Daniel Sehrt

The Effects of Genetic Polymorphisms in the Organic Cation Transporters OCT1, OCT2, and OCT3 on the Renal Clearance of Metformin

Organic cation transporters (OCTs) can mediate metformin transmembrane transport. We explored metformin pharmacokinetics in relation to genetic variations in OCT1, OCT2, OCT3, OCTN1, and MATE1 in 103 healthy male Caucasians. Renal clearance varied 3.8‐fold and was significantly dependent on creatinine clearance (r2 = 0.42, P < 0.0001), age (r2 = 0.09, P = 0.002), and OCT1 polymorphisms. Carriers of zero, one, and two low‐activity OCT1 alleles (Arg61Cys, Gly401Ser, 420del, or Gly465Arg) had mean renal clearances of 30.6, 33.1, and 37.1 l/h, respectively (P = 0.04, after adjustment for creatinine clearance and age). Immunohistochemical staining of human kidneys demonstrated OCT1 expression on the apical side of proximal and distal tubules. Increased renal clearance, in parallel with the known decreased hepatic uptake, may contribute to reduced metformin efficacy in low‐activity genotypes. Renal OCT1 expression may be important not only in relation to metformin but with respect to other drugs as well.

Interindividual Variation in the Pharmacokinetics of Δ9-Tetrahydrocannabinol as Related to Genetic Polymorphisms in CYP2C9

The impact of the CYP2C9 polymorphism on the pharmacokinetics of orally administered Δ9‐tetrahydrocannabinol (THC) was studied in 43 healthy volunteers. THC pharmacokinetics did not differ by CYP2C9*2 allele status. However, the median area under the curve of THC was threefold higher and that of 11‐nor‐9‐carboxy‐9‐tetrahydrocannabinol was 70% lower in CYP2C9*3/*3 homozygotes than in CYP2C9*1/*1 homozygotes. CYP2C9*3 carriers also showed a trend toward increased sedation following administration of THC. Therefore, the CYP2C9*3 variant may influence both the therapeutic and adverse effects of THC.

Carvedilol pharmacokinetics and pharmacodynamics in relation to CYP2D6 and ADRB pharmacogenetics

AIMS Carvedilol is an effective treatment in hypertension and chronic heart failure. The medical impact of polymorphisms in CYP2D6 and in the β-adrenergic receptors ADRB1 and ADRB2 on the pharmacokinetics and pharmacodynamics of carvedilol is controversial. METHODS After carvedilol 25 mg was administered to 110 volunteers, concentrations were enantioselectively quantified and effects on resting and exercise-induced heart rate and blood pressure were analyzed using population pharmacokinetic, pharmacodynamic and pharmacogenetic modeling. RESULTS There were significant CYP2D6 allele-specific differences in carvedilol pharmacokinetics, but the CYP2D6 genotype had no effect on heart rate, blood pressure or adverse effects. ADRB1 Gly49 was associated with higher baseline heart rates and with greater carvedilol effects on exercise heart rates. Carriers of ADRB2 Gln27 had greater reduction in resting blood pressure by carvedilol compared with Glu27. CONCLUSION Carvedilol is a drug where CYP2D6-related pharmacokinetic variation is apparently not carried forward into pharmacodynamic variation. Although current knowledge does not allow utilizing ADRB1 and ADRB2 genotypes for clinical treatment decisions, our data should stimulate further research on the impact of these genotypes in health and disease.

Genetic variation in the renal sodium transporters NKCC2, NCC, and ENaC in relation to the effects of loop diuretic drugs.

There is little data on genetic predictors of loop diuretic efficacy in humans. Therefore, we investigated the diuretic effects of single oral doses of bumetanide, frusemide, and torsemide in a crossover study in 97 healthy Caucasians in relation to genetic variation in the renal sodium transporters NKCC2 (coded by SLC12A1), NCC (SLC12A3), and ENaC (three subunits coded by SCNN1A, SCNN1B, and SCNN1G). The NCC alanine 264 allele (Gly264Ala) and the most frequent SCNN1B haplotype were associated with stronger diuresis, indicating lower reabsorbing function of these alleles. The variant alleles of the tightly coupled polymorphisms rs5723 (Leu649Leu) and rs5729 in SCNN1G were associated with weaker diuresis, indicating higher activity. Extended haplotype homozygosity implied evolutionary selection of the NCC alanine 264 allele. In conclusion, acute diuretic effects of loop diuretics were affected by genetic variation in sodium transporters that, in the nephron, are located distally from NKCC2.

Amelogenin-based sex identification as a strategy to control the identity of DNA samples in genetic association studies

Misassignment between DNA samples and clinical or epidemiological data may compromise the results of genetic association studies. Genotyping in replicates or controlling for Hardy-Weinberg equilibrium cannot identify misassignments caused by sample mix-ups. DNA-based sex identification (sex typing) is currently the best strategy to identify mix-ups. Here we review the available methods and present validated protocols for sex typing. The protocols are based on single-nucleotide differences between the human amelogenin genes, AMELX and AMELY, and are optimized for real-time PCR (TaqMan), primer-extension (SNaPshot) and PCR-RFLP genotyping platforms. In addition, we review the limitations of the sex-typing strategy, including a limited ability to identify single sample mix-ups, the dependence of the power of this approach on the sex distribution in the study population, and rare genetic conditions. Alternative strategies for mix-up identification and possible consequences of mix-up identification are also discussed.

Heritability of metoprolol and torsemide pharmacokinetics

Genetic variation in the pharmacokinetics of metoprolol and torsemide due to polymorphisms in CYP2D6, CYP2C9, and OATP1B1 has been extensively studied. However, it is still unknown how much of the variation in pharmacokinetics of these two clinically important drugs in total is due to genetic factors. Metoprolol and torsemide were intravenously administered to 44 monozygotic and 14 dizygotic twin pairs. Metoprolol area under the curve (AUC) varied 4.7‐fold and torsemide AUC 3.5‐fold. A very high fraction of AUC variations, 91% of metoprolol and 86% of torsemide, were found to be due to additive genetic effects. However, known genetic variants of CYP2D6, ‐2C9, and OATP1B1 explained only 39%, 2%, and 39% of that variation, respectively. Comparable results for genetically explained variation in pharmacokinetics and pharmacodynamics have been found for other substrates of these enzymes earlier. These findings indicate that a substantial fraction of the heritable variability in the pharmacokinetics of metoprolol and torsemide remains to be elucidated.

Bioinformatic and functional analysis of TGFBR1 polymorphisms.

Objectives The transforming growth factor-&bgr; (TGFB) pathway has substantial impact on cellular functions, cell proliferation, and apoptosis. We used bioinformatics, gene expression, and cell biological assays to evaluate the functionality of frequent inherited germline polymorphisms in the TGFB receptor 1 (TGFBR1). Methods In an exploratory (n=55) and confirmatory (n=106) study, we analyzed the TGFB1 pathway after incubation with TGF&bgr;1 ligand and after exposure to X-rays in peripheral blood human mononuclear cells. Expression of TGFB pathway genes was assessed by real-time PCR, and cellular viability was analyzed by flow cytometry. A total of six polymorphisms including the deletion variant (*6A) were identified to tag currently known common genetic variations in TGFBR1 and were analyzed in relation to the phenotypes. Results In accordance with a negative feedback mechanism, incubations with the ligand TGF&bgr;1 was followed by up-regulation of the intracellular SMAD7 and down-regulation of the SMAD3 mRNA molecules. The TGFBR1*6A deletion variant attenuated the suppression of SMAD3 in response to TGF&bgr;1 (P=0.02, in both studies). Moreover, cells harboring *6A were more sensitive toward cytotoxic effects of irradiation (P=0.001 after adjustment for age and sex). Cells were particularly prone toward radiation toxicity when carrying, in addition to *6A, the variant allele of rs11568785, which exhibits a strong genetic selection signature. Conclusion The *6A deletion and the linked rs11568785 polymorphisms seem to attenuate TGFB signaling. This should be considered not only for clinical–epidemiological studies on cancer susceptibility but may also be relevant for side effects from drugs or radiotherapy.

Heritability of Caffeine Metabolism: Environmental Effects Masking Genetic Effects on CYP1A2 Activity but Not on NAT2

Heritability of caffeine pharmacokinetics and cytochrome P450 1A2 (CYP1A2) activity is controversial. Here, we analyzed the pharmacokinetics of caffeine, an in vivo probe drug for CYP1A2 and arylamine N‐acetyltransferase 2 (NAT2) activity, in monozygotic (MZ) and dizygotic (DZ) twins. In the entire group, common and unique environmental effects explained most variation in caffeine area under the curve (AUC). Apparently, smoking and hormonal contraceptives masked the genetic effects on CYP1A2 activity. However, when excluding smokers and users of hormonal contraceptives, 89% of caffeine AUC variation was due to genetic effects and, even in the entire group, 8% of caffeine AUC variation could be explained by a CYP1A1/1A2 promotor polymorphism (rs2470893). In contrast, nearly all of the variations (99%) of NAT2 activity were explained by genetic effects. This study illustrates two very different situations in pharmacogenetics from an almost exclusively genetic determination of NAT2 activity with no environmental modulation to only moderate genetic effects on CYP1A2 activity with strong environmental modulation.

Genetic polymorphisms in the glucuronosyltransferases 1A1, 1A8 and 1A9 in relation to pharmacokinetics of furosemide

About 30% of the loop diuretic furosemide is eliminated via glucuronidation. According to in vitro data, uridine diphosphate glucuronosyltransferase (UGT) 1A8 may catalyze in this reaction but the quantitatively relevant isoenzyme in humans has not been unambiguously identified. We analyzed whether polymorphisms in UGT1A1, 1A8 and 1A9 are associated with the pharmacokinetics of furosemide.

Endpunkte in der Forschung am Menschen

In der klinischen Forschung besteht eine wesentliche Voraussetzung zur Gewinnung auswertbarer Studiendaten in einer vorherigen Festlegung der Forschungsziele, des Forschungsgegenstandes und uberprufbarer Hypothesen. Besonders wichtig ist dabei die Identifikation und Auswahl geeigneter Studienendpunkte. Endpunkte sind Messgrosen, die im Rahmen des Forschungsprojekts systematisch mittels einer standardisierten Methodik erfasst und dokumentiert werden. Die Zuverlassigkeit und Validitat der Endpunkte einschlieslich der geeigneten Messmethodik zu deren Bestimmung sind fur die spatere Aussagekraft der Studienergebnisse eine zwingende Voraussetzung.