Faculty of Pharmacy

 

Recent Submissions

  • Taavitsainen-Wahlroos, Eveliina (Helsingin yliopisto, 2022)
    Bacteria use various mechanisms such as resistance, persistence, and tolerance to survive in unfavorable conditions. Bacterial persistence is a tendency of a bacterial population to adopt a non-growing phenotype and gain the ability to survive exposure to high concentration of antibiotics while hiding from the host immune system. Persisters arise from actively replicating bacterial populations as subpopulations without significant metabolic activity that tolerate traditional antibiotic treatments. Chlamydia pneumoniae is a gram-negative intracellular human pathogen. The ability of C. pneumoniae to convert into a persistent phenotype has been related to several chronic inflammatory diseases, such as atherosclerosis and asthma. At present, antichlamydial treatments against persistent infections are not available for clinical use. Transcriptomic changes in C. pneumoniae and its host cell have been studied previously, but proteomic-level host-chlamydial interactions remain elusive. In this study different cell models were characterized and validated to investigate persistent C. pneumoniae infection and to discover novel drug treatment targets in persisters. Human THP-1 monocyte derived macrophages and A549 human respiratory epithelial cell line were used to examine host-pathogen interactions during persistent C. pneumoniae infection and effects of clinically used antibiotics and the lignans from a widely used dietary supplement Schisandra chinensis. C. pneumoniae was found to adopt a non-replicating and antibiotic tolerant persistent phenotype in THP-1 macrophages soon after inoculation. In addition to persistence, a subpopulation of actively replicating C. pneumoniae was discovered to exist among the persisters. The infection was tolerant to clinically used antichlamydial antibiotic indicating persistent phenotype. As a hallmark of productive infection, infectious progeny production was also observed in the culture, but in a lower level than in permissive host cells. C. pneumoniae infection was concluded to be mixed type, where persistent and productive phenotypes are present at the same time inside macrophages. Label-free quantitative proteome analysis and pathway analysis tools were used to identify changes in THP-1 macrophages upon C. pneumoniae infection. At 48 h post-infection, the inflammatory pathway was induced for example via NF-κB regulation by S100A8 and S100A9 calcium-binding proteins. At 72 h post-infection proinflammatory consequences of the infection were dampened and infection-induced biological processes were related to cell cycle and DNA replication, which were negatively affected by C. pneumoniae. The host macrophage cell cycle control was inhibited by the downregulation of mini chromosome maintenance (MCM) proteins MCM2-7. The relevance of such events for C. pneumoniae replication was demonstrated by the ability of a cyclin-dependent kinase (CDK) 4/6 inhibitor to promote C. pneumoniae infectious progeny production. C. pneumoniae infection was found to suppress retinoblastoma expression and the downregulation was reverted by treatment with a histone deacetylase inhibitor. Together, these findings indicate that C. pneumoniae seems to affect differentiation of host cell macrophages and indicates host cell changes associated with myeloid-derived suppressor cell phenotype. The second persistent infection model was established in the A549 epithelial cell line with beta-lactam treatment. Based on findings by inclusion count and quantitative culture/infectious progeny production, amoxicillin treatment induced persistent phenotype in clinical antibiotic levels. By microscopy, small abnormal inclusions were observed. Persistent infection also reduced the efficacy of azithromycin and doxycycline used in clinically relevant concentrations when the antibiotics were added later or at the same time with amoxicillin treatment. The third persistence model was attempted to be set up in epithelial cells as a continuous infection model. The model has been described before in literature in a different cell line. In contrast to previous reports applying another cell line, bacteria were observed inside host cells by qPCR only for four weeks post-infection. Based on our results, A549 epithelial cells do not support long lasting continuous infection of C. pneumoniae. Schisandra lignans have earlier been reported to have antichlamydial effects against actively replicating C. pneumoniae infection in vitro. The effects of S. chinensis lignans against persistent C. pneumoniae infection were studied in different models. Schisandrin C was found to reduce genome copy numbers and infectious progeny production more than the golden standard antibiotic azithromycin in THP-1 macrophages. Schisandrin did not have bactericidal effect against persistent infection, but instead it activated the resuscitation of persistent bacteria, demonstrated by induced infectious progeny production. The concomitant treatment with schisandrin and azithromycin reduced infectious progeny production and genome numbers of C. pneumoniae more than high-dose azithromycin treatment alone. In contrast to the effects in THP-1 macrophages, similar effect was not observed in the amoxicillin-induced persistence. Moreover, schisandra lignans seem to hinder azithromycin in decreasing bacterial infectious progeny production. Determining the mode of action of schisandra lignans requires further research, but lignans are known to affect mammalian cell glutathione levels. Redox activity of the compounds is suggested to be at least a part of mechanism of action. Overall, these results contribute to the available research methods for persistent C. pneumoniae infection. New findings in pathogen-host relationships may give us new possible antipersister drug targets for future investigations and results with the lignans represent a possible interactive role of redox-active supplements with antimicrobial treatments.
  • Tuunainen, Johanna (Helsingin yliopisto, 2022)
    Absorption, distribution, metabolism, and excretion (ADME) are the main processes that determine the concentration-time profile i.e., the pharmacokinetic (PK) profile of a drug in plasma and tissues. When the PK profile is further connected to relevant information concerning the pharmacodynamics of the drug, the quantitative information can be used to guide drug’s dosing selection. Therefore, it is of utmost importance to try to predict the ADME properties and the PK profile of drugs already in the drug discovery and to refine the understanding during drug development. The ADME processes are complex and dynamic biological events which make the prediction challenging. Despite promising progress during recent years, there is still a lot to do in improving the in silico and in vitro tools for predicting drug PK in human particularly absorption and metabolism. In addition, the ADME properties should not be considered too much in isolation. The ADME properties need to be bound together to produce a quantitative estimate of the drug’s PK profile in human. This requires using the PK modelling techniques. Therefore, continuous development of models predicting individual ADME properties as well as better PK modelling methods are needed. Also, retrospective analysis of the models with in vivo data is an essential part of developing and refining the models. In this thesis, in silico and in vitro models for predicting the passive absorption of drug candidates were developed. Novel quantitative structure property relationship (QSPR) models were built to predict the absorption rate constant (Ka) of drugs in human based on calculated physicochemical molecular descriptors. The paracellular absorption route of in vitro cell line models were characterised and means to scale the Caco-2 in vitro permeability results to human intestine was found. The developed absorption models can be used in the early drug discovery to guide molecule design and to select the most promising compound for further studies. Rapid and complete absorption alone is not enough but metabolism must also be taken into account when predicting the bioavailability of the drug and the possibility of achieving an effective plasma concentration. In this thesis two strategies to overcome extensive drug metabolism were studied namely ocular topical administration route for naloxone and inhibition of metabolism with co-administered drugs for levodopa. All the studies of this thesis produced such data that can be used to develop PK models. As a conclusion, this thesis provided novel models for predicting the passive absorption of drug candidates and proven strategies to overcome the extensive metabolism of drugs. The findings of this thesis support the use of PK modelling techniques to guide drug development from the early discovery to late development.
  • Mahmoudzadeh, Mohammad (Helsingin yliopisto, 2022)
    Systemic administration is the conventional method for administrating drugs. Following injection, ideally, we wish the drug only to locate to the target tissue, however, this is not what occurs; the drug molecules rather distribute throughout the entire body via the blood stream. Regarding some drugs, in particular chemotherapy agents, this often leads to severe dose limiting side effects and unsatisfactory therapeutic results. On the other hand, many drugs as is also the case for the chemotherapy agents, demonstrate low aqueous solubility and suboptimal pharmacokinetic properties. These problems all necessitate the use of drug delivery systems (DDSs) as they decrease the side effects of drugs while also improving drug bioavailability and pharmacokinetics. Although there are different varieties of DDSs, we have focused on those categorized as polymeric or lipidic. Depending on the drug to be delivered and site of action of the drug, polymeric DDSs can be used either locally or systemically. Hydrogels and electrospun polymer fibers are two examples of polymeric DDSs that are used for the local delivery of many drugs, including antibiotics and anticancer drugs. The other form of polymeric DDSs are nanoparticles that are capable of carrying and in some cases targeting drug molecules. These polymeric DDSs are generally injected into the blood stream to reach their target site. Lipidic DDSs mainly are used in the form of nanoparticles that, depending on their lipid composition and method of preparation, would have different characteristics. Liposomes and solid lipid nanoparticles are two examples of lipidic DDSs. Despite the huge number of publications regarding the use of nanoparticles as DDSs, the number of approved drug therapies that make use of nanoparticle-based delivery systems still remains small. One of the reasons for this problem is that formulations of DDSs are complicated and difficult to optimize. Drug delivery systems should be further redesigned and optimized, however, this has proved challenging due to intrinsic and practical experimental limitations. For example, it is difficult to experimentally elucidate the reason many DDSs show promise in vitro but fail in vivo. The limitations to the extent to which mechanistic insight can be gained from experiments regarding DDSs can be compensated by computational molecular modelling techniques that provide detailed information on molecular interactions of drugs and carriers. The insights obtained by the studies performed in this thesis can be used to improve the design of DDSs. In this thesis, two polymeric (studies I and IV) and two lipidic (studies II and III) DDSs were studied by all-atom molecular dynamics (MD) simulations. In each of these studies, a specific property of the DDS was evaluated in detail. These properties are drug release profile (study I), stability (study II), pH-sensitivity (study III) and size (study IV). We evaluated these properties through investigation of the three varieties of interactions DDSs have: interactions of DDSs with the loaded drug, interactions among the components of DDSs and interactions between the DDSs and the medium, namely water and ions. While it is difficult to directly determine an accurate picture of these interactions experimentally at atomic scale resolution, all- atom MD simulation can provide insight into this.
  • Kvarnström, Kirsi (Helsingin yliopisto, 2022)
    Poor medication adherence is a significant barrier to achieving the expected outcomes of a treatment. The prevalence of chronic diseases is increasing globally, and within ageing populations, both morbidity and use of medicines increase. Only about 50% of patients are estimated to treat their chronic diseases according to instructions in developed countries. Patients with multiple conditions and multiple medications are particularly challenging to healthcare professionals. A common feature of poor adherence is a tendency to stop taking the medication within a few months after the provider has prescribed it. In addition, the patients may not take the medicine as regularly as prescribed and skip doses or take lower or higher doses than prescribed. This study aimed to enhance understanding of the complexity of medication adherence as a phenomenon by studying why patients do not take their medication as prescribed. The goal was to collect information on the factors influencing medication adherence by summarizing research evidence obtained using qualitative methods (Study I) and investigating primary care physicians’ and patients’ perspectives (Studies II and III). Study I was a scoping review of qualitative studies (n=89). The literature search for eligible qualitative studies was conducted on September 23, 2019, and updated on June 9, 2021, using MEDLINE (Ovid), Scopus, and the Cochrane Library. The focus was on patients' experiences and attitudes towards medication adherence. The use of qualitative methods both for data collection and data analysis was mandatory. We used the PRISMA-ScR checklist to ensure the quality of the scoping review. Study II applied the focus group discussions (n=4) for primary care physicians (GPs, n=16). The study was conducted in the Kirkkonummi Health Centre, Southern Finland. The qualitative design was chosen to understand GPs' perceptions of medication adherence and the problems GPs thought patients might have experienced following instructions for their medicine taking. An interview guide of semi-structured questions was used to allow the GPs to discuss the topic from a personal point of view. Study III presents a study protocol of a new patient-oriented method to investigate reasons for non-adherence using pharmacist-conducted medication reconciliation in the primary care clinics as a data collection point. The study is based on pharmacist-patient communication during medication reconciliation. It will be carried out in the public primary care clinics in Vantaa, located in the capital region of Finland. By interviewing, the pharmacist will learn how the patient has been taking the prescribed medicines and whether any non-prescription medicines and food supplements have been used for self-medication. Patients aged 55 years or older will be included, as they most commonly have multiple medications and illnesses. Medication reconciliation will occur upon admission to the public outpatient clinic, usually before a physician's appointment. The search (Study I) revealed 4404 studies, of which 89 qualitative studies were included in the scoping review. The studies more often dealt with barriers than facilitators. The factors were classified as patient-specific, illness-specific, medication-related, healthcare and system-related, sociocultural, logistical, and financial. Information and knowledge of diseases and their treatment, communication, trust in patient-provider relationships, support, and adequate resources appeared to be the critical facilitators in medication adherence from the patient perspective. Patients were willing to discuss their concerns about medications. Better communication and better information on medicines appeared to be critical factors for patients. The two main themes that emerged in the focus group discussions with the GPs (Study II) were non-adherence in chronic disease care and increased need for information about medicines. The GPs (n=16) were increasingly confronted with non-adherence in the care of chronic diseases. The medication management challenges identified were related to patient-specific factors, the healthcare system, characteristics of drug therapies and the function and role of healthcare professionals as a team. The GPs offered several solutions such as improved coordination of care, better patient education and IT systems, and enhanced interprofessional involvement in the follow-up of patients. To support medication adherence and self-management, the GPs appreciated pharmacists' assistance, especially with patients with polypharmacy and chronic diseases. Study III will provide quantitative data for descriptive analysis to identify: 1) the number of discrepancies between the physician's prescription orders and the patient's self-reported use of the medicines, 2) what kind of discrepancies there are, 3) which are high-risk medicines in terms of non-adherence, and 4) why medicines were taken differently than prescribed. Based on the results, 5) a preliminary conceptual model of patient-reported reasons for non-adherence will be constructed. There is a wide range of barriers and facilitators to medication adherence, but barriers seem to be better known than facilitators. Better communication and information appear to be the most crucial factors in enhancing medication adherence. Patients wish to discuss their worries about medications. Medication reconciliation could be used more effectively to monitor medication adherence and prevent the inappropriate use of medicines in routine clinical practice. Based on the findings of this doctoral thesis, it is possible to continue developing a theoretical model related to adherence. The model could consider previous theories related to medication adherence, patient perspective and the research evidence constructed by qualitative methods. Developing new interventions should be based on an enhanced understanding of the patient's perspectives on medication adherence.
  • Torrieri, Giulia (Helsingin yliopisto, 2022)
    Cardiovascular diseases (CVDs), and in particular myocardial infarction (MI), represent a huge burden for society. Current therapies for MI are unfortunately unable to fully restore the lost function of the injured heart. In this regard, cell therapies and novel approaches, such as stimulation of cardiomyocytes proliferation and fibroblasts reprogramming, offer appealing alternatives, holding great potential for the regeneration of the infarcted heart. However, they suffer from degradation issues and poor pharmacokinetic properties. Nanomedicines can provide smart solutions to those challenges and have received increasing attention during the last decades. Moreover, they offer tools for minimally invasive treatments, an aspect that is very important for patient compliance and clinical translation. The ability of nanocarriers to target specific sites in the body is the most appealing feature of nanomedicines, which allows to increase the efficacy of treatments and reduce their systemic adverse effects. In the last decades, heart targeting was attempted by both passive strategies and conjugating different moieties on the surface of nanoparticles, e.g. angiotensin II type 1 (AT1) receptor and atrial natriuretic peptide (ANP). However, the constant pumping of the heart and the lack of selectivity of currently known heart targeting moieties, make heart targeting a challenging goal. Therefore, the aim of this thesis was to develop drug-loaded nanoparticles for improved targeting to the infarcted heart and its treatment. Acetalated dextran (AcDX) was chosen due to its pH-responsive properties, biocompatibility, biodegradability and ease of surface functionalization. Firstly, the heart targeting properties of ANP were improved by conjugating on the surface of putrescine modified-AcDX another peptide, lin-TT1. The conjugation of lin-TT1 peptide on the surface of the nanoparticles offered the ability to hitchhike the macrophages that are sequentially accumulating in the heart upon the onset of MI. In vitro studies showed the ability of the system to associate preferentially with M2-like macrophages, which have ant-inflammatory phenotype. Subsequent in vivo studies on a rat model of MI, confirmed the evidence of preferential accumulation of these particles in the infarcted heart after 7 days post-MI, and showed increased heart targeting ability compared to particles conjugated with only ANP peptide. Secondly, spermine-modified nanoparticles were developed and coated with a coordination complex, made of tannic acid (TA) and Fe3+ ions. TA was used for its proven heart targeting capability, which derives from its high affinity for components of the extracellular matrix, in particular collagen and elastin. In vitro studies proved the increased interaction of the system with cardiac cell cultures stimulated with transforming growth factor (TGF)-β, which induced higher collagen I production. The anti-fibrotic properties of TA were also confirmed by in vitro studies, showing the reduced expression of pro-fibrotic genes in cultured fibroblasts treated with the TA coated nanoparticles. Both systems demonstrated in vitro ability to induce cardiomyocytes proliferation due to the encapsulation of two small hydrophobic molecules, which stimulate cardiomyocytes to re-enter the cell cycle. Moreover, biocompatibility and pH-responsive release of the cargos were also evaluated. Overall, two AcDX-based nanoparticulate systems were developed for improved heart targeting and treatment of MI, bringing new insights about potential therapeutic advances in targeted delivery as a minimally invasive therapeutic approach for heart disease.
  • Oinio, Ville (Helsingin yliopisto, 2022)
    Gambling disorder is considered a behavioral addiction that combines reward with executive decision-making processes in a complex and fascinating way. Gambling disorder overlaps with various psychiatric diseases, and comorbidity with alcohol use disorder and other substance use disorders is significant in its etiology. As in drug addictions, the dopaminergic mechanisms play a significant role in the neurobiology of gambling disorder. Clinical studies with opioid antagonist naltrexone also suggest that the opioidergic mechanism may have a role in modulating gambling behavior, especially in the gambler subgroup with a family history of alcoholism. Currently, treatment of gambling disorder mostly relies on psychotherapeutic approaches, and no significant achievements in drug development have been made. The main aim of the research was to validate a preclinical model of probability-based risky decision-making and investigate differences in dopaminergic and opioidergic mechanisms in decision-making behavior between AA (Alko Alcohol) and Wistar rats. The additional aim was to clarify the role of the nucleus accumbens in risky decision-making and develop a model to screen drug targets in gambling disorder. AA rats were used to represent a group of individuals with a genetic preference for alcohol use disorder, and a standard laboratory rat strain, Wistar, was chosen to represent the normal heterogenic population of gamblers. The decision-making of rats was studied in a probabilistic discounting task, which allows us to examine rats’ behavior at different levels of uncertainty. In the task, rats went through operant lever pressing training where different sized sucrose rewards guided the lever choices. The probability of gaining rewards changed slowly to a level where choosing the smaller reward was the most profitable option. After training, the effects of dopaminergic and opioidergic drugs on decision-making behavior were studied. In this research, we completed the aim to validate an animal model for studying the probability-based risky decision-making behavior and showed that D-amphetamine acts as a valid promoter for “gambling-like” behavior in rats. Results indicate that dopaminergic modulation of probability-based risky decision-making is pronounced in AA rats compared to Wistar. In the case of the opioidergic mechanisms, the results were ambiguous, and thus exact predictions of the relevance of opioids could not be made based on this study. The role of nucleus accumbens dopaminergic functions as a modulator of risky decisions was verified, but naltrexone’s effect on reducing risky decisions failed to show any promising results, indicating that the role of the opioid antagonist in the pharmacotherapy of gambling disorder is focused more on decreasing the overall motivation to gamble than modulating the risky decision-making in gambling. These studies create a platform for future studies aiming to point out the specific neurobiological mechanisms that control the behavior of the gambler subgroup with a genetic vulnerability to alcohol use disorder.
  • Kuitunen, Sini (Helsingin yliopisto, 2022)
    Intravenous administration of drugs is associated with the highest medication error frequencies and more serious consequences to the patient than any other administration route. The bioavailability of intravenously administered medication is high, the therapeutic dose range is often narrow, and effects are hard to undo. Many intravenously administered drugs are high-alert medications, bearing a heightened risk of causing significant patient harm if used in error. Smart infusion pumps with dose error-reduction software can be used to prevent harmful medication errors in high-risk clinical settings, such as neonatal intensive care units. This study investigated intravenous medication safety in hospital settings by identifying recent research evidence related to systemic causes of medication errors (Study I) and systemic defenses to prevent these errors (Study II). The study also explored the development of dose-error reduction software in a neonatal intensive care unit (Study III). A systems approach to medication risk management based on the Theory of Human Error was applied as a theoretical framework. The study was conducted in two phases. In the first phase, a systematic review of recent research evidence on systemic causes of intravenous medication errors (Study I) and systemic defenses aiming to prevent these errors (Study II) was carried out. In Study I, 11 studies from six countries were included in the analysis. Systemic causes related to prescribing (n=6 studies), preparation (n=6), administration (n=6), dispensing and storage (n=5) and treatment monitoring (n=2) were identified. Insufficient actions to secure safe use of high-alert medications, lack of knowledge of the drug, failures in calculation tasks and in double-checking procedures, and confusion between look-alike, sound-alike medications were the leading causes of intravenous medication errors. The number of the included studies was limited, all of them being observational studies and graded as low quality. In Study II, 46 studies from 11 countries were included in the analysis. Systemic defenses related to administration (n=24 studies), prescribing (n=8), preparation (n=6), treatment monitoring (n=2), and dispensing (n=1) were identified. In addition, five studies explored defenses related to multiple stages of the medication use process. Defenses including features of closed-loop medication management systems appeared in 61% of the studies, smart pumps being the defense most widely studied (24%). The evidence quality of the included articles was limited, as 83% were graded as low quality, 13% moderate quality, and only 4% high quality. A mixed-methods study was conducted in the second phase, applying qualitative and quantitative methods (Study III). Medication error reports were used to develop simulation-type test cases to assess the suitability of dosing limits in a neonatal intensive care unit’s smart infusion pump drug library. Of all medication errors reported in the neonatal intensive care unit, 3.5% (n=21/601) involved an error or near-miss related to wrong infusion rate. Based on the identified error mechanisms, 2-, 5-, and 10-fold infusion rates and mix-ups between infusion rates of different drugs were established as test cases. When conducting the pump programming for the test cases (n=226), no alerts were triggered with infusion rates responding to the usual dosages (n=32). Of the erroneous 2-, 5-, and 10-fold infusion rates, 73% (n = 70/96) caused an alert. Mix-ups between infusion rates triggered an alert only in 24% (n=24/98) of the test cases. This study provided an overview of recent research evidence related to intravenous medication safety in hospital settings. Current intravenous medication systems remain vulnerable, which can result in patient harm. While in-hospital intravenous medication use processes are developing towards closed-loop medication management systems, combinations of different defenses and their effectiveness in error prevention should be explored. In addition to improved medication safety, implementing new systemic defenses leads to new error types, emphasizing the importance of continuous proactive risk management as an essential part of clinical practice.
  • Rautamo, Maria (Helsingin yliopisto, 2022)
    Oral drug administration to pediatric patients is characterized by a lack of age‐appropriate drug products. Children need personalized doses due to differences in age and weight; therefore, the production of precise doses by means of printing technologies have recently gained growing interest. In addition, hospital pharmacies are envisioned as potential users of novel three-dimensional (3D) printing methods in the production of patient-specific drug formulations. Therefore, the overall objective of this dissertation was to investigate if orally administered age-appropriate formulations for children can be produced at hospital pharmacies via printing technologies. The first part of this research aimed at exploring the current situation of oral drug administration practices to pediatric patients in hospital wards. In addition, we focused on identifying the preferred dosage forms along with deficiencies in currently available dosage forms. This needs assessment study was conducted as focus group discussions among healthcare professionals at a tertiary university hospital. Physicians, nurses, and clinical pharmacists preferred orodispersible tablets or oral liquids with regards to ease of administration and liquid dosage forms to accomplish personalized dosing. Furthermore, they had experienced both dosage form- and patient-related administration challenges associated with oral drug delivery to children. The second study investigated the opinions of healthcare professionals about printed dosage forms and printing technologies for the productions of oral pediatric dosage forms. Focus group discussions are suitable for studying previously unknown subjects; therefore, this qualitative research method was used in Study II, as well. We used combined data collection in Studies I and II, however, the data was analyzed separately. Generally, the pediatric healthcare professionals discussed many positive aspects and opportunities in 3D printing of pharmaceuticals. Personalized doses and dosage forms with appealing shapes and colors are some positive features that were brought up in the conversations. Furthermore, the manufacture of polypills containing several drug substances in one product, were considered beneficial for pediatric patients dealing with polypharmacy. Concerns and prerequisites for the adoption of 3D printed dosage forms in pediatric hospital wards were related to subcategories of drug administration, medication safety, such as stability, dose accuracy and identification, as well as production and delivery on-demand. The final part of the thesis investigated to what extent two different printing methods (inkjet and semi-solid extrusion printing), could meet the requirements identified in Study II for adapting these printing techniques at hospitals while avoiding causing dosage form related challenges that were experienced in Study I. This research question was approached by the comparison of printed orodispersible films to the currently used dose powders in sachets. All dosage forms contained warfarin sodium as model drug. The evaluation of stability, content uniformity and dose accuracy of the studied dosage forms revealed that the printed formulations were potential alternatives to dose powders when personalized doses are requested. All formulations were stable for one month and they were successfully administered through an enteral feeding tube without causing blockage. Identification of printed dosage forms was managed through imprinting of a QR code onto the films by inkjet printing. Both utilized printing techniques involve some formulation aspects and production steps that need to be improved before the methods can be applied to on-demand production of pediatric drug products. These perspectives are suggested to be focus points for future research. In summary, this dissertation gives a holistic picture on different aspects of oral drug administration to pediatric patients and overall patient‐centered drug administration practices. It furthermore demonstrates a positive attitude among healthcare professionals towards 3D printing of oral pediatric formulations. Pharmaceutical industry, academic research groups, hospital pharmacies and community pharmacies can utilize the results of this thesis in the development of age-appropriate pediatric dosage forms along with improving the usability of 3D printing to provide personalized medical treatment close to the patient.
  • Johansson, Niklas (Helsingin yliopisto, 2022)
    Pathogenic protozoan parasites cause devastating diseases, such as malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma spp.), leishmaniasis (Leishmania spp.) and trypanosomiasis (Trypanosoma spp.), which have an enormous health, social and economic impact, particularly in tropical regions of the world. For example, nearly half of the population of the world is at risk of malaria that is responsible for more than 600,000 deaths annually. Interestingly, all of these parasites have in common a homodimeric integral protein, membrane-bound pyrophosphatase (mPPase), consisting of 15 to 17 helices with a molecular weight of 70 to 81 kDa. These mPPase enzymes hydrolyze pyrophosphate, a by-product of many biological processes. Besides taking care of excess pyrophosphate, the energy released through hydrolysis of phosphoanhydride bonds is coupled with the pumping of protons or sodium ions thus creating an ion gradient across the membrane. Consequently, mPPases play an important role in the survival of many organisms under diverse stress situations due to osmotic stress or other energy limitations. Inhibiting the function of mPPases bears promise from a drug discovery perspective, as remarkably these enzymes do not exist in animals or humans. Hitherto, only crystal structures of the hyperthermophilic bacterium Thermotoga maritima mPPase (TmPPase) and mung bean Vigna radiata (L.) R. Wilczek mPPase have been solved and are amenable for structure-based drug design. Still, most known inhibitors, such as pyrophosphate and bisphosphonate derivatives, are not optimal as drugs due to features including poor stability and extensive hydrophilicity. The focus of this study was to explore membrane-bound pyrophosphatase as a potential drug target for pathogenic protozoan parasites, mainly Plasmodium falciparum. Since most of the currently known inhibitors are phosphorus-based, thus limiting their therapeutic usage, the aim of this thesis was to develop new organic mPPase inhibitors with potential as an alternative approach in the treatment of malaria and other related parasitic diseases. By developing nonphosphorus inhibitors targeting mPPase, the intention was to block the essential ion pump of these parasites. The compounds obtained were first tested in a 96-well plate in vitro screening assay using thermostable TmPPase as a model enzyme, resulting in the construction of a small library of drug-like compounds (<500 Da) containing different scaffolds with low micromolar activities. Selected top hits were studied further and evaluated against purified mPPase from P. falciparum (PfPPase-VP1) expressed in baculovirus-infected insect cells. In addition, their ability to inhibit the growth of P. falciparum in a survival assay in erythrocytes was studied. In publication I, the discovery and synthesis of the first nonphosphorus, non-substrate analog inhibitor of Thermotoga maritima mPPase (TmPPase), with a 6-(aminomethyl)benzo[d]thiazol-2-amine core, as well as preliminary structure-activity relationships (SARs) and a X-ray crystal structure of this allosteric inhibitor bound to TmPPase are reported. Unfortunately, the allosteric binding region was not preserved in parasitic mPPase thus indicating that there are alternative ways to inhibit mPPase enzymes. In the subsequent study, publication II, the chemical exploration of phosphate isosteres, which finally led us to 2-bromophenyl 5-arylisoxazole-3-carboxylates, is presented. As some antiparasitic activity was maintained, it is assumed that these inhibitors bind orthosterically in the preserved region of the enzyme even though it has not yet been confirmed by sufficient crystallographic data. Publication III further explores structural analogs of the best isoxazole inhibitor (publication II), which resulted in discovering a new bicyclic scaffold, with a 4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one core, and its related pyrazolo[1,5-a]pyrimidine analogs. These compounds seem promising also against PfPPase and, for that reason further studies thereof are ongoing. Additionally, publication IV describes a cross-project in vitro screening that got us to study azulenes and benzazulenes as potential TmPPase inhibitors. These low micromolar TmPPase inhibitors retained similar activity also in a P. falciparum survival assay in erythrocytes. Altogether, these studies offer a different approach for further development of novel drugs against malaria and other diseases caused by pathogenic protozoan parasites.
  • Auvinen, Vili-Veli (Helsingin yliopisto, 2022)
    Nanoparticles play an essential role in the development of modern drug administration as delivery platforms. They provide specific delivery paths for pharmaceuticals and biomolecules to target tissues and cells. In addition, they allow controlled release via outer activation methods, such as light activation. Traditionally nanoparticles are delivered to patients via injection. However, due to the rapidly growing market and variety of new applications, new delivery methods and carriers should be investigated. Anionic nanocellulose is a hydrogel produced from nanosized cellulose fibers. The aim of this thesis was to prove that nanocellulose hydrogels are suitable for the controlled release of a wide range of pharmaceuticals and nanoparticles. This requires stability from the carrying matrix in vivo, high biocompatibility and preferably clearness for possible light-induction applications. We used the hydrogels to administer model pharmaceuticals and model nanoparticles from standard release well systems and custom-made implant type devices to learn that the concentration of the nanocellulose fiber in the hydrogel affected the drug release rate for most drugs, proteins and nanoparticles. The diffusion coefficients were significantly smaller for large molecules and nanoparticles when higher concentration of nanocellulose was present which indicates that the amount of fiber in the hydrogel can be utilized to control the drug release rates. The implant type devices used in the work were 3D printed with specific geometries to regulate the release rate of the chosen pharmaceuticals. The main benefit of the devices is that the release rate of any nanocellulose compatible drug or particle can be modulated in addition to fiber content by modulating the inner geometry of the PLA capsule. The methodology was shown to be well suited for preclinical development and can be further enhanced to pursue medical applications. In addition, we studied the effects of freeze-drying in the release of human growth factors from the nanocellulose hydrogel. Freeze-drying is an extremely stressful process for any living organism. In the process, the sample is frozen, and water is extracted through sublimation. This is achieved by lowering the air pressure. Moreover, the freeze drying was studied from another perspective to see if the nanocellulose could aid in preserving the outer structures of human cell spheroids. To conclude, we demonstrated the suitability of nanocellulose hydrogels as a delivery platform for a wide range of pharmaceuticals and nanoparticles. In addition, we demonstrated nanocelluloses compatibility with freeze-drying when correct lyoprotectans were implemented which broadens the application development paths for the production of new drug delivery systems.
  • Zhang, Pei (Helsingin yliopisto, 2022)
    Carrier encapsulation of the active ingredient is an efficient strategy to improve the pharmacokinetic and pharmacodynamic properties of peptide and protein therapeutics, for example, by prolonging the half-life of the drug. However, because of the poor miscibility of peptide and protein therapeutics with the hydrophobic carrier materials, it is difficult to realize high loading degree and encapsulation efficiency during the encapsulation process. The poor loading degree may result in carrier materials -related side effects, decreased treatment efficiency and low patient compliance. Due to the low encapsulation efficiency of single peptide and protein therapeutics, it is difficult to reach precise ratiometric control of co-encapsulated peptide and protein molecules. Moreover, as the loading degree of the therapeutics is increased, burst release may occur when in contact with release medium (body fluids), which is economically inefficient and potentially pharmacologically dangerous. Herein, the aim of this dissertation was to develop a strategy to efficiently encapsulate both peptide and protein therapeutics. Based on the efficient encapsulation, microparticles simultaneously featured with ultra-high loading degree and precise ratiometric control or zero-order release of the loaded therapeutics were further prepared. First, a surface adsorption strategy was developed to realize efficient encapsulation of peptide and protein therapeutics. The therapeutic molecules were formulated into nanoparticles and adsorbed with carrier polymers through electrostatic attraction. This adsorbed polymer layer efficiently inhibited the oil-to-water phase transfer of therapeutics nanoparticles, which led to a ultra-high loading degree and encapsulation efficiency of the peptide and protein therapeutics. This strategy was confirmed by efficient encapsulation of insulin, bovine serum albumin, β-lactoglobulin and ovalbumin. Second, as the surface adsorption strategy realized almost 100% encapsulation efficiency of peptide and protein therapeutics, a ratiometric control over the co-encapsulated peptides by simply adjusting their initial weight ratio in the formulation was studied. The fabricated microparticles co-encapsulated with insulin and exenatide effectively reduced the weight gain and glycosylated hemoglobin level of type 2 diabetic rats. Third, based on the results of the surface adsorption strategy, an enhanced interfacial adsorption strategy was further developed. In addition to the adsorption of the carrier polymer to the nanoparticle’s surface, the adsorption of carrier polymer at the oil/water interface was increased to form a thicker interfacial polymer layer. The fabricated microparticles showed not only ultra-high loading degree, but also zero-order release profiles of the encapsulated therapeutics in vitro and in vivo. After encapsulation, the therapeutics’ release profiles were prolonged and the pharmacokinetic and pharmacodynamic properties of the therapeutics were efficiently improved in animal models. In conclusion, this dissertation provides new insights of efficient encapsulation of peptide and protein therapeutics and may facilitate the highly anticipated clinical transition of peptide/protein-loaded microparticles.
  • Hieta, Juha-Pekka (Helsingin yliopisto, 2022)
    Mass spectrometry (MS) imaging (MSI) is an untargeted and label-free chemical imaging method that can map distributions of compounds, such as metabolites, lipids, proteins, and pharmaceutical compounds, in solid samples, such as tissues and cells. Infrared laser ablation ambient MS (IRLA-MS) methods can carry out MSI measurements at atmospheric pressure with minimal sample preparation, which allows straightforward analysis of biological samples in their native state, and which is not feasible with traditional vacuum-based MSI methods that typically require extensive sample preparation and treatment. IRLA-MS methods are best suited for the analysis of small <1500 Da molecules, and they can cover the analysis of small nonpolar, neutral polar, and charged polar molecules depending on the ionization method. IRLA-MS methods with electrospray ionization (e.g., laser ablation electrospray ionization, LAESI) can analyze a wide-range of medium polar, polar, and ionic compounds, whereas IRLA-MS with atmospheric pressure photoionization (i.e., laser ablation atmospheric pressure photoionization, LAAPPI) is suitable for the analysis of nonpolar and polar analytes. However, LAAPPI- and LAESI-MSI have thus far been limited by a relatively poor lateral resolution of 200–400 μm, which has limited the number of structures they can spatially resolve in samples, and which can make them an unappealing choice for biological research. This thesis and its four studies cover the early stage of LAAPPI-MSI research and the development of a novel LAAPPI/LAESI MSI platform for optimized imaging with sub-100 µm lateral resolution. The main objective of the study was to enhance the imaging quality of these IRLA-MS methods for biological research. In the first study, the first in-house built LAAPPI-MSI setup of this work was used to study the distributions of low polarity triterpenoids in the phellem fractions of Betula pendula bark samples with a lateral resolution of 400 µm and provide complementary imaging data to the mass spectra acquired by desorption atmospheric pressure photoionization (DAPPI) MS. LAAPPI-MS offered an efficient tool for sampling and ionization of all the studied triterpenoids: betulin, betulinic acid, betulonic acid, allobetulin, allobetulone, and lupeol. Positive ion LAAPPI-MSI analysis showed that betulinic acid had a relatively high abundance in the lenticels, which are channels that enable the gas exchange through the bark, whereas all other triterpenoids were distributed more evenly in the phellem. The result was consistent with the spectral data acquired by the DAPPI-MS method. The second study demonstrated a simple focusing method for OPO IR laser beams, which are widely used for sampling in IRLA-MS methods. The capability to focus the IR laser beam to the smallest possible spot size typically determines the lateral resolution of the IRLA-MSI analysis. The results showed that the inhomogeneous multimode OPO IR laser beam profiles can be transformed to a better form before focusing by allowing the beam to propagate in free space. The resulting size of the IR laser ablated holes was observed to decrease from 385 to 20 µm as the beam propagation distance in free space increased from 1 to 18 m. Part of the IR laser energy was lost at longer beam propagation distance, but this was compensated by focusing of the radiation to a smaller area on the sample surface. The suitability of the focusing method for MSI was demonstrated in the LAAPPI-MSI of a mouse brain tissue section. In the third study, an enclosed LAAPPI/LAESI MSI platform utilizing the new OPO IR laser focusing method was developed and optimized for the analysis of rodent brain tissue sections with a lateral resolution of 70 µm. The improvement of the imaging resolution below 100 µm was a large step forward in terms of imaging quality, as compared to the first study, and which required a higher sensitivity, repeatability, and stability from the LAAPPI and LAESI methods due to the decreased number of sampled analytes and increased number of measured pixels. After optimization of operational parameters, positive and negative ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods. The stability of the optimized negative ion LAAPPI-MSI for larger sample areas was demonstrated with a successful measurement of a whole rat brain tissue section consisting of nearly 30 000 pixels, which revealed many lipid distributions correlating with different rodent brain structures. The last study applied LAAPPI-MSI with a lateral resolution of 70 µm for the analysis of the untreated and frozen leaves of model organism Arabidopsis thaliana (A. thaliana), which is an important species in fundamental plant research. A. thaliana leaves have a complex, multilayered structure, which can be difficult to analyze with most MSI methods that are confined to the analysis of the sample surface. Negative ion LAAPPI-MSI allowed the analysis of A. thaliana leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. This was also the first time that LAAPPI-MS was shown capable of imaging and analyzing contents of single cells.
  • Piironen, Kati (Helsingin yliopisto, 2022)
    In vitro models of human tissue have advanced during recent years through the development of 3D cell culture models. However, they are not yet able to fully emulate the complexity of the complete cellular microenvironment in tissue. In vitro models can further benefit from microfluidic approaches that allow more intricate control of the cellular microenvironment through microfabrication and microfluidic flow. Microfluidic cell cultures that emulate some aspects of organ function are called organs-on-chips (OOC). Most of the OOC platforms utilize polydimethylsiloxane (PDMS) as a fabrication material, due to its optical clarity, inherent biocompatibility (nontoxicity), gas permeability and cost-effective fabrication. However, PDMS is not always the most desirable material for OOC applications, due to its hydrophobic surface (poor cell adhesion) and unspecific adsorption of small molecules (risk of false positives/negatives). More studies are required to develop alternative materials for OOC applications. The aim of this thesis work was to characterize two emerging classes of polymer-based microfabrication materials for their cell compatibility, namely a set of commercial acrylate resins commonly used in prototyping 3D microdevices by stereolithography and custom off-stoichiometric thiol-ene (OSTE) polymers used in rapid prototyping of planar microdevices by replicamolding techniques. In Publication I, four different commercial 3D printing materials were evaluated for their biocompatibility by studying cell survival on the material surface. Both growth curves and cell staining were used to assess cell proliferation on the surfaces with an emphasis on long-term survival of the cells. Autoclaving was found to be an important factor in making the 3D printing materials bioinert and suitable for cell culture. Furthermore, a microfluidic device using 3D design was developed to study cell adhesion strength on the material surface without any extracellular matrix (ECM) coating. Cell adhesion to the inert 3D printed surfaces under microfluidic flow was shown to be in the range of 3 dyne/cm2. In Publication II, long-term cell survival on differently microfabricated an off-stoichiometric thiol-ene (OSTE) surfaces was studied by evaluating cell proliferation and viability similar to those of the 3D printed materials. Different fabrication and postprocessing methods were used to evaluate and improve the biocompatibility of OSTEs. UV embossing was found to be the most straightforward microfabrication method for making OSTE material surfaces biocompatible and suitable for cell culture. A microfluidic device using a 2D planar design was developed to study the cell adhesion strength on the material surface without any ECM coating. OSTE material surface supported relatively fast adhesion already after 1.5 h incubation and had comparable adhesion strength to the 3D printing materials (after a 4-hour incubation). In Publication III, the previously reported oxygen scavenging property of thiol-rich OSTE was utilized to develop a two-compartment OSTE-PDMS hybrid device. The device incorporated a thin PDMS membrane between two OSTE compartments that were cured using different microfabrication methods to create a biocompatible cell culture compartment via UV embossing and an oxygen scavenging compartment via UV casting. The gas permeable PDMS membrane ensured unrestricted oxygen transfer between the two compartments. The developed device allowed creation of an on-chip oxygen gradient and precise spatiotemporal control of oxygen concentration in the cell culture chamber by only utilizing flow rate regulation. Furthermore, the device facilitated BALB-3T3 fibroblast and human induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) cell cultures in an oxygen-controlled environment. Overall, this thesis demonstrated the need to study the biocompatibility of new microfabrication materials in long term in order to ensure their suitability for cell culture applications. When selecting fabrication materials for OOC applications, more emphasis should be put on their careful characterization prior to use. At best, clever combination of microfabrication materials and methods can enable cell culturing under conditions not achievable for static culture systems, such as the spatiotemporal oxygen control demonstrated in Publication III.
  • Koskela, Maryna (Helsingin yliopisto, 2022)
    Excessive use of alcohol results in about 5.3% of all deaths worldwide (WHO, 2018). Alcohol consumption can have adverse social, legal, occupational, psychological, and medical consequences (WHO, 2018). Harmful alcohol consumption can result in diabetes, cancer, stroke, cardiovascular diseases, and mental and behavioral disorders (Shield et al., 2013). Craving for alcohol refers to an urge or conscious desire to drink alcohol-based solutions. It has been hypothesized to underlie alcohol-seeking and relapse in humans with alcohol use disorder. Only humans have been reported to experience cravings. Associative learning, a memorable event that occurs with drug consumption, is an important factor in craving. To model the craving for alcohol in animals with equivalence to humans is a challenging task. However, the knowledge that animal models provide is pivotal to understanding the mechanism of disease progression. Drug craving models are well established by using laboratory rats. Self-administration and following extinction and relapse paradigms in mice are still poorly developed. However, because of gene-manipulated strains, they still pose a possibility for studies that cannot be conducted in rats. The main aim of this thesis was to clarify whether GDNF and BDNF play a role in alcohol-seeking behavior in group-housed female mice. A novel methodology to study alcohol-seeking behavior in mice was established. The uniqueness of the model is that alcohol drinking is voluntary, and it occurs in a social environment with minimum handling by a human. This model was used to study the role of neurotrophic factors (GDNF, BDNF, MANF, and CDNF) in alcohol-seeking behavior. This thesis project resulted in a novel model to study alcohol-seeking behavior after alcohol withdrawal in group-housed female mice. This model provides the possibility to study the role of neurotrophic factors in alcohol-seeking behavior. The results showed that viral GDNF overexpression decreases alcohol-seeking behavior after withdrawal.
  • Saarukka, Laura (Helsingin yliopisto, 2022)
    Fluoroquinolones are a group of broad-spectrum antibacterial agents that are widely used and indicated for the treatment of genitourinary, respiratory, gastrointestinal, skin, and soft tissue infections. In Finland, fluoroquinolones comprise 5% of all systemic antimicrobial prescriptions. Fluoroquinolones are generally well tolerated and their common adverse reactions, such as nausea and headache, are mostly mild and resolve quickly. However, fluoroquinolones are also associated with more serious adverse reactions, including tendon injuries, Clostridioides difficile infections, and aortic ruptures, which can result in long-term impairment to patients and high healthcare costs. The aim of this study was to estimate the economic impact of fluoroquinolone-related adverse reactions in Finland. A systematic literature review (study I) was conducted to identify health service use and subsequent costs associated with ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin and ofloxacin -related adverse events. A retrospective observational cost of illness study (study II) was conducted to assess costs and health service use associated with tendon injuries after fluoroquinolone use in Finland during 2002-2012 with data from the Finnish Pharmaceutical Insurance Pool's pharmaceutical injury claims. Regression models were used to analyze the impact of patient characteristics on hospital days, as well as the relationship between patient characteristics and tendon ruptures. In study III, a cost of illness decision tree model was built to predict costs and mortality associated with serious adverse drug reactions. Severe Clostridioides difficile infections, severe cutaneous adverse reactions, tendon ruptures, aortic ruptures and liver injuries were included as serious adverse drug reactions in the model. In study I, 19 observational studies, including five case-control studies, fulfilled the inclusion criteria. Length of hospital stay associated with adverse events varied between <5 and 45 days. Clostridioides difficile infections were the adverse event type associated with the longest stays in hospital. The estimated cost of an adverse event treatment episode ranged between 140 € and 18,252 €. In study II, fifty-one percent of the claimants were hospitalized due to tendon injuries, with an average duration of 21 days and an average cost of 9,915 € per hospital episode. Hospital days and direct costs increased with the severity of the injury. Concurrent use of oral glucocorticoids and increasing age were associated with a higher likelihood of tendon ruptures. In study III, a total of 1,831,537 fluoroquinolone prescriptions were filled between 2008 and 2019 in Finland. Severe Clostridioides difficile infections were the most frequent, fatal, and costly serious adverse reactions associated with the use of fluoroquinolones. Because of the wide clinical use of fluoroquinolones, in particular serious fluoroquinolone-related adverse reactions can have substantial economic implications, in addition to imposing potential long-term disability for patients. Accordingly, the risks and benefits of fluoroquinolones should be weighed carefully in antibiotic prescription policies, and additional measures should be developed to prevent and reduce health service use and costs associated with fluoroquinolone-related adverse reactions.
  • Zini, Jacopo (Helsingin yliopisto, 2022)
    Biopharmaceutical products are composed of complex or ordinate combinations of proteins, lipids, sugars, and nucleic acids or living cells or tissues. Due to the intrinsic variability of biological systems and the complexity of the bioprocesses involved in the production of these products, new technologies are required to monitor and characterize, the production processes and the final products. Biophotonic techniques, particularly Raman and Infrared (IR) spectroscopy are rapid, robust, operator independent, non-destructive and label free, thus particularly suitable for these purposes. This dissertation first investigates the use of biophotonic techniques in research of Extracellular Vesicles (EVs). EVs act as intercellular messengers and therefore have considerable potential in drug delivery system, diagnostic biomarker, or therapeutic agent. Subsequently highlighting the need and potential of a new kind of time gated Raman spectrometer to be created. Raman and IR spectroscopy are methods able to characterize and assess the quality of EV suspensions with different degree of purity. These vibrational spectroscopic techniques show different intrinsic advantages, as they are label free and operator independent methods. Particularly, Raman might be the most suitable technology since it is less sensitive to water compared to IR. Raman spectroscopy reveals the information on the chemical composition and physical status of the analyte. However, it does not provide information of the analyte environment which the spectrofluorometer can gather. EVs studied by spectrofluorometer are required to be labelled with a fluorescent dye. Results obtained by fluorescence lifetime imaging spectroscopy underline that the cell uptake of the fluorescently labelled EVs is feasible. Attention must be paid to the efficacy of the labelling and further to the elimination of the unbound dye since the labelling may severely compromise the results or lead to wrong conclusions on EV functionality. The combined advantage of Raman spectroscopy and fluorescence decay are obtained by a previously in-house developed time resolved Raman spectrometer. Thanks to the peculiar sensor of the spectrometer, the width of the time gate can be modified which is used to separate the Raman signal from the fluorescence tail. The modifications can be done even in the data post-processing phase, to obtain the best possible Raman signal-to-noise ratios. The simultaneously detected Raman spectra and time-resolved fluorescence decay curves are used to study the diffusion of small molecular drugs in a hydrogel. The data reveal the chemical composition, physical status, and the interaction with the environment of the samples. Taken together the obtained results suggest that the quality of EV suspensions can be assessed by Raman spectroscopy and their cell uptake detected by fluorescence lifetime spectroscopy. Both Raman spectra and fluorescence decay can be measured simultaneously by a second generation of time-resolved Raman spectrometer.
  • Celikkayalar, Ercan (Helsingin yliopisto, 2022)
    The thesis had the following three objectives: I. To develop and validate a medication safety self-assessment tool (MSSA) in secondary care hospital wards (Study I: organizational level); II. To use clinical pharmacist-conducted collaborative medication reviews (CMRs) in an emergency department (ED) short-term ward to identify inappropriate prescribing (IP) in pre-admission medications; (Study II: health care unit and clinical practice level); III. To investigate how well older people are aware of the major potential risks of benzodiazepines and related drugs (BZD) they are taking and whether the risk awareness changed between 2004 and 2015 (Study III: patient care and medication use level). In Study I (2008-2011), the original MSSA tool (231 items under ten components) was first modified preliminarily and then by the Delphi expert panel (14 panelists) with four rounds. The modified MSSA tool was then pilot tested on 8 hospital wards of various specialties in a regional secondary care hospital. Several safety recommendations were documented, including the development of clinical pharmacy services. In Study II (2016), pre-admission medications of patients were reviewed by the pharmacist. BZD (29%) and antidepressants (28%) were involved in over half of the confirmed IP events. In Study III (2004 and 2015), patients were personally interviewed to determine how well they were aware of the potential risks of the BZD they were taking and whether the risk awareness had changed in the years between the two study periods. The study found that awareness of dependence (p=0.047), interaction with alcohol (p=0.001), dizziness (p=0.002), and developing tolerance (p=0.002) had improved, while awareness of the other potential risks remained unchanged. This thesis found that the modified and validated MSSA tool can be used to support building up safe medication practices in health care organizations, particularly establishing ward-based pharmacotherapy plans (Study I). The pharmacist-led CMR practice was found helpful in ED admissions for older residents (>65 years) in ED admissions (Study II). Older BZD users’ awareness of potential risks related to BZD use was improved between 2004 and 2015. Despite improved patient awareness, no significant change was found in their willingness to discontinue BZD therapy (Study III). National-level coordination is needed to integrate the modified MSSA tool for hospitals as a part of national patient safety policies in Finland (Study I). More research is required to assess whether CMR practice in the ED could impact preventable ED re-admissions (Study II). Future research should also investigate patients’ risk awareness of different high-risk medications, especially in older users (Study III).
  • Merivaara, Arto (Helsingin yliopisto, 2022)
    Freeze-drying is a widely used desiccation method for the preservation of protein drugs and vaccines in a dry state. In freeze-drying, the specimens are first frozen and then dried under a vacuum. The end products are dry products that can be preferably transported without a cold chain and stored at room temperature for years. Despite the common utilization of freeze-drying with proteins and vaccines, optimization of the process for cells and nature-origin biomaterials is problematic. This thesis aimed to change the current paradigm of trial-and-error in the freeze-drying of biomaterials and cells. The research focused on finding new excipients, applying new strategies to improve the results, adapting already existing process analytical technology (PAT) for the new specimen, and improving the current understanding of freeze-drying in general by providing explanations of the mechanism of actions of the excipients. The sub-projects (publications) of this thesis focused on characterizing the freeze-dried nanofibrillated cellulose aerogel, evaluating the effects of nanofibrillated cellulose hydrogel on cells during freeze-drying, setting up old methods for new applications for freeze-drying of nanofibrillated cellulose hydrogel, and studying the applications of freeze-dried nanofibrillated cellulose aerogel in 3D cell culturing. This thesis improved the theoretical understanding of freeze-drying, especially when considering the complex natural biomaterials and cells. The requirements of these specimens were recognized and existing theories of the mechanism of the protection provided by the excipients were applied to the samples. Furthermore, new theoretical aspects of the protection were proposed and the effect of nanofibrillated cellulose as a lyoprotectant was shown. In addition to the theoretical improvements, this thesis provided adaption of already existing PAT into the freeze-drying of biomaterials and an improved method for 3D cell culturing which can be potentially applied later for high-throughput screening of drug candidates.
  • Kari, Heini (Helsingin yliopisto, 2022)
    In Finland, one of the objectives of the government policy which directs ongoing health and social reform for the care of the older people is that people could live longer, and independently, in their own homes. Effective and cost-effective interprofessional care models that support older people to maintain their quality of life (QoL) and physical performance are needed in primary care. However, healthcare organisations are facing challenges in developing and implementing new care models due to traditionally paternalistic, disease-oriented and siloed organisational cultures. Recently, both internationally and in Finland, it has been argued that healthcare should be provided in a more people-centred way, which means holistic approach to care, including identifying of, and responding to, patients’ needs and preferences, shared decision making, and empowering people to participate in their own care. The aim of this multi-method study was to develop an interprofessional people-centred care model (PCCM), including clinical medication reviews and the contribution of a clinically trained pharmacist, for primary care to support care for multimorbid home-living older people (study I). Additionally, the aims were to evaluate how critical older people’s involvement is in identifying clinically significant drug-related problems (DRPs) (study II), and to evaluate the PCCM (study I) and its effectiveness and cost-effectiveness (study III). The study was part of the Care Plan 2100 research project, which was conducted in collaboration with Tornio health centre and the researchers of the Faculty of Pharmacy, University of Helsinki, between 2013 and 2018. Patients eligible to participate in the Care Plan 2100 research project were home-living multimorbid outpatients aged ≥ 75 years living in Tornio. Altogether 831 recruitment letters were sent, and 323 (39 %) patients who fulfilled the inclusion criteria returned the informed consent, after which they were randomised to intervention and usual care groups in a randomised controlled trial. Study I was a qualitative participatory action research study, in which an active involvement of healthcare professionals, i.e. participants (n=18), and researchers (n=3), was utilised to develop the PCCM in the health centre. The PCCM comprised: a self-management evaluation questionnaire sent before a home-visit; a person-centred patient interview at home together by a nurse and a pharmacist; a nurse-led health review and a pharmacist-led clinical medication review with recommendations; an interprofessional (a general practitioner, a pharmacist and a named nurse) case conference meeting; a care plan including health and medication plans; and health support and empowerment interventions delivered by the named nurse. People-centred care and collaborative goal-setting were the key approaches of the PCCM. According to the participating healthcare professionals, interprofessional collaboration in care planning and learning from each other were improved, and they appreciated the advantages of the new enhanced roles and skill-mix, including the involvement of a pharmacist. The PCCM enhanced patient involvement in their own care, and continuity of care, because of the coordinating role of the named nurse. Additionally, it improved identification of the patient’s needs and drug-related problems in the healthcare. The participants found building trust among healthcare professionals and between the professionals and the patients to be essential for implementing the PCCM. Study II evaluated how critical patient involvement is in identifying clinically significant drug-related problems (DRPs) in pharmacist-led clinical medication reviews (n=161), which were conducted for the participants in the intervention group at the baseline. Patient involvement was essential when identifying clinically significant DRPs. The researchers evaluated that of the 111 most significant clinical DRPs identified through patient interviews only 6% (n=7) could have been identified through reviewing the medication list only, and 16% (n=18) through reviewing the medication list and certain patient details (age, sex, medical conditions, laboratory results, drug allergies and contraindications). In study III, the effectiveness, QoL and physical performance, of the PCCM (intervention group n=151) was compared with that of usual care (usual care group n=126) in a randomised controlled trial (RCT) with a two-year follow-up. Additionally, cost-effectiveness of the PCCM was compared to usual care using cost-utility analysis. At the baseline, the mean age of the participants was 81 years and the average number of regular medications on the medication list was 10. At the baseline and at the 1-year and 2-year follow-ups, QoL (SF-36, 36-Item Short-Form Health Survey) and physical performance (SPPB, Short Performance Physical Battery) were measured. The SF-36 data were transformed into SF-6D scores to calculate quality-adjusted life-years (QALYs). Data on healthcare resource use were collected from the health centre patient records and transformed into costs using unit costs. Incremental cost-effectiveness ratio (ICER) was calculated. No statistically significant differences were observed in the QoL or in the physical performance between the groups. However, the cost-utility analysis showed that the PCCM dominates usual care; hence, the mean total costs were lower and generated QALYs higher in the intervention group than in the usual care group. The developed PCCM provides benefits not only for older people (e.g. involvement, continuity of care, better identification of the needs and drug-related problems), but also for healthcare professionals (e.g. advantages of the skill-mix, strengthened interprofessional collaboration, learning from each other, role enhancement), and for the society (cost-effectiveness). The PCCM should be recommended as an option for the care of multimorbid community-living older people in primary care.
  • Parkkinen, Ilmari (Helsingin yliopisto, 2022)
    Successful drug development requires numerous tests to deem a drug safe and efficacious. Before clinical trials, preclinical testing is needed to ensure that the drug can be safely tried out in humans. In preclinical testing, efficacy is also assessed to minimise the risk of a drug failing in clinical trials. Parkinson’s disease (PD) is a common age-associated neurodegenerative disease characterised by distinct debilitating motor symptoms caused by the dysfunction of the dopaminergic nigrostriatal pathway. For PD, a plethora of cellular and animal models have been developed to study the pathophysiology of the disease and to test potential new therapeutic interventions for treating the disease. New models are constantly created. However, methods to study outcomes also need to be developed and refined for reliable and reproducible results, which is pivotal to demonstrating the efficacy of drugs. This dissertation work developed new tools and refined current methods to study PD in preclinical models and studied the characteristics of the cytomegalovirus (CMV) promoter and a primary culture of postnatal dopamine neurons used to model PD. First, we used infrared analysis of optical densities to assess the striatal innervation of tyrosine hydroxylase-positive (TH+) fibres in rat brain sections, a useful alternative to colourimetric optical density analyses. We also developed a novel method based on convolutional neural network algorithms to count dopaminergic neurons from rodent brain sections. The number of neurons counted had a high degree of correlation with results obtained using other counting methods, and counting was substantially faster with the algorithm. Additionally, we developed reporter assays, both reporter plasmids, and cell lines, to measure the activity of a PD-associated drug target, Dicer. These assays, using either exogenous or endogenous fluorescent and bioluminescent indicators, were validated and produced comparable results to previously published similar assays in more physiologically relevant conditions. We also found out that a commonly used promoter in gene therapy, the CMV promoter, could be activated by neurotransmission. We showed in vivo that methamphetamine – a potent dopamine-releasing drug – activated the CMV promoter in the rat brain. Moreover, we observed differences in the distribution of the endoplasmic reticulum between different compartments of cultured mouse TH+ neurons. In summary, the methods and refined tools obtained in these studies expand the toolbox of researchers engaged in studying PD preclinically and may be applicable to other disease areas and human clinical studies as well. Furthermore, our findings on the activation of the CMV promoter are important to consider when designing gene expression systems, reporter assays, or gene therapies for preclinical PD studies utilising amphetamines. And finally, we gained novel insight into the ultrastructural characteristics of cultured postnatal dopamine neurons and provided a valuable resource for the research community.

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