10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Prof. Dr.

Chair

Interaction of dinucleoside polyphosphate, Ap3A and Ap4A, cell cycle regulators with allicin: in silico and electrochemical study

Signal transduction and second messengers

BR03

Thiopolysulfides (TPS) are a group of organic sulfur compounds and are a part of secondary metabolites produced by the plants of the Alliaceae family. A key representing compound of TPS is allicin which has documented bacteriostatic properties by damaging bacterial DNA1. However, in eukaryotic organisms it exhibits no genotoxic effects, except for cancer cells for which the IC50 is significantly higher that of non-cancer ones2,3.The cellular regulators of the cell cycle Ap4A and Ap3A play an essential role in the eukaryotic response to inflammation4. As TPS also exhibit documented anti-inflammatory effects by inducing kinases and inhibiting cytokine production5, the question arises whether they may interact with Ap4A and Ap3A regulators. Such interaction could result in synergistic stronger anti-microbial and anti-tumour effect or converesely decrease the DNA-intercalation and genotoxic effect in eucaryotic cells.Electrochemical techniques allow for the evaluation of not only the antioxidant potential of individual compounds, but also the assessment of xenobiotic interactions with nucleotides6. Therefore, the square-wave voltammetry technique is suitable for investigating the defined research problem.These preliminary studies aim to determine the potential interaction between allicin with cell cycle regulators Ap4A and Ap3A, using the square-wave voltammetry technique and ab initio calculations to confirm the potential interaction found in the electrochemical study.Funding sourceThe research has been co-funded with sources of the National Science Centre of Poland (2024/08/X/NZ3/00519).References1. Tao Z. et al. Microbiology Spectrum 11,e03155-22 (2023).2. Hirsch K. et al. Nutrition and Cancer 38, 245–254 (2009).3. Li C. et al. Mol Med Rep 17, 5976–5981 (2018).4. Marriott AS. et al. PLoS ONE 11, e0154674, (2016).5. Hodge G. et al. Cytometry 48, 209–215 (2002).6. Szczepaniak O. et al. Biomedicine & Pharmacotherapy 152, 113216 (2022).

Basic Research

Short talk

Behavioral and metabolic consequences of an impaired cerebrovascular reactivity

AP04

Cerebrovascular reactivity refers to the ability of the brain´s vasculature to respond to vasodilatory stimuli, particularly carbon dioxide (CO2). CO2 induces a strong increase in brain perfusion, in humans and mice, which is strongly diminished in several diseases such as hypertension, diabetes, or obesity. We generated a mouse line that resembles an isolated cerebral endothelial dysfunction by deleting the Gαq/11 signalling pathway specifically in the endothelium of the brain. These mice lack the cerebrovascular response to CO2 while having normal brain perfusion at baseline. Interestingly, the loss of the cerebrovascular response leads to a strong decline in the brain's pH upon CO2 stimulation indicating a washout function of the vasculature. The vascular effect was accompanied by altered behavioural and metabolic consequences of CO2 such as anxiety-like behaviour, respiratory response, and thermoregulation. In addition, mice lacking the Gαq/11 signalling pathway in brain endothelial cells developed mild cognitive dysfunction with age and showed increased tau phosphorylation. Taken together, these results suggest a previously underestimated role of the cerebrovascular reactivity to CO2 which may protect from some of the acute and long-term neurological impairments in metabolic and vascular diseases.

Applied Pharmacology

Dr.med.

Improving Mechanistic Understanding of Adverse Events by Modelling Biological Mechanisms in CAR-T Cell Therapy

Cancer pharmacology

AP03

Background: Chimeric Antigen Receptor (CAR) T cells are genetically engineered T cells designed to target and destroy cancer cells. Despite their efficacy, CAR-T cells can induce Cytokine Release Syndrome (CRS), a clinical syndrome resulting from generalized immune activation. CRS presents with a spectrum of symptoms ranging from mild to severe. Mild cases are generally self-limiting and manageable with supportive care, while severe cases can be life-threatening, requiring intensive care. Mortality, though rare, can occur. Tocilizumab, an IL-6 receptor antagonist, and steroids are used to mitigate severe CRS.This study utilizes EndogenicsTM, an interactive, patented, mechanism-oriented modeling and simulation framework that is designed for and used by medical experts and biologists. EndogenicsTM offers the ability to model and evaluate the molecular determinants of efficacy and to provide insights into the mechanism of safety findings.Methods: Investigating the mechanistic and predictive safety of CAR-T cells, EndogenicsTM was applied to model cellular kinetics and CRS dynamics, focusing on key cytokines such as Interleukin-6 (IL-6) and Interferon-gamma (IFN-γ).Results: The activation of CAR-T cells leads to the release of pro-inflammatory cytokines, with IL-6 identified as a central mediator in the development and progression of CRS. IL-6 is produced by activated T cells, macrophages, and other immune cells, inducing fever, promoting vascular permeability, and contributing to the activation of the coagulation cascade. Factors affecting the severity of CRS include higher baseline disease burden, higher expansion of CAR-T cells, and elevated levels of IL-6 which were included as factors into the modelling.Conclusion: The application of EndogenicsTM in modeling the biological mechanisms of CRS provides a deeper mechanistic understanding of adverse events associated with CAR-T cell therapy. This approach enhances the predictive safety and efficacy assessment of CAR-T cells, informing therapeutic strategies to mitigate CRS and improve patient outcomes. The study underscores the critical role of CAR-T cell effectiveness, the impact of CAR-T dose on IL-6 levels and timing, and the subsequent tumor response.

Prof.

CD13 inhibitor IP10.C9A decreases colon cancer cell growth in vitro and in vivo

IntroductionColorectal cancer is the third most common malignancy and the second leading cause of cancer related death worldwide. Over the last few years, therapy options have significantly improved patient survival. However, some patients don"t respond to therapeutics or are initially sensitive but quickly become insensitive. CD13, also called Aminopeptidase N, is a promising target for overcoming multiple drug resistance. CD13 is a cancer stem cell marker and highly expressed on drug-resistant and rapid-growing tumor cells. As a zinc-dependent membrane-bound endopeptidase, CD13 is involved in regulating cell motility and angiogenesis. Inhibition of CD13 decreases proliferation in various types of cancer cells. ObjectivesThe aim of our study is to investigate the effects of the CD13 inhibitor IP10.C9A on colon cancer cell growth and tumor progression in vitro and in vivo. Materials & methodsIn a syngeneic subcutaneous tumor model, tumor growth was compared between CD13-knock out (CD13-KO) and wild type (WT) mice. Mice were treated with IP10.C9A over a period of 14 days with a group of untreated mice serving as control. qPCR and Western blot analysis of tumor samples were performed. For in vitro analysis, human (HCT116) and murine (MC38-CEA-1) colon cancer cells were incubated with IP10.C9A, respectively. Spheroid formation assays and MTT viability assays were performed. ResultsCD13-KO mice showed significantly decreased tumor growth in and ex vivo compared to WT mice after 21 days in trial. WT mice treated with IP10.C9A showed a significantly decreased tumor growth rate in vivo compared to untreated WT mice. At the level of both protein and RNA, the expression of CD13 was significantly decreased in tumor samples of CD13-KO mice compared to WT mice. Spheroid formation in MC38-CEA-1 and HCT116 cells was significantly inhibited after incubation with IP10.C9A. Viability of MC38-CEA-1 and HCT116 cells decreased significantly under treatment with IP10.C9A. ConclusionPharmacological inhibition of CD13 as well as genetical knock out of CD13 significantly decreased growth of colon cancer cells in vivo. Increased expression of CD13 in tumor samples of WT mice underlines the tumor promoting properties of CD13. A reduction in cell viability under CD13 inhibition was accompanied by a prevention of spheroid formation. As IP10.C9A inhibits growth of colon cancer cells in vitro and in vivo, IP10.C9A may be a promising new compound for colon cancer therapy.

Prof. Dr. Dr.

Loss of MAPK negative regulator leads to resistance towards apoptotic modulators in acute lymphoblastic leukemia

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy and despite treatment improvements still up to 20% of patients suffer from relapses with dismal outcome. Relapsed ALL is characterized by drug resistance, particularly to glucocorticoid therapy. Although underlying mechanisms are only partly understood, hyperactivity of the mitogenic activated protein kinase (MAPK) signaling pathway clearly contributes to drug resistance. While oncogenic mutation induced pathway hyperactivation is well established, the role of a disrupted negative feedback control remains largely elusive. Thus, the aim of the study is to elucidate whether the dual-specificity phosphatase 2 (DUSP2), one of the major negative feedback regulators of MAPK signaling in immune cells may contribute to drug resistance in ALL.MethodsJurkat T-ALL cells were used as model system to generate cells deficient for DUSP2 (DUSP2KO) or DUSP2/DUSP5 using CRISPR/Cas9 genome editing, while rescue cell lines were made by lentiviral transduction. A high throughput drug sensitivity screening with 528 compounds (FIMM oncology library) was performed to identify drugs causing differential responses depending on the DUSP2 status. Cells were treated for 72 h with various concentrations of respective drugs before cell viability was measured. The results of selected candidates were confirmed for using various methods to determine cell viability including metabolis activity by cell titer glow, direct apaotosis by Annexin V, PI staining and number of living cells using the NucleoCounter.ResultsFor most compounds (~350), the DUSP2 status of cells did not affect the drug response. However, DUSP2KO cells were significantly more resistant towards treatment with specific BH3-mimetics than wild-type and rescue wild-type cells (IC50 KO: 136nM vs. WT: 33nM or resWT: 30nM). These findings were confirmed using various cell viability assays. Thereafter, combination experiments with these BH3-mimetics and MAPK inhibitors were performed which led to overcome the observed drug resistance of the DUSP2KO cells.ConclusionThe findings of this study suggest that not only components of the MAPK pathway itself but also its negative regulators are crucial determinants for drug resistance in ALL. A better understanding of these critical regulators will help to unravel the mechanisms of drug resistance, which may help to identify more effective drug combinations for future treatment.

Changes of the microbiome in an experimental model of functional dyspepsia: Prevention by STW 5

Immunopharmacology / inflammation / anti-infectives

AP02

Introduction: In irritable bowel syndrome (IBS) and functional dyspepsia (FD), STW 5 (Iberogast®) has been shown to be clinically effective, but the involvement of the intestinal microbiota has not been adequately studied.Aim: The aim of the present study was to examine whether changes in gut microbiota induced in an experimental model of functional dyspepsia could be prevented by STW 5.Material and Method: FD was induced in rats by subjecting them to neonatal maternal separation followed by subjecting them to restraint stress for 90 minutes/day for one week. During that week, one group (8 animals) was treated with STW 5, a combination of nine hydro-alcoholic (31 % alcohol) extracts, of Iberis amara, Matricaria recutita, Carum carvi, Melissa officinalis, Mentha piperita, Glycyrrhiza glabra, Silybum marianum, Angelica archangelica and Chelidonium majus (5 ml/Kg) and one with its vehicle (31% alcohol) at the same dose level. Animals were sacrificed 24 h after the last drug administration. Fecal samples were taken from the cecum. Genomic DNA was isolated and changes in selected bacterial phyla and genera were assessed using quantitative Real Time-PCR (qPCR).Results: The main phyla modulated under the influence of stress in the model were the Bacteroidetes, Firmicutes, Fusibacterium and Actinobacteria. FD led to a 50% drop in the Lactobacilli population but to a two-fold rise in Proteobacteria abundance but did not affect significantly the changes in Bacteroidetes. Furthermore, the Methanobrevibacter population was raised more than two-fold while the genomic DNA concentration in Bifidobacterium dropped from ca. 3 ng/g feces to nearly nil. In general, STW 5 tended to ameliorate and protect against the changes induced by the stress model.Discussion and Conclusion: The results lend further support to the use of STW5 in FD and IBS by providing evidence that it acts at least in part through influencing beneficially the changes in intestinal microbiota induced by stress. Furthermore, the findings shed more light on mechanisms involved in potential usefulness of STW 5 in gastro-intestinal disorders.

Hörsaal C

Short talk 6

Immunopharmacology, cancer pharmacology and molecular pharmacology

Short talks

Short talk 6
Immunopharmacology, cancer pharmacology and molecular pharmacology

Termin

Chair

Programm

Short talk 6Immunopharmacology, cancer pharmacology and molecular pharmacology

Termin

Chair

Programm

Short talk 6
Immunopharmacology, cancer pharmacology and molecular pharmacology