Farmasian tiedekunta


Recent Submissions

  • 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.
  • 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.
  • Koskela, Elina (2015)
    Intracranial aneurysms (IAs) affect 2-3% of the population. In Finland, their rupture rate has been exceptionally high. This study aims to evaluate the prevalence and risk factors affecting neuro-ophthalmic findings in patients undergoing treatment for IAs. We also investigate the utility of conventional head computed tomography (CT) in the diagnosis of Terson´s syndrome (TS, vitreous hemorrhage in association with aneurysmal subarachnoid hemorrhage (aSAH)). Patients treated endovascularly or microsurgically for their IAs at the Department of Neurosurgery, Helsinki University Central Hospital, in 2011 prospectively underwent a neuro-ophthalmic examination preoperatively and at 3 days, 14 days, 2 to 4 months, and 6 months postoperatively. Findings suggestive of TS, as independently reviewed by two radiologists, on conventional CT head scans were compared with dilated fundoscopic findings. Participants comprised 121 patients with a ruptured aneurysm and 142 patients with an unruptured intracranial aneurysm (UIA). TS was present on fundoscopy in 13 patients (11%); the overall observed agreement between the two radiologists was 96% (116/121), with a substantial κ of 0.69 (95% CI 0.56-0.82). On average, CT demonstrated sensitivity of 42% and specificity of 97%. Factors independently predicting TS were female gender (OR 5.34, 95% CI 1.05-27.17) and World Federation of Neurosurgical Societies (WFNS) grade (OR 15.05 for grades IV-V vs. I-III, 95% CI 3.07-73.89). For patients with aSAH, the frequencies of a third, fourth, or sixth nerve palsy were 11 (9%; preoperatively), 16 (13%; immediately postoperatively), and 3 (3%; at the last follow-up), compared with corresponding frequencies of 6 (4%), 15 (11%), and 7 (5%) for patients with UIAs. Significant risk factors for postoperative eye movement disorders (EMDs) among patients with UIAs were aneurysm location in the posterior circulation (OR 142.02, 95% CI 20.13-1002.22) and aneurysm size (OR 1.28, for each 1-mm increase in diameter, 95% CI 1.12-1.47). After a follow-up time of 6 months, patients with aSAH presenting with visual field defects (VFDs) for aneurysm- or operation-related reasons numbered 20 (19%); homonymous VFDs were the most prevalent finding, and in logistic regression analysis, they were significantly associated with the Hunt and Hess (H and H) grade (OR 4.45 for grades IV-V vs. I-III, 95% CI 1.21-16.39) and presence of intracranial hemorrhage (OR 8.93, 95% CI 2.14-37.28). By contrast, seven patients (5%) treated for a UIA had VFDs. Poor-grade aSAH (H and H or WFNS grade IV-V) appears to be a strong predictive factor for TS and VFDs. With a high specificity value, conventional head CT scan might prove a useful tool in the diagnosis of TS. Although a common finding in the acute postoperative stage of ruptured and unruptured IAs, EMDs show marked improvement during follow-up.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.