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  • 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.
  • 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.
  • 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.
  • 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.
  • Shahbazi, Mohammad-Ali (2015)
    Porous silicon nanoparticles (PSi NPs) have recently drawn increasing interest for therapeutic applications due to their easily modifiable surface, large pore volumes, high surface area, nontoxic nature, and high biocompatibility. Nevertheless, there is no comprehensive understanding about the role of the surface chemistry of these NPs on the biological interactions and the therapeutic effect of the PSi-based nanosystems. Therefore, extensive attempts are still needed for the development of optimal PSi-based therapeutics. The first step for evaluating the biological activity of the NPs was to investigate the potential toxic effects. Accordingly, the immunotoxicity and hemocompatibility of the PSi NPs with different surface chemistries were assessed at different concentrations on the immune cells and red blood cells, since these are the first biological cells in contact with the NPs after intravenous injection. PSi NPs with positively charged amine functional groups showed higher toxicity compared to negatively charged particles. The toxicity of the negatively charged particles was also highly dependent on the hydrophobic nature of the NPs. Moreover, RBC hemolysis and imaging assay revealed a significant correlation between the PSi NP surface chemistry and hemotoxicity. To further understand the impact of the surface chemistry on the immunological effects of the PSi NPs, the immunostimulatory responses induced by a non-toxic concentration of the PSi NPs were evaluated by measuring the maturation of dendritic cells, T cell proliferation and cytokine secretion. Overall, the results suggested that all the PSi NPs containing higher amounts of nitrogen or oxygen on the outermost surface layer have lower immunostimulatory effects than the PSi NPs with higher amounts of C‒H structures on the NPs surface. Combination cancer therapy by the PSi NPs was then studied by evaluating the synergistic therapeutic effects of the nanosystems. Sorafenib-loaded PSi NPs were biofunctionalized with anti-CD326 monoclonal antibody on their surface. The targeted PSi NPs showed a sustained drug release and increased interactions with the breast cancer cells expressing the CD326 antigen on their surface. These NPs also showed higher antiproliferation effect on the CD326 positive cancer cells compared to the pure drug and sorafenib-loaded PSi NPs, suggesting CD326 as an appropriate receptor for the antibody-mediated drug delivery. In addition, anti-CD326 antibody acted as an immunotherapeutic agent by inducing antibody-dependent cellular cytotoxicity and enhancing the interactions of immune cells with cancer cells for the subsequent phagocytosis and cytokine secretion. Next, the development of a stable PSi NP with low toxicity, high cellular internalization, efficient endosomal escape, and optimal drug release profile was tested by using a layer-by-layer method to covalently conjugate polyethyleneimine and poly(methyl vinyl ether-co-maleic acid) copolymers on the surface of the PSi NPs, forming a zwitterionic nanocomposite. The surface smoothness and hydrophilicity of the polymer functionalized NPs improved considerably the colloidal and plasma stability of the NPs. Moreover, the double layer conjugation sustained the drug release from the PSi NPs and improved the cytotoxicity profile of the drug-loaded PSi NPs. In conclusion, this work showed that the surface modification of the PSi NPs with different chemical groups, antibodies and polymers can affect the toxicological profiles, the cellular interactions and the therapeutic effects of the NPs by modifying the charge, stability, hydrophilicity, the drug release kinetics and targeting properties of the PSi NPs.
  • Palomäki, Jaana (Helsingin yliopisto, 2014)
    Engineered nanomaterials (ENM) are widely used in the industry as their unique characteristics improve competitiveness and potential for innovations in the different sectors of industry: It has been estimated that the market size of nanotechnological innovations will exceed 2 trillion euros in 2015. However, the toxicological hazard and risk assessment of different nanomaterials is far from complete. There is a concern that nanomaterials cause toxic outcomes, and more research efforts are put into hazard assessment of nanomaterials. However, the huge amount of differently modified nanomaterials poses a challenge for toxicologists who will have to develop new, quicker and cheaper, approaches for hazard and risk assessment to guarantee the human health. The human body is protected from foreign material by its immune system. The most common routes for foreign materials to enter the human body are via inhalation, digestion or dermal penetration, and these routes are protected by physical barrier. The physical barrier accompanied with phagocytosing cells are part of the innate immunity: Rapid and non-specific immune system. Phagocytosing cells scavenge foreign material in the body cavities and secrete signaling molecules to further activate other cells of immunity, such as T or B cells, to form a specific immune response. In this thesis, an immunotoxicological approach was used to compare effects of different types of ENM in the phagocytosing cells or in the disease model of atopic dermatitis. Studies performed with metal oxide nanoparticles and carbon nanotubes (CNT) showed that surface treatment or shape of nanomaterial modify their toxic properties in vitro. Mechanistical studies performed with CNT clearly demonstrated that long, rigid carbon nanotubes resemble asbestos fibers in their toxicity whereas other types of CNT showed less toxicity. In addition, the effects of either bulk-sized or nano-sized ZnO particles commonly used in cosmetics were compared in the mouse atopic dermatitis disease model. The results clearly showed that ZnO particles were able to suppress local inflammation, however, there was also an induction of systemic antibody levels. These outcomes were clearly higher if the mice skin had been treated with nano-ZnO shown also to penetrate into the deeper layers on mouse allergic skin. The results suggest that material size, shape and surface modifications are important parameters in their toxicity. Therefore the risk assessment of differently modified nanomaterials shall be performed separately despite the previously performed risk assessment of the bulk-sized form of the same material. These results also underline factors that should be taken into account in the planning of the future studies.
  • Ahonen, Linda (Helsingin yliopisto, 2014)
    Steroids, vitamin D and oxysterols are all part of a group of compounds called lipids, which are generally important in the function of the human body and in many cases the monitoring of these analytes can be of high value. Therefore, the development of a sensitive and universal method, suitable for the simultaneous analysis of these compounds, is of a great importance. In this study, the goal was to develop and test new mass spectrometry based methods for analyzing steroids, vitamin D and oxysterols. First, the feasibility of capillary liquid chromatography-microchip atmospheric pressure photoionization-tandem mass spectrometry was examined for the analysis of anabolic steroids in human urine. The results show that this microchip based method can be feasible for analyzing non-polar and neutral compounds in biological samples. Next, a microfluidic-based liquid chromatography-electrospray ionization-mass spectrometric system (HPLC-Chip/ESI/MS) was compared to a conventional liquid chromatography-electrospray/mass spectrometric system (LC-ESI/MS) for the analysis of steroids. The sensitivity of the HPLC-Chip/MS system was ~50 times higher compared to the conventional LC-MS when injected amounts are compared. The feasibility of the HPLC-Chip/MS system in the analyses of non-derivatized steroids was also tested and the developed method was used in the analysis of mouse plasma samples. An ion mobility mass spectrometry method using a compact traveling wave cell (TWIM-MS) was also developed for the separation of steroid isomers. Three steroid isomer pairs were analyzed in their native form and as their p-toluenesulfonyl isocyanate derivatives. The results show, that the developed TWIM-MS method provides a reliable, fast and repeatable method for separating derivatized steroid isomers. Finally, an ultra-high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometric (UHPLC-APPI-MS/MS) method was developed for the simultaneous analysis of oxysterols and vitamin D related compounds in mouse brain and cell line samples. An UHPLC-APPI-high resolution mass spectrometric method was developed for confirmatory analysis and for the identification of non-targeted oxysterols. Several oxysterols were quantified in the mouse brain and cell line samples. Additionally, 25-hydroxyvitamin D3 was detected in mouse brain samples for the first time. In conclusion, the methods developed in this work are sensitive and selective new methods for the analysis of the selected lipids from biological samples. The methods provide new insights in the analysis of steroids, vitamin D related compounds and oxysterols, and with some additional development some of the presented methods could be implemented e.g. in routine laboratories.
  • Kontturi, Leena-Stiina (Helsingin yliopisto, 2014)
    Cell therapy is defined as cell transplantation into the patient to treat a certain disease state. Therapies utilizing cells can be divided into two main categories, (1) tissue regeneration or engineering and (2) drug delivery. In tissue engineering, the transplanted cells are used to regenerate the functions of a diseased tissue. In drug delivery, the transplanted cells are used as biological factories that produce therapeutic molecules inside the body. For successful cell therapy applications, cells usually must be combined with biomaterials and bioactive factors to mimic the growth environment in vivo. The properties of these scaffolds are important for outcomes of the treatments, because the local environment determines the functionality of the cells. Thus, research on cell-biomaterial interactions is essential for the progress of cell based therapies. Hydrogels are promising cell therapy materials, because their structure resembles the natural tissue environment; they consist of long polymer chains with high water content and elastic properties, thereby enabling cellular functionality. The aim of this study was to investigate hydrogels for cell therapy applications. Firstly, we encapsulated human retinal pigment epithelial cell line (ARPE-19) genetically engineered to secrete an anti-angiogenic protein (1) into alginate-poly-L-lysine-alginate (APA) microcapsules and (2) into a composite hydrogel of cross-linked collagen and interpenetrating hyaluronic acid (HA). A custom-made cell encapsulation device was designed, built and optimized, and pharmacokinetic/pharmacodynamic (PK/PD) model was developed to investigate the intravitreal drug delivery of the anti-angiogenic protein by the encapsulated cells. Secondly, chondrocytes were encapsulated into the cross-linked collagen/HA hydrogel supplemented with transforming growth factor β1 (TGFβ1). Using the cell encapsulation device, cell microcapsules of symmetrical shape and narrow size distribution were produced. The encapsulated ARPE-19 cells remained viable and functional for at least five months. The cross-linked collagen-HA hydrogel was shown to be a suitable encapsulation matrix for ARPE-19 cells; the cells maintained viability and secreted the anti-angiogenic protein at a constant rate for at least 50 days. Moreover, the hydrogel composition could be modified to adjust the properties of the gel structure without compromising cell viability. This approach is suggested to have potential in the treatment of retinal neovascularization. The developed PK/PD model could be used to predict drug levels and therapeutic responses after intravitreal anti-angiogenic drug delivery. The simulations may augment the design of in vivo experiments. The collagen/HA matrix with TGFβ1 was suitable for chondrocyte encapsulation. The hydrogel supported viability and phenotypic cell stability. This hydrogel is strong, stable and biodegradable, and it can be delivered non-invasively as injection. Overall, it is potentially a useful delivery vehicle of chondrocytes for cartilage tissue engineering. In conclusion, ARPE-19 cells maintain viability in different hydrogels for prolonged periods and secrete the therapeutic transgene product constantly, supporting the suitability of ARPE-19 cells for cell therapy. The cross-linked collagen/HA hydrogel appears to be a potential matrix for cell therapy. It is an injectable system that supports functionality of cells, and it is applicable in drug delivery and tissue engineering.
  • Malinen, Melina (Helsingin yliopisto, 2014)
    New organotypic liver cell cultures are needed to predict the metabolism, excretion, and safety of chemical compounds. Liver cell models are particularly important since the liver largely regulates the ultimate fate of compounds in the body. Approximately 70% of the drugs administered to the body are metabolized or excreted by the liver. Animal models, cell cultures, and cell-free assays are the most common liver models. However, animal models and animal cells do not represent humans due to the interspecies differences in drug metabolizing enzymes and transporters. Instead, the most common cell-free methods, microsomes, are appropriate for drug metabolism studies, but the lack of drug transporters and transcription machinery prevents the complete evaluation of compounds. Primary human hepatocytes are capable of both drug metabolism and drug transport, and are, therefore, considered the gold standard to assess metabolism and toxicity of compounds in vitro. Primary hepatocytes, however, suffer limited availability, high functional variability, and difficulty with maintaining differentiated phenotypes and functions in cell cultures. Therefore, continuous human liver cell lines, such as HepG2 and HepaRG, have been widely used to evaluate drugs and chemicals even though they have defects in their biotransformation functions. The advantages of cell lines are their good availability, easy maintenance, and inducible drug metabolism. Generally, these cells are cultured in a two-dimensional (2D) manner that deviates from the physiological morphology and functions of the hepatocytes. The flattened 2D phenotype leads to reduced polarization and loss of important signaling pathways; this is likely to be a major reason for the failure in the prediction of drug metabolism, pharmacokinetics, and hepatotoxicity. It is believed that for more predictive in vitro models, the liver cells should be maintained in a three-dimensional (3D) microenvironment that allows reconstruction of polarization, and cell-cell and cell-extracellular matrix (ECM) contacts. The 3D cell cultures have been generated by different methods, such as cultures in matrices, scaffolds, bioreactors, and microfluidic platforms. Biomaterial hydrogels have demonstrated great potential for 2D liver cell culturing, but their potential to generate functional 3D liver cell cultures is largely unknown. The main goal of this thesis was to establish improved 3D liver cell cultures with biomaterial hydrogels. Particular attention was focused on the effects of 3D hydrogels on drug metabolism and excretion, cytoarchitecture, and cellular differentiation of HepG2 and HepaRG cell lines. As a starting point, we studied the suitability of wood-derived nanofibrillar cellulose (NFC) hydrogel as a cell culture matrix. NFC hydrogel has not been studied in cell culture before; however, as a novel, defined, animal-free, and abundantly available material, it evoked interest for testing. Herein, the wood-derived NFC was proven to own rheological and structural characters that allow 3D cell culture. Moreover, the NFC was compatible with the HepG2 and HepaRG cells, allowing for the formation of 3D multicellular aggregates with increased apicobasal polarity. When compared to commercial hydrogels, the NFC supported the albumin secretion, an indicator of hepatocellular synthetic function, from HepG2 and HepaRG cells as well or even better. These results demonstrate the potential of wood- derived NFC to function as an ECM analogue, and present the first HepaRG aggregate cultures. Next, the effect of the RAD16-I peptide hydrogel on the HepG2 cell line was investigated in more detail. Immunofluorescence staining and vectorial transport showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of canalicular efflux transporters, multidrug resistance-associated protein 2 (MRP2), and multidrug resistance protein 1 (MDR1), in the spherical HepG2 cell aggregates. The study clearly demonstrated that the peptide hydrogel increases the apicobasal polarity and appearance of bile canaliculi structures in HepG2 cell cultures. The plasticity of HepaRG liver cells was exploited to investigate the impact of 3D NFC and hyaluronan-gelatin (HG) hydrogel cultures on the phenotype of both undifferentiated HepaRG cells (early liver progenitors) and differentiated HepaRG cells (hepatocyte-like cells together with cholangiocyte-like cells). Based on the expression and activity of hepatic markers, drug metabolizing enzymes, and drug transporters, the 3D NFC and HG hydrogels promoted the differentiation of HepaRG liver progenitor cells when compared to the standard 2D technique. Instead, the 3D hydrogel cultures could not really improve the properties of differentiated HepaRG cells. In conclusion, these findings reveal the capability of the NFC, RAD16-I peptide, and HG hydrogels to improve the properties of HepG2 and HepaRG human liver cells. The new spheroid cultures of HepG2 and HepaRG cells may represent added value for pharmacokinetic and toxicity predictions, showing a liver-like cytoarchitecture and demonstrating applicability for drug metabolism and transport studies. Overall, the results deepen our knowledge of the 3D liver cell cultures.
  • Liu, Dongfei (Helsingin yliopisto, 2014)
    The co-loading of different therapeutic molecules into a single carrier offers several advantages over individual administration. The aim of this dissertation was to develop a robust platform that would enable precise control over the loading and release of different drug cargos to facilitate combination therapy. Benefiting from numerous attractive features, porous silicon (PSi) materials have emerged as a promising drug delivery system. However, uncontrolled drug release has always been observed on PSi, which can be ascribed to its freely accessible pores. To seal the pores of the PSi materials and to sustain drug release, the drug-loaded PSi particles were embedded into solid lipid nano- and micromatrices. The PSi-encapsulated solid lipid nanocomposites prepared by the traditional emulsion method exhibit superior stability in aqueous solutions and, most importantly, prolonged drug release. Similarly, the microfluidic templated monodisperse PSi solid lipid microcomposites remarkably sustained the release of either hydrophilic or hydrophobic drugs individually. To achieve the stimuli-responsive drug delivery, the drug-loaded PSi particles were assembled into pH-responsive polymeric micro- and nanocomposites. Taking advantage of the high loading degrees of PSi materials for both hydrophilic and hydrophobic therapeutics, multiple drugs with varied physicochemical properties were successfully loaded with ratiometric control. The microfluidic assembled micro- and nanocomposites both exhibited pH-responsive properties and tailored drug release kinetics. In conclusion, PSi-based versatile multicomposites, having the capacity for precisely controlled delivery of simultaneously encapsulated physicochemically distinct cargos, were successfully prepared and characterized. These systems represent a promising platform for future targeted combination therapies.
  • Meinander, Kristian (Helsingin yliopisto, 2014)
    The highly prostate specific serine protease kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen, PSA) is widely used as a biomarker for prostate cancer and it has also been postulated that it may play a part in tumour growth. Especially interestesting is the antiangiogenic effect exerted by proteolytically active KLK3 in cell line models. In order to stimulate the proteolytic activity of KLK3, a series of peptides have been developed by phage display methodology. Even though the peptides are quite potent KLK3 stimulators, they are not directly suitable for in vivo studies or use as drugs. Even though there are many natural and unnatural biologically active peptides, they suffer from rapid clearance via the liver and kidneys and proteolytic degradation of the compounds both in the gastrointestinal tract and other parts of the body. This gives peptides a poor oral bioavailability meaning that they are usually administered as intravenous or intramuscular injections. Several different strategies have been developed in order to access compounds with improved bioavailability including modifications of the peptide structure, development of pseudopeptides and development of small molecular weight peptidomimetics. This thesis concentrates on the further development of the two most potent peptides known to stimulate KLK3, i.e. B-2 and C-4. The main part of the work was concentrated on the replacement of disulphide bridges in the peptides in order to both gain more information on which residues are necessary for obtaining the biological activity and at the same time also gain information on how changes to the geometry of the disulphide bridge affects the activity. A series of different disulphide bridge mimicking building blocks were designed and synthesised with the intention of using them in a protocol for solid-phase synthesis of KLK3 stimulating peptides. Unfortunately, the use of these building blocks in the synthesis of pseudopeptides based on C-4 turned out to be an unsurmountable challenge and the synthesis had to be completed using a different strategy in which the key step was the use of ring-closing metathesis (RCM) for the cyclisation of the partly completed pseudopeptide. Pleasingly, the synthesis of pseudopeptide analogues of the B-2 peptide using the building blocks was more successful. In total three pseudopeptide analogues of C-4 and four of B-2 were synthesised and shown to retain the biological activity of the parent peptides. Based on the information from the synthesised pseudopeptides and a molecular modelling study, a 4-quinolone based peptidomimetic was designed to mimic the C-4 peptide and a synthetic protocol was devised to access this compound. Even though the synthesis of the desired target compound has so far not been successful, the synthetic protocol that was designed has given access to a number of 1,2,8-trisubstituted 4-quinolone derivatives.
  • Magarkar, Aniket (Helsingin yliopisto, 2014)
    Drug delivery is a vital issue in pharmaceutical research; once a drug candidate molecule is identified, it must be delivered to the target area of the body where it can take effect. In addition, non-specific distribution of drug molecules to areas other than the drug target must be decreased to avoid unwanted side effects. To achieve this, nanotechnological drug delivery systems can be used. Nanotechnological drug delivery systems come in a wide variety of forms, including liposomes, dendrimers, nanoparticles, and polymeric micelles. Of these, our research is focused on drug delivery liposomes. Drug delivery liposomes are composed of a membrane that forms a closed spherical sack, with a diameter of approximately 100 nm that can contain drug molecules. The criteria for effectiveness of these drug delivery liposomes (DDLs) are structural stability, its lifetime in the bloodstream, the release rate of the encapsulated content and site specific targeting. Cholesterol is one of the crucial lipid components of the DDL known to increase its stability. They also can have a protective polymer coating such as polyethylene glycol (PEG) that protects the DDL from the body s defense mechanisms. Also the DDL can posses targeting moieties, able to direct the PEGylated liposomes to the specific target. In this study we have investigated surface structure of the DDL and its interactions with elements of the blood stream. While it is difficult to determine an accurate picture of the DDL surface and its interactions with ions and bloodstream proteins with atomistic resolution by experiments alone, computational molecular modelling techniques can provide insights into it. Hence, we have used computational modelling and molecular dynamics simulations to understand the role of each component of the DDL in its structure. The three of the five reported studies in this thesis (I, II, III) are focused on how surface charge plays an important role in the liposome, how it is affected by various components of the DDLs, and how the specific interactions of DDLs and ions present in the blood stream influence it. The chapter IV deals with understanding the properties by systematically varying components such as cholesterol and PEG. Also we have produced the first ever model of the first FDA approved drug delivery liposome (DOXIL ®) at atomistic resolution details. The last study (V) deals with the application of molecular dynamics in targeted drug delivery research. In this study we could identify the reason for failure of specific novel targeting peptide (AETP), which is used to functionalize the DDL, by identifying its interactions with the protective PEG polymer.
  • Subrizi, Astrid (Helsingin yliopisto, 2014)
    Biologics are increasingly used in the treatment of ocular diseases such as age-related macular degeneration (AMD) that cannot be controlled with conventional small molecule drugs. AMD is a multifactorial eye disease that carries significant risk of morbidity and vision loss. In Finland and other western countries, AMD affects one in three people older than 75 years, and until the early 2000s no effective treatment was available for these patients. The marketing approval of anti-VEGF antibodies was a major breakthrough in the management of AMD; indeed these biologics effectively halt choroidal neovascularization and therefore prevent further vision loss in roughly half of the patients with wet AMD. Antibody therapy has been the most successful approach so far, however, other biological therapies such as gene therapy, cell therapy and other therapeutic proteins, may prove beneficial in the treatment of AMD and other vision threatening disorders. This thesis deals with the delivery of biologics, including DNA, cells, proteins and peptides, to the retinal pigment epithelium (RPE), which plays a central role in the development of AMD. Briefly, the main topics and results of this work are presented. New non-viral gene delivery candidates are usually screened for transfection efficiency and toxicity by reading out transgene expression levels relative to a reference formulation after in vitro transfection. The screening protocols, however, can be very different among laboratories, so that comparison of results is often difficult, if not impossible. Our aim was to develop a standardized protocol optimized for the transfection of retinal pigment epithelial cells in vitro. The developed screening protocol provides a relatively simple and reproducible procedure for the pre-selection of potential candidate reagents as non-viral gene delivery systems targeted to the retinal pigment epithelium. The ocular delivery of biologics remains a challenging task due to the barriers of the eye. Short cationic peptides, also known as cell-penetrating peptides (CPPs), have been successfully used as tools to introduce various biologics into cells due to their ability to translocate across the plasma membrane and deliver their cargoes intracellularly. In our work, we have explored the functionality of Tat peptide, one of the most widely studied CPPs. Our results indicate that it is not the sequence of Tat per se that dictates cell uptake, but the cationic charge of the peptide. Moreover no direct penetration was observed; instead all the peptides were endocytosed and, as it is often the case in non-viral gene delivery, ended their journey inside lysosomes. For this reason, we think that the use of Tat peptide for the delivery of biologics to the cytoplasm or nucleus of cells will probably not be very successful. Ocular stem cell therapy holds promise for the reconstruction of the degenerated RPE monolayer in AMD patients; in addition, engineered human RPE constructs may also provide a unique platform for drug discovery and toxicology. We have grown a functional RPE tissue in vitro by using human embryonic stem cells as cell source and the synthetic polymer polyimide as supporting scaffold for the growth and maturation of the cells. The epithelia acquired RPE-like properties, including characteristic RPE phenotype, expression of RPE markers, barrier and phagocytic function. The degeneration of RPE cells in dry AMD is caused by the aggregation of proteins inside RPE cells, and is currently untreatable. We have investigated the cytoprotective properties of heat shock protein 70 kDa (Hsp70) against oxidative damage and the feasibility of rhHsp70 protein therapy as a potential therapeutic approach for dry AMD. This work provides a novel therapeutic option for the treatment of RPE degeneration in AMD.
  • Rosenqvist, Kirsi (Helsingin yliopisto, 2014)
    The effect of systemically and locally administered clodronate on bone quality The aims of this study were to evaluate the effect of clodronate, given systemically or locally, on the quality of bone and to develop a combination product including clodronate and bioactive glass for local administration in the treatment of periodontitis. The beneficial effects of clodronate on bone are known. Clodronate inhibits osteoclasts in bone and reduces bone turnover. It is used in breast cancer patients with non-osseous metastases to reduce the osteolytic complications and bone metastases. It is widely investigated e.g., in healthy women reducing bone loss. The first aim of this thesis was to investigate whether these established effects are similar in women with primary operable breast cancer. Since skeletal bone loss and alveolar bone loss in periodontitis share common mechanisms, the effect of clodronate upon dental application was of interest. Systemic bone conditions impact the periodontium and systemically administered clodronate positively affects the peridontium, but also side effects such as diarrhea, rash and osteonecrosis of the jaw can be problematic. However, clodronate is poorly absorbed from gastrointestinal (GI) tract and the oral bioavailability of clodronate is low. Therefore, systemically administered oral dose of clodronate needs to be high to achieve a therapeutic effect, which in turn leads to increased adverse events. Therefore, the challenge in developing novel drug delivery systems for clodronate is to achieve improved bioavailability and safety. The aim of the second part of this thesis was to develop a new delivery system that would reach the target site in the periodontium, while limiting unwanted side effects and reducing the required dose through local administration. In the first part of this study, the loss of bone mineral density (BMD) was studied in patients with primary operable breast cancer given either clodronate or placebo. Oral clodronate appears to reduce the loss of bone in these patients. In premenopausal patients clodronate significantly reduced the loss of bone after one year and in postmenopausal patients clodronate increased the spinal BMD. Patients receiving clodronate had significantly more incidences of diarrhea than those receiving placebo. This indicates that clodronate is poorly absorbed from GI tract thus causing irritation to the intestine. In addition, the development of bone metastases was compared with patients with primary operable breast cancer given either clodronate or placebo. Clodronate given to these patients was shown to reduce the occurrence of bone metastases. Additionally, there was a significant reduction in mortality. In the second part of the study, a novel combination product of bioactive glass (BAG, SiO2 53%, Na2O 23%, CaO 20% and P2O5 4% (w/w %)) and clodronate was investigated. Firstly, preformulation studies were performed. Clodronate was found to promote the activity of the BAG and a calcium clodronate precipitate formed. Additionally, the bioactivity lasted longer in the combination product than in BAG alone. The optimal ratio for bioactive glass and clodronate and optimal particle size for BAG for local treatment of periodontitis was investigated and selected based on bioactivity of the BAG, safest pH profile of the combination product, as well as highest possible amount of clodronate to achieve the enhanced bioactivity for the BAG. The combination product chosen was 1 g BAG with a particle size of 0.5-0.8 mm and 200 mg of clodronate premoisturized with saline. Finally, the selected combination product was compared to the BAG alone in the treatment of the periodontitis maintenance phase in a pilot study with ten study subjects. Based on the clinical signs and symptoms of inflammation or infection at the site (evaluated by the investigator) and overall satisfaction the combination product is at least as good as bioactive glass alone. Of the two bone remodeling biomarkers (osteoprotegerin and osteocalcin) selected for the study, only osteoprotegerin data indicate that the effect of the novel combination product is beneficial. Osteoprotegerin levels decreased in both combination product and BAG alone treated teeth but less in the combination product treated teeth. However, due to the short time period of investigation as well as the limited number of subjects and treated teeth, the result for bone quality remains only indicative. The positive effect of the combination product on tooth sensitivity may bring additional benefits in comparison to the use of BAG alone in periodontal maintenance therapy.