Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam.

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p-hydroxydiazepam [p-OH-DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p-OH-DZP was significantly less in the horse. Inhibition assays with a CYP-specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos-7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.

Hair analysis to demonstrate administration of amitriptyline, temazepam, tramadol and dihydrocodeine to a child in a case of kidnap and false imprisonment.

Amitriptyline, temazepam, tramadol and dihydrocodeine are prescription-only-medications that are rarely prescribed to children. Each of these drugs has a sedative effect on the central nervous system; their combined use could cause an exacerbation of the sedative effects. Amitriptyline (a tricyclic antidepressant) can be prescribed to treat nocturnal enuresis; temazepam (a hypnotic) can be used as a premedicant in inpatient and day-case surgery; tramadol (a synthetic opioid analgesic) is used to treat moderate or severe pain, though it is not recommended for children under the age of 12 years and dihydrocodeine (opioid analgesic), which is available in combination with acetaminophen (Co-dydramol), is not recommended for children under the age of 4 years; in children over 4 years, a reduced dose is necessary. The North West Forensic Science Service Laboratory, Euxton, Lancashire, was asked by a British police force to analyze three separate hair samples, which had been collected from a young child following their discovery as a result of a large scale kidnap and false imprisonment investigation. After decontamination and segmentation (20 x 1-cm section), two of the three hair specimens were analyzed by liquid chromatography coupled with tandem mass spectrometry after alkaline (pH 9.5) extraction using methylene chloride/isopropanol/n-heptane (25:10:65, v/v/v). The entire length of each hair specimen tested positive for amitriptyline and nortriptyline (7-314 pg/mg amitriptyline; 7-318 pg/mg nortriptyline), temazepam (2-29 pg/mg), tramadol (60-2000 pg/mg) and dihydrocodeine (10-90 pg/mg) demonstrating that the child had ingested these drugs on more than one occasion prior to the kidnap. In this case, the child's mother and the mothers' partner were found guilty of kidnap, false imprisonment and perverting the course of justice. There are very few studies citing the concentrations of these drugs in children - especially children's hair samples. This case demonstrates the added value of hair testing and emphasizes the importance of using hair samples to complement conventional analysis.

Impact of benzodiazepines on posaconazole serum concentrations. A population-based pharmacokinetic study on drug interaction.

OBJECTIVES: Posaconazole is broadly used for antifungal prophylaxis and therapy. Current data suggest a concentration-dependent effect. Unlike other triazoles, cytochrome P450 is not a relevant route of biotransformation for posaconazole but glucuronidation, which might lead to a different spectrum of drug interactions. For benzodiazepines, the major metabolic pathway involves oxidation, but some, including lorazepam and temazepam, undergo conjugation to glucuronic acid. RESEARCH DESIGN AND METHODS: Since 2006 serum levels of posaconazole are determined regularly in all hospitalized patients with intake of this triazole. Here we investigate posaconazole concentration at steady state in relation to the concomitant medication of benzodiazepines. RESULTS: While similar posaconazole concentrations were determined in samples obtained from patients receiving temazepam when compared to samples without any benzodiazepine, a relevant reduction of posaconazole concentration could be observed in patients with concomitant intake of lorazepam. This difference in posaconazole concentration with or without concomittant intake of lorazepam, was consistently significant for analyses of all samples (median 336 ng/ml vs. 585 ng/ml, p 0.001), for the average concentrations (569 ng/ml vs. 276 ng/ml, p 0.039), and for patients receiving a total daily dose of 800 mg posaconazole (292 ng/ml vs. 537 ng/ml, p 0.003). There was also a similar, but not significant trend for patients with a prophylactic dosage of 200 mg posaconazole three times daily (689 ng/ml vs. 512 ng/ml, p 0.186). CONCLUSIONS: In this retrospective study, analyzing blood samples from daily clinical practice of patients in various clinical settings and with different indications for antifungal therapy, concomitant medication of lorazepam was associated with decreased posaconazole concentrations. Therefore, lorazepam but not temazepam might induce posaconazole clearance by glucuronidation.

An in vitro approach to estimate putative inhibition of buprenorphine and norbuprenorphine glucuronidation.

An in vitro inhibition study was performed to investigate potential drug-drug interactions on glucuronidation of buprenorphine (BUP) and norbuprenorphine (NBUP), which represents the major elimination pathway of the drug using cDNA-expressed uridine 5'-diphosphate glucuronosyltransferases (UGTs) and human liver microsomes (HLMs). Following identification of major UGT enzymes for BUP and NBUP glucuronidation, substrates were incubated with drugs (amitriptyline, nortriptyline, lamotrigine, oxazepam, and temazepam), which are extensively cleared by glucuronidation as well as are often used during maintenance treatment. To evaluate the inhibitory potential, the half maximal inhibitor concentration (IC(50)), the inhibition constant (K (i)), and the inhibitor concentration (K (I)) that yield half the maximum rate of inactivation and the enzyme inactivation rate constant (k (inact)) were determined, if appropriate. Amitriptyline and temazepam are inhibitors of NBUP glucuronidation (UGT1A3, HLMs), whereas BUP glucuronidation was affected by amitriptyline (HLMs), oxazepam, and temazepam (UGT2B7). Additionally, BUP inhibits NBUP glucuronidation (UGT1A1, 1A3, HLMs) and vice versa (UGT1A3). A decrease in the metabolic clearance of NBUP may increase the risk of adverse effects such as respiratory depression. Further investigations are needed to evaluate whether inhibition of BUP and NBUP glucuronidation contributes to adverse events.

Impact of melatonin, zaleplon, zopiclone, and temazepam on psychomotor performance.

INTRODUCTION: Modern military operations may require pharmaceutical methods to sustain alertness and facilitate sleep in order to maintain operational readiness. In operations with very limited sleep windows, hypnotics with very short half-lives (e.g., zaleplon, t(1/2) 1 h) are of interest, while with longer sleep opportunities, longer acting agents (e.g., zopiclone, temazepam (t(1/2) 4-6 hours) may be used. This study was designed to compare the effect of a single dose of zaleplon, zopiclone, temazepam, and melatonin on psychomotor performance and to quantify the post-ingestion time required for return to normal performance. METHOD: There were 23 subjects (9 men, 14 women), 21-53 yr of age, assessed for psychomotor performance on 2 test batteries (4 tasks) that included a sleepiness questionnaire. Psychomotor testing was conducted prior to, and for 7 h after, ingestion of a single dose of each of placebo, zaleplon 10 mg, zopiclone 7.5 mg, temazepam 15 mg, and time-released melatonin 6 mg. The experimental design was a double-blind cross-over with counter-balanced treatment order. RESULTS: Zaleplon, zopiclone, and temazepam impaired performance on all four tasks: serial reaction time (SRT), logical reasoning (LRT), serial subtraction (SST), and multitask (MT). Melatonin did not impair performance on any task. The time to recovery of normal performance for SRT during the zaleplon, zopiclone and temazepam conditions were 3.25, 6.25, and 5.25 h respectively; for LRT were 3.25, >6.25, and 4.25 h; for SST were 2.25, >6.25, and 4.25 h, and for MT were 2.25, 4.25, and 3.25 h. The recovery time to baseline subjective sleepiness levels for zaleplon, zopiclone, temazepam, and melatonin were 4.25, >6.25, 5.25, and >4.25 h, respectively. CONCLUSIONS: In spite of a prolonged period of perceived sleepiness, melatonin was superior to zaleplon in causing no impact on performance. The remaining drugs listed in increasing order of performance impact duration are zaleplon, temazepam, and zopiclone.

[Comparative kinetics of 3-hydroxyphenazepam and its metabolism during transdermal and intravenous administration]. / Osobennosti sravnitel'noi kinetiki ékskretsii 3-hydroxyphenazepama i ego metabolitov pri transdermal'nom i vnutrivennom vvedeniiakh.

The kinetics of excretion of 2-(14)C-3-hydroxyphenazepam and its metabolites were studied upon a single transdermal and intravenous administration in mice. The main fraction of the total radioactivity (approximately 80%) was eliminated within approximately 84 h upon intravenous injection and within approximately 360 h after transdermal introduction. In the latter case, the ratio of lipophilic and hydrophilic metabolites is modified as well: the former fraction increases by a factor of 1.75, while the latter decreases by a factor of 1.2 as compared to the case of intravenous drug administration.

Mechanism of increased dissolution of diazepam and temazepam from polyethylene glycol 6000 solid dispersions.

Solid dispersion literature, describing the mechanism of dissolution of drug-polyethylene glycol dispersions, still shows some gaps; (A). only few studies include experiments evaluating solid solution formation and the particle size of the drug in the dispersion particles, two factors that can have a profound effect on the dissolution. (B). Solid dispersion preparation involves a recrystallisation process (which is known to be highly sensitive to the recrystallisation conditions) of polyethylene glycol and possibly also of the drug. Therefore, it is of extreme importance that all experiments are performed on dispersion aliquots, which can be believed to be physico-chemical identical. This is not always the case. (C). Polyethylene glycol 6000 (PEG6000) crystallises forming lamellae with chains either fully extended or folded once or twice depending on the crystallisation conditions. Recently, a high resolution differential scanning calorimetry (DSC)-method, capable of evaluating qualitatively and quantitatively the polymorphic behaviour of PEG6000, has been reported. Unraveling the relationship between the polymorphic behavior of PEG6000 in a solid dispersion and the dissolution characteristics of that dispersion, is a real gain to our knowledge of solid dispersions, since this has never been thoroughly investigated. The aim of the present study was to fill up the three above mentioned gaps in solid dispersion literature. Therefore, physical mixtures and solid dispersions were prepared and in order to unravel the relationship between their physico-chemical properties and dissolution characteristics, pure drugs (diazepam, temazepam), polymer (PEG6000), solid dispersions and physical mixtures were characterised by DSC, X-ray powder diffraction (Guinier and Bragg-Brentano method), FT-IR spectroscopy, dissolution and solubility experiments and the particle size of the drug in the dispersion particles was estimated using a newly developed method. Addition of PEG6000 improves the dissolution rate of both drugs. Mechanisms involved are solubilisation and improved wetting of the drug in the polyethylene glycol rich micro-environment formed at the surface of drug crystals after dissolution of the polymer. Formulation of solid dispersions did not further improve the dissolution rate compared with physical mixtures. X-ray spectra show that both drugs are in a highly crystalline state in the solid dispersions, while no significant changes in the lattice spacings of PEG6000 indicate the absence of solid solution formation. IR spectra show the absence of a hydrogen bonding interaction between the benzodiazepines and PEG6000. Furthermore, it was concluded that the reduction of the mean drug particle size by preparing solid dispersions with PEG6000 is limited and that the influence of the polymorphic behavior of PEG6000 (as observed by DSC) on the dissolution was negligible.

CNS effects of sumatriptan and rizatriptan in healthy female volunteers.

This study investigates the CNS effects of sumatriptan and rizatriptan, with temazepam as an active comparator, in healthy female volunteers. Sixteen volunteers completed a randomized, double-blind, crossover study and on four separate occasions received either 100 mg sumatriptan, 20 mg rizatriptan or 20 mg temazepam. The main parameters were eye movements, EEG, body sway, visual analogue scales and a cognitive test battery. Rizatriptan and sumatriptan decreased saccadic peak velocity by 18.3 (95% CI: 5.7, 30.8) and 15.0 (2.2, 27.9) degrees/sec, respectively, about half the decrease induced by temazepam (35.0 (22.1, 47.8) degrees/sec). Body sway increased (30% for rizatriptan (16%, 45%) and 14% for sumatriptan (1%, 27%), respectively). Temazepam caused larger, similar effects. In contrast to temazepam, sumatriptan and rizatriptan decreased reaction times of recognition tasks and increased EEG alpha power (significant for sumatriptan, 0.477 (0.02, 0.935). Therapeutic doses of sumatriptan and rizatriptan caused CNS effects indicative of mild sedation. For EEG and recognition reaction times the effects were opposite to temazepam, indicating central stimulation.

Comparison of the rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide and tamazepam-glucuronide catalyzed by E. coli beta-D-glucuronidase using the on-line benzodiazepine screening immunoassay on the Roche/Hitachi 917 analyzer.

The catalytic rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide, and temazepam-glucuronide when catalyzed by E. Coli. beta-glucuronidase both in phosphate buffer and buffered drug-free urine were compared as well as the pH dependence of enzyme activity. In 50 mM phosphate buffer pH 6.4, lorazepam-glucuronide has the highest turnover rate of 3.7 s(-1) with an associated Km of about 100 microM, followed by oxazepam-glucuronide, which has a turnover rate of 2.4 s(-1) with an associated Km of 60 microM. Temazepam-glucuronide has the lowest rate of 0.94 s(-1) with an associated Km of 34 microM. In buffered drug-free urine, a similar trend was observed. In addition, an optimal pH for beta-glucuronidase was determined to be between 6 and 7 when the enzyme hydrolyzes the benzodiazepine conjugates in buffered drug-free urine. Effects of temperature and incubation time were also examined. It can be concluded that the electron donating or withdrawing of the individual benzodiazepine structure may play an important role in the reactivity of the lorazepam-glucuronide, oxazepam-glucuronide and temazepam-glucuronide catalyzed by beta-glucuronidase. This is consistent with other observations made for monosubstituted phenyl-beta-glucuronides by Wang et al. (1).

Efficacy of activated charcoal versus gastric lavage half an hour after ingestion of moclobemide, temazepam, and verapamil.

OBJECTIVE: To compare the efficacy of activated charcoal and gastric lavage in preventing the absorption of moclobemide, temazepam, and verapamil 30 min after drug ingestion. METHODS: In this randomized cross-over study with three phases, nine healthy volunteers received a single oral dose of 150 mg moclobemide, 10 mg temazepam, and 80 mg verapamil after an overnight fast. Thirty minutes later, they were assigned to one of the following treatments: 25 g activated charcoal as a suspension in 200 ml water, gastric lavage (10x200 ml), or 200 ml water (control). Plasma concentrations of moclobemide, temazepam, and verapamil were determined up to 24 h. RESULTS: Activated charcoal reduced the area under the plasma concentration time curve from 0 h to 24 h (AUC0-24 h) of moclobemide and temazepam by 55% (P<0.05) and by 45% (P<0.05), respectively. The AUC0-24 h of verapamil was not significantly reduced by charcoal. Gastric lavage decreased the AUC0-24 h of moclobemide by 44% (P<0.05), but had no significant effect on that of temazepam or verapamil. The peak plasma concentration (Cmax) of moclobemide, temazepam, and verapamil was reduced by 40%, 29% (P<0.05), and 16%, respectively, by activated charcoal. Gastric lavage did not significantly decrease the Cmax of any of these drugs. CONCLUSION: The absorption of moclobemide, temazepam, and verapamil can be moderately reduced by activated charcoal given 30 min after drug ingestion, while gastric lavage seems to be less effective.

Drug release from an ensemble of swellable crosslinked polymer particles.

This paper presents a new model suitable to describe the drug release from drug delivery systems constituted by an ensemble of drug loaded crosslinked polymer particles. The model accounts for the main factors affecting the drug release such as the particle size distribution, the physical state and the concentration profile of the drug inside the polymeric particles, the viscoelastic properties of the polymer-penetrant system and the dissolution-diffusion properties of the loaded drug. In order to check the validity of the model, release experiments were performed by using crosslinked polyvinyl-pyrrolidone (PVP) particles and two different model drugs, MAP (medroxyprogesterone acetate) and TEM (Temazepam). MAP and TEM were chosen because of their completely different dissolution behaviours in water. In particular, TEM undergoes a phase transition to the crystalline state upon dissolution when it is loaded in the polymeric network in the amorphous state. The comparison with the experimental results confirms that the most important factors determining the drug release kinetics can be properly accounted for.

Site-to-site variability of drug concentrations in skeletal muscle.

The homogeneity of drug concentrations in skeletal muscle was assessed in eight fatal overdoses. Ten to 30 random samples were taken from leg muscle weighing 1,650 to 7,985 g. For cases involving paracetamol the mean muscle-to-blood ratio ranged from 0.1 to 1.1 (n = 4) for amitriptyline 1.1 to 3.6 (n = 3), and for dothiepin 0.8 to 2.1 (n = 2). The coefficient of variance was large for all drugs, ranging from 10.5 (carbamazepine) to 50 (thioridazine). Skeletal muscle is not homogeneous with respect to drug concentrations in fatal overdose cases. Of 16 instances of drug detection in blood 2 (nortriptyline and promethazine) were not detected in muscle. Muscle-to-blood drug ratios varied significantly among cases, possibly influenced by survival time after drug ingestion. Quantitative interpretations of muscle drug levels present significant difficulties. However, skeletal muscle can be used for qualitative corroboration of blood analyses and is a suitable specimen for drug detection where none other is available.

A study of the effects of long-term use on individual sensitivity to temazepam and lorazepam in a clinical population.

AIMS: The central effects of benzodiazepines may be attenuated after chronic use by changes in pharmacokinetics, pharmacodynamics or both. This attenuation may be influenced by the dosing pattern and the characteristics of the user population. The objectives of this study were to evaluate drug sensitivity in long-term users of temazepam and lorazepam in a clinical population. METHODS: The sensitivity to benzodiazepine effects in chronic users (1-20 years) of lorazepam (n = 14) or temazepam (n = 13) was evaluated in comparison with age and sex matched controls. Drug sensitivity was evaluated by plasma concentration in relation to saccadic eye movement parameters, postural stability and visual analogue scales. RESULTS: Pharmacokinetics of lorazepam and temazepam did not differ between patients and control subjects. Chronic users of lorazepam showed clear evidence of reduced sensitivity, indicated by lack of any pharmacodynamic difference between patients and controls at baseline, when drug concentrations were similar to the peak values attained in the control subjects after administration of 1-2.5 mg of lorazepam. In addition, there was a two- to four fold reduction in the slopes of concentration-effect plots for measures of saccadic eye movements and body sway (all; P < or = 0.01). By contrast, sensitivity in chronic users of temazepam was not different from controls. The difference between the temazepam and the lorazepam group appears to be associated with a more continuous drug exposure in the latter, due to the longer half-life and a more frequent intake of lorazepam. This pattern of use may be partly related to the more anxious personality traits that were observed in the chronic users of lorazepam. CONCLUSIONS: Chronic users of lorazepam show evidence of tolerance to sedative effects in comparison with healthy controls. Tolerance does not occur in chronic users of temazepam. The difference may be related to pharmacological properties, in addition to different patterns of use, associated with psychological factors.

Site to site variability of postmortem drug concentrations in liver and lung.

We evaluated postmortem diffusion of gastric drug residue into tissues and blood in eight suicidal overdoses. Analyses were performed on liver (five sites), lung (four sites), spleen, psoas muscle and kidney (left and right), blood (peripheral and torso), vitreous, pericardial fluid, bile and, urine as well as residual gastric contents. Standard analytical techniques and instrumentation gas chromatograph/mass spectrometer and high performance liquid chromatography (GC-MS and HPLC) were used throughout. These case studies confirm previous studies of an animal and human cadaver model of gastric diffusion, in that in several instances there was drug accumulation in the left posterior margin of the liver and, to a lesser extent, the left basal lobe of the lung. Uncontrollable variables, such as postmortem interval, refrigeration before autopsy, and position of the body appear to influence significantly drug accumulation in a specific site. We suggest that autopsy sampling techniques should be standardized on blood taken from a ligated peripheral (preferably femoral or external iliac) vein, and liver from deep within the right lobe.

Pharmacokinetic aspects of rectal formulations of temazepam.

An in vitro/in vivo study was carried out with different rectal formulations of temazepam. Pharmacokinetic data were determined in a cross-over study in 10 volunteers after rectal administration of 10 mg temazepam as a polyethylene glycol based suppository (selected from in vitro data), a liquid-filled capsule and a micro-enema respectively, using oral administration of a liquid-filled capsule as a reference. Serum levels of temazepam indicate an instantaneous and complete release from the micro-enema (Frel = 0.94 +/- 0.21, Cmax 205 +/- 36.9 micrograms/l, tmax 0.49 +/- 0.31 hour) and a slower but complete release of temazepam from the suppository (Frel = 1.10 +/- 0.25, Cmax 202 +/- 41.3 micrograms/l, tmax 1.48 +/- 0.41 hour). A high interindividual variation in absorption profiles was observed after rectal administration of the liquid-filled capsule (Frel 0.72 +/- 0.36, Cmax 182 +/- 122 micrograms/l, tmax 4.08 +/- 4.28 hour), which makes it less suitable for rectal use. The micro-enema and suppository appear to be useful as rectal formulations for temazepam.

Lack of effect of antimycotic itraconazole on the pharmacokinetics or pharmacodynamics of temazepam.

The azole antimycotics itraconazole and ketoconazole are potent and relatively nonspecific inhibitors of cytochrome P450 enzymes and have a potentially dangerous interaction with midazolam and triazolam. The possible interaction between itraconazole and a short-acting benzodiazepine, temazepam, was investigated in a double-blind, randomized crossover study. Ten healthy volunteers were given placebo or 200 mg itraconazole a day orally for 4 days. The challenge dose of 20 mg of temazepam was ingested on the fourth day, after which plasma samples were collected, and psychomotor performance tests were carried out for 24 h. Despite a statistically significant small increase of the area under the temazepam concentration-time curve, there was no clinically significant interaction, as determined by the psychomotor performance tests. The different metabolic pathways and the lack of significant firstpass metabolism of temazepam explain the great difference in the interaction potential of temazepam compared with midazolam and triazolam. Temazepam, unlike midazolam and triazolam, can be prescribed in usual doses for patients receiving itraconazole and other inhibitors of P450 3A4.

Pharmacokinetics of temazepam in male surgical patients.

The pharmacokinetics of temazepam, the 3-hydroxy1 derivative of diazepam, were studied in nine male surgical patients (age: 28-57 years; weight: 55-87 kg) who had ingested single 40 mg doses, 4 hours prior to minor surgical procedures. Peak plasma temazepam concentrations were achieved rapidly (within 1 h post drug administration) and the estimated volume of distribution (mean: 1.13 1/kg), total clearance (mean: 1.6 ml/min/kg) and terminal elimination half-life (mean: 8 hours) were comparable to previously reported values in healthy subjects. There was no correlation between volume of distribution and either weight or age, and between clearance and age. These findings are broadly consistent with previous reports from studies in healthy subjects. Temazepam can therefore be used as a premedicant in patients requiring minor surgery; the concomitant anaesthetic agents administered and the surgical procedures have no effects on temazepam pharmacokinetics.

Concentration-dependent metabolism of diazepam in mouse liver.

Previous mouse liver studies with diazepam (DZ), N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generated via NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precursors of OZ. In microsomal studies, the Kms and Vmaxs, corrected for binding to microsomal proteins, were 34 microM and 3.6 nmole/min per mg and 239 microM and 18 nmole/min per mg, respectively, for N-demethylation and C3-hydroxylation of DZ. The Kms and Vmaxs for N-demethylation and C3-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 microM and 2.5 nmole/min per mg and 311 microM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 microM, the extraction ratio of DZ (E[DZ]) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disproportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and the in vitro enzymatic parameters provided a poor in vitro-organ correlation. The E[DZ], appearance rates of the metabolites, and the extraction ratio of formed NZ (E[NZ, DZ]) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters improved the correlations and identified NZ as the major contributor of OZ. Saturation of DZ N-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates used in vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poor in vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics.