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  • Backman, Nina (2011)
    Screening of drugs of abuse has to combine sensitivity, selectivity and repeatability. The conventional screening methods include immunoassay screening followed by a more sensitive confirmation method. The aim of the study was to develop a simple, yet sensitive sample preparation method for screening of benzodiazepines and amphetamine derivatives in urine samples with silicon micropillar array electrospray ionization chip (µPESI) coupled to mass spectrometric analysis. Another aim was to evaluate the suitability of µPESI in biological sample analysis. Ideally, the developed method would provide an alternative to immunoassay screening method in forensic urine analysis. The sample preparation methods were separately optimized for benzodiazepines and amphetamine derivatives. Methods used included solid- phase extraction with Oasis HLB cartridge and C18-phase containing ZipTip®-pipette tip, liquid-liquid extraction, and dilution and filtering without prior extraction. Optimization focused, however, on ZipTip®-extraction. The compounds were spiked in blank urine to their cut-off levels, 200 ng/ml for benzodiazepines and 300 ng/ml for amphetamine derivatives. For benzodiazepines, every extraction phase was optimized. The sample pH was adjusted to 5, the ZipTip® phase was conditioned with acetonitrile and washed with a mixture of water (pH 5) and acetonitrile (10 % v/v) and the sample was eluted with a mixture of acetonitrile, formic acid and water (95:1:4 v/v/v). For amphetamine derivatives, pH values of sample and solvents were optimized. The sample pH was adjusted to 10, the ZipTip® phase was conditioned with a mixture of water and ammoniumbicarbonate (pH 10, 1:1 v/v), washed with a mixture of water and acetonitrile (1:5 v/v) and the sample was eluted with methanol. The optimized methods were tested with authentic urine samples obtained from Yhtyneet Medix Laboratories and compared to the results of quantitative GC/MS analysis. Benzodiazepine samples were hydrolyzed prior to extraction to improve recovery. All samples were measured with Q-TOF Micro apparatus and hydrolyzed benzodiazepine samples additionally with microTOF apparatus in Yhtyneet Medix Laboratories. Based on the results the developed method needs more optimization to function properly. The main problems were lack of reproducibility and poor sample ionization. Manual sample preparation and adding to the chip sample introduction spot increased variation. Authentic benzodiazepine samples gave false negative and authentic amphetamine derivative samples false positive results. False negatives may be due to the lack of sensitivity and false positives due to the contamination of sample cone, chips or solvents.
  • Nieminen, Jenni (2016)
    The aged are the biggest age group of using psychotropics. The most used ones of these drugs are hypnotic and sedatives that consists mainly of benzodiazepines and related drugs. However, the aged are extremely sensitive for these drugs that are also noted as potentially inappropriate drugs for the aged in the national, but also in the various international recommendations and care guidelines. Despite the care guidelines, benzodiazepine compounds are usually used for years and often concomitantly. Research material of this longitudinal, observational study with two cohorts was collected from structured interviews at two similar acute wards in Pori City Hospital during one month in 2015. The research protocol of an early similar study which was conducted in 2004 was followed. Results of the two studies (2004 and 2015) were compared. Interviews were conducted among patients aged ≥ 65 years. Users of benzodiazepines or related drugs (2004: n=38, 2015: n=32) were further interviewed. The aim of this study was to compare the characteristics of the usage of benzodiazepine compounds in the aged between the years 2004 and 2015. In particular the medicines information sources and amount of information on these drugs i.e. knowledge on adverse drug reactions was studied and compared. Additionally a systematic review was conducted to explore the current evidence on interventions to rationalize the use of benzodiazepines and related drugs in the aged. In 2004, 54% of the interviewed patients (n=64) were using benzodiazepine compounds. However, in 2015 there were 34% (n=36) using. In 2015 regular usage of these drugs was decreased and irregular usage (given on an as-needed basis) was increased compared to the year 2004. None of the patients used long-acting benzodiazepines in 2015. Medicines information is provided notably more by doctors and pharmacies to 2004, but still the information focused more on benefits of drug other than adverse drug reactions. However, the patients' knowledge about the adverse drug reactions of benzodiazepine compounds has increased. The patients got presented adverse drug reactions known on mean of five in the year 2015, while the same value in 2004 was three. In the both years, the most of the patients were aware of the dependence these drugs may cause. The usage of benzodiazepine and related drugs in the aged has become better, but there is still need to improve multi-professional cooperation and applicate new interventions for rationalize the usage of benzodiazepinecompounds.
  • Saarenpää, Maija (2014)
    The body changes its response to medicine by age. Thus, medicines information for the elderly needs to differ from information targeted to younger adults. Package leaflets (PLs) are among the key sources of medicines information among general public. Although not generally recommended, benzodiazepines are commonly used by the elderly. The aim of this study was to evaluate the usability of benzodiazepine PLs and their information content from the elderly perspective. Additionally, the study aimed to explore how medicines information targeted to elderly in PLs aligns with the information targeted to health care professionals (HCPs). The study focused on PLs of benzodiazepines and their derivatives (diazepam, alprazolam, oxazepam, zopiclone and temazepam) that are commonly used but not recommended for the elderly. The usability of PLs was evaluated by using the Medication Information Design Assessment Scale (MIDAS). The informational content of PLs was studied by identifying all references for the elderly and comparing them to information targeted to HCPs in Database of medication for the elderly, Beer's criteria, Kapseli 35 publication, Martindale, Current Care Guideline for insomnia and the Summaries of Product Characteristics. The usability of the PLs in this study required several improvements. The mean of MIDAS-credits was 6,22 (n = 27; range 5,00-7,00), the scale maximum being 13. Sufficient line spacing and highlighting of important information were among the poorly represented elements. The occurrence of different font-sizes also varied. The most proficiently represented features included headings, contrast and the usage of upper and lower case in text. The PLs included in the content-analysis contained references to the elderly in all cases except one (n = 35). The references were categorized to general warnings, side effects and dose recommendations. They were in line with the information targeted to HCPs, but relatively short and often inadequate. Most PLs did not give an adequate overall picture of the medicine use among the elderly. The PLs for benzodiazepines need to be improved from the elderly perspective both in terms of information content and usability. Attention both from the medicine authorities and the pharmaceutical industry is required. Alternatively, separate drug-specific information leaflets for the elderly may be developed.
  • Sinisalo, Jade (2021)
    Pharmaceutical contaminants in waste and surface waters have been recognized as an emerging risk to environmental health. Bioaccumulation of pharmaceuticals increases the risk of adverse effects in off-target species, as the chemical concentration within the organism exceeds the concentration of the surrounding environment. An organism’s ability to metabolize foreign organic compounds influences the likelihood of bioaccumulation. Current methods for predicting bioaccumulation in aquatic organisms are labour intensive or too simplistic to cover the variety of chemical and physiological processes involved and may lead to over or underestimations of environmental risk. A promising approach to improve bioaccumulation predictions, without the need of excessive animal testing, is to incorporate in vitro biotransformation data into computational models. The primary aim of this study was to assess whether selected pharmaceuticals (diclofenac, gemfibrozil, haloperidol, levomepromazine, levonorgestrel, sertraline and risperidone), that are well metabolized in humans through key biotransformation pathways, are metabolized by rainbow trout (Oncorhynchus mykiss) liver enzymes under physiologically relevant conditions (11°C, pH 7.8). A secondary aim was to produce fish in vitro intrinsic clearance (CLint, in vitro) data, that could potentially be used as input in computational models to predict bioaccumulation. In vitro biotransformation was studied using a single vial approach according to the Organisation for Economic Co-operation and Development (OECD) Test Guideline 319B: Determination of in vitro intrinsic clearance using rainbow trout liver S9 sub-cellular fraction (RT-S9). Depletion of the test compounds were measured during a 3-hour incubation period. High-performance liquid chromatography with ultraviolet detection (HPLC–UV) was used for qualitative and quantitative analysis of the samples. Levomepromazine, levonorgestrel and sertraline showed significant substrate depletion compared to negative controls while gemfibrozil, haloperidol, and risperidone did not seem to be metabolized. The results for verapamil were inconclusive. Levomepromazine displayed a higher in vitro intrinsic clearance rate (26 ml/h/g liver) than diclofenac (6.2 ml/h/g liver). These results are in accordance with previous studies and support the notion that a direct comparability between fish and human metabolism cannot be assumed, highlighting the need of fish in vitro biotransformation studies. The apparent lack of in vitro metabolism of risperidone, haloperidol, and gemfibrozil combined with their lipophilicity suggest that they are more likely to accumulate within rainbow trout, compared with the compounds that showed depletion during the assays, although repetitions and additional studies are needed to confirm this.
  • Falck, Jenni (2019)
    Biological medicines are gaining ground in drug therapy. However, biological medicines are considerably expensive. Top ten drugs that caused the most drug reimbursement expenses included six biological drugs in Finland in 2017. A biosimilar is a biological medicine which is highly similar to another biological medicine (the reference medicine) that has already approved. Biosimilar prices are cheaper than the original medicines because their clinical development program does not have to be as extensive. A wide use of biosimilars save costs for both the patient and society without changing the effectiveness of drug therapy. The aim of this study is to investigate the automatic substitution of biological drugs containing the same active ingredient, especially from the point of view of medication safety. The study was conducted as a systematic literature review. Literature search was carried out by using Pubmed and Scopus databases. The literature was also searched manually from references of the articles and from the industry experts. The literature search produced a total of 454 articles after the deletion of duplicates. A title, abstract and full text screening was conducted by two independent researchers. All in all, 65 articles met the inclusion criteria of the study. As no studies were found on the automatic substitution of biological medicines from the point of view of medication safety, it was decided to include in the study original studies investigating the substitution of biological drugs from the point of view of doctors (n=8), pharmacists (n=3), patients (n=1) and various stakeholders (n=2). The original studies were all surveys except one study. In addition, the review included statements of various medical associations and organizations (n=23), descriptive reviews (n=27), and expert views (n=2) on the automatic substitution of biological drugs. According to the results of the original studies (n=13), it can be stated that automatic substitution is not considered generally acceptable. Doctors consider it is very important that the pharmacist informs them if substitution occurs. They also think it’s critical that doctors should be able to prevent substitution. Patients are also sceptical about the substitution of biological drugs. The quality of the original studies was assessed by the generalizability of the research results. The generalizability of the results of the original studies is weak due to the methodological shortcomings of the studies. Although the automatic substitution of biological drugs is legal in some countries, such as in France and in Australia, it has not been studied from the point of view of medication safety. In order to be safe to implement automatic substitution of biological medicines, more should be investigated on the subject. From the point of view of medication safety, healthcare professionals and patients will need further target group education on biosimilars. In addition, it should be clarified what kind of education the healthcare professionals and patients would need if the automatic substitution of biological medicines was to be realized.
  • Teittinen, Panu (2017)
    Psoriasis (Ps) and Psoriatic Arthritis (PsA) are chronic inflammatory diseases that are associated with profoundly impaired quality of life. Psoriasis is incurable and therefore the treatment aims to relieve patient's symptoms and improve the quality of life. Biologics are an efficacious treatment option for moderate-to-severe Ps and PsA but their relatively high costs limit their use. Health care resources are scarce and therefore economic evaluations provide crucial information for decision-makers. The objectives of this study was to determine 1) What is the incremental cost-effectiveness of biologics for moderate-to-severe Ps and PsA, and 2) What is the quality of cost-utility analyzes examining the subject. The theory section of this Master's thesis considers the current treatment alternatives for Ps and PsA and costs relating their use. The main principles and methodologies conducting economic evaluation and systematic review are also discussed in the theory section. The empirical section concerns the previous systematic reviews regarding the cost-effectiveness of biologics for the treatment of moderate-to-severe Ps and PsA, while also addressing the results of this systematic review and the quality of included cost-utility analyzes. 1425 references were found with the systematic literature search and 17 of them were included in this study. Eight articles concerned the cost-effectiveness of biologics for the treatment of Ps and nine articles for the treatment of PsA. All of the included studies used cost-utility modelling approach. Based on the results of this systematic review, biologics are cost-effective compared standard care for the treatment of severe Ps. Biologics are also cost-effective compared to the standard care for the treatment of moderate-to-severe PsA. However, future studies, independent of influence of pharmaceutical industry, are needed to confirm these results. The quality of cost-utility analyzes included in this study varied substantially. The main shortcomings related to reporting of the data included, modelling methodologies and the arguments for choosing the treatments compared. The strengths of this study are a comprehensive and systematic literature search, careful evaluation of included data and the transparency of methodologies. The main weaknesses relate to generalizability of the results and the possibility of biases. This study updates the current knowledge of cost-effectiveness of biologics for Ps and PsA, while providing a good foundation for the future studies to be conducted.
  • Joensuu, Jaana (2013)
    Rheumatoid arthritis (RA) is a chronic autoimmune disease with prevalence of 0.8% among Finnish adult population. Consequent medical treatment, joint replacement surgery and productivity losses lead to significant expenses for society. While biological treatments for RA are costly, they can improve patients' quality of life and work participation. Economic evaluations provide information on the benefits and costs of these expensive treatments to aid optimal utilization of limited healthcare resources. This master`s thesis comprises the description of the Finnish Current Care Guidelines for RA, the cost of biological treatments and the principles of economic evaluations and health technology assessment. A systematic literature review was performed to identify existing studies examining the cost-effectiveness of biological treatments for RA. Of the 4890 references found with the literature search, 38 original studies and 9 previous systematic reviews were included in the current systematic literature review. Details of the methods as well as information on treatments, costs, benefits and incremental cost-effectiveness were extracted. Quality of the original studies was evaluated using quality assessment tools. Ninety percent (34/38) of the original studies used cost-utility modeling approach. Quality of life estimates were derived from RA specific health assessment questionnaire in a majority of the studies. Based on the current systematic literature review, the evidence on the cost-effectiveness of biological treatments is inconsistent. The incremental cost-effectiveness of the tumor necrosis factor (TNF) blockers was 13 500-772 000 €/ quality adjusted life year (QALY) in comparison to conventional disease modifying anti rheumatic drugs (DMARD) among patients without previous treatment with DMARDs. Several studies reported incremental cost-effectiveness ratios over 100 000 €/QALY in this population. Among patients with insufficient response to DMARDs, TNF blockers provided incremental cost-effectiveness ratios between 6 700 and 317 000 €/QALY. In most studies Rituximab was found to be a cost-effective alternative in contrast to other treatments among patients with insufficient response to TNF blockers. Biological treatments are not cost-effective among patients naïve to conventional DMARDs. Meanwhile, in patients with previous DMARD failure TNF-blockers might be cost-effective. The evidence on the cost effectiveness of biological treatments supports Finnish Current Care Guidelines. The quality assessment of the included studies revealed several sources of bias, consequently reducing the validity of the studies. Only a few of the conference abstracts in current subject has been published later as an article indicating existence of reporting bias. This study has several strengths. First, a comprehensive literature search was performed. Second, the quality of included studies was carefully evaluated. Finally, the methods and reporting are transparent. Weakness of the current study is one person extracting data and assessing the quality of the studies, which may reduce the reliability of this study. This systematic literature review is a basis for future studies examining cost-effectiveness of biological treatments in Finnish healthcare system.
  • Rosqvist, Linn (2021)
    Marine invertebrates are a good and relatively unexplored source of bioactive compounds. These bioactive secondary metabolites can have unique structures and mechanisms of actions, since they are produced by organisms, which means their structures are not limited by the fantasy of chemists. Therefore, bioactive secondary metabolites isolated from marine invertebrates are attractive for drug development. Still, there are challenges regarding bioprospecting marine invertebrates. For example, the amount of material is limited and the environment as well as the biodiversity has to be taken into account when gathering the organisms. The aim of this thesis was to perform the first steps of bioprospecting marine invertebrates; extraction, fractionisation, analysis of bioactivity and identification of bioactive metabolites. The samples used in the experiment, gathered from three different locations, were of the sponge Caulophacus arcticus. The goal was not only to identify one or more bioactive metabolites for eventual further analysis, but also to compare the bioactivity of the samples gathered from different locations. The fractionisation was performed using flash fractionisation, which resulted in eight fractions of each extract. These fractions were tested for anticancer, antibacterial and biofilm inhibiting properties. The bioactivity of the fractions was analysed by performing cell viability assays (MTS assays) on four cell lines, antibacterial growth inhibition assays on five strains of bacteria and biofilm inhibition assays on biofilm of S. epidermidis. The active fractions, the fraction right before and after them and the corresponding fractions of the two other samples were further analysed using UHPLC-HR-MS, in order to identify eventually bioactive compounds and determine the elementary composition of these compounds. The most interesting fractions, from which one or more bioactive compounds were to be identified first, were prioritised based on the bioactivity assays. One compound, which was identified as potentially bioactive with a potentially novel elementary composition, was chosen as a target compound for further analysis. Based on the results, it was also possible to draw the conclusion that there were variations as well as similarities in the bioactivity of samples gathered from different locations. Still, further research is needed to determine if the bioactivity of the same fractions from different samples was caused by the same compounds or not. Even if there are challenges regarding bioprospecting of marine invertebrates, it is still useful to keep studying them in order to find new, bioactive compounds. There is a huge need of new drugs, especially for treating cancer and bacterial infections. Therefore, experiments such as this are relevant also in a bigger perspective. The target compound identified in the experimental part of this thesis might be further analysed in order to determine whether it is bioactive and whether it is profitable to develop it further.
  • Mäkinen, Arttu (2018)
    This is a systematic review aiming to investigate the efficacy, effectiveness, and safety of biosimilars in the treatment of inflammatory bowel diseases. Biosimilar drugs used to treat inflammatory bowel diseases include biosimilar infliximab and biosimilar adalimumab. Biosimilar infliximab has been authorized by the European Medicines Agency (EMA) in 2013 and by the US Food and Drug Administration (FDA) in 2016. Biosimilar adalimumab has been authorized by EMA and FDA in 2017 and, at the time the literary search for this systematic review was conducted no studies were found regarding the treatment of adalimumab biosimilar for inflammatory bowel diseases. To acquire marketing authorization for biosimilars, it must be proven that the biosimilar is biologically similar to the original medicinal product. Bioequivalence is demonstrated through physicochemical trials and clinical trials. However, clinical trials do not have to be performed with all of the indications for which the original medical product is registered. After proving bioequivalence with one or more indication it is possible to extrapolate the biosimilar to be used in all of the original medical products indications. This has raised the question of whether biosimilars are really comparable to the originator in indications for which no clinical trials have been conducted. This systematic review was implemented using the Cochrane Handbook for Systematic Reviews and Interventions. Systematic literature searches were made in Cochrane, Medline (Ovid®), PubMed and Scopus databases on 12.05.2017. 14 observational studies, one systematic review and a randomized clinical trial that met the inclusion criteria were included in the systematic review. The quality of the publications was evaluated using the STROBE-, NOS- and CONSORT-checklists and information regarding the efficacy, effectiveness and safety of biosimilars was extracted. CD-patients receiving tumor necrosis factor alpha inhibitors for the first time, the clinical response was achieved in 50.0 % to 97.2 % of patients depending on patient population and the duration of treatment. Similarly, for UC-patients, the clinical response was achieved in 62.2 % to 100.0 %. The clinical remission was achieved among 28.9 % to 84.4 % of CD-patients and among 28.9 % to 84.4 % of UC-patients, depending on patient population and treatment follow-up. After the switch from original infliximab to biosimilar, the proportion of patients in clinical remission during follow-up ranged from 62.3 % to 100.0 % in CD-patients and from 45.5 % to 100.0 % in UC-patients. Clinical remission was sustained throughout the whole follow-up in 70 % to 100 % of CD-patients and 66.7 % to 92.0 % of UC-patients. The incidence of adverse events leading to the discontinuation of drug treatment was between 0.0 % and 25.0 %, and the incidence of all adverse events ranged from 0.0 % to 93.6 % in CD- and UC-patients. Biosimilar infliximab seems to be comparable to the original product regarding the efficacy, effectiveness and safety. This result is supported by the systematic literature review published earlier. Conducting a meta-analysis of the information contained in this systematic literature review could have led to a more final decision considering efficacy, effectiveness and safety of biosimilar-infliximab in the treatment of inflammatory bowel diseases.
  • Luukkanen, Saana (2021)
    Pharmaceutical costs have been rising globally every year. A significant portion of drug costs is caused by biological drugs, which are often very expensive, yet essential in the treatment of many chronic diseases. Biosimilars are clinically equivalent to biological originator products and are expected to alleviate the increase in drug costs. The biosimilar development process does not need to repeat the complete development process of the originator product, allowing the biosimilar to enter the market at a lower price than the originator after the patent and data protection period for the originator ends. The aim of this study was to find out what impact the market entry of biosimilars has on the prices of the reference products in outpatient care in Finland, and to investigate whether biosimilars create price competition for biological drugs. In addition, the study examined how the prices and market shares of outpatient biosimilars have developed in Finland. The study examined the development of price and market shares for adalimumab, etanercept, insulin glargine, insulin lispro, enoxaparin, filgrastim, pegfilgrastim, somatropin, follitropin alfa, teriparatide and epoetin biosimilars and their reference products. The data for the study was acquired from IQVIA and it covered pharmacy wholesale data between 1.1.2009–31.8.2020 for products under investigation. The weighted average wholesale price and monthly wholesale amounts were determined for each product, and the development of the price and market shares were analyzed. In addition, a linear segmented regression analysis was performed to examine the impacts of market entry of biosimilars on the prices of the reference products. According to the study, the prices of the reference products mainly decreased after the biosimilar entered the market. If the price of the reference product did not fall, it lost its reimbursement under the Health Insurance Act. The market shares of the reference products were marginal when they were no longer reimbursed. The prices of biosimilars did not change as much as the prices of reference products, and for most active substances biosimilar prices remained stable or decreased. The use of biosimilars varies widely between different biologics. The study found that prices of reference products were decreasing mainly as a result of various changes in drug policies. Therefore, biosimilars were not seen to generate genuine price competition between biological products. In many of the drug groups examined, the market shares of biosimilars had future growth potential.
  • Heiskanen, Vilma (2021)
    Binge eating disorder (BED) is the most common eating disorder characterized by compulsive recurrent binge eating episodes with the sense of a lack of control. During a binge eating episode, one eats a larger amount of food, typically high in fat and/or sugar, than would normally be eaten in a discrete period of time. After the episode, negative emotions, such as shame and self-disgust, are present. However, BED does not include compensatory behavior, such as vomiting or excessive exercise. Due to compulsive and uncontrollable eating behavior, it has been suggested that BED represents a food addiction. Eating energy-dense food activates the dopamine, opioid, and endocannabinoid systems in the brain. This elicits the activation of the reward process. Some drugs and medications affect the same neurotransmitter systems, which may produce neuronal alterations in the reward process, leading to an addiction. Several studies have found that cannabidiol (CBD) reduces the self- administration of cocaine, morphine, alcohol, and sucrose in rodents, suggesting an effect on the reward-response. Some of these effects have been shown to be mediated by cannabinoid receptor 2 and TRPV1 receptor. However, the effects of CBD on bingeing behavior have not been studied up to date. The aim of the study was to investigate the effect of CBD on homeostatic feeding and binge eating behavior in C57BL/6 mice. Five separate experiments were conducted. The first experiment investigated the effect of CBD (15, 50, and 150 mg/kg, i.p.) on locomotor activity in a modified open field test over a 2-hour period. In the second test, the effect of CBD (15, 50, and 150 mg/kg, i.p.) on homeostatic feeding was monitored in non-bingeing mice. Next, a limited intermittent access binge eating model without food deprivation or stressors was inducted. Mice had access to laboratory chow ad libitum, but a high energy diet (high in fat, HED) was presented in 24-hour periods every 5-8 days. Then the effect of CBD (15, 50, and 150 mg/kg, i.p.) on HED and chow intake in bingeing mice was investigated. In the fourth experiment, seven days following the administration, the after effect of CBD was studied by monitoring food intake without CBD treatment. Finally, it was investigated whether the effect of CBD can be inhibited by TRPV1 receptor antagonist AMG9810 (1 mg/kg, i.p). In each test, the food intake was monitored at the time point 0,5, 2,5, and 24 h after CBD treatment. Also, water consumption was measured in each experiment. The results revealed that CBD does not affect locomotor activity or homeostatic feeding at a dose of 15, 50, or 150 mg/kg (i.p). However, the results showed that CBD reduces the intake of HED in a dose-dependent manner (15, 50, or 150 mg/kg; i.p.) and, possibly, increases chow intake. No after effect was observed seven days following the administration. Most likely, TRPV1 does not mediate the effect of CBD on HED intake. Furthermore, no significant effects on water intake were observed. In this study, the core aims were to evaluate whether CBD affects homeostatic feeding or binge eating behavior in mice. The results provided a novel insight into the effects of CBD. The findings indicate that the acute systemic administration of CBD reduces HED intake, and possibly, simultaneously increases chow intake, suggesting a balancing effect on feeding in bingeing mice. However, the role of TRPV1 in this effect remains unclear, and further studies are needed.
  • Paukkonen, Heli (2013)
    Casein based formulations are promising materials for controlled drug release. Caseins are the major milk proteins, and their biocompatibility, low toxicity and natural metabolism in physiological systems make caseins extremely suitable materials for pharmaceutical formulations. Polyelectrolyte complex nanoparticles can be prepared under very mild conditions, and they are stable in the gastrointestinal tract, which makes them suitable carrier materials for oral delivery and controlled release of peptide and protein drugs. Aim of this work was to synthesize casein-poly(acrylic acid) polyelectrolyte complex nanoparticles in different mass ratios, and to study the release profile of a model compound rhodamine 6G from these nanoparticles. The casein shell of the nanoparticles was crosslinked with two different crosslinkers, because the objective was to study the effect of surface modification on size of nanoparticles as well as on the release profile of the model compound. The goal was to achieve controlled release of the model compound by modifying the thickness and the density of the casein shell structure. Size and size distribution of nanoparticles was studied by dynamic light scattering. Surface charge was studied by electrophoretic mobility measurements. Morphology was characterized with electron microscopy, and the effect of the casein shell thickness on the release of rhodamine 6G was studied with dialysis method. The synthesized nanoparticles had spherical morphology, but the size distribution was wide. The release of rhodamine 6G was slower from the nanoparticles when compared to the release of reference free rhodamine 6G, but the effect of casein shell thickness on the release of loaded rhodamine 6G remained partially unclear. However, it seems possible to achieve controlled release of encapsulated compounds from casein-poly(acrylic acid) nanoparticles with optimal surface modification in the future.
  • Silmu, Veera (2021)
    Parkinsonin tauti on hitaasti etenevä hermorappeumasairaus, jossa mustatumakkeen dopamiinihermosolut tuhoutuvat. Taudille on tyypillistä dopamiinihermosoluissa esiintyvät Lewyn kappaleet, jotka koostuvat pääasiassa väärin laskostuneesta ja kasautuneesta alfasynukleiiniproteiinista. Myös neuroinflammaation uskotaan olevan osa Parkinsonin taudin patofysiologiaa. Nykyiset lääkkeet vaikuttavat ainoastaan taudin oireisiin, joten tarve uusille lääkkeille on suuri. Pilottikokeen tarkoituksena oli selvittää aiheuttaako adenoassosioidun virus- (AAV) vektorin alfasynukleiinin ja alfasynukleiinifibrillien yhdistelmämalli rotilla liikehäiriöitä ja tyrosiinihydroksylaasi- (TH) positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa sekä saadaanko mallilla aikaan neuroinflammatorinen vaste. Varsinaisen pitkän kokeen tarkoituksena oli selvittää aivojen dopamiinihermokasvutekijän (CDNF) mahdollinen neurorestoratiivinen vaikutus tässä mallissa. Alfasynukleiinin kasautumispatologian tasoa ja CDNF:n neurorestoratiivista vaikutusta selvitettiin käyttäytymiskokeilla sekä mustatumakkeen ja aivojuovion TH-vasta-ainevärjäyksillä. Yhdistelmämallista aiheutuvaa neuroinflammatorista vastetta selvitettiin ionisoidun kalsiumia sitovan adapterimolekyylin 1 (Iba1) ja gliaalisen fibrillaarisen happaman proteiinin (GFAP) vasta-ainevärjäyksillä. Pilottikokeen sylinterikokeessa yhdistelmämalli ei indusoinut liikehäiriötä, mutta pitkän kokeen askel- ja sylinterikokeessa mallin osoitettiin aiheuttavan unilateraalille leesiolle tyypillinen liikehäiriö. Pilottikokeen ja pitkän kokeen TH-vasta-ainevärjäyksissä mallin osoitettiin aiheuttavan TH-positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa. Nämä tulokset osoittavat, että yhdistelmämallilla saadaan aikaan alfasynukleiinin kasautumispatologiaa. Pilottikokeessa osoitettiin myös, että yhdistelmämallilla saadaan aikaan neuroinflammatorinen vaste, mikä osoittaa, että malli soveltuu hyvin uusien lääkkeiden vaikutuksen tutkimiseen Parkinsonin tautiin liittyvässä neuroinflammaatiossa. Pitkän kokeen sylinterikokeessa AAV-CDNF:llä ei ollut vaikutusta mallista aiheutuvaan liikehäiriöön. Sen sijaan askeltestissä kämmenen suunnan mittauksessa AAV-CDNF korjasi liikehäiriötä. AAV-CDNF ei kuitenkaan suojannut TH-positiivisia hermosoluja mustatumakkeessa tai hermopäätteitä aivojuoviossa, minkä perusteella johtopäätöstä CDNF:n neurorestoratiivisesta vaikutuksesta ei voida tehdä.
  • Tallberg, Thomas (2017)
    Transactive DNA Response Element Binding Protein 43 (TDP-43) is a RNA binding protein participating in gene expression on a transcriptional level. It is localized in the cell nucleus. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons. In most ALS patients TDP-43 becomes localized into the cytoplasm of neurons and glia cells. The TDP-43 rat ALS model provide insight in ALS disease progression and molecular mechanisms. This animal model has been characterized previously in the literature. Cerebral Dopamine Growth Factor (CDNF) is a neuroprotective and restorative protein in rat animal model of Parkinson's disease. CDNF may have an impact on disease progression in ALS. One of the goals in this work was to recharacterize the TDP-43 rat ALS model and to try repeat published data. The other aim of this work was to treat TDP-43 rats with intraventricular chronic infusion of CDNF, and to compare symptom progression with TDP-43 rats treated with phosphate buffered saline. Behavioral assays were done trice a week and when rats reached endpoint, spinal cords were removed. Motor neuron counting and detection of stress granule formation were investigated in spinal cords with immunohistochemistry. Also, the volume of CDNF diffusion in rat brain after chronic intraventricular CDNF infusion was investigated with immunohistochemistry. In the characterization part, symptom progression was repeated in a similar manner as it has been reported previously. CDNF treatment could not stop the symptom progression nor slow down the progression of symptoms in TDP-43 rats. Motor neuron counting revealed a heavy loss of motor neurons in the lumbal part of the spinal cord in both treatment groups. Diffusion of CDNF was very poor in the rat brain. Higher doses of CDNF and proper administration depth in the brain or route of administration should be reconsidered in the future.
  • Montonen, Ella (2015)
    Endoplasmic reticulum stress (ER-stress) is the result of accumulation of unfolded and misfolded proteins in the ER. The unfolded proteins activate the unfolded protein response (UPR), which seeks to reduce the protein load in the ER and reduces ER-stress. When ER-stress is prolonged, the UPR will activate apoptosis. Amyotrophic lateral sclerosis (ALS) is a rare, progressive neurodegenerative disease that affects lower and higher motorneurons. The cause of ALS is unknown but ER-stress is known to play a role in the disease progression. CDNF is a new neurotrophic factor, which is known to play a role in protein folding in the ER. CDNF is neuroprotective and neurorestorative in animal models of Parkinson's disease. Thus, CDNF is a potential new drug candidate for treating ALS. The aim of this work was to examine the effect of CDNF on disease state and life span in transgenic SOD1(G93A)-mice. CDNF or PBS was injected into the mouse's ventricle in stereotaxic surgery when the mice were about 90 days old. Clinical status and motor coordination was monitored twice a week throughout the study. The mice were dissected when they reached the end point that was set for the study. Deepfrozen gastrocnemius muscles were stained with antibodies, to examine the integrity of the neuromuscular junctions (NMJ). Quantitative PCR (qPCR) was executed on deepfrozen spinal cord and motor cortex samples to measure the expression of ER-stress genes. The results showed that CDNF improves motor coordination and delays disease progression in SOD1 female mice. The NMJs were notably more damaged in SOD1 mice than in wild type mice, but CDNF did not have any significant effect on NMJ integrity. ER-stress could be observed in the spinal cord and motor cortex of SOD1 mice and CDNF decreased ER-stress in the motor cortex. CDNF did not decrease ER-stress in the spinal cord where the expression of apoptosis related genes was increased. Thus, CDNF is a potential new drug candidate for treating ALS and it should be studied further.
  • Järvinen, Erkka (2016)
    UDP-glucuronosyltransferases (UGTs) catalyse glucuronidation reactions between glucuronic acid and drug molecules, which contain nucleophilic groups, mostly hydroxyls, amines or carboxylic acids. Glucuronidation is the most important reaction in the conjugative drug metabolism. Because these conjugates are not usually able to cross cell membranes passively, they need active efflux transport. Efflux transporters mostly belong to superfamily of ATP-binding cassette transporters (ABC). Subfamily C of ABC transporters (ABCC) are known to be involved in efflux transport of glucuronides. Especially MRP2 (ABCC2) and MRP3 (ABCC3) play key roles in the elimination of glucuronide conjugates of drugs. MRP2 is localized in the apical membranes of hepatocytes and enterocytes, whereas MRP3 is localized in the basolateral membranes of the respective cells. On the other hand, UGT1A1 and UGT2B7 are highly expressed in liver and small intestine and are the most important UGTs in drug metabolism. It is known, that UGTs and efflux transporters work together forming interplay to eliminate drugs. Therefore, studying both of them in the same in vitro system is in important focus of drug metabolism studies. The Madin Darby canine kidney cell line (MDCK) is one of the standard in vitro tools in drug metabolism studies. In this study, MDCK was chosen for a cell line to co-express UGTs (UGT1A1 or UGT2B7) and efflux transporters (MRP2 and MRP3 simultaneously. Therefore, cloning of the UGT2B7 cDNA and the ABCC3 cDNA encoding MRP3 was aimed in this study. On the other hand, the UGT1A1 cDNA was already cloned in-house and MRP2 expressing MDCK cells were established earlier. Cloning of the UGT2B7 cDNA was not successful in this study despite of several different strategies such as PCR-amplification of the cDNA fragment using kidney or liver sscDNA as template. Cloning of the ABCC3 cDNA encoding MRP3 was achieved and a mammalian expression vector containing this cDNA was constructed. In addition, the mammalian expression vector containing the UGT1A1 cDNA was used to establish MDCK-UGT1A1 cells and this cell line was characterized regarding the expression of UGT1A1 mRNA and UGT1A1 protein amount. Furthermore, establishment of MDCK-UGT1A1-MRP2 cell line was attempted in this study without success. The mammalian expression vector containing the ABCC3 cDNA encoding MRP3 could be used for future experiments to achieve novel cell lines such as MDCK-UGT1A1-MRP3 and MDCK-UGT1A1-MRP2-MRP3 for drug metabolism studies. In addition, the novel cell line MDCK-UGT1A1 could be used for drug metabolism studies in further experiments, but also as a cell line for further establishment of above cell lines. On the other hand, the cloning of the UGT2B7 cDNA needs optimization and several different strategies should be used to achieve the mammalian expression vector containing this cDNA.
  • Holvikari, Kira (2015)
    MRP2 is an efflux-transporter from the group of ABC-transporters located in the apical side of cell membranes mainly in the liver, intestine, kidneys and lungs. This transporter is associated with multidrug resistance, a phenomenon where the absorption of a drug to the cell is prevented by the transporter as it transports the compound out of the cell. To overcome this phenomenon, inhibitors and substrates for MRP2 are constantly studied. Several flavonoids have been presented of being inhibitors and the research of these compounds continues. Pharmaceutical excipients are also another major group of compounds that possess inhibitory effects towards MRP2. Excipients, as well as flavonoids, are an increasingly studied section of drug interactions and today it may be evaluated that excipients are not thought as inert compounds as has been presented for several years. For now the research of MRP2 interactions focuses mainly on in vitro studies. In the experimental part of this thesis the effects of natural compounds and pharmaceutical excipients are studied towards MRP2 with the vesicular transport assay (VT-assay) with MRP2- Spodoptera frugiperda 9 (Sf9)-membrane vesicles using 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF) as probe. A total of 157 compounds are screened using this in vitro method and hits are further experimented studying IC50 and Ki values. Potential compounds are also tested with two types of particle size measurements (Dynamic light scattering and nephelometer) to evaluate inhibition caused by microaggregates. Some compounds are also studied with liquid chromatograph-mass spectrometry (LC-MS) to determine possible substrates for MRP2. 19 (12%) hits were found from the library of 157 compounds. These hits included 6 stimulators (CDCF transport increased ≥ 150%) and 13 inhibitors (CDCF transport decreased ≤ 50%). IC50 determination was conducted for 12 inhibitors with best-fit values of: Ellagic acid 10.4 µM, gossypin 17.4 µM, morin dihydrate 19.4 µM, myricetin 27.1 µM, nordihydroguaiaretic acid (NDGA) 36.2 µM, octyl gallate 20.3 µM, silybin 52.3 µM, pluronic ®F98 6.9 µM, lutrol F127 ~ 8.2 µM and tannic acid 1.99 µM. Ki determination was conducted for 3 compounds where best-fit values were myricetin 42.9 ± 47.4 µM, gossypin 19.4 ± 12.5 µM and tannic acid 0.0538 ± 0.0398 µM. Ki determination allowed determination of inhibition type: competitive inhibition for tannic acid and gossypin, noncompetitive inhibition for myricetin. Particle sizes studied with dynamic light scattering (DLS) and a nephelometer did not show any significant aggregate formation and inhibition by that mechanism can be ruled out granted that the measurement method should be optimised. Stimulators baicalein, baicalin, digitoxigenin and inhibitors myricetin, gossypin and tannic acid were studied finally with the VT-assay with LC-MS as detector in search of substrates for MRP2. With significant changes in ‚àíATP and +ATP at 50 µM was gossypin. To conclude, gossypin possesses competitive inhibition towards MRP2 and exhibits sings of being a substrate for the transporter as well. Further studies need to be performed to confirm these findings.
  • Sainio, Janne (2011)
    Lactose is probably the most used tablet excipient in the field of pharmacy. Although lactose is thoroughly characterized and available in many different forms there is a need to find a replacer for lactose as a filler/binder in tablet formulations because it has some downsides. Melibiose is a relatively unknown disaccharide that has not been thoroughly characterized and not previously used as an excipient in tablets. Structurally melibiose is close to lactose as it is also formed from the same two monosaccharides, glucose and galactose. Aim of this research is to characterize and to study physicochemical properties of melibiose. Also the potential of melibiose to be used as pharmaceutical tablet excipient, even as a substitute for lactose is evaluated. Current knowledge about fundamentals of tableting and methods for determinating of deformation behavior and tabletability are reviewed. In this research Raman spectroscopy, X-ray powder diffraction (XRPD), near-infrared spectroscopy (NIR) and Fourier-transform infrared spectroscopy (FT-IR) were used to study differences between two melibiose batches purchased from two suppliers. In NIR and FT-IR measurements no difference between materials could be observed. XPRD and Raman however found differences between the two melibiose batches. Also the effects of moisture content and heating to material properties were studied and moisture content of materials seems to cause some differences. Thermal analytical methods, differential scanning calorimetry (DSC) and thermogravimetry (TG) were used to study thermal behaviour of melibiose and difference between materials was found. Other melibiose batch contains residual water which evaporates at higher temperatures causing the differences in thermal behaviour. Scanning electron microscopy images were used to evaluate particle size, particle shape and morphology. Bulk, tapped and true densities and flow properties of melibiose was measured. Particle size of the melibiose batches are quite different resulting causing differences in the flowability. Instrumented tableting machine and compression simulator were used to evaluate tableting properties of melbiose compared to α-lactose monohydrate. Heckel analysis and strain-rate sensitivity index were used to determine deformation mechanism of melibiose monohydrate in relation to α-lactose monohydrate during compaction. Melibiose seems to have similar deformation behaviour than α-lactose monohydrate. Melibiose is most likely fragmenting material. Melibiose has better compactibility than α - lactose monohydrate as it produces tablets with higher tensile strength with similar compression pressures. More compression studies are however needed to confirm these results because limitations of this study.
  • Autzen Virtanen, Anja (2023)
    Poorly water-soluble drugs are challenging to formulate as solid oral dosage forms because of their inadequate solubility in the gastro-intestinal tract. Amorphous solid dispersions (ASDs) are a proven method of increasing the oral bioavailability of poorly water-soluble drugs through drug supersaturation. Downstream processing of ASDs into oral tablets has gained academic interest in recent years. However, minitablets, which are tablets smaller than 4 mm in size, have not received the same level of attention. Minitablets have been cited as a promising dosage form for children, the elderly and in veterinary use because of their good compliance, flexible dosing, and ease of swallowing. In this work, 15 different blends of microcrystalline cellulose and lactose have been characterized for their suitability in the formulation of an ASD of spray-dried poorly soluble indomethacin in PVP K 29-32 or HPMCAS MF as minitablets. Minitablets were compressed at the compression forces ~1000 N and ~1500 N. The flowability of the blends were evaluated based on the Carr’s indices, Hausner ratios and angles of repose. From the most promising blends, 3.0 mm placebo minitablets were manufactured. A mixing test using colored beetroot powder was used to determine the optimal mixing time. The finished tablets were tested for their uniformity of mass, crushing strength, height, and disintegration. Based on their Carr’s indices and Hausner ratios, Vivapur 105, Vivapur 200, Pharmatose 200M and Pharmatose 80M had the best flowabilities. Placebo minitablets were successfully manufactured from blends of these excipients except for the 1:1 ratio of Vivapur 105/Pharmatose 80M. The mixing test indicated that the optimal mixing time is 20 to 25 minutes. The mass variation for all placebo batches except the 1:3 ratio of Vivapur 105/Pharmatose 80M was less than 10 percent from the average mass and most batches therefore fulfilled the uniformity of mass requirement of the European Pharmacopoeia. For five of the batches, the variation was within 2.80 percent. The average crushing strengths were between 32.4 N and 79.7 N and increased with increasing compression force. All batches of placebo minitablets disintegrated within 6 to 19 seconds on average except the 1:3 ratio of Vivapur 105/Pharmatose 80M which took 90 seconds to disintegrate. Minitablets filled in capsules disintegrated within 124 to 167 seconds on average except for the previously mentioned slower disintegrating batch which disintegrated in 477 seconds. All placebo minitablets, individual or loaded into capsules disintegrated within 15 minutes thereby fulfilling the requirement of the European Pharmacopeia. When considering the results obtained for placebo minitablets, the 3:1 ratio blend of Vivapur 200/Pharmatose 200M with 0.5 % (w/w) magnesium stearate was found to be the most promising candidate for ASD formulation. This formulation was subsequently used as the basis for the manufacture of 3.0 mm minitablets containing 6.22 % (w/w) of a spray-dried dispersion of indomethacin and PVP K 29-32. Except for one outlier, the mass variation of these minitablets fell within 2.37 % of the average mass, thereby fulfilling the requirement of the European Pharmacopoeia. Single indomethacin-PVP minitablets disintegrated within 6 minutes and 38 seconds, and capsules containing twelve minitablets disintegrated within 10 minutes and 37 seconds, which also is accordance with the pharmacopoeia. At 80.3 to 80.4 N the crushing strength was at the upper end of the targeted range, but still adequate. Thus, the formulation developed in this study appears promising for the manufacture of minitablets containing 6.22 % of an amorphous indomethacin-PVP dispersion. This study demonstrated that minitablets could be manufactured from a spray-dried solid dispersion despite its poor flowability.