Saarbrücken, Germany
Saarbrücken, Germany

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Rizner T.L.,University of Ljubljana | Brozic P.,University of Ljubljana | Doucette C.,Life Technologies | Turek-Etienne T.,Life Technologies | And 6 more authors.
Steroids | Year: 2011

Dydrogesterone is widely used for menstrual disorders, endometriosis, threatened and habitual abortion and postmenopausal hormone replacement therapy. Although progestins have a promiscuous nature, dydrogesterone does not have clinically relevant androgenic, estrogenic, glucocorticoid or mineralocorticoid activities. To date, systematic biochemical characterization of this progestin and its active main metabolite, 20α-dihydrodydrogesterone, has not been performed in comparison to progesterone. The objective of this study was to evaluate the selectivity and potential androgenic/antiandrogenic effects of dydrogesterone and its metabolite in comparison to progesterone and medroxyprogesterone acetate by analyzing their interference with AR signaling in vitro. We characterized dydrogesterone and its metabolite for their binding and transactivation of androgen and other steroid hormone receptors and for their potential inhibitory effects against androgen biosynthetic enzymes, 17β-hydroxysteroid dehydrogenase types 3 and 5 and 5α-reductase types 1 and 2. We found that dydrogesterone resembled progesterone mainly in its progestogenic effects and less in its androgenic, anti-androgenic, glucocorticoid and antiglucocorticoid effects; whereas, 20α- dihydrodydrogesterone showed reduced progestogenic potency with no androgenic, glucocorticoid and mineralocorticoid effects. Effects on the androgen and glucocorticoid receptor differed depending on the technology used to investigate transactivation. Progesterone, but not dydrogesterone and 20α- dihydrodydrogesterone, exerted anti-androgenic effects at the pre-receptor level by inhibiting 5α-reductase type 2. Dydrogesterone, 20α- dihydrodydrogesterone and progesterone inhibited the biosynthesis of testosterone catalyzed by 17β-hydroxysteroid dehydrogenase types 3 and 5; however, due to their micromolar Ki values, these activities appeared to be not of relevance at therapeutic levels. Overall, our data show that the anti-androgenic potential of dydrogesterone and 20α-dihydrodydrogesterone is less pronounced compared to progesterone. © 2011 Elsevier Inc. All rights reserved.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2010.2.3.3-4 | Award Amount: 7.82M | Year: 2010

Influenza viruses cause a highly contagious respiratory disease in both humans and animals. Typically, influenza spreads worldwide in seasonal epidemics resulting in an estimated 3 to 5 million cases of severe illness and 250,000 to 500,000 deaths annually. In addition to these seasonal epidemics there have been several pandemics since the early 1900s, where highly virulent strains emerged, the most devastating being the Spanish Flu of 1918, which caused 20-40 million deaths globally. Vaccination is currently the primary means of controlling the spread of influenza virus infections but due to the viruss notorious ability to mutate, new vaccines must be developed each year. There are a few antiviral drugs that are currently on the market; however, their therapeutic potential is restricted through rapid appearance of drug-resistant viruses during treatment. Thus, the need for novel effective drugs against influenza is evident. The FLUCURE project aims at developing innovative, first-in-class therapeutics against influenza by targeting the viral ribonucleoprotein complex, which is replication core of the virion and a major contributor to viral virulence. The high level of conservation combined with slow mutation rates of the ribonucleoprotein complex should result in therapeutics with broad viral strain specificity associated with a reduced risk for developing resistance. FLUCURE builds further on two successful EU-FP7 drug discovery projects, FLUINHIBIT and FluDrugStrategy, both targeting specific but different protein-protein interactions of the viral ribonucleoprotein complex with small molecule inhibitors. A consortium of 10 partners with the required complementary skills will progress the lead candidates from these two projects synergistically through lead optimization and preclinical development phases, with the final objective to deliver one or more drug candidates suitable for entering clinical development within 4 years.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2013.2.3.1-1 | Award Amount: 6.11M | Year: 2013

Invasive fungal diseases are estimated to kill 1.5 million people each year. The incidence of mortality has risen significantly across the EU over the last 20 years due to an expansion of at risk patient populations. Given the obvious importance of these diseases it is perhaps surprising that only four classes of drug are available to treat systemic fungal infection. The azole class of antifungals provide the front line role for most disease treatment but recently resistance has emerged and it is of growing concern that levels are rising dramatically. European researchers have led the world in identifying the extent of the problem with some centres reporting itraconazole resistance in the Aspergillus species as high as 20% and in Candida, resistance to posaconazole upto 30%. Additionally the epidemiology of serious fungal infections is changing with more intrinsically resistant organisms now being seen more frequently. This represents a major problem for clinicians who are increasingly treating infections for which there is no current effective therapy. This proposal brings together leading European SMEs and academics to address this problem through the development of novel classes of antifungals and the identification of novel drug targets. The NOFUN consortium has identified potent novel broad spectrum antifungal molecules that are active against multi-resistant fungal pathogens and intends to qualify these as drug candidates. One of these assets is already at the lead identification stage. Cutting edge fungal genomics will be used to identify novel druggable targets and advance these to develop qualified tractable chemical inhibitors. With its wide ranging expertise across medicinal chemistry, ADMET, fungal biology, chemical genomics and drug development the partners will build and progress a broad pipeline of agents that have the potential to reach the clinic within 5 years.


Hoffmann S.A.,Charité - Medical University of Berlin | Muller-Vieira U.,Pharmacelsus GmbH | Biemel K.,Pharmacelsus GmbH | Knobeloch D.,Cytonet GmbH | And 6 more authors.
Biotechnology and Bioengineering | Year: 2012

Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4μM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230±57pmol/h/106 cells followed by a period of stable conversion of about 100pmol/h/106 cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver. © 2012 Wiley Periodicals, Inc.


Lubberstedt M.,Charité - Medical University of Berlin | Muller-Vieira U.,Pharmacelsus GmbH | Biemel K.M.,Pharmacelsus GmbH | Darnell M.,Karolinska Institutet | And 8 more authors.
Journal of Tissue Engineering and Regenerative Medicine | Year: 2015

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions. © 2012 John Wiley & Sons, Ltd.


Schyschka L.,TU Munich | Sanchez J.J.M.,University of Tübingen | Wang Z.,TU Munich | Burkhardt B.,University of Tübingen | And 6 more authors.
Archives of Toxicology | Year: 2013

Primary human hepatocytes (PHH) are the "gold standard" for in vitro toxicity tests. However, 2D PHH cultures have limitations that are due to a time-dependent dedifferentiation process visible by morphological changes closely connected to a decline of albumin production and CYP450 activity. The 3D in vitro culture corresponds to in vivo-like tissue architecture, which preserves functional characteristics of hepatocytes, and therefore can at least partially overcome the restrictions of 2D cultures. Consequently, several drug toxicities observed in vivo cannot be reproduced in 2D in vitro models, for example, the toxic effects of acetaminophen. The objective of this study was to identify molecular differences between 2D and 3D cultivation which explain the observed toxicity response. Our data demonstrated an increase in cell death after treatment with acetaminophen in 3D, but not in 2D cultures. Additionally, an acetaminophen concentration-dependent increase in the CYP2E1 expression level in 3D cultures was detected. However, during the treatment with 10 mM acetaminophen, the expression level of SOD gradually decreased in 3D cultures and was undetectable after 24 h. In line with these findings, we observed higher import/export rates in the membrane transport protein, multidrug resistance-associated protein-1, which is known to be specific for acetaminophen transport. The presented data demonstrate that PHH cultured in 3D preserve certain metabolic functions. Therefore, they have closer resemblance to the in vivo situation than PHH in 2D cultures. In consequence, 3D cultures will allow for a more accurate hepatotoxicity prediction in in vitro models in the future. © 2013 Springer-Verlag Berlin Heidelberg.


Mueller D.,Saarland University | Tascher G.,Saarland University | Muller-Vieira U.,Pharmacelsus GmbH | Knobeloch D.,Charité - Medical University of Berlin | And 4 more authors.
Journal of Tissue Engineering and Regenerative Medicine | Year: 2011

As the major research focus is shifting to three-dimensional (3D) cultivation techniques, hollow-fiber bioreactors, allowing the formation of tissue-like structures, show immense potential as they permit controlled in vitro cultivation while supporting the in vivo environment. In this study we carried out a systematic and detailed physiological characterization of human liver cells in a 3D hollow-fiber bioreactor system continuously run for > 2 weeks. Primary human hepatocytes were maintained viable and functional over the whole period of cultivation. Both general cellular functions, e.g. oxygen uptake, amino acid metabolism and substrate consumption, and liver-specific functions, such as drug-metabolizing capacities and the production of liver-specific metabolites were found to be stable for > 2 weeks. As expected, donor-to-donor variability was observed in liver-specific functions, namely urea and albumin production. Moreover, we show the maintenance of primary human hepatocytes in serum-free conditions in this set-up. The stable basal cytochrome P450 activity 3 weeks after isolation of the cells demonstrates the potential of such a system for pharmacological applications. Liver cells in the presented 3D bioreactor system could eventually be used not only for long-term metabolic and toxicity studies but also for chronic repeated dose toxicity assessment. © 2011 John Wiley & Sons, Ltd.


Yang W.,University of Tübingen | Yang W.,Huazhong University of Science and Technology | Burkhardt B.,University of Tübingen | Fischer L.,University of Tübingen | And 8 more authors.
EXCLI Journal | Year: 2015

Aging is characterized by a progressive decrease of cellular functions, because cells gradually lose their capacity to respond to injury. Increased oxidative stress is considered to be one of the major contributors to age-related changes in all organs including the liver. Our study has focused on elucidating whether important antioxidative enzymes, the mTOR pathway, and MAPKs exhibit age-dependent changes in the liver of rats during aging. We found an age-dependent increase of GSH in the cytosol and mitochondria. The aged liver showed an increased SOD enzyme activity, while the CAT enzyme activity decreased. HO-1 and NOS-2 gene expression was lower in adult rats, but up-regulated in aged rats. Western blot analysis revealed that SOD1, SOD2, GPx, GR, γ-GCL, and GSS were age-dependent up-regulated, while CAT remained constant. We also demonstrated that the phosphorylation of Akt, JNK, p38, and TSC2Ser1254 decreased while ERK1/2 and TSC2Thr1462 increased agedependently. Furthermore, our data show that the mTOR pathway seems to be activated in livers of aged rats, and hence stimulating cell proliferation/regeneration, as confirmed by an age-dependent increase of PCNA and p-eIF4ESer209 protein expression. Our data may help to explain the fact that liver cells only proliferate in cases of necessity, like injury and damage. In summary, we have demonstrated that, age-dependent changes of the antioxidant system and stress-related signaling pathways occur in the livers of rats, which may help to better understand organ aging. © 2015, Leibniz Research Centre for Working Environment and Human Factors. All rights reserved.


PubMed | University of Tübingen, Karolinska Institutet, Charité - Medical University of Berlin, Cytonet GmbH and 2 more.
Type: Journal Article | Journal: Journal of tissue engineering and regenerative medicine | Year: 2015

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.


PubMed | University of Tübingen, Charité - Medical University of Berlin, Pharmacelsus GmbH and Huazhong University of Science and Technology
Type: | Journal: EXCLI journal | Year: 2016

Aging is characterized by a progressive decrease of cellular functions, because cells gradually lose their capacity to respond to injury. Increased oxidative stress is considered to be one of the major contributors to age-related changes in all organs including the liver. Our study has focused on elucidating whether important antioxidative enzymes, the mTOR pathway, and MAPKs exhibit age-dependent changes in the liver of rats during aging. We found an age-dependent increase of GSH in the cytosol and mitochondria. The aged liver showed an increased SOD enzyme activity, while the CAT enzyme activity decreased. HO-1 and NOS-2 gene expression was lower in adult rats, but up-regulated in aged rats. Western blot analysis revealed that SOD1, SOD2, GPx, GR, -GCL, and GSS were age-dependent up-regulated, while CAT remained constant. We also demonstrated that the phosphorylation of Akt, JNK, p38, and TSC2(Ser1254) decreased while ERK1/2 and TSC2(Thr1462) increased age-dependently. Furthermore, our data show that the mTOR pathway seems to be activated in livers of aged rats, and hence stimulating cell proliferation/regeneration, as confirmed by an age-dependent increase of PCNA and p-eIF4E(Ser209) protein expression. Our data may help to explain the fact that liver cells only proliferate in cases of necessity, like injury and damage. In summary, we have demonstrated that, age-dependent changes of the antioxidant system and stress-related signaling pathways occur in the livers of rats, which may help to better understand organ aging.

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