Tobias Klaassen

Toxicokinetics of Acrylamide in Humans after Ingestion of a Defined Dose in a Test Meal to Improve Risk Assessment for Acrylamide Carcinogenicity

High amounts of acrylamide in some foods result in an estimated daily mean intake of 50 μg for a western style diet. Animal studies have shown the carcinogenicity of acrylamide upon oral exposure. However, only sparse human toxicokinetic data is available for acrylamide, which is needed for the extrapolation of human cancer risk from animal data. We evaluated the toxicokinetics of acrylamide in six young healthy volunteers after the consumption of a meal containing 0.94 mg of acrylamide. Urine was collected up to 72 hours thereafter. Unchanged acrylamide, its mercapturic acid metabolite N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), its epoxy derivative glycidamide, and the respective metabolite of glycidamide, N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA), were quantified in the urine by liquid chromatography-mass spectrometry. Toxicokinetic variables were obtained by noncompartmental methods. Overall, 60.3 ± 11.2% of the dose was recovered in the urine. Although no glycidamide was found, unchanged acrylamide, AAMA, and GAMA accounted for urinary excretion of (mean ± SD) 4.4 ± 1.5%, 50.0 ± 9.4%, and 5.9 ± 1.2% of the dose, respectively. Apparent terminal elimination half-lives for the substances were 2.4 ± 0.4, 17.4 ± 3.9, and 25.1 ± 6.4 hours. The ratio of GAMA/AAMA amounts excreted was 0.12 ± 0.02. In conclusion, most of the acrylamide ingested with food is absorbed in humans. Conjugation with glutathione exceeds the formation of the reactive metabolite glycidamide. The data suggests an at least 2-fold and 4-fold lower relative internal exposure for glycidamide from dietary acrylamide in humans compared with rats or mice, respectively. This should be considered for quantitative cancer risk assessment. (Cancer Epidemiol Biomarkers Prev 2006;15(2):266–71)

Validated quantitation method for a peptide in rat serum using liquid chromatography/high-field asymmetric waveform ion mobility spectrometry

The analysis of peptides presents serious challenges for bioanalytical scientists including low total ion current and non-selective fragmentation during tandem mass spectrometry (MS/MS). During method validation of a peptide in rat serum matrix some interferences could not be easily removed and thus prevented accurate and precise measurement. These problems associated with peptide quantitation were resolved by using FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry). This selectivity-enhancing technique filters out matrix interferences, and the resulting pseudo-selected reaction monitoring (pseudo-SRM) chromatograms were nearly free from interferences. Control blank matrix samples contained an acceptable level of interference (only 7% signal as compared to the lower level of quantitation). Chromatographic peaks were easily, accurately and precisely integrated resulting in a validated liquid chromatography (LC)/FAIMS-MS/MS method for the analysis of a peptide drug in rat serum according to United States Food and Drug Administration (US FDA) bioanalytical guidelines. These results confirm that new selectivity-enhancing technologies aid the pharmaceutical industry in reliably producing acceptable pharmacokinetic data.

Effect of an Antiretroviral Regimen Containing Ritonavir Boosted Lopinavir on Intestinal and Hepatic CYP3A, CYP2D6 and P‐glycoprotein in HIV‐infected Patients

This study aimed to quantify the inhibition of cytochrome P450 (CYP3A), CYP2D6, and P‐glycoprotein in human immunodeficiency virus (HIV)‐infected patients receiving an antiretroviral therapy (ART) containing ritonavir boosted lopinavir, and to identify factors influencing ritonavir and lopinavir pharmacokinetics. We measured activities of CYP3A, CYP2D6, and P‐glycoprotein in 28 patients before and during ART using a cocktail phenotyping approach. Activities, demographics, and genetic polymorphisms in CYP3A, CYP2D6, and P‐glycoprotein were tested as covariates. Oral midazolam clearance (overall CYP3A activity) decreased to 0.19‐fold (90% confidence interval (CI), 0.15–0.23), hepatic midazolam clearance and intestinal midazolam availability changed to 0.24‐fold (0.20–0.29) and 1.12‐fold (1.00–1.26), respectively. In CYP2D6 extensive metabolizers, the plasma ratio AUCdextromethorphan/AUCdextrorphan increased to 2.92‐fold (2.31–3.69). Digoxin area under the curve (AUC)0–12 (P‐glycoprotein activity) increased to 1.81‐fold (1.56–2.09). Covariates had no major influence on lopinavir and ritonavir pharmacokinetics. In conclusion, CYP3A, CYP2D6, and P‐glycoprotein are profoundly inhibited in patients receiving ritonavir boosted lopinavir. The covariates investigated are not useful for a priori dose selection.

Effect of propiverine on cytochrome P450 enzymes: a cocktail interaction study in healthy volunteers*

The present study was conducted to assess a possible in vivo effect of propiverine, an anticholinergic drug to treat urinary incontinence and related disorders, on the activity of intestinal CYP3A4 and of hepatic CYP3A4, CYP2C9, CYP2C19, and CYP1A2. The activity of the respective cytochromes P450 was measured using the following metrics of selective substrates given as a tailored low-dose phenotyping cocktail: intestinal availability of midazolam (2 mg orally), clearance of midazolam (1 mg i.v.), apparent clearance of tolbutamide (125 mg orally), urinary excretion of 4′-hydroxymephenytoin 0 to 8 h postdose (50 mg of mephenytoin orally), and the paraxanthine/caffeine plasma ratio 6 h postdose (150 mg of caffeine orally). These metrics were determined in 16 healthy young men at the end of 7 days of treatment with 15 mg of propiverine (test) or placebo (reference) twice daily. All phenotyping drugs were quantified by liquid chromatography-tandem mass spectrometry. Chronic propiverine treatment reduced hepatic and intestinal CYP3A4 activity slightly to 0.89-fold and 0.80-fold, respectively [90% confidence interval (CI) for test/reference ratios 0.85–0.93 and 0.72–0.89], with the combined effect resulting in a 1.46-fold increase in area under the curve of oral midazolam (90% CI 1.36–1.57). Propiverine had no relevant effect on CYP2C9, CYP2C19, and CYP1A2 (90% CI for test/reference ratios 0.93–1.00, 0.84–0.96, and 0.97–1.07, respectively). All study drugs were well tolerated. In conclusion, propiverine has a minor potential to cause drug-drug interactions.

Assessment of Activity Levels for CYP2D6*1, CYP2D6*2, and CYP2D6*41 Genes by Population Pharmacokinetics of Dextromethorphan

The pharmacokinetics of dextromethorphan (DM) is markedly influenced by cytochrome P450 2D6 (CYP2D6) enzyme polymorphisms. The aim of this study was to quantify the effects of the CYP2D6*1, *2, and *41 variants on DM metabolism in vivo and to identify other sources of pharmacokinetic variability. Concentrations of DM and dextrorphan (DO) in plasma and urine were evaluated in 36 healthy Caucasian men. These volunteers participated in three clinical studies and received a single oral dose of 30 mg DM‐HBr. Data were modeled simultaneously using the population pharmacokinetics NONMEM software. A five‐compartment model adequately described the data. The activity levels of the alleles assessed differed significantly. The clearance attributable to an individual CYP2D6*1 copy was 2.5‐fold higher as compared with CYP2D6*2 (5,010 vs. 2,020 l/h), whereas the metabolic activity of CYP2D6*41 was very low (85 l/h). Urinary pH was confirmed as a significant covariate for DM renal clearance. These results refine genotype‐based predictions of pharmacokinetics for DM and presumably for other CYP2D6 substrates as well.

Do activities of cytochrome P450 (CYP)3A, CYP2D6 and P-glycoprotein differ between healthy volunteers and HIV-infected patients?

BACKGROUND In inflammation and infection, cytochrome P450 (CYP) enzyme activities are down-regulated. Information on possible discrepancies in activities of CYP enzymes and drug transporters between HIV-infected patients and healthy people is limited. METHODS We used midazolam, dextromethorphan and digoxin as in vivo phenotyping probes for CYP3A (CYP3A4/5), CYP2D6 and P-glycoprotein activities, respectively, and compared these activities between 12 healthy Caucasian volunteers and 30 treatment-naive HIV-infected patients. RESULTS Among the HIV-infected patients, the overall CYP3A activity (apparent oral midazolam clearance) was approximately 50% of the activity observed in healthy volunteers (point estimate 0.490, 90% confidence interval [CI] 0.377-0.638). The CYP2D6 activity (plasma ratio area under the curve [AUC]; AUC(dextromethorphan)/AUC(dextrorphan)) was essentially unchanged (point estimate 1.289, 90% CI 0.778-2.136). P-glycoprotein activity was slightly lower in patients (digoxin maximum concentration point estimate 1.304, 90% CI 1.034-1.644). CONCLUSIONS The overall CYP3A activity was approximately 50% lower in HIV-infected patients than in healthy volunteers. The CYP2D6 activity was highly variable, but, on average was not different between groups, whereas a marginally lower P-glycoprotein activity was observed in treatment-naive HIV-infected patients.

No role for the CYP3A5*3 polymorphism in intestinal and hepatic metabolism of midazolam

Kharasch et al. [1] recently published a study in Clinical Pharmacology and Therapeutics on the role of CYP3A5 polymorphisms in the pharmacokinetics and pharmacodynamics of the CYP3A probe drugs alfentanil and midazolam, both CYP3A4 and CYP3A5 substrates. These researchers reported that there was no relevant impact of CYP3A5 alleles *3, *6 and *7 on systemic or apparent oral clearances of alfentanil and midazolam, although these polymorphisms did confer decreased CYP3A5 expression and activity. We have assessed data on midazolam pharmacokinetics following semi-simultaneous administration of midazolam orally and intravenously (i.v.) to 59 healthy male volunteers and 30 human immunodeficiency virus (HIV)-infected patients who had participated in six phenotyping studies performed earlier (see Table 1). Our methodology allowed for separate assessment of intestinal and hepatic CYP3A activity, and the results were related to the main CYP3A5 polymorphism CYP3A5*3. All studies were approved by the Ethics Committee of the Medical Faculty of the University of Cologne and conducted in accordance with the Declaration of Helsinki; all participants gave their written informed consent. In all studies, midazolam was given orally and i.v. as part of a phenotyping cocktail, whereas the other components of the cocktails varied between the respective studies (see Table 1). In study A, the cocktail was given only once, while in studies B–F (interaction studies), phenotyping was performed in two separate study periods, with and without co-medication. CYP3A5*3 genotyping was done by LightCycler melting curve analysis using Primer R5′ (TAgTTgTACgACACA CAgCAACC), Primer F5′ (TTTgCCTCTTTgTACTTCTT CATC), Sensor 5′ (gAgCTCTTTTgTCTTTCAATATCTCTFL) and Anchor 5′ (LC Red640-CCCTgTTTggACCACA TTACCCTT–PH). Midazolam in the plasma was quantified by liquid chromatography/tandem mass spectrometry [2] Total clearance (Cl) and intestinal availability (Fi) of midazolam, which were used as metrics of hepatic and intestinal CYP3A activity, respectively, were assessed by population analysis using NONMEM software ver. 1.1 (NONMEM Project Group, University of California at San Francisco, 1998). A two-compartment model gave the best fit, allowing for the determination of Cl and oral bioavailability, whereas for the calculation of Fi an additional equation was implemented [3]. The results of the pharmacokinetic evaluation sorted by study and CYP3A5 genotype are shown in Table 1. The Eur J Clin Pharmacol (2008) 64:1033–1035 DOI 10.1007/s00228-008-0503-9

PI-56Influence of ritonavir boosted lopinavir on cytochrome P450 2D6 activity in HIV infected and healthy individuals

Ritonavir (RTV) is used as an inhibitor of CYP3A4 to increase the bioavailability of lopinavir (LPV). In addition, we examined the effect of RTV boosted LPV on CYP2D6 activity in 30 HIV infected patients and 12 healthy controls.