Leszek Wojnowski

Clinical implications of CYP3A polymorphisms

Due to their enormous substrate spectrum CYP3A4, -3A5 and -3A7 constitute the most important drug-metabolising enzyme subfamily in humans. CYP3As are expressed predominantly, but not exclusively, in the liver and intestine, where they participate in the metabolism of 45 60% of currently used drugs and many other compounds such as steroids and carcinogens. CYP3A expression and activity vary interindividually due to a combination of genetic and nongenetic factors such as hormone and health status, and the impact of environmental stimuli. Over the past several years, genetic determinants have been identified for much of the variable expression of CYP3A5 and -3A7, but not for CYP3A4. Using these markers, an effect of CYP3A5 expression status has been demonstrated beyond doubt for therapies with the immunosuppressive drug tacrolimus. Further associations are likely to emerge for drugs metabolised predominantly by CYP3A5 or -3A7, especially for individuals or tissues with concomitant low expression of CYP3A4. However, as exemplified by the controversial association between CYP3A4*1B and prostate cancer, the detection of clinical effects of CYP3A gene variants will be difficult. The most important underlying problems are the continuing absence of activity markers specific for CYP3A4 and the strong contribution of nongenetic factors to CYP3A variability.

Antitumor Effects of a Combined 5-Aza-2′Deoxycytidine and Valproic Acid Treatment on Rhabdomyosarcoma and Medulloblastoma in Ptch Mutant Mice

Patched (Ptch) heterozygous mice develop medulloblastoma (MB) and rhabdomyosarcoma (RMS) resembling the corresponding human tumors. We have previously shown that epigenetic silencing of the intact Ptch allele contributes to tumor formation in this model. Here, we investigated whether targeting of epigenetic silencing mechanisms could be useful in the treatment of Ptch-associated cancers. A reduction of endogenous DNA methyltransferase1 (Dnmt1) activity significantly reduced tumor incidence in heterozygous Ptch knockout mice. A combined treatment with the Dnmt inhibitor 5-aza-2deoxycytidine (5-aza-dC) and the histone deacetlyase (HDAC) inhibitor valproic acid (VPA) efficiently prevented MB and RMS formation, whereas monotherapies with either drug were less effective. Wild-type Ptch expression was efficiently reactivated in tumors by 5-aza-dC/VPA combination therapy. This was associated with reduced methylation of the Ptch promoter and induction of histone hyperacetylation suggesting inhibition of HDACs in vivo. However, the treatment was not effective in clinically overt, advanced stage tumors. This is a first in vivo demonstration that targeting of Dnmt and HDAC activities is highly effective in preventing formation of Ptch-associated tumors. The results suggest a novel clinical strategy for consolidation therapy of corresponding tumors in humans after completion of conventional treatment. Our data also suggest that epigenetic therapy may be less effective in treating advanced stages of tumors, at least in this tumor model.

Genotyping the risk of anthracycline-induced cardiotoxicity

Anthracyclines belong to the most successful antineoplastic drugs, but they are cardiotoxic, which may result in congestive heart failure (CHF). The CHF risk increases with the cumulative anthracycline dose, but it seems also to be modified by individual factors. A role of the individual genetic background is consistent with the altered sensitivity to anthracyclines observed in many transgenic and knockout mouse strains. First clinical data obtained in humans suggest the existence of predisposing variants in genes involved in the oxidative stress, and in the metabolism and transport of anthracyclines. These data will have to be verified in further clinical trials before any attempts of their application in the individual cardiotoxicity prediction can be undertaken. In the meantime, anthracycline-induced cardiotoxicity can be best reduced by application of liposomal anthracycline formulations or by a co-medication with the cardioprotective iron chelator dexrazoxane.

Variability in PXR-mediated induction of CYP3A4 by commercial preparations and dry extracts of St. John’s wort

St. John’s wort (SJW, Hypericum perforatum) is a well-tolerated herbal medicine widely used for the treatment of mild and moderate depressions. In the last 5xa0years, SJW has been implicated in drug interactions, which are largely mediated by the induction of the drug metabolizing enzymes, especially CYP3A4. There is still some controversy regarding the exact mechanism of induction and the identity of the SJW constituents involved. We investigated in LS174T cells the induction of CYP3A4 by ten SJW extracts, six commercial preparations, and the purified SJW constituent hyperforin. The content of hyperforin among the commercial preparations of SJW varied 62-fold (range 0.49–30.57xa0mg/dose). The CYP3A4 induction was mediated by PXR, but not by CAR. The magnitude of the induction correlated statistically significantly with the content of hyperforin in commercial SJW preparations (Ru2009=u20090.87, pu2009=u20090.004) and in dry extracts (Ru2009=u20090.65, pu2009=u20090.03), but not with their content of flavonoids or hypericin. Most of the CYP3A4 induction response occurred in the hyperforin range encountered in the blood of patients treated with SJW preparations. A temperature-induced decrease in the hyperforin content of a selected dry SJW extract abolished the induction of CYP3A4. In conclusion, commercial SJW preparations still exhibit an enormous variability in CYP3A4 induction, which is mediated by hyperforin and PXR. SJW preparations with lower hyperforin content should reduce the frequency of clinical interactions involving this herbal drug.

6-mercaptopurine and 9-(2-phosphonyl-methoxyethyl) adenine (PMEA) transport altered by two missense mutations in the drug transporter gene ABCC4.

Multiple drug resistance protein 4 (MRP4, ABCC4) belongs to the C subfamily of the ATP‐binding cassette (ABC) transporter superfamily and participates in the transport of diverse antiviral and chemotherapeutic agents such as 6‐mercaptopurine (6‐MP) and 9‐(2‐phosphonyl methoxyethyl) adenine (PMEA). We have undertaken a comprehensive functional characterization of protein variants of MRP4 found in Caucasians and other ethnicities. A total of 11 MRP4 missense genetic variants (nonsynonymous SNPs), fused to green fluorescent protein (GFP), were examined in Xenopus laevis oocytes for their effect on expression, localization, and function of the transporter. Radiolabeled 6‐MP and PMEA were chosen as transport substrates. All MRP4 protein variants were found to be expressed predominantly in the oocyte membrane. A total of four variants (Y556C, E757u2009K, V776I, and T1142u2009M) exhibited a 20% to 40% reduced expression level compared to the wild type. Efflux studies showed that 6‐MP is transported by MRP4 in unmodified form. Compared to wild‐type MRP4, the transmembrane variant V776I, revealed a significant lower activity in 6‐MP transport, while the amino acid exchange Y556C in the WalkerB motif displayed significantly higher transport of PMEA. The transport properties of the other variants were comparable to wild‐type MRP4. Our study shows that Xenopus oocytes are well suited to characterize MRP4 and its protein variants. Carriers of the rare MRP4 variants Y556C and V776I may have altered disposition of MRP4 substrates. Hum Mutat 29(5), 659–669, 2008.

Time-point and dosage of gene inactivation determine the tumor spectrum in conditional Ptch knockouts

Mutations in Patched (PTCH) have been associated with tumors characteristic both for children [medulloblastoma (MB) and rhabdomyosarcoma (RMS)] and for elderly [basal cell carcinoma (BCC)]. The determinants of the variability in tumor onset and histology are unknown. We investigated the effects of the time-point and dosage of Ptch inactivation on tumor spectrum using conditional Ptch-knockout mice. Ptch heterozygosity induced prenatally resulted in the formation of RMS, which was accompanied by the silencing of the remaining wild-type Ptch allele. In contrast, RMS was observed neither after mono- nor biallelic postnatal deletion of Ptch. Postnatal biallelic deletion of Ptch led to BCC precancerous lesions of the gastrointestinal epithelium and mesenteric tumors. Hamartomatous gastrointestinal cystic tumors were induced by monoallelic, but not biallelic Ptch mutations, independently of the time-point of mutation induction. These data suggest that the expressivity of Ptch deficiency is largely determined by the time-point, the gene dose and mode of Ptch inactivation. Furthermore, they point to key differences in the tumorigenic mechanisms underlying adult and childhood tumors. The latter ones are unique among all tumors since their occurrence decreases rather than increases with age. A better understanding of mechanisms underlying this ontological restriction is of potential therapeutic value.

Accuracy and Completeness of Drug Information in Wikipedia: A Comparison with Standard Textbooks of Pharmacology

The online resource Wikipedia is increasingly used by students for knowledge acquisition and learning. However, the lack of a formal editorial review and the heterogeneous expertise of contributors often results in skepticism by educators whether Wikipedia should be recommended to students as an information source. In this study we systematically analyzed the accuracy and completeness of drug information in the German and English language versions of Wikipedia in comparison to standard textbooks of pharmacology. In addition, references, revision history and readability were evaluated. Analysis of readability was performed using the Amstad readability index and the Erste Wiener Sachtextformel. The data on indication, mechanism of action, pharmacokinetics, adverse effects and contraindications for 100 curricular drugs were retrieved from standard German textbooks of general pharmacology and compared with the corresponding articles in the German language version of Wikipedia. Quantitative analysis revealed that accuracy of drug information in Wikipedia was 99.7%±0.2% when compared to the textbook data. The overall completeness of drug information in Wikipedia was 83.8±1.5% (p<0.001). Completeness varied in-between categories, and was lowest in the category “pharmacokinetics” (68.0%±4.2%; p<0.001) and highest in the category “indication” (91.3%±2.0%) when compared to the textbook data overlap. Similar results were obtained for the English language version of Wikipedia. Of the drug information missing in Wikipedia, 62.5% was rated as didactically non-relevant in a qualitative re-evaluation study. Drug articles in Wikipedia had an average of 14.6±1.6 references and 262.8±37.4 edits performed by 142.7±17.6 editors. Both Wikipedia and textbooks samples had comparable, low readability. Our study suggests that Wikipedia is an accurate and comprehensive source of drug-related information for undergraduate medical education.

Evolutionary History and Functional Characterization of the Amphibian Xenosensor CAR

The xenosensing constitutive androstane receptor (CAR) is widely considered to have arisen in early mammals via duplication of the pregnane X receptor (PXR). We report that CAR emerged together with PXR and the vitamin D receptor from an ancestral NR1I gene already in early vertebrates, as a result of whole-genome duplications. CAR genes were subsequently lost from the fish lineage, but they are conserved in all taxa of land vertebrates. This contrasts with PXR, which is found in most fish species, whereas it is lost from Sauropsida (reptiles and birds) and plays a role unrelated to xenosensing in Xenopus. This role is fulfilled in Xenopus by CAR, which exhibits low basal activity and pronounced responsiveness to activators such as drugs and steroids, altogether resembling mammalian PXR. The constitutive activity typical for mammalian CAR emerged first in Sauropsida, and it is thus common to all fully terrestrial land vertebrates (Amniota). The constitutive activity can be achieved by humanizing just two amino acids of the Xenopus CAR. Taken together, our results provide a comprehensive reconstruction of the evolutionary history of the NR1I subfamily of nuclear receptors. They identify CAR as the more conserved and remarkably plastic NR1I xenosensor in land vertebrates. Nonmammalian CAR should help to dissect the specific functions of PXR and CAR in the metabolism of xeno- and endobiotics in humans. Xenopus CAR is a first reported amphibian xenosensor, which opens the way to toxicogenomic and bioaugmentation studies in this critically endangered taxon of land vertebrates.

Dystrophin-deficiency increases the susceptibility to doxorubicin-induced cardiotoxicity.

The clinical use of doxorubicin (DOX) and other anthracyclines is limited by a dosage‐dependent cardiotoxicity, which can lead to cardiomyopathy. The role of the individual genetic makeup in this disorder is poorly understood. Alterations in genes encoding cardiac cytoskeleton or sarcolemma proteins may increase the susceptibility to doxorubicin‐related cardiotoxicity.

The SGLT2 inhibitor empagliflozin improves the primary diabetic complications in ZDF rats

Hyperglycemia associated with inflammation and oxidative stress is a major cause of vascular dysfunction and cardiovascular disease in diabetes. Recent data reports that a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), empagliflozin (Jardiance®), ameliorates glucotoxicity via excretion of excess glucose in urine (glucosuria) and significantly improves cardiovascular mortality in type 2 diabetes mellitus (T2DM). The overarching hypothesis is that hyperglycemia and glucotoxicity are upstream of all other complications seen in diabetes. The aim of this study was to investigate effects of empagliflozin on glucotoxicity, β-cell function, inflammation, oxidative stress and endothelial dysfunction in Zucker diabetic fatty (ZDF) rats. Male ZDF rats were used as a model of T2DM (35 diabetic ZDF‐Leprfa/fa and 16 ZDF-Lepr+/+ controls). Empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 6 weeks. Treatment with empagliflozin restored glycemic control. Empagliflozin improved endothelial function (thoracic aorta) and reduced oxidative stress in the aorta and in blood of diabetic rats. Inflammation and glucotoxicity (AGE/RAGE signaling) were epigenetically prevented by SGLT2i treatment (ChIP). Linear regression analysis revealed a significant inverse correlation of endothelial function with HbA1c, whereas leukocyte-dependent oxidative burst and C-reactive protein (CRP) were positively correlated with HbA1c. Viability of hyperglycemic endothelial cells was pleiotropically improved by SGLT2i. Empagliflozin reduces glucotoxicity and thereby prevents the development of endothelial dysfunction, reduces oxidative stress and exhibits anti-inflammatory effects in ZDF rats, despite persisting hyperlipidemia and hyperinsulinemia. Our preclinical observations provide insights into the mechanisms by which empagliflozin reduces cardiovascular mortality in humans (EMPA-REG trial).