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. The projects for example serve to elucidate the mode of action in respect to desired but also to adverse effects of therapeutic drugs, using suitable in-vitro methods.

Leader of the projects

Dr. Roland Frötschl, Phone: +49-(0)228-99-307-3441, roland.froetschl@bfarm.de

PD Dr. Peter Mayer, Phone: +49-(0)228-99-307-3843, peter.mayer@bfarm.de

Dr. Bernd-Bodo Haas, Phone: +49-(0)228-99-307-3476, bodo.haas@bfarm.de

The Research group

Projects

Direct effect of sulfonylureas on the differentiation of fat cells

Project leader: Peter Mayer, Bodo Haas

Sulfonylureas (e.g. glibenclamide [also called glyburide] and glimepiride) are a very old substance class for the treatment of type 2 diabetes. They are still in use today, but their therapeutic value is questioned due to an unfavourable mode of action. A newer class of oral antidiabetics are the glitazones. The latter also under discussion because of an unfavourable pharmacologic profile. They lead to increased formation of fat cells (from precursors) and thereby to an increase in body fat mass. The underlying mechanism is the activation of the nuclear receptor PPARγ.
In this project we could show that sulfonylureas directly activate PPARγ in the fat cell precursors (called preadipocytes), independent from their established action on the insulin-secreting cells in the pancreas. Therefore, they mimic the effect of the glitazones, which most likely contributes to the unfavourable therapeutic profile; like glitazones, they lead to an increase in body fat mass.

Publication:
Mayer P, Haas B, Celner J, Enzmann H, Pfeifer A. Glitazone-like action of glimepiride and glibenclamide in primary human adipocytes. Diabetes Obes Metab. 2011 Sep;13(9):791-9.

The role of DPP4 (the target structure of gliptins) in fat cells

Project leader: Peter Mayer

A newer, and meanwhile widely used, therapeutic principle in type 2 diabetes treatment is the administration of DPP4 inhibitors, also called gliptins (e.g. sitagliptin). Their established pharmacological target is the enzyme dipeptidyl peptidase 4 (DPP4) which degrades the peptide hormone GLP1 from gut mucosa. By the action of the gliptins, the level of GLP1 circulating in the blood increases. This affects insulin secretion from pancreatic islet cells in a favourable way.
DDP4 is also found in adipose tissue. In this project, it could be shown by selective knock-down of DPP4 in preadipocytes (fat cell precursors) and consecutive genome-wide expression profiling that DPP4 affects proliferation of preadipocytes as well as their differentiation to mature fat cells. However, gliptins had no noticeable effect on the fat cell precursors. The reason for this observation most likely is the fact that DPP4 is a multi-functional protein. The gliptins inhibit the protease activity of DPP4, which obviously plays no role for the observed effects on proliferation and differentiation.

Publication:
Zilleßen, P., Celner, J., Kretschmann, A. et al. Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes. Sci Rep 6, 23074 (2016). https://doi.org/10.1038/srep23074

Complex mode of action of glitazones at their target site (PPARγ)

Project leader: Bodo Haas

The well-known mode of action of sulphonylureas such as Glimepiride and Glibenclamide is inhibition of an ATP-dependent potassium channel on the plasma membrane of pancreatic ß-cells which subsequently leads to insulin secretion. It has been published that sulphonylureas, apart from their classical mode of action, can activate PPARγ and increase murine adipocyte differentiation similar to glitazones (extrapancreatic effect of sulphonylureas). Using in vitro cultivated cells and in silico models, we could show that sulphonylureas not only exhibit agonistic effects but also influence phosphorylation at Serine 273 of PPARγ. We concluded that sulphonylureas are very similar to glitazones in terms of effects and binding characteristics.

The influence of inhibition of intracellular signalling cascades on chemoresistance of brain tumour cells

Project leader: Bodo Haas

In several projects with in vitro cultured brain tumour cells (glioblastoma cells, a very aggressive form of brain tumours), we characterized two signalling pathways which could be responsible for chemoresistance of glioblastoma cells towards the standard therapeutic drug temozolomide. On the one hand we found the PI3K signalling cascade as potential target to increase temozolomide efficacy and on the other hand we identified thioredoxin as possible drug target.
Literature data indicate that the opioid methadone has anti-tumour effects in glioblastoma cells and is able to reverse resistance towards chemotherapeutics. In two finished projects with in vitro cultured glioblastoma cells we could demonstrate that methadone is cytotoxic at clinically not relevant concentrations. Under our experimental conditions we were not able to show an enhancement of temozolomide effects by methadone.

Publications:

• Haas B, Ciftcioglu J, Jerma S, Weickhardt S, Eckstein N, Kaina B. Methadone-mediated sensitization of glioblastoma cells is drug and cell line dependent. J Cancer Res Clin Oncol. 2020 Dec 14.
• Kaina B, Beltzig L, Piee-Staffa A, Haas B (2020). Cytotoxic and Senolytic Effects of Methadone in Combination with Temozolomide in Glioblastoma Cells. Int. J. Mol. Sci. 2020, 21(19):E7006.
• Haas B, Schütte L, Wos-Maganga M, Weickhardt S, Timmer M, Eckstein N (2018). Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells. Int. J. Mol. Sci. 2018, 19(10), 2874.
• Haas B, Klinger V, Keksel C, Bonigut V, Kiefer D, Caspers J, Walther J, Wos-Maganga M, Weickhardt S, Röhn G, Timmer M, Frötschl R, Eckstein N (2018). Inhibition of the PI3K but not the MEK/ERK Pathway Sensitizes Human Glioma Cells to Alkylating Drugs. Cancer Cell Int. 2018, 18:69.

Immunogenicity of biologicals

Project leader: Peter Mayer

Biological drugs are large molecules, which can trigger an immune response in the human body, partly with serious sequels. Examples are heparin (immune response can result in thrombosis and thrombocytopenia, the so-called HIT syndrome), epoetin (disturbed formation of red blood cells) and insulin (loss of efficacy). To date, no non-clinical tests are available which can estimate the risk of immunogenicity of a biological drug and the consequences thereof. For the development of vaccines, in-vitro tests exist which measure the activation of T-lymphocytes.
For the immunogenicity of biologicals, mainly a special population of B-lymphycytes, localised in the spleen, appears to be responsible instead of T-lymphocytes. These B-lymphocytes hardly can be cultured in vitro so that development of an activation assay is difficult. In this project, possible alternative approaches for an in-vitro B-cell immunogenicity assay are being evaluated. It is investigated whether from human spleen RNA a pool of antigen-binding B-cell receptors (BCR) can be retrieved by PCR and expressed in B-cells that can be permanently cultured.

Inter-individual differences of cellular response to genotoxic stress induced by Fluoroquinolones

Project leader: Roland Frötschl

Fluoroquinolones are an important class of antibiotics. Generally, fluoroquinolones are well tolerated. Rare but serious and mainly idiopathic side effects (e.g. tendonitis/tear, peripheral neuropathies) in some patients have led to EMA referral procedure (EMA/175398/2019) in the last years. The cause of these side effects is unknown. Fluoroquinolones are inhibitors of bacterial gyrases. The human homologs, topoisomerase are also targets of fluoroquinolones, but with orders of magnitude less activity. This project aims to investigate the hypothesis whether the cause of the rare side effects may be a genetically determined hypersensitivity caused by inter-individual differences in the primary stress response to DNA damage. Such differences may be indicative of toxicity caused by treatment in particularly sensitive cells. An exaggerated response to fluoroquinolone-induced DNA damage in certain cell clones could provide evidence of individual factors for hypersensitivity, which can then be further analysed for biomarkers at the transcriptional level. Positive findings would also have implications for thresholds of toxicity in sensitive individuals. To this end, a collection of B lymphoblastoid cells from different donors will be examined for individual differences in stress response.

Publications:

• EMA/175398/2019 https://www.ema.europa.eu/en/documents/referral/quinolone-fluoroquinolone-article-31-referral-disabling-potentially-permanent-side-effects-lead_en.pdf
• Guérard, M., Baum, M., Bitsch, A., Eisenbrand, G., Elhajouji, A., Epe, B., Habermeyer, M., Kaina, B., Martus, HJ., Pfuhler, S., Schmitz, C., Sutter, A., Thomas, A.D., Ziemann, C., and Frötschl, R., (2015) Assessment of mechanisms driving non-linear dose-response relationships in genotoxicity testing. Mutation Research Reviews 763:181-201.
• Li, H-H., Chen, R., Hyduke D.R., Williams, A., Frötschl, R., Ellinger-Ziegelbauer, H., O’Lone, R., Yauk, C.L., Aubrecht, J., and Fornace, A.L. Jr. (2017) Development and validation of a high-throughput transcriptomic biomarker to address 21st century genetic toxicology needs PNAS 2017 ; published ahead of print December 4, 2017, doi:10.1073/pnas.1714109114
• Li HH, Yauk C, Chen R, Hyduke DR, Williams A, Frötschl R, Ellinger-Ziegelbauer HC, Pettit S, Aubrecht J, Fornace Jr. AJ (2019). TGx-DDI, a transcriptomic biomarker for genotoxicity hazard assessment of pharmaceuticals and environmental chemicals. Frontiers in Big Data 2019, 08 October 2019, Vol 2, doi: 10.3389/fdata.2019.00036