Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms.

Interindividual variability in acetaminophen (APAP) glucuronidation may contribute to differences in susceptibility to APAP intoxication in humans. The purpose of this study was to identify the relevant UDP-glucuronosyltransferase (UGT) isoforms mediating APAP-UGT activity in human liver microsomes (HLMs). APAP-UGT activities and enzyme kinetics were determined using HLMs from 56 donors and nine recombinant human UGTs. Activities mediated by UGT1A1, UGT1A4, UGT1A9, and UGT2B7, and relative UGT1A6 protein content were quantified using 20 livers. More than 15-fold variation in liver microsomal APAP-UGT activities was observed with a distribution skewed toward lower activities. Although most UGTs could glucuronidate APAP, UGT1A1, UGT1A6, and UGT1A9 were most active. UGT1A6 was a relatively high-affinity (K(m) = 2.2 mM), low-capacity enzyme; UGT1A1 was intermediate in affinity (K(m) = 9.4 mM) and capacity; and UGT1A9 was a low-affinity (K(m) = 21 mM), high-capacity enzyme. K(m) values were similar to UGT1A1 in high- and intermediate-activity HLMs (6-10 mM) and UGT1A9 in low-activity HLMs (10-55 mM). APAP-UGT activities correlated best with propofol-UGT (r = 0.85; UGT1A9) and bilirubin-UGT (r = 0.66; UGT1A1) activities, but poorly with UGT1A6 protein (r = 0.30). A kinetic model was constructed from these data that identified UGT1A9 as the predominant APAP-UGT (>55% total activity) in HLMs over a clinically relevant APAP concentration range (50 microM-5 mM). UGT1A1 was also predicted to contribute substantially at toxic concentrations (>1 mM; >28% activity), whereas UGT1A6 was most active at relatively low concentrations (<50 microM; >29% activity).     

True single-molecule DNA sequencing of a pleistocene horse bone

Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first "true single molecule sequencing" of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3' ends of ancient templates, indicating the presence of 3'-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second- and third-generation sequencing approaches.     

A genome-wide scan in affected sibling pairs with idiopathic recurrent miscarriage suggests genetic linkage

Previously, siblings of patients with idiopathic recurrent miscarriage (IRM) have been shown to have a higher risk of miscarriage. This study comprises two parts: (i) an epidemiological part, in which we introduce data on the frequency of miscarriage among 268 siblings of 244 patients with IRM and (ii) a genetic part presenting data from a genome-wide linkage study of 38 affected sibling pairs with IRM. All IRM patients (probands) had experienced three or more miscarriages and affected siblings two or more miscarriages. The sibling pairs were genotyped by the Affymetrix GeneChip 50K XbaI platform and non-parametric linkage analysis was performed via the software package Merlin. We find that siblings of IRM patients exhibit a higher frequency of miscarriage than population controls regardless of age at the time of pregnancy. We identify chromosomal regions with LOD scores between 2.5 and 3.0 in subgroups of affected sibling pairs. Maximum LOD scores were identified in four occurrences: for rs10514716 (3p14.2) when analyzing sister-pairs only; for rs10511668 (9p22.1) and rs341048 (11q13.4) when only analyzing families where the probands have had four or more miscarriages; and for rs10485275 (6q16.3) when analyzing one sibling pair from each family only. We identify no founder mutations. Concluding, our results imply that IRM patients and their siblings share factors which increase the risk of miscarriage. In this first genome-wide linkage study of affected sibling pairs with IRM, we identify regions on chromosomes 3, 6, 9 and 11 which warrant further investigation in order to elucidate their putative roles in the genesis of IRM.     

Inhibition of CYP2C9 by selective serotonin reuptake inhibitors in vitro: studies of phenytoin p-hydroxylation

Aims Inhibition of cytochrome P450 (CYP) activity by selective serotonin reuptake inhibitors (SSRIs) has frequently been reported with regard to pathways mediated by CYP2D6, CYP3A4/5, and CYP1A2. Little data exist on the capability of SSRIs to inhibit CYP2C9.
Methods We investigated the effect of SSRIs on p -hydroxylation of phenytoin (PPH), an established index reaction reflecting CYP2C9 activity, in an in vitro assay using liver tissue from six different human donors.
Results In control incubations (without inhibitor), 5-( p -hydroxy-phenyl)-5phenylhydantoin (HPPH) formation rates were: V _max 0.023  nmol min⁻¹  mg⁻¹; K _m 14.3  &mgr;m. Average inhibition constants ( K _i ) differed significantly among the SSRIs, with fluvoxamine having the lowest K _i (6  &mgr;m ) followed by R-fluoxetine (13  &mgr;m ), norfluoxetine (17  &mgr;m ), RS-fluoxetine (19  &mgr;m ), sertraline (33  &mgr;m ), paroxetine (35  &mgr;m ), S-fluoxetine (62  &mgr;m ), and desmethylsertraline (66  &mgr;m ). Thus, assuming comparable molar concentrations at the site of inhibition, fluvoxamine can be expected to have the highest probability of interfering with the metabolism of CYP2C9 substrates. S-fluoxetine is on average a 5 fold weaker CYP2C9 inhibitor than either R-fluoxetine or the racemic mixture.
Conclusions These findings are consistent with published case reports describing SSRI-related increments in plasma phenytoin levels. Because phenytoin has a narrow therapeutic index, plasma levels should be closely monitored when SSRIs are coadministered.     

Human cytochromes mediating N-demethylation of fluoxetine in vitro

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 K_m = 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 (K_i = 2.8 μM), an inhibitory probe for P450-2C9, and by ketoconazole (K_i = 2.5 μM) and fluvoxamine (K_i = 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. Received: 23 December 1996 / Final version: 4 March 1997     

Human cytochromes P450 mediating phenacetin O-deethylation in vitro: validation of the high affinity component as an index of CYP1A2 activity

Phenacetin O-deethylation, widely used as an index reaction for cytochrome P450 1A2 (CYP1A2) activity, displays biphasic kinetics in human liver microsomes. CYP1A2 has been identified as contributing to the high affinity component, but is not verified as the sole contributor to the high affinity phase. In addition, the human CYP isoforms accounting for the low affinity phase have not been identified. We have used heterologously expressed human CYP isoforms to identify, kinetically characterize, and predict the relative contribution of the major human liver CYP isoforms mediating phenacetin O-deethylation. CYP1A2 (Km 31 microM) is the only high affinity phenacetin O-deethylase in human liver microsomes, while CYPs 2A6 (Km 4098 microM), 2C9 (Km 566 microM), 2C19 (Km 656 microM), 2D6 (Km 1021 microM), and 2E1 (Km 1257 microM) all contribute to the low affinity phase of the reaction. Considering the relative abundance of the various CYPs in human liver, CYP1A2 accounts for 86% of net reaction velocity at a substrate concentration of 100 microM, while CYP2C9 becomes the primary phenacetin O-deethylase at substrate concentrations of 865 microM and higher and accounts for 31% of the net Vmax of the reaction. Predictions from kinetic studies on heterologously expressed CYPs are consistent with chemical inhibition studies on human liver microsomes with sulfaphenazole and alpha-naphthoflavone that suggest a greater role for CYP2C9, and a smaller role for CYP1A2, at higher substrate concentrations. Thus CYP1A2 is the only high affinity human liver phenacetin O-deethylase, thereby validating the use of the high affinity component as an index of CYP1A2 activity in human liver microsomes.     

Analysis of ritonavir in plasma/serum and tissues by high-performance liquid chromatography

A method has been developed to quantify ritonavir concentrations in human plasma and in mouse serum, liver, and brain using high-performance liquid chromatography. Extraction recoveries for ritonavir and its internal standard averaged greater than 95%. Within-day variability, expressed as a coefficient of variation, averaged 6% over the concentration range 0.5 μg/mL to 15 μg/mL ritonavir, and between-day variability averaged 5.6% over 5 μg/mL to 15 μg/mL ritonavir. The method was applied to quantitation of ritonavir in mouse serum and tissue. Measured values deviated less than 5% from the actual values in mouse serum, liver, and brain samples containing 5 μg/mL ritonavir. The slopes of calibration curves for extracted calf serum, mouse serum, mouse liver and mouse brain standards were nearly identical to the calibration slope of standards which were not extracted. All curves were linear through zero, and r² was no less than 0.998 for any form of calibration. In addition, there was no chromatographic interference from commonly prescribed medications.     

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 (C(max)) 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 zolpidem's sedative and performance-impairing effects, and cannot be considered as an antidote to benzodiazepine agonists.     

Ethanol inhibits in-vitro metabolism of nifedipine, triazolam and testosterone in human liver microsomes

Although extended exposure to ethanol induces CYP3A metabolism in-vivo, the acute effects of ethanol on CYP3A metabolism have not been fully evaluated in-vitro. We assessed the effect of ethanol on CYP3A-mediated biotransformation using human liver microsomes in-vitro with three prototypic CYP3A-mediated reactions: nifedipine to oxidized nifedipine, triazolam to its 1-hydroxy (1-OH TRZ) and 4-hydroxy (4-OH TRZ) metabolites, and testosterone to 6beta-hydroxytestosterone (6beta-OH TST). Ethanol inhibited metabolism of nifedipine (oxidized nifedipine IC50 3 mg dL(-1), where the IC50 value is the inhibitor concentration corresponding to a 50% reduction in metabolite formation velocity), triazolam (1-OH TRZ IC50 1.1 mg dL(-1), 4-OH TRZ IC50 2.7 mg dL(-1)) and testosterone (6beta-OH TST IC50 2.4 mg dL(-1)). The inhibitory potency of ethanol was similar for the three substrates representing the three hypothetical CYP3A substrate categories. The IC50 values obtained were lower than clinically relevant blood alcohol concentrations. In conclusion, ethanol is an inhibitor of human CYP3A metabolism and may contribute to clinically important interactions.