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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.

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.

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.

Adoptive cell therapy utilizes the patient's own immunological system in the treatment of cancer. T cells expressing the chimeric antigen receptor (CAR) are produced from the patient's own T-cells. The CAR gene is introduced into the T cells by a gene transfer vector, which results in the T cells expressing the CAR molecule that recognizes the antigen on the surface of the cancer cell. When CAR-T cells are returned to the body, they recognize the cancer cell with the CAR molecule and destroy it. CAR-T cell therapy has shown promising results in the treatment of malignant hematological cancers. The white blood cells used as starting material for CAR-T cells are collected from the patient using a specially designed leukapheresis device. The collected leukapheresis product is transported to the CAR-T cell manufacturing site as soon as possible, either fresh or frozen. The aim of this stability study of leukapheresis products was to determine the effect of storage time and temperature on the quality of fresh cell products regarding cell number, viability and composition. In addition, the goal was to determine the optimal storage temperature and the shelf life of leukapheresis product to ensure high quality cell starting material for CAR-T cell production. The study was performed by dividing the leukapheresis products into two cell bags immediately after collection, one stored at +15–25 °C and the other at +2–8 °C for five days. The leukapheresis products were examined at five different time points (0, 25, 49, 73 and 121 h) for white blood cell count, viability, apoptosis and white blood cell composition. The microbiological purity of the cell products was examined after leukapheresis. The leukocyte composition was stable, viability and cell yield over 80 % for at least 72 hours at +2–8 °C storage temperature. Although small proportions of cells were apoptotic after the 48 hours of storage +2–8 °C, the leukapheresis products contained more than 80 % viable leukocytes after 72 hours and over 70 % after 120 hours. Leukapheresis products remained stable for 48 hours at +15–25 °C, after which their leukocyte composition changed, leukocyte viabilities and yields decreased. The viabilities of the leucocytes were above 90 % for 48 hours at +15–25 °C, but at the 73 h time point, only half of the cells were viable. The optimum storage temperature for leukapheresis products was +2–8 °C, at which white blood cells remained in good quality for 72 hours. These results can be used to set quality requirements for the cell source material of CAR-T cell product and to plan the transport from the collection site of the leukapheresis to the CAR-T cell production site.

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.

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.

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.

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.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease in which both upper and lower motor neurons degenerate gradually. The disease leads to a total paralysis of almost all skeletal muscles and to death within 3-5 years after onset. At the moment there are two disease modifying medicines available, riluzole and edaravone. Neither is able to cure the disease or even to stop or remarkably slow down its progression. Endoplasmic reticulum (ER) stress has been proposed as one of the pathophysiological mechanisms underlying ALS. During ER stress misfolded of unfolded proteins accumulate in ER lumen. As a defense mechanism, the cell launches unfolded protein response (UPR). UPR response aims to reduce the protein load in ER and restore cell’s normal functions. If the damage is already beyond repair, UPR signal cascades lead to programmed cell death. Neurotrophic factors (NTFs) regulate the growth of nervous tissue and participate in repairing processed. Many of the known NTFs have first seemed promising in the preclinical models of ALS but however failed in clinical trials. Cerebral dopamine neurotrophic factor (CDNF) differs drastically both in structure and function from conventional NTFs. CDNF has seen to relieve ER stress and improve motor behavior in the animal models of Parkinsons’s disease. Recently CDNF entered clinical trials in Parkinson’s patients. Since ER stress is believed to be present not only in ALS but also in Parkinson’s disease and other neurodegenerative diseases, it might have an effect in treating ALS patients. SOD1-G93A is a well-established animal model of ALS in which the animals show typical motor impairments comparable to human disease. In this study we used a novel mouse line obtained from crossing traditional SOD1-G93A model and CDNF knock out models. The study aimed to evaluate the effect of endogenic CDNF loss in survival, onset of symptoms, motor behavioral and spinal motor neuron degeneration in the new line. ER-stress and autophagy marker levels were studied with quantitative polymerase chain reaction (CNDF) and western blotting techniques. Spinal motor neuron loss was examined by anti-choline acetyltransferase antibody (ChAT) stainings. SOD1-G93A CDNF knock out animals were observed to have more severe motor impairments in the early stages of the disease compared to the traditional SOD1-G93A mice. In addition, the degeneration of spinal motor neurons appeared to be more severe in the new line. There were no statistically significant differences in ER stress between the genotypes although a trend of increased ER stress was observed. Endogenous CDNF loss had no effect on the healthy animals. The results suggest that CNDF is a potential treatment for ALS and it might have only little side effect since it does not seen to affect healthy tissue. In medical usage, CDNF might be most effective when administered immediately after disease onset. However, this might be difficult because of the challenges in ALS diagnosis.

Chilit ovat Capsicum-sukuun kuuluvia yleensä korkean kapsaisiinipitoisuuden omaavia paprikalajeja. Niitä käytetään mausteena. Lisäksi chilien sisältämillä kapsaisinoideilla on todettu olevan useita farmakologisia ominaisuuksia, kuten analgeettisia ja antioksidanttisia vaikutuksia. Niiden antimikrobisia ominaisuuksia on myös hieman tutkittu, mutta tutkimuksia on vielä verrattain vähän. Tämän työn tarkoituksena oli selvittää muutamien eri chililajikkeista valmistettujen uutteiden antimikrobisia vaikutuksia Escherichia colia ja Staphylococcus aureusta vastaan. Uutteet testattiin dimetyylisulfoksidiin (DMSO) ja veteen liuotettuina. Lisäksi testattiin myös kahden puhdasaineen, kapsiaatin ja solaniinin, vaikutuksia kyseisiä bakteereita vastaan. Antimikrobiakokeet suoritettiin 96-kuoppalevyllä noudattaen aseptisia työtapoja. Testattuja chiliuutteita oli 19. Uutteita valmistettiin eri chililajikkeiden versoista (1 kpl) siemenistä (3 kpl), lehdistä (10 kpl) ja hedelmistä (5 kpl). Dimetyylisulfoksidiin liuotetut uutteet testattiin pitoisuuksilla 2,0 mg/ml ja 4,0 mg/ml. Veteen liuotetut uutteet testattiin pitoisuudella 4,0 mg/ml. Solaniini- ja kapsiaattiuutteet testattiin kahdeksalla eri pitoisuudella (0,001172–0,15 μg/ml). Tutkimuksen tuloksena on, että testatut chiliuutteet eikä solaniini- ja kapsiaattiuutteet estäneet E. colin tai S. aureuksen kasvua. DMSO:iin liuotetuista uutteista korkeimmat estoprosentit kumpaakin bakteeria vastaan saatiin nuorilla Pimento-lehdillä. Veteen liuotetuista uutteista korkein estoprosentti E. colia vastaan saatiin Dulcen versoilla (30 % esto) ja S. aureusta vastaan Dulcen hedelmillä (50 % esto). Aiemmat tutkimustulokset chilien antimikrobisista vaikutuksista ovat ristiriitaisia, joten yhteneviä johtopäätöksiä chilien vaikutuksista bakteereihin ei voida tehdä. Johtopäätöksenä voidaan todeta, että chileillä on lukuisia terveysvaikutuksia. Antimikrobisen tehon varmistamiseksi tarvittaisi kuitenkin lisää tutkimuksia. Antibioottiresistenssi on maailmanlaajuinen ongelma, koska yhä useammat bakteerit ovat resistenttejä käytetyille antibiooteille. Tulevaisuudessa onkin erittäin tärkeää löytää uusia yhdisteitä bakteerien tappamiseksi, joten tutkimuksia uusien antimikrobisten aineiden löytämiseksi tarvitaan jatkuvasti lisää