Pharmacelsus GmbH

Saarbrücken, Germany

Pharmacelsus GmbH

Saarbrücken, Germany
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Lubberstedt M.,Charité - Medical University of Berlin | Muller-Vieira U.,Pharmacelsus GmbH | Mayer M.,Pharmacelsus GmbH | Biemel K.M.,Pharmacelsus GmbH | And 6 more authors.
Journal of Pharmacological and Toxicological Methods | Year: 2011

Introduction: Primary human hepatocytes are considered as a highly predictive in vitro model for preclinical drug metabolism studies. Due to the limited availability of human liver tissue for cell isolation, there is a need of alternative cell sources for pharmaceutical research. Methods: In this study, the metabolic activity and long-term stability of the human hepatoma cell line HepaRG were investigated in comparison to primary human hepatocytes (pHH). Hepatocyte-specific parameters (albumin and urea synthesis, galactose and sorbitol elimination) and the activity of human-relevant cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were assayed in both groups over a period of 14 days subsequently to a two week culture period in differentiated state in case of the HepaRG cells, and compared with those of cryopreserved hepatocytes in suspension. In addition, the inducibility of CYP enzymes and the intrinsic clearances of eleven reference drugs were determined. Results: The results show overall stable metabolic activity of HepaRG cells over the monitored time period. Higher albumin production and galactose/sorbitol elimination rates were observed compared with pHH, while urea production was not detected. CYP enzyme-dependent drug metabolic capacities were shown to be stable over the cultivation time in HepaRG cells and were comparable or even higher (CYP2C9, CYP2D6, CYP3A4) than in pHH, whereas commercially available hepatocytes showed a different pattern The intrinsic clearance rates of reference drugs and enzyme induction of most CYP enzymes were similar in HepaRG cells and pHH. CYP1A2 activity was highly inducible in HepaRG by β-naphthoflavone. Discussion: In conclusion, the results from this study indicate that HepaRG cells could provide a suitable alternative to pHH in pharmaceutical research and development for metabolism studies such as CYP induction or sub-chronic to chronic hepatotoxicity studies. © 2010 Elsevier Inc.


Mueller D.,Saarland University | Muller-Vieira U.,Pharmacelsus GmbH | Biemel K.M.,Pharmacelsus GmbH | Tascher G.,Saarland University | And 2 more authors.
European Journal of Pharmaceutical Sciences | Year: 2012

In vitro repeated dose testing for the assessment of chronic drug-induced effects is a huge challenge in preclinical pharmaceutical drug development. Chronic toxicity results in discontinuation of therapy or post-marketing withdrawal of drugs despite in vivo preclinical screening. In case of hepatotoxicity, due to limited long term viability and functionality of primary hepatocytes, chronic hepatic effects are difficult to detect. In this study, we maintained primary human hepatocytes in a serum-free cultivation medium for more than 3 weeks and analyzed physiology, viability and drug metabolizing capacities of the hepatocytes. Moreover, we assessed acute (24 h) diclofenac toxicity in a range of (10-1000 μM) concentrations. The chronic (9 repeated doses) toxicity at one clinically relevant and another higher concentration (6.4 and 100 μM) was also tested. We investigated phase I and II metabolism of diclofenac upon repeated dose exposure and analyzed effects on the cellular exometabolome. Acute 24 h assessment revealed toxicity only for the highest tested concentration (1 mM). Upon repeated dose exposure, toxic effects were observed even at a low, clinically relevant concentration (6.4 μM). Biotransformation pathways were active for 3 weeks and diclofenac- acylglucuronide was detected as the predominant metabolite. Dose dependent diclofenac-induced effects on exometabolome, such as on the production of lactate and 3-hydroxybutyric acid as well as glucose and galactose metabolism, were observed upon nine repeated doses. Summarizing, we show that repeated dose testing on long-term functional cultures of primary human hepatocytes may be included for the assessment of long term toxic effects in preclinical screening and can potentially help replace/reduce in vivo animal testing. © 2012 Elsevier B.V. All rights reserved.


Mandenius C.-F.,Cellartis | Steel D.,Cellartis | Noor F.,Saarland University | Meyer T.,MultiChannel Systems GmbH | And 7 more authors.
Journal of Applied Toxicology | Year: 2011

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given. © 2011 John Wiley & Sons, Ltd.


Beckers S.,Saarland University | Noor F.,Saarland University | Muller-Vieira U.,Pharmacelsus GmbH | Mayer M.,Pharmacelsus GmbH | And 2 more authors.
Toxicology in Vitro | Year: 2010

A dynamic respiration assay based on luminescence decay time detection of oxygen for high throughput toxicological assessment is presented. The method uses 24-well plates (OxoDishes) read with the help of a sensor dish reader placed in a humidified CO 2-incubator. Adherent primary rat hepatocytes and the human hepatic cell line Hep G2 were exposed to known toxic compounds. Dissolved oxygen concentration, a measure of respiration, was measured with an oxygen sensor optode immobilized in the centre of each well. The cells were maintained in the dishes during the assay period and can afterwards be processed for further analyses. This dynamic, non-invasive measurement allowed calculation of 50% lethal concentrations (LC 50) for any incubation time point giving concentration-time-dependent responses without further manipulation or removal of the cells from the incubator. Toxicokinetic profiles are compared with Sulforhodamine B assay, a common cytotoxicity assay. The novel assay is robust and flexible, very easy to carry out and provides continuous online respiration data reflecting dynamic toxicity responses. It can be adapted to any cell-based system and the calculated kinetics contributes to understanding of cell death mechanisms. © 2009 Elsevier Ltd. All rights reserved.


Grant
Agency: European Commission | 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: European Commission | 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.


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.


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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