Scientific progress and innovative development with regard to medicinal products always entail new challenges for the prevention of health risks. In the case of possible risks, the BfArM will always have to make decisions in a foresighted manner. Therefore, the BfArM's research on special indications focusses on various current and safety-relevant issues as well as on regulatory tasks.
Leader of the projects
|Dr. Roland Frötschl||Phone: +49-(0)firstname.lastname@example.org|
|Dr. Cornelia Lipperheide||Phone: +49-(0)email@example.com|
|PD Dr. Peter Mayer||Phone: +49-(0)firstname.lastname@example.org|
|Dr. Josef Zündorf||Phone: +49-(0)email@example.com|
|Prof. Dr. Bernd-Joachim Zünkler||Phone: +49-(0)firstname.lastname@example.org|
|Prof. Dr. Werner Knöss||Phone: +49-(0)email@example.com|
|Dr. Harald Enzmann||Phone: +49-(0)228-99-307-3315||Harald.Enzmann@bfarm.de|
|Dr. Karsten Spicher||Phone: +49-(0)firstname.lastname@example.org|
|Dr. Susanne Brendler-Schwaab||Phone: +49-(0)email@example.com|
|Dr. Bernd-Bodo Haas||Telefon: +49-(0)firstname.lastname@example.org|
The Research group
Role of Dipeptidyl-Peptidase 4 (DPP4)
Pia Zilleßen, Peter Mayer
Gliptins (DPP4 inhibitors) are a new class of antidiabetics . The enzyme DPP4 is involved in many biological processes, indirectly also in the glucose homeostasis . Recently DPP4 was also described as an adipokine . Certain adipokines and inflammation mediators are associated with the manifestation of a diabetes. Direct involvement of DPP4 is still under discussion. Therefore the study is to investigate the functions of DPP4 in human adipocytes, later also in other cell types.
Use of protein and gene expression analyses (Western Blot, qPCR) in in-vitro systems and DNA array analyses; lentiviral transduction of siRNA and measurement of cell proliferation and migration.
Outcome. Lentiviral transduction was used to generate a specific knock-down of the DPP4 gene for further analyses. The DPP4 knock-down changed the expression of numerous other genes in the adipocytes. Detailed investigations will follow.
Understanding the role of DPP4 and related enzymes is of great importance for the targeted and selective administration of antidiabetics. The knowledge gained here is of enormous benefit in detecting the so-called off-target-effects of gliptins.
1. Dobrian AD, Ma Q, Lindsay JW et al (2010) Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice. Am J Physiol Endocrinol Metab 300(2):E410–E421
2. Drucker DJ (2007) Dipeptidyl peptidase-4 inhibition and the treatment of type 2 diabetes. Diabetes Care 30(6):1335–1343
3. Lamers D, Famulla S, Wronkowitz N et al (2011) Dipeptidyl peptidase 4 is a novel adipokine potentially linking obesity to the metabolic syndrome. Diabetes 60(7):1917–1925
4. Yu DM, Wang XM, McCaughan GW, Gorell MD (2006) Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis. FEBS J 273(11):2447–2460
Effects of Sulfonyl urea on (pre-)dipocytes
Moritz Hass, Bernd-Bodo Haas
The transcription factor PPARγ (peroxisome proliferator activated receptor γ) is substantially involved in fat cell differentiation. The project is to find out whether sulfonyl urea (SU) and structurally related substances change the phosphorylation pattern of PPARγ, as the glitazones are known to do , and what their impact is on the insulin sensitivity of adipocytes. Our own preliminary investigations have shown that SU influences PPARγ and the expression of cytokines . In this connection we intend to explain the mechanism of cytokine expression and the impact of test substances on it.
Studies are conducted with human primary adipocytes. The phosphorylation status of PPARγ is investigated by phosphor specific Western Blots and specially developed ELISAs. Cytokine expression is investigated in knockdown studies and over-expression via lentiviral gene transfer and mutagenesis of the phosphorylation sites, using CHIP analyses and realtime-PCR; the effect of SU on cytokine expression is investigated. Direct interactions of PPARγ and active substances are detected in co-localisation studies and in-vitro tests. First results suggest effects of SU on the phosphorylation of PPARγ. A change in phosphorylation is associated with a positive antidiabetic expression profile.
Glitazones are looked at increasingly critically because of their adverse side effects. The antidiabetic action and the desired reduction of insulin resistance appear to be correlated with the phosphorylation status of PPARγ . Elucidation of the mechanism is expected to lay the foundation for antidiabetic agents with a more favourable benefit-risk-ratio.
1. Choi JH, Banks AS, Estall JL et al (2010) Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5. Nature 466(7305):451–456
2. Mayer P, Haas B, Celner J et al (2011) Glitazone-like action of glimepiride and glibenclamide in primary human adipocytes. Diabetes Obes Metab 13(9):791–799
3. Choi JH, Banks AS, Kamenecka TM (2011) Antidiabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation. Nature 477(7365):477–481
Interactions of protein-containing parenteralia with elastomer components in primary containers
Carolin Richter, Cornelia Lipperheide, Uwe Lipke, Thomas Zapf, Alf Lamprecht*
Finding out whether liquid parenteralia get contaminated during storage with leachables diffusing into the formulation from the rubber stopper in contact with it. Qualitative and quantitative determination of the leachables and investigation of their effect on the stability of protein-containing medicinal products.
For obtaining a maximum leachable profile of rubber stoppers, commercially available stoppers were extracted under reflux using isopropanol. Besides, the migration of the leachables into aqueous formulations was tested under ICH storage conditions. To determine the effect of the leachables on the stability of proteins the rubber stopper extract, extracted by isopropanol, was charged with IgG as a model protein.
The leachable profiles of the investigated rubber stoppers showed a marked qualitative and quantitative variability although all of them complied with pharmacopoeial quality. Presence of polysorbate solutiser in the aqueous formulation enhanced the migration also of unpolar substances from the rubber stopper. Various analytical methods were used to examine IgG for potential structural changes.
The developed test procedure is suitable for the selection of compatible rubber stoppers for protein-containing drug solutions.
*Rheinische Friedrich-Wilhelms-Universität Bonn
New methods of characterising herbal medicinal products
Annika Orland, Werner Knöss
Identification and quality of medicinal plants is fundamental to the safeguarding of therapeutical efficacy and safety. The project pursues the question whether modern methods based on Omics techniques can be reasonably applied.
NMR fingerprint, HPLC, PCR, qPCR, microarray, methods of cell biology.
Previous investigations have shown that a lot of European medicinal plants can be identified using PCR based techniques. Different extracts can be distinguished using NMR fingerprints followed by the analysis of the main components; patterns in gene expression profiles were observed.
Beside PCR based methods  and metabolic fingerprints , Omics based technologies can be employed in identification and authentication. Gene expression profiles may be of additional benefit in the evaluation of the toxic potential of medicinal plants. Examining the applicability of the present examples should be continued.
1. Kersten T, Daniel C, König GM, Knöß W (2008) Das Potential PCR-basierter Markermethoden zur Identifizierung von Arzneipflanzen. Z Phytother 29(3):122–128
2. Daniel C, Kersten T, Kehraus S et al (2008) Identifizierung und Charakterisierung von Arzneipflanzen mit „Metabolic Fingerprinting“. Z Phytother 29(6):270–274
Irradiation of granulocytes
Ines Preuth, Josef Zündorf, Dirk von Mallek, Stephan Garbe*
Neutrophil granulocytes (PMN) are the primary effector cells in the immune system responsible for the prevention of bacterial infections. In therapeutical terms, anti-inflammatory efficacy of low-dosed radiotherapy is empirically well documented . In a radiotherapy, e.g. of facial furuncles, single doses of 0.2 and 0.5 Gy were reported to cause the disease process to recede . The underlying mechanism is not yet known and shall be discussed in detail.
Homogeneous irradiation of heparinised human full blood and determination of its impact on activation, phagocytosis and the killing of pathogenic micro-organisms by PMN.
Outcome and conclusions:
The activation (CD11b and CD66b expression) of PMN is slightly reduced after irradiation with F-18 fluoride. The same effect occurs in the phagocytosis of S. aureus by PMN. The changes in the antibody expression following radiation is likely to influence other PMN functions as well. This will be investigated in the further course of the project. In regulatory terms, the results of the project can possibly be used to derive criteria applicable in the assessment of incidents with radioactive medical devices.
1. Freund F (1927) Zum Wirkungsmechanismus der Röntgenstrahlen bei entzündlichen Erkrankungen. Klin Wochenschr 6(31):1462–1465
2. Hess F (1987) Die Strahlentherapie gutartiger Erkrankungen. In: Scherer E (Hrsg) Strahlentherapie Radiologische Onkologie, 3. Auflage. Springer, Berlin, S 354–369
* Rheinische Friedrich-Wilhelms-Universität Bonn
Function and regulation of the granulocytory unique feature, CD66b, and its importance for the
phagocytosis of pathogenic microorganisms
Alva Brodesser, Thomas Schmidt*, Norbert Schnitzler**, Thomas Grüger, Josef Zündorf
The function of the surface receptor CD66b on granulocytes is not yet known . It has been supposed that CD66b expression takes place in co-expression with CD11b/CD18, whereby complement-opsonised bacteria are phagocyted. It is asked whether CD66b expression on granulocytes takes place independently of other receptors; further, what is the relevance of CD66b for phagocytosis and can the latter be influenced by modulation of CD66b.
Granulocytes are stimulated in human full blood by Staphylococcus aureus supernatant. In subsequent experiments receptor expression is determined using fluorescence labelled antibody, and phagocytosis of S. aureus using PMN. Flow cytometry (FACSCalibur, ImageStreamX) is used for the analysis.
Bacteria supernatant induces an overshooting CD66b expression. The expression of other receptors, e.g. CD11b, is hardly affected. The degree of CD66b expression correlates with granulocyte aggregation and has a phagocytosis inhibiting effect.
CD66b is important in cell aggregation and is controlled independently of CD11b. Further experiments will investigate to what extent phagocytosis can be controlled by modulation of CD66b.
1. Schröder AK, Uciechowski P, Fleischer D, Rink L (2006) Crosslinking of CD66b on peripheral blood neutrophils mediates the release of Interleukin-8 from intracellular storage. Hum Immunol 67(9):676–682
**Gesundheitsamt des Kreises Düren
Identification of mode-of-action specific biomarkers für (geno-)toxic carcinogens
Florian Engel, Roland Frötschl
Investigation of class specific effects of substances from various genotoxic action classes on p53 modification, on expression of p53-pathway-associated genes, on cell cycle and apoptosis. The analysis is expected to identify potential class specific biomarkers for a better evaluation of the mutagenic potential, in three cell culture systems from blood (TK6), liver (HepG2) and CNS (SH-SY5Y).
In cytotoxicity tests the treatment concentrations are determined for ten substances in cell culture systems (six genotoxic and two non-genotoxic carcinogens, two neurotoxins); they are examined in Comet and micronucleus tests, expression profiling, ELISA and Western Blot.
Our investigations have demonstrated the expected genotoxicity of the substances used. First expression profile data display specific deviations in the expression pattern as a function of time and concentration. First Western Blot and ELISA tests confirm these results also for the p53 modification and protein synthesis.
This in-vitro procedure is intended to contribute essentially to improving the risk assessment of medicinal products and the screening of new substances, thus increasing the pharmaceutical safety and reducing the need for animal experiments.
Gallium 68 and Fluorine 18 labelled Annexin-V-Analog for imaging vulnerable plaques in coronary
vessels by means of pet/ct
Yasmin Hashlamun, Stefan Guhlke*, Dirk von Mallek
Synthesis of suitable annexin-V-analogs that may be labelled with 68Ga and [18F] fluoride. The tracers shall be used in the detection of vulnerable plaques, since annexin-V is able to identify the apoptotic cells present therein .
Fluoride labelling of annexin-V-analogs is done by chelating, which is a very promising method. The time factor, because of the short half-life of radionuclides, and the physiological conditions play a crucial role in the investigations.
Preparation of the precursor from protein and a bifunctional chelator, e.g. NOTA-NCS, yields the best result after 24 h at room temperature. By subsequent purification in a PD10 column it is possible to obtain a pure protein chelator fraction, which may then be labelled with gallium-68 or fluorine-18.
First, aluminium and fluorine-18 is mixed to the aluminium fluoride complex (Al-18F)x+ , which is then bound to the appropriate chelators, as already described by McBride .
Outcome and conclusion:
The method was able to obtain good yields of radiolabelled annexin-V-analog, which could be reacted with apoptotic cells in vitro in a possible further step.
1. Murakami Y, Takamatsu H, Taki J et al (2004) 18F-labelled annexin V: a PET tracer for apoptosis imaging. Eur J Nucl Med Mol Imaging 31(4):469–474
2. Martin RB (1996) Ternary complexes of Al3+ and F− with a third ligand. Coord Chem Rev 141:23–32
3. McBride WJ, Sharkey RM, Karacay H et al (2009) A novel method of 18F radiolabeling for PET. J Nucl Med 50(6):991–998
4. Laverman P, McBride WJ, Sharkey RM (2010) A novel facile method of labeling octreotide with (18)F-fluorine. J Nucl Med 51(3):454–461
*Rheinische Friedrich-Wilhelms-Universität Bonn