A national centre of pharmaceutical drug research

Abstract

Schizophrenia is a severe psychiatric disorder affecting approximately 20 million people worldwide. The disease consists of positive symptoms e.g. hallucinations, negative symptoms such as anhedonia, and cognitive deficits, e.g. impaired episodic memory. Most of the currently available treatment options for schizophrenia only target the positive symptoms, possess severe side effects and do not work for a large group of patients. In this thesis, the unique antipsychotic drug lumateperone and adjunctive treatment of sodium nitroprusside (SNP) to sub-maximal doses of conventional antipsychotic drugs are investigated in preclinical tests as novel treatment options for schizophrenia 

In paper I we showed that SNP enhances the antipsychotic-like effect of a sub-effective dose of risperidone in the conditioned avoidance response (CAR) test in rats. Moreover, by using microdialysis we showed that SNP significantly enhances risperidone-induced dopamine release in the rat medial prefrontal cortex (mPFC) but not in the nucleus accumbens, indicating that adjunct SNP could be used to improve the efficacy of antipsychotic drugs, while reducing their dose and subsequently lower the risk of side effects.

In paper II we used microdialysis combined to the behavioral novel object recognition test in rats to show that the release of both dopamine and norepinephrine is increased in the ventral hippocampus in response to a novel object, suggesting that dopamine and norepinephrine may play a crucial role in recognition memory. 

In paper III we showed that SNP significantly enhanced the antipsychotic-like effect of sub-effective doses of olanzapine in the CAR test, but not of clozapine, this could be explained by the developed tolerance towards clozapine after repeated administrations.

In paper IV we used enzyme-coated microelectrode arrays to show that lumateperone significantly increased cortical glutamate release in the mPFC of anaesthetized rats. By using electrophysiology, we also show that lumateperone facilitates NMDA and AMPA-mediated currents in a dopamine D1 dependent manner in layer V/VI pyramidal neurons in the mPFC of rats. Moreover, lumateperone increases dopamine release in the mPFC of freely moving rats as shown by using microdialysis. These mechanisms may improve cognitive deficits and contribute to the clinically demonstrated antidepressant effects of lumateperone. 

Taken together, these results show that lumateperone is a promising novel treatment option for schizophrenia, and that adjunct SNP treatment may allow for improved efficacy at maintained or even reduced dosage of conventional antipsychotic medication.

Abstract

The platelet-derived growth factor receptors (PDGFRs) play important roles in multiple cellular processes including cell survival, cell growth and cell migration. Dysregulation of PDGFRs causes aberrant PDGF signaling, which leads to various diseases. Insights from the research about PDGF/PDGFR signaling have enlightened new opportunities to understand the molecular mechanisms of cancer and other diseases. The goal of this thesis is to further investigate the mechanisms of the modulation of PDGF/PDGFR signaling to identify new ways of controlling aberrant signaling of these RTKs in various diseases, including cancer.

Ubiquitination is an important post-translational modification related to protein degradation, receptor internalization, intracellular trafficking, cell proliferation, and other cellular processes. It can be reversed by DUBs, and the overexpression of DUBs is involved in various diseases including cancers. In paper I, we identified two main DUBs working on PDGFRβ, USP17 and USP4. They affected the timing of STAT3 activation and trafficking via different mechanisms, thus fine-tuning its transcriptional activity, which further regulated the proliferative response induced by PDGF-BB.

SUMOylation is another post-translational modification that is important for the regulation of protein subcellular localization, protein stability, protein-DNA interactions, protein-protein interactions, genome organization, DNA repair and transcriptional regulation. In paper II, we have identified PDGFRα as a SUMOylation substrate and performed a characterization of the functional role of SUMOylation in PDGFRα signaling and cell proliferation.

Proteolytic cleavage of RTKs regulates their downstream signaling pathways by affecting their structure, stability, subcellular localization and interaction with other proteins. In paper III, we have identified that PDGFRβ is cleaved in the region Y579-Y857 upon ligand stimulation by a Ca2+-dependent protease, which is dependent on its internalization. The proteasomal inhibitor bortezomib blocked the internalization, as well as the cleavage of PDGFRβ, and also affected its downstream signaling.

Apart from the classic degradation in lysosomes and proteasomes, several RTKs have also been found to undergo autophagy and to be targeted in autophagosomes which further fuse with lysosomes. In liver hepatocytes (LX2) cells, it has been reported that PDGFRα, but not PDGFRβ, undergo selective autophagy. In paper IV, we identified that in certain types of cells, PDGFRβ may be involved in the autophagy pathway, which may affect the synthesis of new PDGFRβ.

To conclude, in this study, we investigated how the turnover and signaling of the RTKs PDGFR isoforms α and β are modulated by ubiquitination, SUMOylation, proteolytic cleavage and degradation.

Abstract

Globally, hepatocellular carcinoma (HCC) is the most common form of liver cancer and a leading cause of cancer death. One important risk factor is liver cirrhosis and the disease progression is characterized by deposition of extracellular matrix proteins that form a fibrous network, which increases liver stiffness and may limit the effectiveness of different treatment strategies. 

The overall aim of this thesis was to investigate the anticancer drug doxorubicin (DOX) and its clinically relevant drug delivery systems from an in vitro perspective. The focus was on developing and using qualitative and quantitative methods to better understand formulation performance, intracellular uptake and molecular diffusion. The experimental in vitro findings were then translated to clinical scenarios using physiologically based pharmacokinetic (PBPK) modelling.

The performance of clinically employed emulsion formulations containing DOX and the tumor accumulating oil Lipiodol® were evaluated in terms of their stability (Paper I). The most stable emulsion (> 72 h) was achieved when using an aqueous phase containing the contrast agent iohexol and with an aqueous to lipid phase ratio of 1:4 to assure formation of a water-in-oil emulsion.  This was followed by a cell-based study (Paper II) where nanoformulated DOX was compared to DOX in solution in terms of tumor cell toxicity, intracellular DOX uptake and intracellular formation of the main active metabolite doxorubicinol (DOXol). DOX in solution was more potent in all investigated cell lines, where the most sensitive cells (HepG2) displayed IC50 values that were approximately 100 times lower than the most resistant cell line (SNU449). This was explained by the rapid intracellular uptake in HepG2 cells which was also confirmed with a complimentary miniaturized chip technique in Paper IV.  In papers III and IV the focus was on molecular diffusion across biomimetic hydrogels mimicking tissue properties of cirrhotic liver and early stage HCC. The diffusion of DOX was significantly reduced in biomimetic gels as compared with more commonly used agarose gels, however the presence of human liver tumor cells did not significantly influence diffusion. Simulations using a developed PBPK and spatio-temporal tissue concentration model suggested that a liver tumor resembling SNU449 cells would not reach therapeutic exposure levels in a clinical scenario while the diffusion of DOX required further reduction by the tumor extracellular matrix in order to generate tumor concentration-time curves consistent with in vivo observations.

This thesis contributes to an increased understanding of using DOX and its drug delivery systems as a treatment option for HCC. The approach of translating in vitro experimental data to clinical scenarios using modelling will grow in relevance as methods become more complex and data more bio-relevant.