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  • Shrestha, Neha (Helsingin yliopisto, 2016)
    Regardless of the considerable efforts, there have been no major breakthroughs in the development of effective oral protein/peptide delivery. When compared to parenteral administration, oral delivery can significantly improve the patients quality of life, especially in chronic conditions, such as diabetes mellitus (DM), which requires multiple injections daily. However, oral absorption of proteins/peptides is severely limited by their physico-chemical properties and various physiological barriers in the gastrointestinal tract. Porous silicon (PSi) has emerged as a promising drug delivery system, owing to its beneficial properties, such as top-down production, customizable particle and pore morphology, easy surface modification, simple drug loading, biodegradability and biocompatibility. Thus, the aim of this dissertation was to develop a multifunctional PSi based platforms that would be able to overcome the physiological barriers and efficiently deliver insulin and glucagon-like peptide-1 (GLP-1) orally. First, the influence of different PSi surface chemistries was evaluated on the intestinal transport of insulin. Due to the negatively charged surface of PSi, there was minimal interactions with the intestinal cells. Thus, chitosan, a polycationic mucoadhesive biopolymer with permeation enhancing effect, was used to modify the surface of the PSi microparticles. When comparing different surface modification techniques, chemical conjugation of chitosan to PSi exhibited strongest cellular interaction, and the highest insulin permeation and uptake across the intestinal cell monolayers. Secondly, three different nanoparticles (NPs) were developed based on lipids, polymers and PSi, with and without chitosan coating, and evaluated as potential oral GLP-1 delivery system. The results showed that the chitosan-modified PSi NPs were the most efficient nanosystem with the best loading degree and the highest GLP-1 permeation across the cellular monolayer. To overcome several physiological barriers, the next step was to develop a multistage nanocomposite comprising of chitosan-conjugated PSi NPs that were coated with a pH responsive polymer, in order to deliver GLP-1 and dipeptidylpeptidase-4 (DPP4) inhibitor simultaneously via the oral route. This multistage nanosystem showed enhanced GLP-1 transport across the intestinal cell monolayers and across the rat intestinal tissue. Furthermore, the nanosystem also demonstrated hypoglycemic effect in vivo after the oral administration in diabetic rats. The efficacy of the nanosystem could be attributed to the combined effect of the permeation enhancing chitosan-modified PSi NPs, the presence of DPP4 inhibitor that prevented GLP-1 degradation, and the pH responsive coating that helped in avoiding premature GLP-1 release/degradation in the stomach. Moreover, it was shown that the mucoadhesivity and permeation enhancing ability of chitosan-modified PSi NPs could be significantly increased by further surface modification of NPs with either L-cysteine or cell-penetrating peptide (CPP). It was disclosed that electrostatic interactions between the NPs and the glycocalyx were the most prominent pathway for the transport and uptake of insulin from the NPs, together with the contribution of active transport, adsorptive endocytosis and clathrin-mediated endocytosis. Overall, advanced PSi-based systems were developed which successfully overcame several limitations associated with the oral delivery of biomacromolecules, and thus, showed high clinical potential as oral protein/peptide delivery systems for DM therapy
  • Hirvinen, Mari (Helsingin yliopisto, 2016)
    Cancer is the leading cause of death worldwide creating a need for novel cancer treatments that are more efficient but also safer and more specific. Oncolytic viruses (OVs) have shown a solid safety profile in clinical trials. OVs are nowadays considered immunotherapies because of to their ability to stimulate the host immune system to fight against cancer. Promising efficacy has been seen in some trials, however, efficacy is often seen only in a small group of patients. The purpose of the thesis was to improve the efficacy of OV therapies by boosting the immunogenicity of the viruses, and to optimize the therapeutic efficacy by selecting favorable patient populations and by developing a method to tailor the drug individually for each patient. In the first study, an oncolytic adenovirus (OAd) was modified to express human tumor necrosis factor alpha (hTNFα), a potent immunomodulatory cytokine. The TNFα-virus showed effective tumor cell killing associated with signs of immunogenic cell death and enhanced recruitment of immune cells to the infection site. We also saw potential for combining the TNFα-virus therapy with radiation. In another study the immunogenicity of an oncolytic vaccinia virus was enhanced by modifying it to express DNA-dependent activator of interferon-regulatory factors (DAI), a potent inducer of innate immune responses during virus infection. We showed that the DAI-virus induces expression of genes involved in immune responses, and treatments with the virus showed improved cancer-killing efficacy and immunogenicity in murine and human melanoma models, suggesting applicability also in vaccine design. Response rates after virotherapies vary between patients, and there is a lack of markers that would help predict the patient cohorts who would benefit from the therapy. We screened over 200 cancer patients treated with OAds for two Fc gamma receptor (FcγR) polymorphisms to determine if these polymorphisms would affect the responsiveness to the treatments. We observed a certain FcγR genotype combination (FcγRIIIa-VV + FcγRIIa-HR) to be predictive of poor overall survival after OAd treatments. To tailor the OV therapy for enhanced specificity, we developed a novel platform (PeptiCRAd) to coat a virus with tumor-specific antigens (peptides) for improved induction of cancer-specific immunity. Efficacy and immunogenic potency of the PeptiCRAd were shown in several in vivo models. Our results suggest that administration of tumor-specific peptides on the surface of OVs increases the anti-tumor efficacy compared to treatments with viruses or peptides alone. This platform has potential to be used as a carrier and adjuvant for patient-specific peptides to trigger anti-tumor immunity in a personalized manner.
  • Montalvão, Sofia (Helsingin yliopisto, 2016)
    Marine environment is prolific in organisms with unique properties. Seas and oceans contain a wide diversity of species with biologically active metabolites that represent a valuable source with great potential for the development of novel therapeutic agents. This dissertation is focused on the biological study of synthetic compounds based on marine scaffolds as well as on marine natural product extracts originating from the Aegean Sea. Furthermore, it offers an introduction on the importance of marine natural products in the search of new bioactive compounds, the use of natural products as scaffolds for the synthesis of new drugs, and a general overview on bioactivity screening and the current status of marine-derived bioactive compounds as therapeutic agents. The potential of oroidin and clathrodin as parent structures for synthesis of novel compounds was explored. Antimicrobial and antiproliferative studies were conducted and it was concluded that 4-phenyl-2-aminoimidazoles 6g(I) and 6h(I) showed the best antimicrobial effect against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), while compound 6j(I) showed the most interesting IC50 in antiproliferative studies. Compound 7(II), a synthetic derivative of 2-aminobenzothiazole, showed IC50 of 16 μM and 71 μM against a cancer cell line and a normal cell line, respectively. The selectivity index showed selectivity towards cancerous cells. In addition, okadaic acid was used as inspiration for the synthesis of crown ether acyl compounds. Compound (1,4,7,10,13,16-hexaoxacyclooctadecan-2-yl)methyl 3-(pyren-1-yl)propanoate) 1o(III) was found to be the most active in antimicrobial studies against Gram-positive Staphylococcus aureus with a MIC50 of 7.2 μM. The importance of bioprospecting the rich marine biodiversity in the Aegean Sea was also studied in this thesis. Biological activities of extracts from cyanobacteria, micro- and macroalgae were evaluated, and microalgae extracts (Amphora cf capitellata and Nitzschia communis) showed the most interesting antimicrobial results against Staphylococcus aureus and fungus Candida albicans. The results of the biological studies conducted in this thesis demonstrated antimicrobial and antiproliferative activity of several marine natural products and their synthetic derivatives. Further studies and structural optimization should be done to fully explore their potential for the development of therapeutic agents.
  • Kapp, Karmen (Helsingin yliopisto, 2015)
    Mentha plants are used in pharmaceutical and food industries. The genus Mentha has been classified into 18 species, 11 hybrids and hundreds of subspecies, varieties and cultivars. The medicinal and culinary properties of Mentha plants are bounded to the composition of polyphenolic compounds and essential oils. In the present study, the polyphenolic and essential oil composition of 47 Mentha plants. Similar profiling was conducted for 27 commercial peppermint tea samples. This was the first more detailed study to present the composition of mint flavourings isolated from 45 candies and food supplements. For the first time, the effects of peppermint teas water extracts, mint flavouring hydrodistilled extracts and terpenoidic reference substances against respiratory tract pathogen Chlamydia pneumoniae were investigated in vitro. Mentha plants water extracts and essential oils, mint flavouring hydrodistilled extracts and terpenoidic reference substances were tested in vitro for their antimicrobial properties against the potential pathogens Escherichia coli and Staphylococcus aureus. The antimicrobial effects of M. × villosa Huds., M. suaveolens Ehrh., M. × gracilis Sole, M. arvensis L. water extracts and mint flavouring hydrodistilled extracts were studied for the first time on E. coli and S. aureus. The polyphenolics of Mentha plants and peppermint teas water extracts were rather similar. The most abundant compounds found were rosmarinic acid and eriocitrin. Nine polyphenolic compounds were detected for the first time in the plants. The total content and composition of the essential oils in the Mentha plants and commercial peppermint teas samples varied. The essential oil analyses of peppermint teas showed that three tea samples may contain M. spicata L., different from that claimed on the package. The isolated mint flavouring hydrodistilled extract content was higher in mint flavoured sugar candies, pastilles and tablets than in chocolates. The three most abundant flavouring compounds were limonene, menthol and menthone. Peppermint teas water extracts were active against C. pneumoniae. The antichlamydial activity was often related to high content of luteolin and apigenin glycosides. The mint flavouring hydrodistilled extracts and reference substances decreased the infectivity of C. pneumoniae elementary bodies. Antichlamydial activity could be related to the high menthol content in extracts. Mentha plants water extracts showed antibacterial activity against S. aureus. Essential oils and mint flavouring hydrodistilled extracts inhibited the growth of E. coli and S. aureus. Linalool acetate and (-)-carvone were the most active reference substances against both bacteria. This study showed that consumption of Mentha plants may be beneficial for human health.
  • Matikainen, Minna (Helsingin yliopisto, 2015)
    The pharmaceutical industry has a vital reliance on successful new product launches (NPL), which are a critical driver of a company s performance. The prevailing literature on NPLs is fragmented and has mainly concentrated on a product s superiority as well as the strategic and tactical launch activities largely omitting the importance of customer relationships. The aim of this thesis is to provide a comprehensive overview on the key determinants of a successful NPL in the Finnish pharmaceutical industry. In practice, this study considers the extent to which a NPL and getting physicians to prescribe a new drug is relational activity. The role and relative impact of a company s strategic orientations and their mediating mechanisms were studied with survey data collected from the pharmaceutical companies operating in Finland. Partial least squares (PLS) path modeling revealed that the relationship orientation had the strongest positive impact on both customer acceptance and financial launch success. The company s accumulated market-based assets represented an alternative mediator in addition to product advantage. Sales force management and relationship marketing activities transformed a relationship-oriented organizational culture into launch performance. PLS regression modeling combined with target projection identified the diversity of determinants affecting launch performance at different stages of NPL. Product advantage and relationship marketing activities contributed to gaining the acceptance of key opinion leaders in the early phase of launch, while market-based assets and a company s relationship orientation largely determined the acceptance of the majority of target customers in the later phase. The buyer s perspective focused on the physician-pharmaceutical industry relationship and was studied by means of theme-interviews among a randomized sample of physicians. The positive relationship orientation of the physicians towards the pharmaceutical industry and whether they actively interacted with pharmaceutical companies were reflected in their early adoption of new drugs, especially when a product had a unique advantage and the physician s own personal interest accelerated the adoption of a new drug. In comparison, physicians who were negatively oriented towards the pharmaceutical industry and interacted passively adopted a new drug later based on evidence- and experience-based reasoning and the opinions of colleagues. In conclusion, this thesis calls for a relationship approach in order to complement the traditional sales and marketing approach regarding the launch of new pharmaceutical products. A successful pharmaceutical product launch should focus on appropriate relationship marketing activities that are conducted in a timely manner to achieve customer acceptance and financial launch performance.
  • Haavikko, Raisa (Helsingin yliopisto, 2015)
    Betulin is a triterpene class natural product present, for example, in the outer layer of birch bark, which is a low-value waste product of the forestry industry in Northern Hemisphere. Oxidation products of betulin, namely betulinic acid and betulonic acid, have been shown to have several biological activities. This makes betulin an interesting starting point for drug discovery projects. In this work, mainly ring A-fused heterocyclic derivatives of betulin were synthesized, and their structure-activity relationships against a protozoan parasite Leishmania donovani, prostate cancer cell lines, a serine hydrolase enzyme (ABHD12), and inflammatory factors were studied. The heterocycles used included pyridine, pyrazine, pyrazole, indole, isoxazole, and oxazole rings. Also, positions C-28 and C-20 of these compounds were modified and the resulting structure-activity relationships (SAR) were studied. Among the tested compounds, two heterocyclic betulin derivatives showed significant inhibition against L. donovani amastigotes growth. These compounds showed improved activity compared to the heterocyclic betulin derivatives tested previously. Also, a betulinderived potent anti-HIV compound, bevirimat, showed L. donovani growth inhibition at a similar level to the ring A-fused heterocyclic betulin derivatives. However, further optimization is needed to get more potent betulin derivative activity against L. donovani. In prostate cancer studies some of the betulin derivatives displayed dose-dependent anti-invasive activity at nanomolar concentrations with negligible cytotoxicity. The most potent compounds were betulin derivatives with a ring A-fused heterocycle. Also, the carboxyl group at C-28 seemed to be important for activity. These compounds showed considerably improved anti-invasive effects compared to betulinic acid. Betulin derivatives were found to selectively inhibit the ABHD12 serine hydrolase enzyme without inhibiting other endocannabinoid hydrolases and without activity towards cannabinoid receptors. In mechanistic studies, the inhibition type was shown to be reversible. Important structural features required for ABHD12 inhibition were revealed based on our SAR studies. Heterocyclic betulin derivatives were shown to suppress the expression of several inflammation mediators, such as iNOS, IL-6, and MCP-1, a pyrazole derivative being the most potent compound. With further improvement and development, more potent betulin derivatives could be found. The betulin derivatives show different activity with different targets, which means they are selective. Selectivity and potency could be further improved to obtain a potential betulin-derived compound for further development. In this work, betulin has been shown to be a good starting point for several different drug discovery projects.
  • Savolainen, Mari (Helsingin yliopisto, 2015)
    Parkinson's disease (PD) is characterized by a slow and gradual loss of neurons and subsequent loss of neurotransmitter dopamine in the movement-related nigrostriatal brain tracts. This causes symptoms of PD, which are resting tremor, rigidity and slowness of movement. Genetic and environmental factors are linked to PD, but its etiology is still largely unknown. Current drug therapies can only relieve the symptoms of PD but treatment to stop or delay the disease progression does not exist at the moment. The main findings in neuropathological characterization of the brain regions that are influenced in PD, are abnormal intraneuronal protein inclusions, called Lewy bodies. Insoluble, aggregated α-synuclein (aSyn) protein is the most abundant component of Lewy bodies. Pathological aggregation and accumulation of aSyn is associated with neuronal death in PD. Therefore, approaches to target aSyn as a potential disease-modifying treatment for PD have been under investigation. The aim of this study was to examine the role of prolyl oligopeptidase (PREP) and pharmacological inhibition of its enzymatic activity by KYP-2047 in aSyn aggregation. Using purified proteins and cell culture model, we showed that PREP forms direct protein-protein interaction with aSyn, thus enhancing its aggregation. KYP-2047 reduced the PREP-mediated aSyn aggregation in cell culture. Then the effects of PREP inhibition on aSyn aggregation were examined in vivo. The purpose was to first characterize a genetic mouse model, carrying mouse A30P mutated aSyn, which is linked to early-onset PD in humans, in order to find out if the mutated mouse aSyn is more prone to aggregate; and to cause PD-like phenotype. The effects of KYP-2047 treatments were assessed in the model. The main findings were that the A30P mutation in mouse aSyn protein caused minor hyperactive behaviour but did not change the brain dopamine levels, and A30P aSyn accumulated in the brain more than wildtype aSyn by age. KYP-2047 treatment reduced the amount of A30P aSyn in immunohistochemical analysis, and the reduction was more specific for high-molecular weight aSyn oligomers in Western blot analysis. We also observed increased autophagy markers in brain tissue. Therefore, PREP inhibition was further studied in cell culture, where it was shown to enhance macroautophagic protein clearance pathway, which is an important pathway in the degradation of high-molecular weight aSyn forms. In the last study, the effects of KYP-2047 were examined in a mouse model of PD that was based on lactacystin-induced inhibition of ubiquitin-proteasome protein degradation pathway. Proteasome inhibition was shown to induce rapid PD-like neurodegeneration recapitulating the cardinal features of PD. KYP-2047 treatment partially protected dopaminergic neurons in the brain and had beneficial effect on motor behaviour, but did not have an effect on aSyn amount. Taken together, this study has provided new insights into the role of PREP in aSyn aggregation and suggest that PREP inhibition has beneficial effects on reducing the aggregation process via two mechanisms. PREP inhibition could be promising and further assessed in the treatment of PD, other α-synucleinopathies and possibly other protein accumulation diseases.
  • del Amo Páez, Eva María (Helsingin yliopisto, 2015)
    Drug discovery and development is a long process: it takes usually 12 to 15 years before a drug candidate reaches the market. The pharmacokinetics of the drug is an important aspect of drug discovery and development, because the drug must reach its target site and exert the therapeutic response. The pharmacokinetic parameters of new compounds should be investigated early in drug discovery. Pharmacokinetic predictions can be made with Quantitative Structure-Property Relationships (QSPR) which are computational models that correlate chemical features with pharmacokinetic properties. The correlations are based on in vivo or in vitro pharmacokinetic data and molecular descriptors. QSPR models can be used to predict the pharmacokinetic parameters even before any actual drug synthesis and can be exploited to guide drug discovery. Pharmacokinetic models can also simulate concentration profiles of drugs during the drug discovery and development process. It was decided to develop QSPR models of pharmacokinetic parameters of drugs to be delivered by the systemic or ocular routes. A combination of Principal Component Analysis and Partial Least Square multivariate statistical methods was used to obtain QSPR equations for volume of drug distribution and fraction of unbound drug in plasma. Parallel modelling of these parameters resulted in acceptable R2 (0.58 - 0.77) and Q2 values (0.55 - 0.58). These models are based on a large set of structurally unrelated compounds, they are open and they have a defined applicability domain. Charge and lipophilicity related descriptors were the relevant ones which influenced the volume of distribution and free fraction of drug in plasma. Pharmacokinetics is an important factor in the development of ocular medications, because the ocular drug targets are difficult to reach, particularly in the posterior tissues such as retina and choroid. Therefore, drugs need to be injected intravitreally in the treatment of retina and choroid diseases (e.g. in exudative age-related macular degeneration) and thus prediction of intravitreal pharmacokinetics would be especially advantageous in ocular drug discovery and development. The first comprehensive collection of intravitreal volume of distribution and clearance values of compounds was collated based on extensive rabbit eye data from the literature. Moreover, predictive QSPR models for intravitreal clearance and half-life were created which had R2 and Q2 values of 0.62 0.84 for clearance and 0.61 - 0.80 for half-life. LogD7.4 and hydrogen bonding capacity defined the intravitreal clearance and half-life of compounds with a molecular weight below 1500 Da. The intravitreal volumes of drug distribution lay within a narrow range (80% within 1.18 - 2.28 ml). The QSPR models for intravitreal clearance and the typical values for intravitreal volumes of distribution were implemented in pharmacokinetic simulation models; the simulated profiles based on the real and predicted pharmacokinetic parameter values were similar. Thus, a combination of QSPR and pharmacokinetic models can be used in drug discovery and development to aid in the design of drugs and drug delivery systems. A comprehensive comparison of intravitreal pharmacokinetic data between rabbit and human was carried out to clarify the translational value of the rabbit model. The analysis revealed that the rabbit can be considered as a clinically predictive animal model for intravitreal pharmacokinetics of small molecules (18 Da - 1500 Da) and macromolecules (7.1 kDa - 149 kDa). There was a correlation between the intravitreal clearance values in human patients and healthy rabbits; they showed similar, but not identical, absolute values. The intravitreal pharmacokinetics of small molecules is mainly governed by permeability-limited clearance across blood-ocular barriers and occurs via the posterior route, whereas large molecules are cleared mostly via the anterior route. Although the literature contains some claims about the significance of the viscosity of the vitreous, it seems that this is not a major factor in drug elimination from the eye. In conclusion, new in silico tools were generated for systemic and ocular pharmacokinetics and drug delivery. These models can be exploited in industrial drug discovery and will hopefully speed up the development of new medications.
  • Ilina, Polina (Helsingin yliopisto, 2015)
    Gene therapy offers promise for the treatment of both inherited and acquired diseases through the introduction of genetic material into target cells. The primary challenge for gene therapy is to develop a safe and efficient method for the delivery of therapeutic genetic material to the specific intracellular target. Non-viral carriers have received significant attention because of their potential to overcome the limitations of viral-based systems. However, their relatively low efficacy is a major obstacle to their clinical application. A thorough understanding of the key factors affecting the gene delivery process will provide clues on how to develop more effective carriers. This thesis focuses on the role of the carrier and the endocytic pathway in non-viral gene delivery, and also suggests improvements in the experimental methodology that would make it possible to obtain more reliable results in nanoparticle uptake studies. More effective carriers for gene delivery are very much needed. We tested the novel pentaspheric lysine-based dendrimer and its analog, modified with fatty acid residues, for their gene delivery capacity. We demonstrated that despite their relatively low in vitro transfection efficacy, lysine dendrimers have good plasmid DNA (pDNA) binding and protective properties, and can therefore be used as the basis for the development of more effective carriers. A detailed understanding of the cellular kinetics of gene delivery systems is critical to the further development of more effective carriers. We studied the impact of the carrier and of the endocytic pathway on cell uptake and the intracellular processing of genetic material (pDNA). The highly sensitive method of quantitative real-time PCR was applied to the study of the intracellular kinetics of pDNA introduced by the cationic polymer PEI, cationic lipid DOTAP, and CaP precipitates at multiple time points after transfection. The results obtained indicate that the carrier affects the cell uptake and the intracellular kinetics, and therefore predetermines the main transfection-limiting step. Furthermore, we demonstrated the important role of the post-nuclear processes in efficient non-viral gene delivery. The impact of a specific endocytic pathway was studied by the inhibition of either the clathrin- or dynamin-mediated endocytosis. Selective blockage of endocytosis was achieved by two approaches, namely the genetically manipulated cell lines and the chemical inhibitors of endocytosis. Analysis of the intracellular kinetics of pDNA in the genetically blocked cells revealed that neither the amount of pDNA taken up by the cell, nor the intracellular pDNA elimination, but the amount of pDNA delivered to the nucleus was indicative of the significance of the particular pathway in the resulting efficacy of the carrier. A comparison of chemical and genetic means for blocking endocytosis revealed the limitations of both these methods. A careful optimization of the method and the use of several alternative approaches is recommended in order to obtain more reliable data. We suggest that the characterization of in vitro cell models for the expression and activity of specific endocytic pathways (endocytic profiling) would facilitate the interpretation of the data obtained in nanoparticle uptake studies. Endothelial and epithelial cells are widely utilized in such studies because they form substantial barriers en route from the administration site to the target tissue. We performed endocytic profiling of the epithelial CaCo-2 cells and the endothelial hCMEC/D3 cells at different stages of differentiation. The expression of genes involved in specific endocytic pathways was analysed at the mRNA level by quantitative real-time PCR, and at the protein level by Western blotting. The endocytic activities of the cells were analyzed by flow cytometry. We concluded that the mRNA expression of the endogenous proteins involved in particular endocytic pathways can be indicative of the expression and activity of these pathways. Furthermore, we showed that the differentiation status of the cells affects their endocytic activity, and must therefore be taken into account when designing nanoparticle uptake and transcellular permeability experiments. A comparison of the endocytic profiles of cell lines with primary cells revealed clear discrepancies, pointing to the importance of careful selection of in vitro models for endocytosis-related studies. Overall, our study has improved the mechanistic understanding of the non-viral gene delivery process. We have described how the carrier and endocytic pathway both affect intracellular kinetics and the efficacy of gene transfer. Furthermore, we have demonstrated the importance of method optimization and the endocytic profiling of in vitro cell models in improving the quality of the obtained data.
  • Keurulainen, Leena (Helsingin yliopisto, 2015)
    The aim of this thesis was to synthesize 1H-benz[d]imidazole- and benzo[c][1,2,5]oxadiazole-derived compounds active against intracellular bacterium Chlamydia pneumoniae and protozoan parasites Leishmania donovani, and Plasmodium falciparum, and to find new potent compounds as hit molecules for further development. A number of issues dictate the importance of the pursuit of this work. First, C. pneumoniae contributes to human health by being a widespread bacterium and causing respiratory infections such as pneumonia. In addition, atherosclerosis has been shown to be connected to the bacterium s persistent form. Second, a neglected tropical disease, visceral leishmaniasis in turn is caused by a protozoan parasite L. donovani and can be fatal if left untreated. Its current treatments suffer from toxicity, poor compliance and prevalent parasite resistance. Third, another tropical disease, malaria is caused by protozoan parasites belonging to the genus Plasmodium. P. falciparum resistance to recent antimalarial drugs is an ever growing and alarming issue, and there is an unmet medical need for new antimalarial chemotypes targeting the different parasite forms present in various stages of the Plasmodium life cycle. Heterocyclic chemical structures are widely used in the early drug discovery process and in compound screening. At the outset of this study, a series of 2-arylbenzimidazole derivatives was designed to target C. pneumoniae and L. donovani. Further development of these 2-arylbenzimidazoles resulted in a set of 2-aminobenzimidazoles against P. falciparum. Benzoxadiazole derivatives were designed against L. donovani. Facile and general synthesis routes for the preparation of both benzimidazole and benzoxadiazole derivatives were developed. In order to study structure-activity relationships of the antichlamydial and antileishmanial 2-arylbenzimidazoles, the left, right and central parts of the core molecular structure were modified and different substitution patterns were employed. Antichlamydial, antileishmanial and antimalarial inhibition activities were related to the different structural modifications carried out. Antichlamydial and antileishmanial 2-arylbenzimidazoles or benzoxadiazole derivatives inhibited target pathogens at the micromolar level. Furthermore, 2-aminobenzimidazoles were studied as antimalarial compounds. The best derivative from this study inhibits growth of P. falciparum (IC50 94 nM) and has a good pharmacokinetic profile. The compound turned out to be efficacious in vivo against P. falciparum upon once a day oral administration. In this study, selectivity of the 2-arylbenzimidazoles against selected intracellular parasites over free living (planktonic) pathogens e.g.Escherichia coli was observed. This is a great advantage from the antimicrobial drug discovery point of view. In spite of the mechanisms of action of the studied derivatives remaining elusive, it was possible to show in this study that antimicrobial compound design can be successful even in the case of unknown macromolecular targets of C. pneumoniae, L. donovani, and P. falciparum.
  • Tuomela, Annika (Helsingin yliopisto, 2015)
    Low oral bioavailability and general delivery problems related to poorly water soluble drugs are major challenges in pharmaceutical formulation development. Nanocrystal technologies have been introduced as advantageous formulation approaches for these molecules. Nanocrystals, with greater surface to volume ratio, can effectively increase both the dissolution rate and saturation solubility of active ingredients. The aim of this thesis was to obtain detailed knowledge about the dissolution characteristics of drug nanocrystals, and to develop feasible nanocrystal applications for ocular and oral drug delivery purposes. Hence, develop-ability issues from the analytical difficulties of the drug dissolution behavior to the scale-ability of the nanocrystals were covered. A new approach in dissolution testing of nanocrystals, UV imaging, was introduced and applied in order to obtain detailed understanding of the real-time dissolution behavior of different nanosized particle fractions. Moreover, both liquid and solid nanocrystal dosage forms were formulated and studied in vitro and in vivo. High-quality nanocrystal suspensions were prepared using the rapid and industrially relevant top-down wet milling technique. Detailed, real-time dissolution analyses of the nanocrystals enabled a close level insight into the occurring phenomena. With regards to the formulation development, an in vivo effective nanocrystal suspension formulation was developed for ocular delivery, to treat elevated intraocular pressure. Furthermore, nanocrystal suspensions were converted into dry powders, by both freeze-drying and granulating technique, and further processed into tablet and capsule formulations. By screening both powder and tablet properties, optimal compositions for nanocrystalline solid formulations were indicated with regards to the higher scale process-ability. The formulations were studied both in vitro and in vivo. As a result, the great difficulty of predicting the in vivo behavior based on the in vitro results and the importance of in vivo studies in the early development phase, were addressed. It was concluded that the methods applied and studied provided valuable knowledge and important tools for the pharmaceutical formulation development in order to solve the current problems related to the delivery of poorly soluble drugs.
  • Kurko, Terhi (Helsingin yliopisto, 2015)
    Nicotine Replacement Therapy (NRT) products are the most commonly used smoking cessation (SC) pharmacotherapy. This study explored the deregulation of NRT products from pharmacy-only distribution to general sales in Finland which took place in 2006. The overall aim of this study was to assess the reasons for the NRT deregulation and its reflections on SC practices in Finland. NRT deregulation was explored from three perspectives: 1) policy-making; 2) community pharmacists as health care professionals providing guidance on NRT use; and 3) NRT users perceptions of NRT in SC. The NRT deregulation from the policy-making perspective was assessed by inductive content analysis of all the publicly available documents and interviews of 12 Members of the Parliament (Study I). A nationwide representative survey to every second pharmacy owner and staff pharmacist (n=2291) was conducted a year after the deregulation in 2006-2007 (Studies II and III). The NRT users perspective was assessed by inductive content analysis of smokers and quitters postings (n=24 481) in five internet discussion areas in the national SC support Forum, STUMPPI, in 2007-2012 (Study IV). The NRT deregulation was politically communicated as a safe and evidence-informed decision promoting public health by advancing SC in Finland (I). However, two of the most important motives for the deregulation, poor NRT availability and the effectiveness of sole NRT use in SC, were largely based on assumptions instead of scientific evidence. At the time of the pharmacists survey, nearly half of the respondents reported being familiar with the Finnish SC Guideline. The familiarity with the Guideline was directly reflected in the level SC actions taken by pharmacist (II). Due to the NRT deregulation, in particular pharmacy owners , motivation towards counselling NRT customers decreased (III). The analysis of smokers and quitters internet-based discussions (IV) revealed that many Finnish smokers and quitters saw NRT as less important in SC or held negative perceptions towards NRT use. From smokers and quitters perspective, the most highlighted factors of successful quitting were quitters own psychological empowerment and peer support from the discussion community. This study found a great variety of NRT usage patterns. Of these, the most commonly emerged one was the use of lower dosage or shorter period compared with the instructions on NRT use. The findings of this study suggest that the sole increased NRT use may not be so crucial for SC as suggested during the political decision-making process. This study provided also evidence that in Finland NRT products are used in a way which may not be optimal for treatment success. Therefore many smokers and quitters could benefit from personalized support for NRT use which would include behavioural components and a plan for NRT use to optimize the treatment. In future it is important to guarantee that community pharmacist are not lost as an important public health resource to provide such individualized NRT counselling and managing personalized SC plans. In addition, there is a need to develop more advanced SC services fitting to the needs of individual quitters. These services could utilize internet-based services and peer support. The findings of this study highlight the need to further evaluate the public health benefits gained from the deregulation.
  • Nybond, Susanna (Helsingin yliopisto, 2015)
    Due to the emergence of multidrug resistant bacteria, bacterial infections are still a major healthcare problem. Many factors have led to a discovery void of new antibacterial agents, rendering the current antibiotic pipeline inadequate for future medical needs. For example, the outcomes from pure biochemical high-throughput screening (HTS) assays have, in many cases, not led to successful clinical compounds. Therefore cell-based assays might be a better choice for primary screening. However, the antibacterial cell-based assays in the current use often require long incubation times and they are not always amenable for miniaturization and automation for HTS. In this work, two screening assays based on recombinant bioluminescent E. coli strains were optimized and implemented in the screening of chemical libraries and natural products in antibacterial drug discovery. One of the recombinant bacterial strains was a strain which is sensitive towards transcriptional and translational inhibitors. The assay based on this strain was successfully miniaturized into 384-format using automatized liquid handling and was validated with a proof-of-concept library containing known drugs. This provided a means to perform a larger scale high throughput screen of a compound library. Based on the HTS hit structures, a ligand-based in silico screening of a virtual chemical library was employed for hit enrichment. The most active hits and the in silico selected compounds were further investigated in more detail. Natural products have been an important source in drug discovery, especially in the discovery of antibiotics in the current use. However, matrix effects such as colour or turbidity of natural product extracts can potentially cause interference in conventional absorbance based microbial growth inhibition assays. Also, conventional antibacterial assays are usually not sensitive enough for detecting very small concentrations in fractionated natural product extracts. The feasibility of bioreporter -based assays in antimicrobial screening of natural products was demonstrated by screening an in-house natural product library. One of the assays was also implemented for investigating the antibacterial properties of an extract from a fungal culture filtrate, which demonstrated the sensitivity of the assay for identification of active components from fractionated samples. In conclusion, sensitive and reproducible bioassays amenable for further miniaturization and automation were developed for antibacterial drug discovery. Compared to conventional antimicrobial testing, the bioreporter-based methods offer important improvements such as simultaneous data acquirement on antimicrobial activity, first indication of mode of action and significant reduction of assay time to 2-4 h compared to 24 h in standard susceptibility assays. The developed bioluminescent assays led to the improvement of compound throughput in antimicrobial screening: from hundreds of samples (natural product extracts and fractions) in manually performed assays in 96-well plates, to thousands of test compounds (synthetic compound libraries) in 384-well format using automated liquid handling.
  • Wissel, Gloria (Helsingin yliopisto, 2015)
    The main goal of this dissertation is to identify novel modulators acting on ATP Binding Cassette subfamily C member 2 (ABCC2) transporters and α2-adrenoceptors subtypes. With the purpose of identifying novel modulators and their mode of action, a combination of experimental and computational approaches have been used. The first protein presented in this dissertation is the ABCC2 transporter, also known as the multidrug resistance associated protein 2 (MRP2), an efflux transporter expressed in polarized cells where it effluxes a variety of both endogenous and exogenous molecules out of the cell. The most common way to study the interactions between small molecules and ABCC2 transporter is by a vesicle transport assay. Three assays are commercially available, which use different probes to define the ABCC2- transport. With the intent to define the different assays and identify the effect that small molecules have on the ABCC2-transport, a small set of eight compounds and, subsequently a larger library of compounds were tested with the different assays. Additionally, the aim was to identify and characterise novel ABCC2 inhibitors, 16 inhibitors have been identified from the larger library and classification models were built to identify important descriptors that were able to discriminate inhibitors from inactive molecules. Instant structure-activity relationships (SAR) of four scaffolds of ABCC2 modulators are also presented. In addition, some unpublished results are presented, the homology model of ABCC2 and further insights into the SAR of ABCC2 modulators. The other proteins included in this dissertation are the three subtypes of the α2-adrenoceptors, G-protein coupled receptors, involved in the signalling pathway of adrenaline and noradrenaline. A clear subtype characterization/profile of these proteins is not available. Selective molecules could be used in treatment of high blood pressure, in the alleviation of withdrawal symptoms, and as anaesthetic with fewer side effects than the current drugs. To define the affinity of a small set of antagonists and outline the involvement of the first transmembrane helix in ligand binding, a competition binding assay has been used with chimera receptors where the first transmembrane helix has been swapped between the three subtypes. Molecular modelling has been used to explain the different binding affinities to the chimera receptors. Additionally, the aim was to identify novel α2B-adrenoceptor selective compounds, thus a mid-sized library has been screened using a miniaturized binding assay. Hierarchical classification and chemoinformatics analysis has been used to visualize and analyse the screening results.
  • Akhgari, Amir (VTT Technical Research Centre of Finland Ltd, 2015)
    Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in the Middle East and South Asia. It produces a large number of terpenoid indole alkaloids(TIAs), some of which possess important pharmacological properties. This study focused on the establishment of biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities it was necessary first to develop different analytical methods to determine the alkaloid contents and changes in their profiles in transgenic cultures and after various treatments. Targeted and non-targeted analyses from cell andorgan cultures were carried out using gas chromatography-mass spectrometry(GC-MS), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Callus cultures were successfully initiated from five different explants onmodified B5 medium containing phytohormones. The phenotypes of the calli varied, but as was expected the callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems had elevated levels of strictosidine lactam compared to other cultures. Transformation experiments revealed that only leaves but not cotyledons, hypocotyls or stem segments were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. Wild type and gus hairy root clones contained twofold higher amounts of alkaloids than adventitious roots. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS analysis allowed the separation of the most volatile and non-polar alkaloids in a single run. The composition of typical non-polar alkaloids indicated the occurrence of 20 TIAs belonging to nine different groups. The quantities of these alkaloids varied between clones in the order eburenine, vincanine, vallesiachotamine and yohimbine isomer II. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GC-MS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Methyl jasmonate, a well-known elicitor, caused a significant increase in the total alkaloid content of wild type hairy roots as determined by NMR analyses. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased compared to non-elicited cultures. Another studied elicitor, chitosan, did not have any effect on individual alkaloid contents. Transgenic hairy root clones did not exhibit phenotype differences. Multivariate analysis from NMR data showed a clear discrimination between transformed and wild type/gus cultures. This was most probably due to differences in primary metabolites, as the total alkaloid content did not vary between different hairy roots and controls. In general, the production of individual TIAs, analysed by HPLC, was repressed in hairy roots transformed with geraniol synthase(ges) and geraniol 8-oxidase (g8o) genes compared to the wild types. Overexpression of the strictosidine synthase (str) gene resulted in a higher accumulation of serpentine, whereas the production of strictosidine lactam was decreased. There were no significant differences in the contents of other alkaloids compared to the wild type hairy roots. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabled comprehensive investigation of the alkaloids. These data might serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.
  • Aaltonen, Kalle (Helsingin yliopisto, 2015)
    Rheumatoid Arthritis (RA) is an autoimmune disease, which is treated with anti-inflammatory and immunosuppressive medication comprising synthetic disease-modifying anti-rheumatic (sDMARDs) and biologic drugs. In this thesis all published randomized controlled trials studying the efficacy and safety of biologic drugs based on the inhibition of tumor necrosis factor (TNF) were identified, evaluated and pooled in using a systematic review including a meta-analysis. Then we pursued a cross-sectional overview on disease activity and medical treatment of patients with RA treated wthin the Finnish specialized healthcare. Finally, we executed two cohort studies in which we combined longitudinal patient data with information on the incidence of serious infections, malignancies and joint replacement operations retrieved from national registers. Forty-one articles reporting on 26 RCTs of TNF-inhibitors were included in the systematic review and meta-analysis. TNF-inhibitors as a monotherapy were more efficacious than placebo but comparable to methotrexate (MTX). TNF-inhibitor and MTX combination was superior to either MTX or TNF-inhibitor alone. TNF-inhibitors were relatively safe as compared to either MTX or placebo. The cross-sectional study revealed 91%, 58% and 21% of patients as concurrent users of (sDMARDs), glucocorticoids and biologics, respectively. The cohort studies showed that the adjusted incidence rate ratios (aIRRs) of infections compared to sDMARD users were 0.9 (95% CI 0.6-1.4) and 1.1 (95% CI 0.59-1.9) for the users of TNF-inhibitors and rituximab, respectively. The aIRRs of malignancies were similar between the sDMARD and biologics users. There were more primary joint replacement operations per 100 patient years among the users of biologic drugs (3.89, 95% CI 3.41 4.41) vs. DMARD (2.63, 2.35 2.94) users but slightly fewer revisions (0.65, 0.46 0.88 vs. 0.83, 0.68 1.01). Efficacy and safety of TNF-inhibitors are comparable to MTX and only few differences were observed between individual agents. Currently, more than 20% of Finnish RA patients are using biologic drugs, with a majority of them in combination therapy with sDMARDs. The incidence of serious infections and malignancies is comparable between the users of sDMARDs, TNF-inhibitors and rituximab. Compared to sDMARD users, biologic drugs users had a higher incidence of joint replacement operations while the durability of the prostheses were similar.
  • Suominen, Tina (Helsingin yliopisto, 2015)
    Neurotransmitters and neurosteroids are compounds that regulate the functions of the brain. The neurotransmitters dopamine (DA) and serotonin (5-HT) play a role in several psychological conditions, including schizophrenia, depression and anxiety. DA also has an important role in Parkinson s disease. Neurosteroids are involved in neurodegenerative diseases. In Alzheimer s disease and multiple sclerosis, the levels of neurosteroids are decreased in certain areas of the brain. Neurosteroids differ from classical neurotransmitters in that they are lipid-soluble and can easily cross the blood-brain barrier (BBB). Neurotransmission can be studied in vivo by microdialysis, and as the concentrations of neurotransmitters in the microdialysates are very low, sensitive analytical methods are needed for their analysis. In this work an UPLC-MS/MS method was developed for the determination of 5-HT, DA, their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfonate and glucuronide conjugates. The method was validated and applied for analyzing human brain microdialysis and cerebrospinal fluid (CSF) samples. Intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain. The origin of the determined phase II metabolites in the brain is unknown. Even though sulfonate-conjugated compounds such as dopamine sulfonate (DA-S) and 5-HIAA-S were detected in the human brain, it is unclear whether they were locally formed or transported into the brain through the BBB from peripheral sources. The BBB permeation of DA-S was studied by administration of isotope (13C6)-labelled DA-S, which can be distinguished from endogenous DA-S by mass spectrometry, subcutaneously (s.c.) while brain microdialysis samples were collected and analyzed by UPLC-MS/MS. The fate of 13DA-S in brain was followed by monitoring 13C6-labelled DA-S metabolites and hydrolysis products. The results proved that DA-S permeates through the BBB, and indicated that DA-S finally either permeates through the BBB back to the peripheral circulation or is dissociated or metabolized by unknown mechanisms. While the hydrophilic neurotransmitters DA and 5-HT are well suited for analysis by liquid chromatography coupled to atmospheric pressure ionization, the neurosteroids have more commonly been analyzed by methods based on gas chromatography (GC) coupled to ionization in vacuum. Recently GC has been combined to atmospheric pressure photoionization utilizing heated nebulizer microchips (μAPPI). We now constructed a simpler interface for combining GC to mass spectrometry (MS) using dopant-assisted atmospheric pressure photoionization (APPI), utilizing commercially available hardware. The neurosteroids were analyzed as trimethylsilyl (TMS) derivatives, and the effect of different dopants (chlorobenzene, toluene and anisole) on the ionization and on the sensitivity of the method was investigated. Chlorobenzene was chosen as the best dopant, as the neurosteroid-TMS derivatives formed intense molecular ions with minimal fragmentation, while with toluene and anisole also protonated molecules were observed. The molecular ions of the steroids formed by APPI ionization showed fragmentation patterns in their MS/MS spectra similar to the patterns seen in corresponding spectra obtained by electron impact ionization (EI). Therefore the use of EI libraries could be possible, thus enabling the identification of a wide range of unknown compounds.
  • Soppela, Ira (Helsingin yliopisto, 2015)
    The manufacturing of the most common pharmaceutical dosage forms, tablets, requires good mass flowability and uniform particle size distribution. Granulation is often needed to improve these properties prior to tablet compression. Thus, rapid methods for analysing the key powder and granule properties, such as particle size, flowability and moisture content are needed. Until recently, the development and control of pharmaceutical unit operations was based on an empirical approach rather than process understanding. To be able to build quality into the products, improved understanding of materials and processing is needed. This can be reached by developing complementary automated analytical methods that are suitable for continuous on-line or in-line process monitoring. The aim of this thesis was to investigate whether modern analytical tools can provide rapid and reliable real-time insight into powder performance during solid dosage form processing. The first study evaluated the impact of paracetamol loading and the physical characteristics of powders on the flowability of microcrystalline cellulose and paracetamol mixtures. A novel small-scale flow device proved to be suitable for rapid flowability screening of different formulations. Particle size distribution and drug loading had the largest impact on the flowability. The main focus of this thesis was on the utilisation of image analysis, near infrared (NIR) spectrocopy and process measurements as complementary process analytical tools during granulation. In addition to particle size distribution, the images revealed batch specific granule growth and attrition behaviour in real time. The changes in granule size were clearly linked to the continuously measured process conditions. Moreover, changes in image brightness during drying reflected the removal of water from the granules. The continuous moisture measurements based on process air moisture content and NIR spectroscopy provided real time information on the moisture content as well as the batch moisture profile during processing. The comparison of the methods also enabled the evaluation of the location of water in the process. The combination of on-line photometric imaging and near-infrared spectroscopy with continuous in-line process measurements facilitated continuous evaluation of key product properties during fluid bed granulation and provided insight into batch performance. The powder characterisation and process analytical technology (PAT) tools applied in this work enabled rapid and non-destructive determination of key powder and granule quality attributes. Even small changes in the material properties during processing were detected using the continuous and complementary process analytical measurements.
  • Nordman, Nina (Helsingin yliopisto, 2015)
    Analytical microsystems are attractive in modern bioanalysis where sample amounts often are low and fast analyses are required. Microsystems also provide the prospect of integrating several functional elements on a single platform. The aim of this work was to develop analytical microsystems for fast analysis of bio- and drug molecules. For this, microchips with separation- and injection channels and monolithically integrated electrospray ionization (ESI) emitter were fabricated of epoxy photoresist SU-8 by photolithography and adhesive bonding. For peptide mass fingerprinting and protein sequencing characteristic tryptic peptides were fast and easily separated and detected by microchip capillary electrophoresis (MCE)-ESI/mass spectrometry (MS). Additionally, protein identification based on tandem MS fragmentation data of a single tryptic peptide was achieved. Finally, this rapid (total analysis time below ten minutes) microchip method permitted analysis of human muscle cell lysates. For online coupling of microchip capillary isoelectric focusing (cIEF) to ESI/MS a bilateral sheath flow interface or a two-dimensional separation unit was integrated on-chip. Rapid focusing of peptides by their isoelectric points (pI) was achieved without pretreatment of the SU-8 surface. After focusing the peptides were electrokinetically mobilized toward ESI/MS. The two-dimensional chip design enabled unique separation selectivity for peptides based on both pI values and intrinsic electrophoretic mobilities by multiplex-cIEF-transient-isotachophoresis. Rapid metabolic profiling was demonstrated from urine after intake of tramadol or paracetamol. Both phase I- and II metabolites were separated and detected by MCE-ESI/MS within 35 s. In addition, Michaelis-Menten kinetics was successfully determined for the CYP450-mediated oxidation of bufuralol to 1-hydroxybufuralol. Sample preconcentration (pretreatment) was integrated on-chip by solid-phase extraction (SPE) and liquid-phase microextraction (LPME). For SPE, a monolith zone was firmly anchored at the injection cross of the MCE-ESI/MS microchip by laser induced photopolymerization. The monolith was selective toward hydrophobic and hydrophilic sample molecules and enrichment factors as high as 23-fold was achieved with a loading time as short as 25 s. In addition, LPME was easily downscaled to low volume applications and offered selectivity in the analysis of phase I metabolites compared to SPE. In contrast to previous research in the same field this work offers bioanalysis with several on-chip integrated steps (preconcentration, injection, separation, and analysis) without considerably increasing the short analysis times characteristic of microchip assays.
  • Wang, Chang-Fang (Helsingin yliopisto, 2015)
    Anticancer drugs inhibit the cancer growth by killing the rapidly dividing cancer cells. However, anticancer drugs also kill the dividing healthy cells and cause severe damage to healthy tissues. More specific delivery of the cancer drugs to the cancer tissue can increase the drug delivery efficiency and reduce the drug s side effects. Nanocarriers can increase the solubility of poorly-water soluble anticancer drugs and be modified for targeted drug delivery and theranostic applications. For efficient drug delivery, the drug loading capacity has been one of the key issues for the development of nanoparticle (NP)-based drug delivery systems. The biocompatible and biodegradable porous silicon (PSi) nanomaterial presents high drug loading capacity and tunable surface chemistry which renders it an ideal candidate as a drug delivery carrier. Chemical surface modification, which is one of the approaches to improve the nanomaterials properties, can lead to a stable nanosystem for further drug delivery applications. The main aim of this dissertation was to employ chemical approaches and surface modified PSi nanoparticles (NPs) to improve the drug delivery efficiency for potential cancer therapy applications. Incorporating targeting moieties to the surfaces of the nanocarriers, such as targeting peptides, can increase the nanocarrier s accumulation into the cancer tissue after the intravenous administration. In this thesis, surface modification of amine-terminated PSi NPs was achieved with targeting peptides (RGDS and iRGD) via strain-promoted azide-alkyne cycloaddition click reaction. The functionalization of the PSi NPs with the targeting peptides did not comprise the drug loading capacity, but enhanced the cellular uptake and the drug delivery efficacy of the PSi NPs in vitro. In addition to the targeting NP surface modifications, a multifunctional nanosystem was prepared with simultaneous fluorescence- and radio-labeling, and iRGD surface modification of the carboxylic acid-terminated PSi NPs. Both labelings were accessible for the in vivo biodistribution evaluation in mice by single-photon emission computed tomography and X-ray computed tomography, and ex vivo by immunofluorescence staining, respectively. The iRGD modification enhanced the tumor uptake of the PSi NPs after the intravenous administration. In order to reduce the plasma protein adsorption onto the PSi NPs, five bioactive molecules (peptides and hydrophilic anti-fouling polymers) were used to modify the surface of alkyne-terminated PSi NPs using copper-catalized click chemistry. Dextran 40 kDa modified PSi NPs presented enhanced cellular uptake and the least protein adsorption of all the tested NPs. Furthermore, the chemical conjugation of drug molecules was studied. The targeting peptides were successfully conjugated to antisense interleukin-6 via copper-catalyzed [3+2] azide-alkyne cycloaddition for targeted angiogenic anti-inflammation in cancer. Finally, anticancer drug methotrexate (MTX) was chemically conjugated to the cationic PSi NPs and demonstrated to increase the cellular uptake of MTX with up to 96 h sustained drug release. A hydrophobic anti-angiogenic drug, sorafenib, was also loaded to the MTX-conjugated PSi NPs, and the dissolution rate of this drug was considerably increased. In conclusion, in this thesis different chemical approaches were used to biofunctionalize PSi NPs and to prepare drug-conjugates formulations for potential anti-cancer applications.