In vitro prediction of clinical drug interactions with CYP3A substrates: we are not there yet.

In 1973, Malcolm Rowland and associates described an approach to predicting clinical pharmacokinetic drug-drug interactions (DDIs) using an inhibition constant determined in vitro (Ki) together with anticipated inhibitor exposure in vivo ([I]). Despite numerous modifications and refinements of the core model over the following 40 years, we still have not achieved a predictive paradigm having accuracy sufficient to justify bypassing all, or even most, clinical DDI studies in the course of drug development.

Pomegranate juice and pomegranate extract do not impair oral clearance of flurbiprofen in human volunteers: divergence from in vitro results.

Nutrient interactions with prescription drugs are a topic of ongoing basic and clinical research. Pomegranate juice and a 1-g capsule containing pomegranate extract were evaluated in vitro and in vivo as inhibitors of cytochrome P450 2C9 (CYP2C9), with flurbiprofen serving as the index substrate. Fluconazole was the positive control inhibitor. The in vitro 50% inhibitory concentration (IC(50)) values for pomegranate juice and extract were below 1% (vol/vol), with no evidence of mechanism-based (irreversible) inhibition. In clinical studies, flurbiprofen pharmacokinetics were unchanged by pomegranate juice or extract as compared to a low-polyphenol placebo control beverage. However, fluconazole significantly reduced the oral clearance of flurbiprofen. Despite inhibition of CYP2C9 in vitro, pomegranate juice and extract had no effect on CYP2C9 activity in human subjects, and can be consumed by patients taking CYP2C9 substrate drugs with negligible risk of a pharmacokinetic interaction.

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.

Short-term clarithromycin administration impairs clearance and enhances pharmacodynamic effects of trazodone but not of zolpidem.

The kinetic and dynamic interactions of 5 mg zolpidem and 50 mg trazodone with 500 mg clarithromycin (4 doses given over 32 h) were investigated in a 5-way double crossover study with 10 healthy volunteers. The five treatment conditions were: placebo + placebo; zolpidem + placebo; zolpidem + clarithromycin; trazodone + placebo; and trazodone + clarithromycin. Coadministration of clarithromycin increased trazodone area under the curve, prolonged elimination half-life, increased peak plasma concentration (C(max)), and reduced oral clearance. In contrast, clarithromycin had no significant effect on any kinetic parameter for zolpidem. Clarithromycin did not potentiate sedation caused by zolpidem. However, clarithromycin coadministered with trazodone significantly increased self- and observer-rated sedation and ratings of feeling "spacey." Thus, short-term clarithromycin coadministration significantly impairs trazodone clearance, elevates plasma concentrations, and enhances sedative effects. However, clarithromycin has no significant kinetic or dynamic interaction with zolpidem.

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.

Lorazepam concentrations, pharmacokinetics and pharmacodynamics in a cohort of mechanically ventilated ICU patients.

OBJECTIVES: To evaluate plasma concentrations, pharmacokinetics and pharmacodynamics of lorazepam in a cohort of mechanically ventilated patients. INTERVENTIONS: Patients underwent simultaneous measurement of lorazepam concentration and sedation assessments using the Sedation-Agitation Scale (SAS) and Bispectral Index (BIS). Lorazepam administration was classified as either continuous intravenous infusion (CIVS) or bolus. MAIN RESULTS: A total of 124 observations were made in 13 patients. The median concentration was 59 ng/ml, interquartile range 23 - 93 ng/ml, range 0 - 1,072 ng/ml. Clearance was preserved at 92 +/- 71 ml/min. Higher concentrations were associated with deeper sedation determined by both SAS and BIS. Two patients were managed with CIVS and received more lorazepam than those managed without (288 +/- 53.5 versus 55 +/- 25.2 mg, p-value < 0.005). CIVS administration was associated with higher concentrations (629 +/- 36 versus 49 +/- 15 ng/ml, p-value < 0.001) and deeper sedation by both SAS and BIS. CONCLUSIONS: Lorazepam clearance was preserved with a wide range of concentrations. Higher concentrations were associated with deeper sedation and use of CIVS. Elevated concentrations during CIVS were attributable to administration of larger doses.

Role of CYP3A enzymes in the biotransformation of triazolam in rat liver.

1. Triazolam (TRZ) has been used extensively in rat to evaluate its benzodiazepine agonist central nervous system effects. However, the pharmacokinetics of TRZ in the male rat are not well understood. 2. To characterize further TRZ biotransformation across species, the NADPH-dependent biotransformation of TRZ was examined in rat and human liver microsomes. The role of specific cytochrome P450s (CYPs) in the biotransformation of TRZ in the rat were also determined using both rat cDNA-expressed CYPs and chemical and antibody inhibition techniques. 3. The formation of TRZ's primary hydroxylated products, alpha-OH- and 4-OH-TRZ, was consistent with a single-enzyme Michaelis-Menten model in humans. 4. Although 4-OH-TRZ formation in the male rat liver was also approximated by a single-enzyme model, a second low-affinity component was identified as contributing to alpha-OH-TRZ formation in the rat. 5. The K(m) values for the primary metabolic pathway differed between the two species. However, the net intrinsic clearances were similar for the rat and human. 6. As observed previously for humans, chemical and antibody inhibition studies suggested that CYP3A enzymes contribute significantly to TRZ hydroxylation in the rat. This finding was further supported by the use of rat cDNA-expressed CYPs. 7. The male rat might serve as a useful model for evaluating mechanisms regulating TRZ metabolism in vivo.

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.

Differential metabolism of midazolam in mouse liver and intestine microsomes: a comparison of cytochrome P450 activity and expression.

1. Although multiple cytochrome P450s (CYP) contribute to hepatic phase I metabolism, CYP3A is the principal subfamily present in human and mouse small intestine. 2. Differences in phase I metabolism were investigated using midazolam (MDZ) hydroxylation in mouse liver and intestinal microsomes. The net MDZ metabolite formation rate in intestinal microsomes was approximately 30% that of liver microsomes (at 250 micro M MDZ). 3. Quantitative Western blotting with anti-CYP3A1 antibody detected two bands of immunoreactive protein in both liver and intestinal samples, 2.24 +/- 0.27 (mean +/- SD, n = 3) and 0.64 +/- 0.08 pmol mg(-1) protein, respectively. Qualitative Western blotting with anti-CYP2C11 antibody detected a single band of immunoreactive protein in liver microsomes and no signal in intestinal samples (1 micro g sample). 4. Ketoconazole potently inhibited formation of both alpha- and 4-OH-MDZ metabolites in intestinal microsomes (IC(50)' of 0.126 +/- 0.010 and 0.0955 +/- 0.014 micro M, respectively) and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) of 0.041 +/- 0.003 micro M). However, ketoconazole (5 micro M) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Inhibition by ritonavir (5 micro M) produced similar results. 5. MDZ hydroxylation is predominately CYP3A dependent in mouse intestine (compared with mouse liver) since CYP2C is not expressed in the intestine. The importance of CYP3A in the mouse intestine appears to mirror that in humans.

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 beta-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.

Molecular and pharmacokinetic evaluation of rat hepatic and gastrointestinal cytochrome p450 induction by tamoxifen.

Tamoxifen (TAM) is a first-line endocrine treatment for all stages of postmenopausal breast cancer. The cytochrome P450 (CYP) enzymes catalyze the majority of TAM's primary metabolism, producing N-desmethyltamoxifen (DMT) and 4-hydroxytamoxifen (4-OH-TAM) in both humans and rats. CYP 3A isoforms are the predominant subfamily involved in the formation of DMT and recent studies have shown that TAM induces hepatic forms of these enzymes. TAM's inductive effect on gastrointestinal CYP 3A has not been previously reported. The current studies investigated TAM's induction of CYP isoforms (3A and 2B) in female rat gastrointestinal and hepatic tissue at the mRNA, protein, and catalytic level. Since previous studies have not addressed whether TAM induction causes changes to the overall pharmacokinetics (PKs), a rat PK model was used to determine if TAM induced its own metabolism, and/or the metabolism of a CYP 3A substrate, midazolam (MDZ). Phenobarbital (PB) and/or dexamethasone (DEX) were used as positive controls for all studies. TAM significantly induced, or caused a trend towards induction of all studied parameters for hepatic CYP 3A and 2B, whereas intestinal CYP 3A and 2B analysis did not show significant induction by TAM at any level. A study evaluating time-dependent alterations in the PK profile of TAM showed no change in apparent oral clearance (Cl(app)) during two weeks of chronic dosing with TAM. However, the Cl(app) for MDZ was shown to trend towards an increase after two weeks of dosing with TAM, in a second PK study. These combined investigations suggest that TAM is an inducer of rat hepatic CYP 3A and 2B isoforms, and this agent has the potential of influencing the PK of coadministered 3A substrates.

Ritonavir induces P-glycoprotein expression, multidrug resistance-associated protein (MRP1) expression, and drug transporter-mediated activity in a human intestinal cell line.

The present study characterized the response of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) to chronic ritonavir (RIT) exposure by assessing increases in P-gp and MRP1 protein expression and activity. LS-180V intestinal carcinoma cells were exposed for 3 days to 1-100 microM RIT concurrently with controls. P-gp and MRP1 protein was quantified by Western blot analysis. Cell accumulation assays, using the P-gp substrate rhodamine 123 (RH123), the P-gp/MRP1 substrate doxorubicin (DOX), and the MRP substrate carboxyfluorescein (CBF), were performed as a measure of transporter activity. RIT strongly induced P-gp and MRP1 expression (maximum 6-fold and 3-fold increases, respectively) in a concentration-dependent fashion. Following extended exposure to RIT (> 10 microM), cells accumulated < 50% of the RH123 and DOX compared with controls, whereas accumulation of CBF was decreased by 30% at 30 microM. Differences in cell accumulation of RH123 could be eliminated with verapamil (100 microM; a P-gp inhibitor), whereas decreased DOX cell accumulation was only partially reversed by verapamil. Indomethacin (100 microM; an MRP1 inhibitor) had no significant effect on RH123 or DOX accumulation, suggesting limited MRP1-mediated activity. Thus, RIT induced protein expression of P-gp and MRP1 and increased cellular drug exclusion of RH123, DOX, and CBF. Similar in vivo phenomena may occur during anti-HIV drug therapy, explaining potential decrements in therapeutic efficacy due to decreases in bioavailability or alterations in drug distribution.

Saint John's wort: an in vitro analysis of P-glycoprotein induction due to extended exposure.

1. Chronic use of Saint John's wort (SJW) has been shown to lower the bioavailability for a variety of co-administered drugs including indinavir, cyclosporin, and digoxin. Decreases in intestinal absorption through induction of the multidrug resistance transporter, P-glycoprotein (P-gp), may explain decreased bioavailability. 2. The present study characterized the response of P-gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system. Experiments were performed with 3 to 300 microg ml(-1) of methanol-extracted SJW and 0.03 to 3 microM HYP, representing low to high estimates of intestinal concentrations. 3. In induction experiments, LS-180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir). P-gp was quantified using Western blot analysis. P-gp expression was strongly induced by SJW (400% increase at 300 microg ml(-1)) and by HYP (700% at 3 microM) in a dose-dependent fashion. Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P-gp substrate, that was reversed with acute verapamil, a P-gp inhibitor. Fluorescence microscopy of intact cells validated these findings. In Caco-2 cell monolayers, SJW and HYP caused moderate inhibition of P-gp-attributed transport at the maximum concentrations tested. 4. SJW and HYP significantly induced P-gp expression at low, clinically relevant concentrations. Similar effects occurring in vivo may explain the decreased bioavailability of P-gp substrate drugs when co-administered with SJW.

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).

Human drug metabolism and the cytochromes P450: application and relevance of in vitro models.

The cytochromes P450 (CYPs) constitute a superfamily of hemoprotein enzymes that are responsible for the biotransformation of numerous xenobiotics, including therapeutic agents. Studies of the biochemical and enzymatic properties of these enzymes and their molecular genetics and regulation of gene expression and activity have greatly enhanced our understanding of several aspects of clinical pharmacology such as pharmacokinetic variability, drug toxicity, and drug interactions. This review evaluates the major human hepatic drug-metabolizing CYP enzymes and their clinically relevant substrates, inhibitors, and inducers. Also discussed are the molecular bases and clinical implications of genetic polymorphisms that affect the CYPs. Much of the information on the specificity of substrates and inhibitors of the CYP enzymes is derived from in vitro studies using human liver microsomes and heterologously expressed CYP enzymes. These methods are discussed, and guidelines are provided for designing enzyme kinetic and reaction phenotyping studies using multiple approaches. The strengths, weaknesses, and discrepancies among the different approaches are considered using representative examples. The mathematical models used in predicting the pharmacokinetic clearance of a drug from in vitro estimates of intrinsic clearance and the principles of quantitative in vitro-in vivo scaling of metabolic drug interactions are also discussed.

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, p.o.) 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.

Pharmacokinetics and pharmacodynamics of vecuronium in children receiving phenytoin or carbamazepine for chronic anticonvulsant therapy.

The pharmacokinetics and time course of action of vecuronium in normal children and children receiving anticonvulsant drugs for prolonged periods were characterized. A bolus dose of vecuronium 0.15 mg kg(-1) was administered i.v. to 10 non-epileptic children and to 10 children on phenytoin and 10 children on carbamazepine, who were matched for age and weight. Plasma concentrations of vecuronium, 3-OH desacetylvecuronium (the primary metabolite of vecuronium) and alpha1-acid glycoprotein (AAG) were determined. Pharmacokinetic variables were derived from plasma samples collected before and after administration of vecuronium. Neuromuscular transmission was monitored by evoked compound electromyography. Recovery of the first twitch of the train-of-four (T1/T0) and the recovery index (RI), the time for 25-75% recovery of T1/T0, were determined. The elimination half-life of vecuronium was significantly reduced in both anticonvulsant groups compared with control [control 48.2 (SD 40.3), phenytoin 23.5 (13.1), carbamazepine 18.4 (16.6) min, P<0.05]. Vecuronium clearance was increased in both anticonvulsant groups [control 9.0 (3.6), phenytoin 15.1 (8.9), carbamazepine 18.8 (13.1) ml kg(-1) min(-1), 0.05