Thomas Gerloff

Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects

P‐glycoprotein, the gene product of MDR1, confers multidrug resistance against antineoplastic agents but also plays an important role in the bioavailability of common drugs in medical treatment. Various polymorphisms in the MDR1 gene were recently identified. A silent mutation in exon 26 (C3435T) was correlated with intestinal P‐glycoprotein expression and oral bioavailability of digoxin.

Modulation of steady‐state kinetics of digoxin by haplotypes of the P‐glycoprotein MDR1 gene

We investigated the effect of polymorphisms in the P‐glycoprotein (P‐gp) MDR1 gene on steady‐state pharmacokinetics of digoxin in Caucasians. According to earlier data, homozygous TT of the exon 26 complementary deoxyribonucleic acid (cDNA) 3435C>T polymorphism was associated with low P‐gp expression in the human intestine.

Evidence for Inverse Effects of OATP-C (SLC21A6) *5 and *1b Haplotypes on Pravastatin Kinetics

We compared the pharmacogenetic effects of OATP‐C (organic anion transporting polypeptide C) *1a, *1b (A388G), and *5 (T521C) haplotypes on single‐dose pharmacokinetics of pravastatin in white subjects.

Differential expression of basolateral and canalicular organic anion transporters during regeneration of rat liver

BACKGROUND & AIMS Liver regeneration in response to various forms of injury or surgical resection is a complex process resulting in restoration of the original liver mass and maintenance of liver-specific functions such as bile formation. However, liver regeneration is frequently associated with cholestasis, whose molecular pathogenesis remains unknown. METHODS To study the molecular mechanisms leading to cholestasis, expression of all major hepatic organic anion transporters contributing to bile formation was determined for up to 2 weeks in rats after 70% partial hepatectomy. RESULTS Inversely related to serum bile acid levels, basolateral transporters including the sodium-taurocholate cotransporter (Ntcp) and the organic anion transporting polypeptides Oatp1 and Oatp2 were markedly down-regulated at both protein and steady-state mRNA levels by 50%-60% of controls (P < 0.05) during early replicative stages of regeneration (12 hours to 2 days) with a slightly delayed time course for Oatp2. Expression of all basolateral transporters returned to control values between 4 and 4 days after partial hepatectomy. In contrast, protein and mRNA expression of both the canalicular ATP-dependent bile salt export pump (Bsep) and the multiorganic anion transporter Mrp2 remained unchanged or were slightly increased during liver regeneration, but also returned to control values 7-14 days after partial hepatectomy. CONCLUSIONS The data suggest a differential regulation of basolateral and canalicular organic anion transporters in the regenerating liver. Unaltered expression of Bsep and Mrp2 provides a potential molecular mechanism for regenerating liver cells to maintain or even increase bile secretion expressed per weight of remaining liver. However, down-regulation of basolateral organic anion transporters might protect replicating liver cells by diminishing uptake of potentially hepatotoxic bile salts, because the remaining liver initially cannot cope with the original bile acid pool size.

Hepatobiliary organic anion transporters are differentially regulated in acute toxic liver injury induced by carbon tetrachloride

BACKGROUND/AIMS Hepatobiliary transporters are down-regulated in cholestasis, but their expression in acute, non-cholestatic, cytokine-mediated liver injury is unknown. Thus we studied the molecular mechanisms, by which sodium taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, multidrug-resistance protein 2 (Mrp2) and bile salt export pump (Bsep) are regulated in liver injury induced by carbon tetrachloride (CCl(4)). METHODS mRNA and protein levels were determined in rats 24 and 72h after CCl(4) injection. Transporter gene transcription and binding activities of Ntcp and Mrp2 transactivators were assessed by nuclear runoff and electrophoretic mobility shift assays. RESULTS mRNA levels significantly declined to 41+/-44% for Ntcp, 65+/-41% for Oatp1 and 64+/-28% for Oatp2, but remained unchanged for Oatp4, canalicular Mrp2 and Bsep. Protein levels declined only for Oatp4 (-50+/-17%) and Ntcp (-23+/-13%) at 24h. Reduced mRNA levels (Ntcp, Oatp1, Oatp2) were associated with decreased transcriptional activities. Binding activity of Ntcp transactivators (hepatocyte nuclear factor 1 alpha (HNF1alpha) and CAAT enhancer binding protein alpha (C/EBPalpha) were reduced by 24h, whereas retinoid X receptor alpha (RXRalpha):retinoid acid receptor alpha (RARalpha) as transactivator of both Ntcp and Mrp2 remained unaltered. Recovery of acute hepatitis and changes in gene expression occurred after 72h. CONCLUSIONS Acute liver injury results in down-regulation of basolateral organic anion transporters similar to liver regeneration after partial hepatectomy, but in contrast to endotoxin-induced cholestasis. Maintained binding activity of RXRalpha:RARalpha may explain differences in Mrp2 expression.

Regulation of basolateral organic anion transporters in ethinylestradiol-induced cholestasis in the rat

BACKGROUND/AIMS Estrogen-mediated cholestasis is an important clinical entity, but its molecular pathophysiology is still not fully understood. Impaired sodium-dependent uptake of bile acids has been associated with diminished expression of a basolateral Na(+)/bile acid cotransporter (Ntcp), whereas sodium-independent uptake is maintained despite a down-regulation of the organic anion transporter Oatp1. Thus, expression of the two other rat Oatps (Oatps2 and -4) was determined in estrogen-induced cholestasis. In addition, known transactivators of Oatp2 and Ntcp were studied to further characterize transcriptional regulation of these transporter genes. METHODS Hepatic protein and mRNA expression of various Oatps (1, 2, 4) in comparison to Ntcp were analyzed after 0.5, 1, 3 and 5 days of ethinylestradiol (EE) treatment (5 mg/kg) in rats. Binding activities of Oatp2 and Ntcp transactivators were assessed by electrophoretic mobility shift assays. RESULTS All basolateral Oatps (1, 2 and 4) were specifically down-regulated at the protein level by 30-40% of controls, but less pronounced than Ntcp (minus 70-80%). In contrast to unaltered Oatp4 mRNA levels, Oatp1 and Oatp2 mRNAs were reduced to various extents (minus 40-90% of controls). Binding activity of known transactivators of Ntcp and Oatp2 such as hepatocyte nuclear factor 1 (HNF1), CAAT enhancer binding protein alpha (C/EBPalpha) and pregnane X receptor (PXR) were also diminished during the time of cholestasis. CONCLUSIONS Estrogen-induced cholestasis results in a down-regulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, -2 and -4 may explain the unchanged sodium-independent transport of bile acids due to overlapping substrate specificity. Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF1, C/EBP and PXR by estrogens.

Comparison of SLCO1B1 sequence variability among German, Turkish, and African populations

BackgroundOATP1B1 is one of the key hepatocellular uptake transporters providing extraction of diverse compounds, including bile acids, xenobiotics, and a variety of drugs, from portal venous blood into the liver. Polymorphisms of the SLCO1B1 gene have been demonstrated to influence in vitro transport function and the pharmacokinetic profile of compounds.ObjectiveThe goal of our study was the comparison of SLCO1B1 gene sequence variability in three ethnic groups as a basis for future genetic association studies.MethodsEighteen exonic SLCO1B1 single nucleotide polymorphisms (SNPs) were genotyped by PCR and RFLP analysis in 300 German, 94 Turkish, and 115 African subjects. Calculation of pairwise linkage disequilibrium and estimation of population haplotype frequencies were carried out, and haplotype block structure was determined.ResultsOnly eight genotyped SNPs (c.388A>G, c.411G>A, c.463C>A, c.521T>C, c.571C>T, c.597C>T, c.1463G>>C, c.1929A>C) were found in at least one of our German, Turkish, or African samples. A total of 12 haplotypes with a frequency ≥1% in at least one of the three populations could be inferred. Between the Caucasian and African samples, significant differences in sequence variability were observed leading to a different haplotype profile in these populations.ConclusionOur results demonstrate a high sequence variability of OATP1B1 within different popuations. In the future, distinct haplotypes should be taken into account when studying the effect of OATP1B1 on drugs in different populations.

Pharmacogenetics-based new therapeutic concepts.

Pharmacogenetics, one of the fields of clinical pharmacology, studies how genetic factors influence drug response. If hereditary traits are taken into account appropriately before starting drug treatment, the type of drug and its dosage can be tailored to the individual patients needs. Pharmacogenetics adds a considerable amount of stringency to the doctors therapeutic approach. Today, it is the relationship between dosage requirements and genetic variations in drug metabolizing enzymes like cytochrome P450 (CYP) 2D6 and CYP2C19, or in drug transporters like p‐glycoprotein, that is substantiated best. A standard dose will bring about more adverse effects than usual if enzymatic activity is lacking or feeble. Sometimes, however, therapeutic response might be better due to higher concentrations: proton pump inhibitors for eradication of Helicobacter pylori are more efficacious in carriers of a deficient CYP2C19 variant. The drugs interaction with its target (e.g. receptor) also depends on genetic factors. In some cases genetic tests can help distinguish between responders and non‐responders of a specific drug treatment. The first pharmacogenetic tests are already on the market.

Pharmakogenetik als Basis neuer Therapiekonzepte

ZusammenfassungPharmakogenetik als Teilgebiet der Klinischen Pharmakologie befasst sich mit dem Einfluss genetischer Faktoren auf die Arzneimittelwirkung. Durch Berücksichtigung der individuellen erblichen Eigenschaften des jeweiligen Patienten sollen die Dosis und auch die Art des Arzneimittels quasi nach Maß dem Patienten angepasst werden. Die Pharmakogenetik verhilft dem therapeutischen Vorgehen des Arztes zu mehr Stringenz. Gegenwärtig weitaus am besten belegt ist der Zusammenhang zwischen unterschiedlichem Dosisbedarf und genetischen Polymorphismen in arzneimittelmetabolisierenden Enzymen, wie z. B. dem Cytochrom P450 (CYP) 2D6 oder CYP2C19, oder in den Arzneimitteltransportern wie dem P-Glykoprotein. Bei reduzierter Aktivität dieser Enzyme oder Transporter sind bei unveränderter Dosis vermehrt Nebenwirkungen zu erwarten, der Therapieerfolg kann aber auch aufgrund höherer Konzentrationen verbessert sein. So ist die Eradikationstherapie bei Ulcus ventriculi und duodeni mit Protonenpumpenhemmern erfolgreicher bei Trägern einer Defizienz von CYP2C19. Auch die Effektivität der Arzneimittelwirkung am Wirkort (z.B.Rezeptor) unterliegt genetischen Einflüssen. So können in manchen Fällen Responder und Non-Responder einer spezifischen Arzneitherapie durch genetische Untersuchungen erkannt werden. Erste pharmakogenetische Teste sind bereits auf dem Markt erhältlich.AbstractPharmacogenetics, one of the fields of clinical pharmacology, studies how genetic factors influence drug response. If hereditary traits are taken into account appropriately before starting drug treatment, the type of drug and its dosage can be tailored to the individual patient’s needs. Pharmacogenetics adds a considerable amount of stringency to the doctor’s therapeutic approach. Today, it is the relationship between dosage requirements and genetic variations in drug metabolizing enzymes such as cytochrome P450 (CYP) 2D6 and CYP2C19, or in drug transporters such as p-glycoprotein, that is substantiated best. A standard dose will bring about more adverse effects than usual if enzymatic activity is lacking or feeble. Sometimes, however, therapeutic response might be better due to higher concentrations: proton pump inhibitors for eradication of Helicobacter pylori are more efficacious in carriers of a deficient CYP2C19 variant. The drug’s interaction with its target (e. g., receptor) also depends on genetic factors. In some cases genetic tests can help distinguish between responders and nonresponders to a specific drug treatment. The first pharmacogenetic tests are already on the market.

Clinical drug investigations. Definition of terms

ZusammenfassungIm Rahmen der 12. Novelle des Arzneimittelgesetzes wurden erstmals Definitionen für die klinische Prüfung und die nichtinterventionelle Prüfung im Gesetzestext verankert. Andere Begriffe, wie der systematische Heilversuch, der individuelle Heilversuch sowie Studien zur Post-Marketing-Surveillance oder der Versorgungsforschung sind weniger klar definiert. Dieser Artikel erläutert die verschiedenen Begriffe und geht dabei sowohl auf die Unterschiede als auch Gemeinsamkeiten ein.AbstractImplementation of the European Directive 2001/20/EC led to the inclusion of definitions for clinical trial and non-interventional trial in German Drug Law. Other terms, such as non-commercial clinical trial, single patient use, post marketing surveillance and public health study are less well defined. This article explains the various terms by comparing their differences and similarities.