Computer assisted modeling of ethyl sulfate pharmacokinetics.

For 12 volunteers of a drinking experiment the concentration-time-courses of ethyl sulfate (EtS) and ethanol were simulated and fitted to the experimental data. The concentration-time-courses were described with the same mathematical model as previously used for ethyl glucuronide (EtG). The kinetic model based on the following assumptions and simplifications: a velocity constant k(form) for the first order formation of ethyl sulfate from ethanol and an exponential elimination constant k(el). The mean values (and standard deviations) obtained for k(form) and k(el) were 0.00052 h(-1) (0.00014) and 0.561 h(-1) (0.131), respectively. Using the ranges of these parameters it is possible to calculate minimum and maximum serum concentrations of EtS based on stated ethanol doses and drinking times. The comparison of calculated and measured concentrations can prove the plausibility of alleged ethanol consumption and add evidence to the retrospective calculation of ethanol concentrations based on EtG concentrations.

The urinary disposition of intravenously administered 11-nor-9-carboxy-delta-9-tetrahydrocannabinol in humans.

The objective of this study was to investigate the fraction of an administered dose of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH) that is actually excreted into urine and to determine its urinary half-life independent of the parent compound. Ten healthy, male marijuana nonusers who were enrolled in the study were administered a single dose of 5 mg THCCOOH by the intravenous route. Urine specimens were collected up to 96 hours after administration. Samples were extracted before and after alkaline hydrolysis. The concentration of unconjugated and total THCCOOH was determined using gas chromatography-mass spectrometry. Most of the THCCOOH found in urine was conjugated, with only 0.14 +/- 0.08% of the dose present as unconjugated THCCOOH. The amount of conjugated THCCOOH ranged from 149.3 to 559.8 (mean +/- SD, 342.8 +/- 117.3) microg, representing a recovery of 3% to 11% of the administered dose. The measured amounts of total THCCOOH were low and highly varied among individuals. Renal excretion does not appear to be the preferred elimination pathway for THCCOOH. Urinary elimination half-life of unconjugated and conjugated THCCOOH ranged from 9.0 to 27.4 (mean +/- SD, 17.3 +/- 5.3) hours and from 10.7 to 27.6 (mean +/- SD, 16.0 +/- 5.0) hours, respectively. Although preliminary in nature, the actual urinary elimination half-life of THCCOOH appears to be significantly shorter than its apparent or terminal half-life reported from single or multiple dosing of delta-9-tetrahydrocannabinol (THC).

High clozapine concentrations in leukocytes in a patient who developed leukocytopenia.

Up to now direct toxic effects or immunological processes have been said to explain clozapine-induced agranulocytosis. However, more recent studies may suggest that not yet metabolized clozapine is taken up by leukocytes and transformed by oxidative processes to apoptosis-inducing metabolites. To verify this hypothesis the concentrations of clozapine were measured in the plasma and the leukocytes of a patient receiving clozapine who developed clozapine-induced leukocytopenia and in 10 patients receiving clozapine who did not show any serious adverse side effects. The patient who developed leukocytopenia showed clozapine concentrations in the leukocytes that were about 8 times higher than the mean clozapine concentrations in the leukocytes in the group of 10 patients receiving clozapine with no changes in the leukocyte count in the history. However, no major difference was found in the clozapine plasma concentrations. The results may suggest that patients at risk of developing clozapine-induced leukocytopenia show increased clozapine concentrations in the leukocytes although the clozapine plasma concentration is in the therapeutic range. It is assumed that changes or abnormalities of clozapine uptake at the cell membrane might play a role in the development of clozapine-induced leukocytopenia and/or agranulocytosis.

Pharmacokinetics of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (CTHC) after intravenous administration of CTHC in healthy human subjects.

After cannabis consumption there is only limited knowledge about the pharmacokinetic (PK) and metabolic properties of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (CTHC), which is formed by oxidative breakdown from Delta(9)-tetrahydrocannabinol (THC). Despite widely-varying concentrations observed in smoking studies, attempts have been made to interpret consumption behavior with special regard to a cumulated or decreasing concentration of CTHC in serum. Ten healthy nonsmoking white male individuals received 5 mg CTHC intravenously over 10 min. Highest serum concentrations of CTHC were observed at the end of the infusion (336.8+/-61.7 microg/l) followed by a quick decline. CTHC concentration could be quantified up to 96 h after administration, with a terminal elimination half-life of 17.6+/-5.5 h. Total clearance was low (91.2+/-24.0 ml/min), with renal clearance having only a minor contribution (0.136+/-0.094 ml/min). This first metabolite-based kinetic approach will allow an advanced understanding of CTHC PKs data obtained in previous studies with THC.

Effect of psychotropic medication on the in vitro metabolism of buprenorphine in human cDNA-expressed cytochrome P450 enzymes.

OBJECTIVE: The aim of the present study was to estimate the drug interaction potential of psychtropic medication on buprenorphine (BUP) N-dealkylation using cDNA-expressed cytochrome P450 (CYP) enzymes. METHODS: BUP was incubated with psychotropic drugs and cDNA-expressed CYP 3A4 and CYP 2C8 enzymes. Seven substances were screened for their inhibition potency. To check for a mechanism-based component in inhibition, all substances were tested with and without preincubation, respectively. Norbuprenorphine (NBUP) concentrations were determined by liquid chromatography/tandem mass spectrometry, following liquid/liquid extraction. RESULTS: Midazolam and zolpidem demonstrated greatest inhibition in screening experiments. As expected, IC(50) values without preincubation were higher than those after 30-min preincubation, with zolpidem 113.1 microM and midazolam 20.25 microM. Following a 30-min preincubation period in the absence of the probe substrate BUP, the apparent IC(50) values for zolpidem and midazolam were 20.17 microM and 3.5 microM. CONCLUSION: Both midazolam and zolpidem showed a distinct inhibitory potency towards NBUP formation by CYP 3A4, implicating a decreased conversion of BUP. When preincubated, the inhibitory potency was increased, which strongly suggests a metabolically activated component in inhibition.

Tissue distribution of mirtazapine and desmethylmirtazapine in a case of mirtazapine poisoning.

An ingestion of an unknown quantity of mirtazapine in a suicide attempt leading to death is described. Sertraline and amitriptyline have been co-ingested. Because mirtazapine is reported to be relatively safe in overdose, body fluids and tissues were investigated for both mirtazapine and desmethylmirtazapine by high-pressure liquid chromatography/tandem mass spectrometry following liquid-liquid extraction. The limit of detection was sufficiently low to also apply the assay in pharmacokinetic studies. The levels of amitriptyline and nortriptyline were very low (38 and 19 ng/mL femoral venous blood) and the amount of sertraline in blood taken from the femoral vein (880 ng/mL) was considerably lower than those seen in overdosage. Accumulation of mirtazapine and N-desmethylmirtazapine was evident in fluids and tissues involved in enterohepatic circulation and excretion. The concentration determined in a brain sample suggests a contribution of the metabolite to the drug's pharmacodynamic activity. Based on literature data, significant adverse or synergistic effects among the drugs detected as well as adverse reactions such as a serotonin reaction appeared less probable. Mirtazapine exhibits alpha(1)-antagonistic properties on the cardiac-vascular system and may cause hyponatraemia. In the face of the cardiac findings at autopsy and the lack of an apparent cause of death, these effects of mirtazapine may have initiated a process leading to death.

Affinities of dihydrocodeine and its metabolites to opioid receptors.

Dihydrocodeine is metabolized to dihydromorphine, dihydrocodeine-6-O-, dihydromorphine-3-O- and dihydromorphine-6-O-glucuronide, and nordihydrocodeine. The current study was conducted to evaluate the affinities of dihydrocodeine and its metabolites to mu-, delta- and kappa-opioid receptors. Codeine, morphine, d,1-methadone and levomethadone were used as controls. Displacement binding experiments were carried out at the respective opioid receptor types using preparations of guinea pig cerebral cortex and the specific opioid agonists [5H]DAMGO (mu-opioid receptor), [3H]DPDPE (delta-opioid receptor) and [3H]U69,593 (K-opioid receptor) as radioactive ligands at concentrations of 0.5, 1.0 and 1.0 nmol/l, respectively. All substances had their greatest affinity to the mu-opioid receptor. The affinities of dihydromorphine and dihydromorphine-6-O-glucuronide were at least 70 times greater compared with dihydrocodeine (Ki 0.3 micromol/l), whereas the other metabolites yielded lower affinities. For the delta-opioid receptor, the order of affinities was similar with the exception that dihydrocodeine-6-O-glucuronide revealed a doubled affinity in relation to dihydrocodeine (Ki 5.9 micromol/l). In contrast, for the K-opioid receptor, dihydrocodeine-6-O- and dihydromorphine-6-O-glucuronide had clearly lower affinities compared to the respective parent compounds. The affinity of nordihydrocodeine was almost identical to that of dihydrocodeine (Ki 14 micromol/l), whereas dihydromorphine had a 60 times higher affinity. These results suggest that dihydromorphine and its 6-O-glucuronide may provide a relevant contribution to the pharmacological effects of dihydrocodeine. The O-demethylation of dihydrocodeine to dihydromorphine is mediated by the polymorphic cytochrome P-450 enzyme CYP2D6, resulting in different metabolic profiles in extensive and poor metabolizers. About 7% of the caucasian population which are CYP2D6 poor metabolizers thus may experience therapeutic failure after standard doses.

Excretion profiles of ethyl glucuronide in human urine after internal dilution.

Ethyl glucuronide (EG) is a useful marker of alcohol consumption because its presence in urine can be detected up to five days. We investigated the impact of diuresis on the urinary excretion of EG, a minor ethanol metabolite. Seven healthy volunteers drank 250 mL of wine (25 g ethanol) in 15 min and, 240 min later, ingested 1 L of water within 15 min. Urine was voided before the drinking started and every 30-60 min for 400-550 min thereafter. Urinary ethyl glucuronide (UEG), creatinine, and ethanol were determined using liquid chromatography-tandem mass spectrometry, Jaffé's method, and the enzymatic ADH method, respectively. The maximum diuresis coincided with the lowest values of the UEG concentrations of 2 mg/L and the lowest creatinine values of 10 mg/dL 250-400 min after drinking. After drinking the wine, the urinary creatinine decreased slowly. After a short period of increasing, it decreased to minimum values caused by the water intake. After the intake of 1 L water, the diuresis increased within 60 min to its maximum. The amount of ethyl glucuronide excreted in urine was 10 mg (SD 5 mg) corresponding to 0.04% (SD 0.02%) of the dose administered. In successive voids during the elimination phase, the UEG and the diuresis were influenced after the subjects drank 1 L of water. Minimum UEG values of 0.5 mg/L could still be measured. Measuring UEG provides a reliable way to monitor recent drinking of alcohol. However, urinary creatinine needs to be measured additionally. Establishing a cutoff value of 25 mg/dL for urinary creatinine in diluted samples, like for the analysis of illicit drugs, is recommended. If the creatinine value is too low, the analyst has to decide about the further procedure.

A kinetic model describing the pharmacokinetics of ethyl glucuronide in humans.

The glucuronide conjugation is a minor pathway of ethanol metabolism. The determination of ethyl glucuronide (EG) in serum and urine has gained importance in forensic and other legal decisions. To prospectively calculate the serum concentration of this non-oxidative ethanol metabolite, the computer program developed includes a parameter fitting routine. Multiple ethanol doses can be handled. The mathematical modeling was based on the following assumptions and simplifications, respectively. A single enzyme system is responsible for ethanol conjugation at one distinct site; the distribution of EG into the systemic circulation is delayed; the elimination of EG follows first-order kinetics. The concentration of EG was calculated using three kinetic parameters: a rate constant for the first-order formation of EG from serum ethanol, a transfer constant for an obstructed transfer of EG from the formation site (FS) into the central compartment (CC) and an exponential elimination constant. The program was applied to the data collected from 21 drinking experiments. The fitting algorithm optimized the three kinetic parameters, until the sum of concentration error squares of the data points was minimized. The means+/-standard deviation of the rate constant for the first-order formation of EG from serum ethanol was 0.0011+/-0.0006 h(-1), the transfer constant for an obstructed transfer of EG from the FS into the CC was 0.43+/-0.1996 h(-1) and the exponential elimination constant was 3.0+/-1.45 h(-1). Using the range of these parameters, it is now possible to calculate minimum and maximum serum concentrations of EG based on ethanol doses and drinking times. The comparison of calculated and measured concentrations can prove the plausibility of an alleged ethanol consumption. This can be crucial when the serum ethanol concentration (SEC) itself is not meaningful.

Preliminary immunochemical test for the determination of ethyl glucuronide in serum and urine: comparison of screening method results with gas chromatography-mass spectrometry.

Ethyl glucuronide is a highly specific metabolite of ethanol that is formed by enzymatic conjugation of ethanol with glucuronic acid. Because of its suitability as a marker of excessive alcohol consumption in serum with low blood-alcohol concentration and as a consumption marker in serum and urine, especially after the breakdown of ethanol, demand exists for a simple and fast analytical procedure, which is rarely possible using mass spectrometric determination methods. For this reason, we developed an immunochemical screening procedure (ELISA) in which polyclonal antibodies are bound to the walls of microtiter plates. To test suitability, 335 authentic serum and 186 urine samples were examined using immunochemistry and gas chromatography-mass spectrometry (GC-MS). The serum (urine) samples with cutoff values of 0.31 mg/L (1.33 mg/L) yielded false-negative results in 9.5% (24.3%) and false positives in 8.4% (23.2%) of cases. Specificity was calculated at 91.6% (76.8%) and sensitivity at 90.5% (75.7%). Test efficiency was 90.8% (76.3%). The study shows that ethyl glucuronide and therefore alcohol consumption can be detected in immunochemical screening of serum in a similar manner as current drugs, but the method is of limited value for urine. A GC-MS confirmation continues to remain a necessity.