroxy and 4-hydroxy metabolites by human liver microsomes in vitro was used as an index of human cytochrome P450 3A (CYP3A) activity. Results: The reaction was strongly inhibited by co-incubation with the viral protease inhibitors ritonavir (IC50=0.14 μM) and amprenavir (IC50=2.5–2.9 μM), and by the azole derivative ketoconazole (IC50 = 0.07 μM). Pre-incubation of microsomes with ritonavir or amprenavir increased inhibitory potency (IC50 reduced to 0.07 μM and 1.4 μM, respectively). This was not the case with ketoconazole. Conclusions: Thus, ritonavir and amprenavir are highly potent mechanism-based inhibitors of human CYP3A isoforms.

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.

Ritonavir and dexamethasone induce expression of CYP3A and p-glycoprotein in rats

1. The consequences of extended exposure to the human immunodeficiency viral protease inhibitor ritonavir (RIT) on the expression and function of CYP3A isoforms in the liver and in enteric mucosal cells, and on the expression of the efflux transport protein P-glycoprotein (P-gp) in enteric mucosa and in brain microvessel endothelial cells, were evaluated in rat. Dexamethasone (DEX), a known inducer of CYP3A and P-gp in rodents, served as a positive control. 2. Male CD-1 rats received RIT (20 mg kg−1), DEX (80 mg kg−1) or vehicle by oral/duodenal gavage once daily for 3 days. 3. Compared with vehicle control, CYP3A activity in liver microsomes (intrinsic clearance for triazolam hydroxylation in vitro) was increased by a factor of 2–4 by RIT, and by 10–14-fold by DEX. Similar increases were observed in expression of immunoactive CYP3A protein. Overall, maximum reaction velocity and immunoactive protein were highly intercorrelated (r2 = 0.89). Both RIT and DEX also increased function and expression of enteric CYP3A, although to a more modest extent (about 1.7-fold for RIT, about 3.3-fold for DEX). 4. Enteric P-gp expression was equally induced (by 2.8-fold) by both RIT and DEX. P-gp expressed in brain microvessel endothelial cells was increased by a factor of 1.3 by both compounds. 5. Thus, increased expression of CYP3A isoforms and of P-gp occurs with 3 days of exposure to RIT in rats. Qualitatively similar changes occur in human cell culture models and in clinical studies, and might contribute to drug interactions involving RIT (and other antiretroviral agents) in humans.

Pharmacokinetic and pharmacodynamic interactions between zolpidem and caffeine

The kinetic and dynamic interaction of caffeine and zolpidem was evaluated in a double‐blind, single‐dose, six‐way crossover study of 7.5 mg zolpidem (Z) or placebo (P) combined with low‐dose caffeine (250 mg), high‐dose caffeine (500 mg), or placebo. Caffeine coadministration modestly increased maximum plasma concentration (Cmax) and area under the plasma concentration‐time curve of zolpidem by 30–40%, whereas zolpidem did not significantly affect the pharmacokinetics of caffeine or its metabolites. Compared to P+P, Z+P significantly increased sedation, impaired digit‐symbol substitution test performance, slowed tapping speed and reaction time, increased EEG relative beta amplitude, and impaired delayed recall. Caffeine partially, but not completely, reversed most pharmacodynamic effects of zolpidem. Thus, caffeine only incompletely reverses zolpidems sedative and performance‐impairing effects, and cannot be considered as an antidote to benzodiazepine agonists.

Serotonin (5-hydroxytryptamine) glucuronidation in vitro : assay development, human liver microsome activities and species differences

1. The main purpose was to develop a high-performance liquid chromatography (HPLC)-based method to assay serotonin glucuronidation activity using liver microsomal fractions. Application of this method was then demonstrated by determining serotonin UDP-glucuronosyltransferase (UGT) enzyme kinetics using human liver microsomes and recombinant human UGT1A6. Interspecies differences were also evaluated using liver microsomes from 10 different mammalian species. 2. Incubation of liver microsomes with serotonin, UDP-glucuronic acid and magnesium resulted in the formation of a single product peak using HPLC with fluorescence and ultraviolet absorbance detection. This peak was confirmed as serotonin glucuronide based on sensitivity to β-glucuronidase and by obtaining the expected mass of 352 with positive-ion mass spectrometry. 3. Following a preparative HPLC isolation, the structure of this metabolite was established as serotonin-5- O -glucuronide by 1 H-NMR spectroscopy. 4. Enzyme kinetic studies showed apparent K m and V max of 8.8 ±0.3 mM and 43.4 ±0.4 nmoles min <1 mg <1 protein, respectively, for human liver microsomes, and 5.9 ±0.2 mM and 15.8 ±0.2 nmoles min <1 mg <1, respectively, for recombinant UGT1A6. 5. The order of serotonin-UGT activities in animal liver microsomes was rat > mouse > human > cow > pig > horse > dog > rabbit > monkey > ferret. Cat livers showed no serotonin-UGT activity. Heterozygous and homozygous mutant Gunn rat livers had 40 and 13%, respectively, of the activity of the normal Wistar rat, indicating a significant contribution by a rat UGT1A isoform to serotonin glucuronidation. 6. This assay provides a novel sensitive and specific technique for the measurement of serotonin-UGT activity in vitro.

Identification of polymorphisms in the 3′-untranslated region of the human pregnane X receptor (PXR) gene associated with variability in cytochrome P450 3A (CYP3A) metabolism

Single nucleotide polymorphisms in the 3′-untranslated region (3′UTR) of the human pregnane X receptor (PXR) gene might contribute to interindividual variability in cytochrome P450 3A (CYP3A) activity. Genotype–phenotype associations involving PXR-3′UTR single nucleotide polymorphisms were investigated through in vitro (53 human livers from primarily White donors) and in vivo (26 mainly White or African-American volunteers) studies using midazolam 1′-hydroxylation and midazolam apparent oral clearance (CL/F), respectively, as CYP3A-specific probes. PXR-3′UTR resequencing identified twelve single nucleotide polymorphisms, including two that were novel. Although none of the single nucleotide polymorphisms evaluated were associated with altered midazolam 1′-hydroxylation in the liver bank, both rs3732359 homozygotes and rs3732360 carriers showed 80% higher (p < 0.05) CL/F compared with homozygous reference individuals. These differences in CL/F were even larger (100% and 120% higher, respectively; p < 0.01) when only African-American subjects (n = 14) were considered. Five major haplotypes were identified containing the PXR-3′UTR single nucleotide polymorphisms and previously identified intron single nucleotide polymorphisms. Although CL/F differences were not statistically significant within the entire study cohort, African-American carriers of Haplotype-1 (which includes both rs3732359 and rs3732360 variants) exhibited 70% higher median CL/F compared with African-American non-carriers (p = 0.036). The results identify rs3732359 and rs3732360 as PXR-3′UTR single nucleotide polymorphisms associated with higher CYP3A activity in vivo in African-Americans.

Gepirone and 1-(2-pyrimidinyl)-piperazine in vitro: human cytochromes mediating transformation and cytochrome inhibitory effects

Abstract Biotransformation of gepirone to its principal metabolite, 1-(2-pyrimidinyl)-piperazine (1-PP), was studied in human liver microsomes and in microsomes from cDNA-transfected human lymphoblastoid cells. Formation of 1-PP from gepirone in liver microsomes proceeded with a mean apparent Km ranging from 335 to 677 μM. Coincubation with 1 μM ketoconazole reduced reaction velocity to less than 5% of control values at a gepirone concentration of 250 μM. Three other metabolites, presumed to be hydroxylated products, were also formed from gepirone. Formation of all three products was reduced to approximately 20% of control values by 1 μM ketoconazole; quinidine at 1 μM produced a small reduction in formation (91–94% of control) of two of the metabolites. 1-PP was formed from gepirone exclusively by pure P450-3A4 with a Km of 849 μM; Km values for the other metabolites were 245, 240, and 415 μM. Two of the products were also formed by P450-2D6. The results indicate that 3A4 is the principal cytochrome mediating 1-PP formation, as well as formation of the other metabolites. The properties of gepirone and 1-PP themselves as cytochrome inhibitors were tested in human liver microsomes using index reactions representing activitiy of P450-1A2, -2C9, -2C19, -2D6, -2E1 and -3A. Gepirone and 1-PP produced negligible inhibition of all these reactions. Thus gepirone at therapeutic doses in humans has a low likelihood of inhibiting P450-mediated drug metabolism involving these cytochromes.