Faculty of Pharmacy

 

Recent Submissions

  • Stepanova, Polina (2021)
    Parkinson’s disease (PD) and Huntington’s disease (HD) are characterized by loss of function or death of definite cell populations in the basal ganglia. HD is triggered by an expanded polyglutamine tract (glutamine repeats) in the huntingtin protein, leading to misfolding of the protein and subsequent accumulation of mutant huntingtin (mHtt) in the nuclei of different types of neurons. In contrast, in PD, dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) degenerate; moreover, there is also the accumulation of misfolded proteins associated with the neuropathology of PD. Additionally, endoplasmic reticulum (ER) stress has been detected in both of these diseases. Currently, no effective treatment for PD or HD is available to slow, stop, or reverse the progression of neurodegeneration. Cerebral dopamine neurotrophic factor (CDNF) is an evolutionarily conserved protein with neurotrophic properties. CDNF protects and restores the function of DA neurons in preclinical models of PD more effectively than other neurotrophic factors (NTFs), making it a promising drug candidate as a disease-modifying treatment of PD. Additionally, CDNF was safe and well-tolerated, showing therapeutic effects in some PD patients in phase 1/2 clinical trials. This thesis aimed to investigate the potential of CDNF as a drug candidate in cellular and rodent models of both HD and PD. We focused on studying the effect of coadministration of CDNF with another NTF, glial cell line-derived neurotrophic factor (GDNF), in a 6-hydroxydopamine (6-OHDA) rat model of PD. Moreover, we wanted to evaluate the difference in the mode of action between the two factors in this model. We found an additive neurorestorative effect after intrastriatal CDNF and GDNF coadministration in the 6-OHDA toxin model of PD. CDNF alone and in combination with GDNF showed a trend toward an increase in the density of tyrosine hydroxylase (TH)-positive fibers in the lesioned striatal area, and moreover, the combination of the two factors significantly protected TH-positive cells in the SNpc area. CDNF activated the PI3K/AKT pathway, whereas GDNF activated two signaling pathways: PI3K/AKT and MAPK/ERK. We found a delay in activating the prosurvival pathway by CDNF compared to GDNF. Additionally, in comparison with GDNF, CDNF alone significantly enhanced the phosphorylation of ribosomal protein S6 downstream of the PI3K/AKT pathway in lesioned rats. Finally, CDNF, but not GDNF, reduced striatal levels of some ER stress markers in the above model. PD and HD show a wide range of commonalities, and as CDNF has been successful in preclinical trials and demonstrated a positive effect in clinical trials of PD, we expected to observe some beneficial effects of CDNF administration in the experimental HD models. We studied the neuroprotective and neurorestorative potential after different delivery paradigms of CDNF in preclinical models of HD to find the optimal paradigm for possible future application in clinical trials. First, we investigated the protective effects of CDNF in in vitro and in vivo quinolinic acid (QA) toxin models of HD. We demonstrated that CDNF improved motor coordination in QA-lesioned animals, which could be explained by the neuroprotective and neurorestorative effects of CDNF in the cell populations that are vulnerable in HD. Moreover, we found a protective effect after a single intrastriatal injection of CDNF in an in vivo QA-lesion model of HD. Second, we tested the therapeutic efficacy of CDNF in a transgenic mouse model of HD. CDNF was delivered as a chronic intrastriatal infusion using Alzet minipumps for N171-82Q mice. Chronic CDNF administration ameliorated the behavioral deficits and showed a trend toward reduced the nuclear staining and intranuclear inclusions in N171-82Q transgenic mice. Additionally, chronic delivery of CDNF demonstrated a trend toward a decrease in ER stress markers in the striatum in the above model. Furthermore, we found a significant increase in hippocampal BDNF mRNA levels after chronic CDNF administration in N171-82Q mice. In conclusion, CDNF shows a unique beneficial effect in several models of HD independent of the etiology of the disease. Notably, this thesis reports the first beneficial effects of CDNF in different models of HD.
  • De Lorenzo, Francesca (2020)
    Neurodegenerative diseases are characterized by the dysfunction and death of specific neuronal populations. Parkinson’s disease (PD) is caused by the progressive loss of dopamine neurons in the substantia nigra, whereas motor neurons (MNs) in the motor cortex, brain stem, and spinal cord degenerate and die in amyotrophic lateral sclerosis (ALS). Accumulation of misfolded proteins and endoplasmic reticulum (ER) stress are some common hallmarks in the pathophysiology of neurodegenerative diseases. ER stress triggers the unfolded protein response (UPR), a physiological response that aims at restoring the ER homeostasis by degrading misfolded proteins, attenuating protein translation, and increasing the expression of ER chaperones important for protein folding. Initially the UPR is protective, but, upon prolonged ER stress, the UPR switches from an adaptive to a pro-apoptotic response. Cerebral dopamine neurotrophic factor (CDNF) is an ER resident protein with neurotrophic properties that is protective and restorative in preclinical models of PD. The mechanism underlying CDNF’s action is still unclear, but experimental data suggest a possible involvement of CDNF in the ER homeostasis. The aim of this thesis work was to study the therapeutic potential of CDNF in PD and ALS rodent models and investigate CDNF mode of action, with a special focus on the ER stress response. Herein, we report that co-administration of CDNF and glial cell line-derived neurotrophic factor (GDNF) showed an additive neurorestorative effect in the unilateral 6-hydroxydopamine rat model of PD, suggesting a different mechanism of action for these two proteins. We found that GDNF activated the pro-survival MAPK/ERK and PI3K/AKT pathways in the striatal dopamine neurons within 1 hour from protein administration. In contrast, CDNF activated only the PI3K/AKT pathway and at 4 hours upon treatment. Furthermore, CDNF, but not GDNF, reduced the expression of UPR markers ATF6, p-eIF2α, and GRP78. Therefore, the ability of CDNF to regulate ER stress was thoroughly investigated in three rodent models of ALS with different genetic etiology and disease progression. We showed that CDNF decreased the ER stress response specifically in MNs, by attenuating all three branches of the UPR, initiated by transducers inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6). CDNF treatment was effective in all three models, indicating that CDNF’s therapeutic effect was independent of disease etiology. CDNF rescued MNs from ER-stressed induce cell death, halting the progression of the disease and ameliorating the motor deficit in the SOD1-G93A mouse model and in the TDP43-M337V rat model. Finally, we identified that depleting endogenous CDNF from the SOD1-G93A model worsened the motor symptoms in the mice, but did not affect their lifespan. The ER stress response in the Cdnf -/- SOD1-G93A mice was especially exacerbated in the skeletal muscle, where CDNF is normally highly expressed, and an overexpression of homologous protein mesencephalic astrocyte-derived neurotrophic factor (MANF) was detected in the same tissue. We observed a reduction in the number of lumbar MNs in Cdnf -/- SOD1-G93A compared to classical SOD1-G93A mice, which would explain the aggravated motor impairment. At this point, however, we could not determine whether the increase in MNs loss was caused by CDNF depletion in MNs, or rather a consequence of CDNF-deficiency in the degenerating muscle cells, targets of MNs. It was previously reported that, in mice, endogenous CDNF is important for the development and maintenance of enteric submucosal neurons, as well as for the regulation of gastrointestinal transit. Remarkably, we found that Cdnf -/- mice had less lumbar MNs at 4 months, compared to WT littermates, although this decrease did not result in any motor deficit. These findings suggest that CDNF may also have a role in the development and/or survival of MNs. Altogether, these studies indicate that ER stress is an important therapeutic target for neurodegenerative diseases, such as PD and ALS, and that CDNF is a promising drug candidate, due to its ability to attenuate all three pathways of UPR.
  • Miettinen, Ilkka (Helsingin yliopisto, 2020)
    Treatment-resistant bacterial infections pose a major challenge to healthcare. In order to establish a chronic infection, bacteria must tolerate antimicrobial treatment and evade clearance by host immune system. The ability of bacteria to form biofilms, which are sessile communities of adherent microbes encapsulated in self-produced matrix of extracellular polymeric substances, contributes crucially to both of these traits. Biofilm bacteria produce numerous virulence factors that facilitate their adherence, invasion into host tissue, and evasion of the host immune system. Variable microenvironments within the biofilm give rise to metabolically sedentary subpopulations of bacteria, known as persisters, which can transiently tolerate antimicrobial chemotherapy. The biofilm matrix provides additional mechanical barrier against a selection of therapeutics. This thesis presents the optimization and validation of a Staphylococcus aureus model for persistence. Bacteria were monitored past the stationary phase transition to detect metabolic switching and a culture with halted energy metabolism and replication was confirmed to withstand extreme levofloxacin concentrations in a manner that was reversible by resurrecting growth. This model culture was used to study the effect of the metabolic status of the inoculum on biofilm characteristics. A label-free proteomics-based analysis of surface-associated proteins was carried out, and persister model-derived biofilms were shown to display boosted tolerance traits, such as oxidative stress defense and immune evasion, when compared to biofilms created from metabolically active cultures. This was reflected to increased survival in macrophage-like cells. A mixed biofilm model based on S. aureus and Pseudomonas aeruginosa, common co-habitants in chronic wounds and cystic fibrosis lung infections, was also established to study the effect of interspecies interaction on biofilm virulence and tolerance. A label-free proteomics approach was applied to concurrently compare surface-associated and extracellular protein profiles between mono- and co-cultured biofilms. This study provided proteomics-level insight that simultaneously covered multiple facets of virulence and tolerance in mixed-species biofilms. Several of the key findings here were found to be aligned with previously published functional studies. The proteomics studies also involved the compilation and improved annotation of S. aureus and P. aeruginosa theoretical proteomes, which are now fully accessible for future studies involving these or related bacterial strains. Altogether, this work demonstrates the applicability of label-free proteomics approaches in two different, clinically relevant settings involving bacterial biofilms. It also proposes models, methods, and proteomic workflows for the study of bacterial virulence and tolerance.  
  • Shawesh, Amna Mohammed (Helsingin yliopisto, 2015)
    Indomethacin (IND) is a potent non-steroidal anti-inflammatory drug used in the treatment of rheumatoid arthritis, osteoarthritis, acute gout and other disorders. IND is available worldwide mostly in the form of capsules and suppositories, however, these formulations usually create side effects. Consequently, an alternate route of administration to avoid or minimize side effects may be found in the form of semisolid dermatological formulations, now available in few countries. The specific goals of this study were: (I) to determine the solubility of IND using different co-solvents: hexylene glycol (HG), propylene glycol (PG), polyethylene glycol 300 (PEG), butylene glycols (1,2 BG; 1,3 BG and 1,4 BG) and ethanol (ETOH). 1% (w/w) Tween® 80 or polyvinyl pyrrolidone (PVP-25) were used as enhancers; (II) to develop suitable topical gel preparations using 20% (w/w) Pluronic® (PF-127) or 1% (w/w) Carbopol ETD® 2001 (C2001) as gelling agents and HG or PEG 300 as solvents (1% (w/w) Tween® 80 and PVP-25 were added as excipients); (III) To evaluate the effect of composition of prepared gel formulations on the following parameters: appearance, crystallization, pH and rheological behaviour and (IV) to investigate the influence of storage time and storage conditions on the characteristics of the gels. These results indicate that all the solvents tested increased the solubility of IND to varying degrees. Tween® 80 and PVP-25 only slightly enhanced the solubility of IND. 1% (w/w) IND was able to form a structural gel with both PF-127 and C2001. Storing the INDPF-127 gels at 6°C resulted in the precipitation of IND. All gels stored at room temperature exhibited good stability. The gels stored at 45°C developed a dark yellow colour. Gels with C2001 and PF-127/PEG had slightly decreased viscosities with increasing storage time, while the gels with PF-127/HG showed increase in viscosities with time. In conclusion, the water solubility of IND was increased by the addition of co-solvents. 1% (w/w) IND gel can be suitable for using as a gel formulation and it is stable at room temperature. The search for suitable gels for IND topical formulation needs to be continued with more stability studies. Moreover, in-vitro and in-vivo experiments will be necessary for providing data on bioavailability.
  • Zhou, Fang (Helsingin yliopisto, 2012)
    We propose network abstraction as a research area. It is motivated by the growth of networks in many areas of life. Consider, for instance, networks of thousands of genes, millions of people, or billions of web pages. They are too large to be directly analyzed by users. The aim of network abstraction is to summarize a large network as a smaller one. An abstracted network can then help users to see the overall topology of a large network, or to understand the connections of distant nodes. The general network abstraction task is: given a large network, transform it into a smaller one, which contains in some well-specified sense the most relevant information. In this thesis, we analyze this research area and propose methods to solve some instances of the problem. The methods also provide different trade-offs between the graph quality and simplicity, as well as between result quality and efficiency. More specifically, we propose two approaches to abstracting a network. The first one is to simplify a weighted network by removing edges under the constraint that distances between all pairs of nodes are preserved. We first empirically show that a number of edges can be removed from real biological networks without losing any graph connectivity. We next relax the constraint of fully preserving original graph connectivity, extend lossless network simplification to lossy network simplification, and demonstrate that many more edges can be removed with little loss of quality. The second approach we give for network abstraction is to compress a weighted network by grouping nodes and edges. We propose novel methods and experimentally show that real graphs can be compressed efficiently with relatively little error. We next consider graphs with weights also on the nodes, and utilize them as node importances to extend the definition of weighted graph compression. We present new compression operations and demonstrate that the compressed graph can preserve more information related to more important nodes. Furthermore, we propose the idea of using node weights and compression to summarize the metabolisms in a set of organisms, and apply the methodology to better understand the metabolic biodiversity between Archaea and Eubacteria, the two most fundamental branches of life.
  • Vellonen, Kati-Sisko (2010)
    Drug discovery and development from its very onset up to market approval is a long process lasting 10-15 years. New research tools are needed to accelerate and rationalize this process. Ocular drug research still relies heavily on animal testing with rabbits and other rodents. Computational methods and cell culture models are promising tools for early pharmacokinetic studies and may partly replace the animals in pharmacokinetic and toxicological studies. Computational methods are initially based on experimental data, but thereafter their application is straightforward and they can be used to reduce, partly replace and refine further experimental studies. Similarly, cell culture models may enable absorption and toxicity testing of drug candidates with continuously growing cells of human origin, and thereby reduce the need for animal experiments. The cornea is the main route of ocular drug absorption after topical administration, and the corneal epithelium is the most important barrier to drug permeation. Membrane transporter proteins play an important role in the general pharmacokinetics and toxicology. However, their role in ocular pharmacokinetics is still poorly understood. Based on literature analysis many ocular drugs seem to be substrates of transporters, but the expression of these proteins in the eye is largely unknown. The goal of this work was to develop and evaluate cellular and computational tools for ocular pharmacokinetics and toxicology, and to characterise the active drug transporters in the corneal epithelium. The expression of monocarboxylate transporters and ATP-binding cassette (ABC) class efflux proteins was studied in the corneal epithelium and human corneal epithelial (HCE) cell model. Human corneal epithelium expressed monocarboxylate transporters 1 and 4 (MCT1 and MCT4), efflux transporters multidrug resistance-associated protein 1 and 5 (MRP1 and MRP5), and breast cancer resistance protein (BCRP). Cultured human corneal epithelial cells over-expressed several ABC class efflux proteins and MCT1 and MCT4. The functionality of efflux and monocarboxylate transport was demonstrated in HCE cells and in the rabbit cornea ex vivo. The MTT test is a widely used cytotoxicity test in cell research. It was demonstrated that substrates and inhibitors of ABC class efflux proteins may interfere with the MTT test, presumably by inhibiting dye efflux from the cells. This may lead to an underestimation of toxicity in the MTT test. Quantitative structure property relationship (QSPR) models are commonly used in early drug discovery to predict ADME properties of novel compounds. Multivariate analysis was used to develop QSPR models for in silico prediction of the corneal permeability. Two factors, the distribution coefficient (logD7.4 /logD8.0) and hydrogen binding potential, were shown to be the parameters that determine the transcorneal permeability of a compound. These models were able to predict intracameral steady state drug concentrations in rabbit eyes. In conclusion, the new in silico QSPR model can make reliable predictions for passive drug permeability in the cornea, while the HCE model seems to over-express some membrane transporters as compared to the normal human corneal epithelium. Even if these investigated methods have some restrictions they are still very useful tools for drug discovery purposes.