Strategies for personalized medicine-based research and implementation in the clinical workflow.

The decoding of the human genome, paralleled by the development of high-throughput technologies to obtain large-scale molecular data (e.g., information on genetic variations, transcription levels, and metabolite concentrations) is providing new insights into the molecular basis of common diseases and the causes of variability in drug response. This article presents strategies to incorporate this new knowledge into research and clinical workflow.

Population imaging as valuable tool for personalized medicine.

Personalized medicine is commonly regarded as an extension of genomic medicine. However, a personalized treatment should not [corrected] be based solely on the presence or absence of genetic factors. Complex imaging methods supplement the diagnostic picture of an individual patient. Comprehensive imaging in population-based settings provides information on reference intervals, the predictive value of subclinical findings, and the complex interrelationships among risk factors, subclinical imaging phenotypes, and diseases.

Compartment-specific gene regulation of the CAR inducer efavirenz in vivo.

Nuclear receptors such as the constitutive androstane receptor (CAR) are central factors that link drug exposure to the activities of drug metabolism and elimination. In order to determine the in vivo effects of efavirenz, a CAR activator, the expression of target genes was determined in duodenal biopsies obtained from 12 healthy volunteers before treatment and after 10 days of treatment with efavirenz; concomitant administration of the cholesterol inhibitor ezetimibe produced no significant difference. However, in in vitro studies, efavirenz significantly increased CYP2B6 expression in several cell types, suggesting that the drug transactivates CAR. This hypothesis is supported by our findings that there is significant induction of CAR target genes in in vivo peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers treated with multiple doses of efavirenz. The impact of efavirenz on hepatic metabolism in vivo was confirmed by significant changes in plasma 4ß-hydroxycholesterol and bilirubin levels and the area under the curve (AUC) of efavirenz. Induction of CYP2B6 mRNA expression correlated with the decrease in the AUC of efavirenz (r = 0.61; P = 0.036). Taken together, our results provide evidence that efavirenz exerts compartment-specific inductive capacity in vivo.

Impact of efavirenz on intestinal metabolism and transport: insights from an interaction study with ezetimibe in healthy volunteers.

Hypercholesterolemia frequently occurs in patients treated with efavirenz who cannot be treated adequately with statins because of drug interactions. These patients may benefit from cholesterol-lowering therapy with ezetimibe. This study determined the influence of single-dose and multiple-dose efavirenz (400 mg/day for 9 days) on the pharmacokinetics and sterol-lowering of ezetimibe (10 mg) in 12 healthy subjects. In addition, the influence of efavirenz on genome-wide intestinal expression and in vitro function of ABCB1, ABCC2, UGT1A1, and OATP1B1 was studied. Efavirenz (multiple dose) had no influence on the pharmacokinetics and lipid-lowering functions of ezetimibe. Intestinal expression of enzymes and transporters (e.g., ABCB1, ABCC2, and UGT1A1) was not affected by chronic efavirenz. Efavirenz (single dose) slightly increased ezetimibe absorption and markedly decreased exposure to ezetimibe-glucuronide (single dose and multiple dose), which may be explained by inhibition of UGT1A1 and ABCB1 (in vitro data). Ezetimibe had no effect on the disposition of efavirenz. Consequently, ezetimibe may be a safe and efficient therapeutic option in patients with HIV infection.

Regulation of interferon-inducible proteins by doxorubicin via interferon γ-Janus tyrosine kinase-signal transducer and activator of transcription signaling in tumor cells.

Activation of the immune system is a way for host tissue to defend itself against tumor growth. Hence, treatment strategies that are based on immunomodulation are on the rise. Conventional cytostatic drugs such as the anthracycline doxorubicin can also activate immune cell functions of macrophages and natural killer cells. In addition, cytotoxicity of doxorubicin can be enhanced by combining this drug with the cytokine interferon-γ (IFNγ). Although doxorubicin is one of the most applied cytostatics, the molecular mechanisms of its immunomodulation ability have not been investigated thoroughly. In microarray analyses of HeLa cells, a set of 19 genes related to interferon signaling was significantly over-represented among genes regulated by doxorubicin exposure, including signal transducer and activator of transcription (STAT) 1 and 2, interferon regulatory factor 9, N-myc and STAT interactor, and caspase 1. Regulation of these genes by doxorubicin was verified with real-time polymerase chain reaction and immunoblotting. An enhanced secretion of IFNγ was observed when HeLa cells were exposed to doxorubicin compared with untreated cells. IFNγ-neutralizing antibodies and inhibition of Janus tyrosine kinase (JAK)-STAT signaling [aurintricarboxylic acid (ATA), (E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG490), STAT1 small interfering RNA] significantly abolished doxorubicin-stimulated expression of interferon signaling-related genes. Furthermore, inhibition of JAK-STAT signaling significantly reduced doxorubicin-induced caspase 3 activation and desensitized HeLa cells to doxorubicin cytotoxicity. In conclusion, we demonstrate that doxorubicin induces interferon-responsive genes via IFNγ-JAK-STAT1 signaling and that this pathway is relevant for doxorubicin's cytotoxicity in HeLa cells. Immunomodulation is a promising strategy in anticancer treatment, so this novel mode of action of doxorubicin may help to further improve the use of this drug among different types of anticancer treatment strategies.

[Strategies to improve medication adherence]. / Strategien zur Verbesserung der Einnahmetreue von Medikamenten.

Up to 50 % of patients with chronic diseases do not take their medication regularly. Poor adherence to drug therapy is associated with higher morbidity and mortality. A selective literature search using the terms adherence, compliance, concordance, persistence, medication management, and pharmaceutical care was performed. Evidence for improving adherence has been provided for the following principles: individual counselling of patients and care givers, medication management including simplifying dosing and use of combination tablets as well as the use of individual unit doses, e. g. blister cards. The effectiveness has only been shown for the duration of the interventions. The improvement of medication adherence represents an area of research with high impact on outcomes and cost. Measures to improve adherence may be as important as the development of novel therapies. However, prospective clinical evaluations with clinical endpoints are missing especially for the German health care system in order to develop recommendations for clinical practice. Joint efforts of physicians and pharmacists are needed.

[Pharmacogenomics: hype or hope?]. / Pharmakogenomik: Hype oder Hope?

Pharmacogenomics follows the concept of individualized medicine via targeted and tailored therapy for each patient. This is aided by current progress in human genome research that provided the basis to link genetic markers with pathogenesis and progression for the definition of new therapy options. To achieve this it will be necessary to show the validity and clinical utility of pharmacogenomic tests prior to release. The implementation of pharmacogenomics into clinical practise is a momentous future challenge that requires the establishment of interdisciplinary networks and professional organizations.

The growth hormone--IGF-I axis as a mediator for the association between FTO variants and body mass index: results of the Study of Health in Pomerania.

CONTEXT: Risk alleles of the fat mass- and obesity-associated gene (FTO) are related not only to increased body mass index (BMI) values but also to mortality. It was speculated that cellular effects of the FTO gene affect most organs, especially their ability to maintain or regenerate proper function when afflicted by various diseases. FTO is highly expressed in the hypothalamus and also in the pituitary gland. The decrease in growth hormone (GH) secretion is known to cause a decrease in lean body mass in older subjects. OBJECTIVE: We hypothesized an association of rs9926289 with insulin-like growth factor (IGF)-I. DESIGN AND SETTING: Cross-sectional data from the Study of Health in Pomerania, a population-based study in the northeastern part of Germany, were used. PARTICIPANTS: For the final analyses, 3882 subjects aged 20-79 years were available. MAIN OUTCOME MEASURES: Continuous IGF-I, low IGF-I according to clinically meaningful age- and gender-specific reference values, and BMI were used as outcome measures. RESULTS: Over all age groups, a statistically significant relationship between FTO and IGF-I was found. In subjects younger than 55 years of age, homozygous carriers of the FTO risk allele exhibited lower serum IGF-I levels adjusted for 5-year age groups, gender and IGF-I binding protein 3 levels (linear regression, coefficient±s.e. for FTO AA genotype:-8.6±2.8; P=0.002). Further adjustments for obesity and diabetes did not suspend this association (coefficient:-7.8; P=0.005). As expected, the FTO AA genotype effect on BMI was reduced from 0.76 to 0.62 kg m(-2) by including IGF-I. No relationship between FTO and IGF-I levels was found in subjects aged 55 years or older (-2.7±2.4; P=0.260 for FTO AA genotype adjusted for age, gender and IGF-I binding protein 3 levels). CONCLUSION: We propose that the GH-IGF-I axis is a mediator for the relationship between FTO and BMI.

MDR1-P-glycoprotein (ABCB1)-mediated disposition of amyloid-ß peptides: implications for the pathogenesis and therapy of Alzheimer's disease.

The accumulation of neurotoxic amyloid-ß (Aß) peptides within the brain represents a hallmark of Alzheimer's disease (AD). It is proposed to be partly due to reduced elimination of Aß from the brain into the blood. Diverse mechanisms of Aß clearance out of the brain have been suggested. As discussed here, several lines of evidence suggest a significant role of the MDR1-P-glycoprotein (ABCB1), which is a major component of the blood-brain barrier (BBB).

Doxorubicin-induced cell death requires cathepsin B in HeLa cells.

The cysteine protease cathepsin B acts as a key player in apoptosis. Cathepsin B-mediated cell death is induced by various stimuli such as ischemia, bile acids or TNFα. Whether cathepsin B can be influenced by anticancer drugs, however, has not been studied in detail. Here, we describe the modulation of doxorubicin-induced cell death by silencing of cathepsin B expression. Previously, it was shown that doxorubicin, in contrast to other drugs, selectively regulates expression and activity of cathepsin B. Selective silencing of cathepsin B by siRNA or the cathepsin B specific inhibitor CA074Me modified doxorubicin-mediated cell death in Hela tumor cells. Both Caspase 3 activation and PARP cleavage were significantly reduced in cells lacking cathepsin B. Moreover, mitochondrial membrane permeabilization as well as the release of cytochrome C and AIF from mitochondria into cytosol induced by doxorubicin were significantly diminished in cathepsin B suppressed cells. In addition, doxorubicin associated down-regulation of XIAP was not observed in cathepsin B silenced cells. Lack of cathepsin B significantly modified cell cycle regulatory proteins such as cdk1, Wee1 and p21 without significant changes in G(1), S or G(2)M cell cycle phases maybe indicating further cell cycle independent actions of these proteins. Consequently, cell viability following doxorubicin was significantly elevated in cells with cathepsin B silencing. In summary, our data strongly suggest a role of cathepsin B in doxorubicin-induced cell death. Therefore, increased expression of cathepsin B in various types of cancer can modify susceptibility towards doxorubicin.

Fcgamma-receptor IIa polymorphism and the role of immunoadsorption in cardiac dysfunction in patients with dilated cardiomyopathy.

In patients with dilated cardiomyopathy (DCM), cardiac autoantibodies are able to bind with their Fab fragment to epitopes on cardiomyocytes, but thereafter they crosslink through their Fc fragment to cardiac Fc(gamma)-receptor IIa. Polymorphic variability of the Fc(gamma)-receptor IIa is associated with modified affinity of immunoglobin G (IgG) binding and may influence therapeutic effects. In this study, 103 consecutive DCM patients were treated with immunoadsorption (IA) therapy with subsequent IgG substitution (IA/IgG). Echocardiography was performed at baseline and again at 3 and 6 months after IA/IgG. Fc(gamma)-receptor IIa polymorphism R/H131 was genotyped using a nested sequence-specific primer polymerase chain reaction (PCR). Patients with the Fc(gamma)-receptor IIa genotype R/R131 showed significantly greater improvement in left ventricular (LV) function than patients with the R/H131 or H/H131 genotypes did. Irrespective of the Fc(gamma)-receptor polymorphism, patients with shorter disease duration and a more impaired LV function responded with a greater increase in LV ejection fraction (LVEF). Therefore, the Fc(gamma)-receptor polymorphism influences the efficacy of immunomodulatory therapy involving IA/IgG.

[Pharmacogenomics in routine medical care]. / Pharmakogenomik in der Praxis.

Pharmacogenomics investigates inherited differences in drug responses including beneficial and adverse reactions. While a considerable amount of evidence for genetic influences on drug responses has been accumulated within the last decade, predominantly in small studies, its value in routine therapy is still a matter of debate. The aim of this review is to discuss well established examples where pharmacogenomic techniques can improve routine treatment. Examples include genotyping of CYP2D6 in the context of antidepressant therapy, analysis of TPMT variants for the prediction of mercaptopurine-induced bone marrow depression, VKORC1 and CYP2C9 analyses for a better control of anticoagulant administration and the SLCO1B1 variant in the context of statin-induced myopathies.

A piece in the puzzle of personalized medicine.

Predicting individual drug response based on inherited factors remains a major challenge. Some examples of significant contributions of single genetic variants to overall therapeutic success of drugs in complex diseases are currently emerging. For most compounds, however, multiple genetic and nongenetic factors will modify drug action. Comprehensive integration of these factors will determine the role of pharmacogenomics for personalized medicine.

[Pharmacokinetic problems in clinical practice: role of drug transporters]. / Pharmakokinetische Probleme in der Praxis: Rolle von Arzneimitteltransportern.

Drug disposition is controlled by drug metabolism and drug transport. In the last decade numerous drug transporters have been identified and characterized in the context of drug uptake, efflux and interactions. This article reviews major advancements in this field. Efflux pumps like the multidrug resistance protein 1 (MDR1, ABCB1) are expressed in the intestine where they secrete drugs back into the intestinal lumen. Inhibitors of ABCB1 can increase the bioavailability of such drugs due to an increased absorption. Inducers of metabolism (rifampicin, carbamazepine, St. John's Wort) also induce the expression of drug transporters like ABCB1. Subsequently, an increased intestinal secretion in addition to an increased metabolism can diminish plasma levels of drugs, for example ciclosporin. The relevance of uptake transporters is increasingly recognized. SLCO1B1 is a hepatic uptake transporter involved in the absorption of statins. Inhibition of SLCO1B1 as well as common genetic variants can lead to increased bioavailability and to adverse reactions, ultimately culminating in rhabdomyolysis.

Vardenafil protects isolated rat hearts at reperfusion dependent on GC and PKG.

BACKGROUND AND PURPOSE: The type-5 PDE inhibitor vardenafil reduces myocardial infarct size in situ, following ischemia/reperfusion, when applied at reperfusion in animal models. Little is known about the underlying protective signaling. Here, we test whether vardenafil is protective in rat isolated hearts and in a cell model of calcium stress. EXPERIMENTAL APPROACH: Infarct size in rat isolated hearts was measured after a 30 min regional ischemia and 120 min reperfusion. Vardenafil (1 nM-1 microM) was infused during reperfusion. HL-1 cardiomyocytes were loaded with tetramethylrhodamine ethyl ester (TMRE), a fluorescent marker of mitochondrial membrane potential (psi m). KEY RESULTS: Vardenafil at reperfusion reduced infarct size as percentage of the ischemic zone from 45.8+/-2.0% in control hearts to 26.2+/-2.7% (P<0.001) only at 10 nM, whereas higher or lower dosages failed to protect. This protective effect was blocked by co-administration of either the GC inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or the PKG inhibitor, KT-5823. HL-1 cardiomyocytes, loaded with TMRE, were treated for 80 min with the calcium ionophore, calcimycin, to induce calcium stress. This reduced the mean cell fluorescence to 63.3 +/- 3.8% of baseline values and vardenafil protected against this fall (78.6 +/- 3.6%, P<0.01). The vardenafil-induced protection of HL-1 cells was blocked by ODQ, KT-5823 or the PKG-inhibiting peptides DT-2 and DT-3, confirming a role for GC and PKG. CONCLUSIONS AND IMPLICATIONS: These results further support the hypothesis that PDE-5 inhibitors are protective in ischemic hearts, in addition to their known clinical effects in the treatment of erectile dysfunction in men.

Variability in human hepatic MRP4 expression: influence of cholestasis and genotype.

The multidrug resistance protein 4 (MRP4) is an efflux transporter involved in the transport of endogenous substrates and xenobiotics. We measured MRP4 mRNA and protein expression in human livers and found a 38- and 45-fold variability, respectively. We sequenced 2 kb of the 5'-flanking region, all exons and intron/exon boundaries of the MRP4 gene in 95 patients and identified 74 genetic variants including 10 non-synonymous variations, seven of them being located in highly conserved regions. None of the detected polymorphisms was significantly associated with changes in the MRP4 mRNA or protein expression. Immunofluorescence microscopy indicated that none of the non-synonymous variations affected the cellular localization of MRP4. However, in cholestatic patients the MRP4 mRNA and protein expression both were significantly upregulated compared to non-cholestatic livers (protein: 299+/-138 vs 100+/-60a.u., P<0.001). Taken together, human hepatic MRP4 expression is highly variable. Genetic variations were not sufficient to explain this variability. In contrast, cholestasis is one major determinant of human hepatic MRP4 expression.

Transporter-mediated uptake into cellular compartments.

Active transport across biological membranes represents a critical step in the disposition of many drugs. It is now well-established that different efflux and uptake transporters such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) or organic anion transporting polypeptides (OATPs) are involved in the overall disposition and efficacy of numerous compounds. These proteins are mainly expressed at physiological sites of drug absorption and elimination, thus leading to diminished absorption and/or increased transporter-facilitated excretion. Moreover, drug transporters are known to be of protective significance in blood-organ barriers. On the contrary, only little is known about the relevance of transporter function on drug levels within tissues and cellular compartments, i.e. the site of action for many substances. Moreover, the pharmacokinetic processing inside the cell is characterized by uptake, metabolism and elimination. It is gradually being recognized that active uptake and/or efflux transporters may modify target concentrations at the subcellular receptor sites which in turn may have an influence on drug effects. This review will summarize current knowledge about the impact of transporter proteins on drug availability within pharmacologically relevant cellular compartments and tissues as hepatocytes, enterocytes, different blood cell types, brain, and the heart with emphasis on the potential clinical significance of these transporters.