Andreas Lazar

Impact of P-glycoprotein on clopidogrel absorption.

The antiplatelet activity of clopidogrel is characterized by considerable interindividual differences. Variable intestinal absorption is suggested to contribute to the inconsistencies in response. We tested the hypothesis that the intestinal efflux transporter P‐glycoprotein (P‐gp) limits the oral bioavailability of clopidogrel and that variance in the MDR1 gene encoding P‐gp predicts absorption variability.

Pharmacokinetics of clopidogrel after administration of a high loading dose

The adenosine diphosphate (ADP) receptor P2Y12 blocking agent clopidogrel is clinically proven to be efficient in preventing thrombotic events. However, its therapeutic value is limited by an, as yet poorly explained, interindividual heterogeneity in platelet inhibition. To evaluate possible pharmacokinetic determinants of this response variability, we developed a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantification of unmodified inactive clopidogrel, its inactive carboxyl metabolite, and its active thiol metabolite in plasma. Analyte concentrations and platelet aggregation were assessed in ten healthy volunteers receiving an oral load of 600 mg clopidogrel. Subjects showed marked inter-individual differences in maximal platelet inhibition and in plasma pharmacokinetics. Univariate regression revealed linear correlations between maximal antiplatelet effect and peak plasma concentrations (cmax) of unchanged clopidogrel (r=0.76; p=0.01), of the carboxyl metabolite (r=0.70; p=0.03), and of the thiol metabolite (r=0.73; p=0.02), as well as linear correlations between cmax values of clopidogrel and its metabolites. This indicates that the response variability is predominantly caused by individual differences in clopidogrel absorption and that other factors, such as ADP receptor reactivity or differences in bioactivation of clopidogrel, do not play a major role.

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)

Pharmacogenetics of Oral Anticoagulants

Coumarin derivatives, including warfarin, acenocoumarol and phenprocoumon, are the drugs of choice for long-term treatment and prevention of thromboembolic events. The management of oral anticoagulation is challenging because of a large variability in the dose-response relationship, which is in part caused by genetic polymorphisms. The narrow therapeutic range may result in bleeding complications or recurrent thrombosis, especially during the initial phase of treatment. The aim of this review is to systematically extract the published data reporting pharmacogenetic influences on oral anticoagulant therapy and to provide empirical doses for individual genotype combinations. To this end, we extracted all data from clinical studies of warfarin, phenprocoumon and acenocoumarol that reported genetic influences on either the dose demand or adverse drug effects, such as bleeding complications. Data were summarized for each substance, and the relative effect of each relevant gene was calculated across studies, assuming a linear gene-dose effect in Caucasians. Cytochrome P450 (CYP) 2C9, which is the main enzyme for rate-limiting metabolism of oral anticoagulants, had the largest impact on the dose demand. Compared with homozygous carriers of CYP2C9*1, patients homozygous for CYP2C9*3 were estimated to need 3.3-fold lower mean doses of warfarin to achieve the same international normalized ratio, with *2 carriers and heterozygous patients in between. Differences for acenocoumarol and phenprocoumon were 2.5-fold and 1.5-fold, respectively. Homozygosity of the vitamin K epoxide reductase complex subunit 1 (VK0RC1) variant C1173T (*2) allele (VKORC1 is the molecular target of anticoagulant action) was related to 2.4-fold, 1.6-fold and 1.9-fold lower dose requirements compared with the wild-type for warfarin, acenocoumarol and phenprocoumon, respectively. Compared with CYP2C9 and VKORC1 homozygous wild-type individuals, patients with polymorphisms in these genes also more often experience severe overanticoagulation. An empirical dose table, which may be useful as a basis for dose individualization, is presented for the combined CYP2C9/VKORC1 genotypes. Genetic polymorphism in further enzymes and structures involved in the effect of anticoagulants such as γ-glutamylcarboxylase, glutathione S-transferase A1, microsomal epoxide hydrolase and apolipoprotein E appear to be of negligible importance.Despite the clear effects of CYP2C9 and VKORC1 variants, these polymorphisms explain less than half of the interindividual variability in the dose response to oral anticoagulants. Thus, while individuals at the extremes of the dose requirements are likely to benefit, the overall clinical merits of a genotype-adapted anticoagulant treatment regimen in the entire patient populations remain to be determined in further prospective clinical studies.

Extraneuronal monoamine transporter and organic cation transporters 1 and 2: a review of transport efficiency.

The extraneuronal monoamine transporter (EMT) corresponds to the classical steroid-sensitive monoamine transport mechanism that was first described as uptake2 in rat heart with noradrenaline as substrate. The organic cation transporters OCT1 and OCT2 are related to EMT. The three carriers share basic structural and functional characteristics. Hence, EMT, OCT1 and OCT2 constitute a group referred to as non-neuronal monoamine transporters or organic cation transporters. After a brief general introduction, this review focuses on the critical analysis of substrate specificity. We calculate from the available literature and compare consensus transport efficiency (clearance) data for human and rat EMT, OCT1 and OCT2, expressed in transfected cell lines. From the plethora of inhibitors that have been tested, the casual observer likely gets the impression that these carriers indiscriminately transport very many compounds. However, our knowledge about actual substrates is rather limited. 1-Methyl-4-phenylpyridinium (MPP+) is an excellent substrate for all three carriers, with clearances typically in the range of 20-50 microl min(-1) mg protein(-1). The second-best general substrate is tyramine with a transport efficiency (TE) range relative to MPP+ of 20%-70%. The TEs of OCT1 and OCT2 for dopamine, noradrenaline, adrenaline and 5-HT in general are rather low, in the range relative to MPP+ of 5%-15%. This suggests that OCT1 and OCT2 are not primarily dedicated to transport these monoamine transmitters; only EMT may play a significant role in catecholamine inactivation. For many substrates, such as tetraethylammonium, histamine, agmatine, guanidine, cimetidine, creatinine, choline and acetylcholine, the transport efficiencies are markedly different among the carriers.

Dermal absorption of permethrin following topical administration

ObjectivePermethrin is an insecticide used in the treatment of lice and scabies infections. Although its efficacy and safety have been well documented, pharmacokinetic data are sparse. The objective of this study was to determine the systemic exposure of permethrin and the duration of residence in the human body following topical administration.MethodsThe study consisted of three parts. In six young healthy men (part 1), 50xa0ml of an ethanolic solution containing 215xa0mg permethrin (cis/trans: 25/75) was administered to the hair of the head. In another six young healthy men (part 2) and in six male or female scabies patients (part 3), 60xa0g of cream containing 3xa0g permethrin was administered to the skin of the whole body. Urine was collected up to 168xa0h post-dose. Urinary excretion of the main metabolite of permethrin, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, and its conjugates was measured using a gas chromatography/electron capture detection method.ResultsPharmacokinetics were similar in all study parts. The time of maximal urinary excretion rate was 12.3, 20.0 and 14.6xa0h, terminal elimination half-life was 32.7, 28.8 and 37.8xa0h and urinary recovery of the metabolite reached 0.35, 0.47 and 0.52xa0M percent of the permethrin dose, respectively, in parts 1, 2 and 3 (means). The treatment was well tolerated.ConclusionsThe extent of systemic exposure following external therapeutic administration of permethrin is very low compared with doses used for preclinical toxicity studies, and elimination is virtually complete after 1xa0week. These data provide the pharmacokinetic basis for the clinical safety of topical permethrin.

Cytochrome P450 2C9 phenotyping using low-dose tolbutamide

ObjectivesThe hypoglycaemic drug tolbutamide is used for assessment of CYP2C9 activity in vivo. However, therapeutically active doses of 500xa0mg bear the risk of hypoglycaemia, and a tolbutamide-derived parameter based on a single plasma or urine concentration reflecting CYP2C9 activity accurately is lacking.MethodsWe examined tolbutamide and its metabolites 4′-hydroxy-tolbutamide and carboxytolbutamide in plasma and urine of 26 healthy, male volunteers up to 24xa0h after intake of 125xa0mg tolbutamide using liquid chromatography–tandem mass spectrometry. CYP2C9 genotypes were determined by sequencing of exons 3 and 7. Raw plasma and urine data were compared with pharmacokinetic parameters, CYP2C9 genotypes, and data from a study in 23 volunteers with all six CYP2C9*1–*3 combinations who received 500xa0mg tolbutamide.ResultsPlasma clearance and tolbutamide plasma concentrations 24xa0h after drug intake reflected the genotypes: 0.85xa0l/h and 1.70xa0µg/ml (95% confidence interval, CI, 0.80–0.89xa0l/h and 1.50–1.90xa0µg/ml) for CYP2C9*1 homozygotes (n=15), 0.77xa0l/h and 2.14xa0µg/ml (95%CI, 0.67–0.88xa0l/h and 1.64–2.63xa0µg/ml) for *1/*2 genotypes (n=7), 0.60xa0l/h and 3.13xa0µg/ml (95%CI, 0.58–0.62xa0l/h and 2.68–3.58xa0µg/ml) for *1/*3 genotypes (n=3), and 0.57xa0l/h and 3.27xa0µg/ml in the single *2/*2 carrier. Natural logarithms of tolbutamide plasma concentrations 24xa0h after intake correlated to plasma clearance (r2=0.84, P<0.0000001). This correlation was confirmed in the comparison data set (r2=0.97, P<0.0000001).ConclusionsA low dose of 125xa0mg tolbutamide can safely and accurately be used for CYP2C9 phenotyping. As a simple metric for CYP2C9 activity, we propose to determine tolbutamide in plasma 24xa0h after drug intake.

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.

In vivo Role of Cytochrome P450 2E1 and Glutathione-S-Transferase Activity for Acrylamide Toxicokinetics in Humans

Acrylamide, a potential food carcinogen in humans, is biotransformed to the epoxide glycidamide in vivo. Both acrylamide and glycidamide are conjugated with glutathione, possibly via glutathione-S-transferases (GST), and bind covalently to proteins and nucleic acids. We investigated acrylamide toxicokinetics in 16 healthy volunteers in a four-period change-over trial and evaluated the respective role of cytochrome P450 2E1 (CYP2E1) and GSTs. Participants ingested self-prepared potato chips containing acrylamide (1 mg) without comedication, after CYP2E1 inhibition (500 mg disulfiram, single dose) or induction (48 g/d ethanol for 1 week), and were phenotyped for CYP2E1 with chlorzoxazone (250 mg, single dose). Unchanged acrylamide and the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA) accounted for urinary excretion [geometric mean (percent coefficient of variation)] of 2.9% (42), 65% (23), and 1.7% (65) of the acrylamide dose in the reference period. Hemoglobin adducts increased clearly following the acrylamide test-meal. The cumulative amounts of acrylamide, AAMA, and GAMA excreted and increases in AA adducts changed significantly during CYP2E1 blockade [point estimate (90% confidence interval)] to the 1.34-fold (1.14-1.58), 1.18-fold (1.02-1.36), 0.44-fold (0.31-0.61), and 1.08-fold (1.02-1.15) of the reference period, respectively, but were not changed significantly during moderate CYP2E1 induction. Individual baseline CYP2E1 activity, CYP2E1*6, GSTP1 313A>G and 341T>C single nucleotide polymorphisms, and GSTM1-and GSTT1-null genotypes had no major effect on acrylamide disposition. The changes in acrylamide toxicokinetics upon CYP2E1 blockade provide evidence that CYP2E1 is a major but not the only enzyme mediating acrylamide epoxidation in vivo to glycidamide in humans. No obvious genetic risks or protective factors in xenobiotic-metabolizing enzymes could be determined for exposed subjects. (Cancer Epidemiol Biomarkers Prev 2009;18(2):433–43)

The localisation of the extraneuronal monoamine transporter (EMT) in rat brain.

The extraneuronal monoamine transporter plays an important role in the inactivation of monoamine transmitters. A basal extraneuronal tissue expression of this transporter has been reported, but it is also expressed in CNS glia. As little is known about the expression pattern and the function of the extraneuronal monoamine transporter in the brain, we performed a detailed investigation. Firstly, a northern blot analysis of different rat organs revealed that the transporter is strongly expressed in placenta, lung and heart and less prominently in the whole brain, brain stem, intestine, testis, epididymis, stomach, kidney and skeletal muscle. It was not expressed in cerebellum, liver and embryo. Using an in situ hybridization to the rat brain, we detected a marked and highly confined expression of the extraneuronal monoamine transporter in the area postrema, but in no other brain areas. These findings were confirmed by polyclonal antibodies against rat extraneuronal monoamine transporter showing an intensive signal in the area postrema, although a few cells in the cerebellum and the brain stem also showed a signal. Additionally, a partly overlapping expression pattern of the monoamine oxidase‐B was detected. Summarizing, we firstly describe a marked and highly confined expression of the extraneuronal monoamine transporter in the rat area postrema by in situ hybridisation which may play a role in physiological functions of this circumventricular organ such as emesis, food intake and the regulation of cardiovascular functions.