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(2014)Parkinson's disease is a progressive neurodegenerative disease. The incidence of the disease is 1.5-2 per cent after age 60. Typical symptoms are tremor, rigidity and bradykinesia. At the late stage of the disease patients have psychic disorders, for example dementia, anxiety and depression. Motor impairment is caused by degenerative loss of dopamine cells in nigrostriatal tract. Current treatment of the disease relieves the symptoms but it cannot stop or slow the progress of the disease. Neurotrophic factors and gene therapy have been trialled to improve the treatment of Parkinson's disease and the results have been encouraging. Neurotrophic factors are proteins that regulate actions of neurons. It has been discovered that they are neuroprotective and neurorestorative. The results with glial cell line-derived neurotrophic factor (GDNF) have been encouraging in in vivo studies of Parkinson's disease. There has been variability in success of clinical trials though. GDNF degrades quickly in vivo but overexpression of GDNF in cells can be produced with viral vector adeno-associated virus. Two different forms of GDNF, pre-α-pro-GDNF (α-GDNF) and pre-β-pro-GDNF (β-GDNF), are produced as precursors and they are activated proteolytically. Based on in vitro studies, some differences in secretion of precursors have been discovered. α-GDNF is secreted constitutively and secretion of β-GDNF is dependent on physiological stimulation. Previous in vitro studies have focused on α-GDNF, but β-GDNF might be a better solution for treating Parkinson's disease based on physiological regulation system. Cerebral dopamine neurotrophic factor (CDNF) is recently discovered and less studied than GDNF. It has been discovered that CDNF also has neuroprotective and neurorestorative effects in animal models of Parkinson's disease. The aim of the first part of this study was to discover the neurorestorative effect of single injection of CDNF injected above substantia nigra for rats that received injection of 6-hydroxydopamine (6-OHDA) into medial forebrain bundle. One week later rats received PBS, GDNF or CDNF injection. The degree of the lesion was estimated with apomorphine (0.1 mg/kg s.c.) or d-amphetamine sulphate (2.5 mg/kg) induced rotation test. The rats were perfused nine weeks post-lesion and their brains were sliced. Tyrosine hydroxylase (TH) positive dopamine cells were stained by immunohistochemistry. The amount of TH positive cells in substantia nigra was counted and optical density of TH positive fibres in striatum was measured. The aim of the second part of the study was to research the neuroprotective effect of two different precursors of GDNF, dsAAV1-pre-α-pro-GDNF and dsAAV1-pre-β-pro-GDNF, given with viral vectors. The dopamine cells in nigrostriatal tract were destroyed with a 6-OHDA injection into striatum and viral vectors were injected two weeks later. Rats in control group received injection of dsAAV1-GFP. The degree of the lesion was evaluated with d-amphetamine sulphate (2.5 mg/kg) induced rotation tests and cylinder test. The rats were perfused eight weeks post-lesion and their brains were processed for immunohistochemistry. The results of the study were interesting and supporting previous studies. The success of the neurotrophic factor treatment is dependent on a successful injection of protein or viral vector, and the dose is dependent on the size of the lesion. Neurotrophic factors and gene therapy needs to be studied more before wide clinical usage.
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(2012)ommunity pharmacies and medicines information centers increasingly provide e-mail medication counseling services to consumers and health care professionals. As e-mail medication counseling services are fairly new type of pharmaceutical service, only little is known about the use and users of these services, content of e-mail enquiries and quality of these services. Objective of this study was to analyze utilization of e-mail medication counseling services in the University Pharmacy. The special focus was on e-mail enquiries concerning human and veterinary medicines and health issues. All e-mail enquiries sent to the University Pharmacy Medicines Information Center in 2009 and 2010 were recorded by using a structured data collection instrument and content-analyzed both qualitatively and quantitatively. The total numbers of e-mail enquiries were 3 376. Of those 383 (11 %) were related to human medicines, 45 (1 %) to health and diseases and 33 (1 %) veterinary medicines. The majority of e-mail enquiries (72 %) were come via web page of the University Pharmacy. Of the human medicines related e-mail enquiries 92 % were sent by consumers and 65 % by woman. The most commonly e-mail enquiries concerned sleeping pills (5 %), antiepileptics (3 %), cholesterol medicines (3 %) and virus vaccines (3 %). E-mail enquiries fell commonly into the categories of availability (28 %), cost and reimbursements questions (13 %), medicines interactions (11 %) and adverse effects (5 %). Some background information was missing from e-mail enquiries, for example user of medicine (39 %), gender of medicine user (50 %) or age of medicine user (94 %). However, only part (3 %) of e-mail enquiries were sent adjunct questions. After improvement of usability and quality of e-mail medication counseling services response rapidity was increased in the University Pharmacy. Consumers appear to have medicines-related information needs. Additionally they are actively using e-mail services for multiple functions. However, the use of these services for medicines information is still fairly uncommon. The results of this study can be utilized when designing and developing e-mail medication counseling services in community pharmacies, medicines information centers and online pharmacy services. Additionally they can be utilized in pharmacy education in universities and in continuing education. More information is needed about the quality of pharmacists' responses and consumers' needs, experiences and perceptions on e-mail medication counseling services.
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(2019)Pitkäaikaiset lääkitykset lisääntyvät jatkuvasti kroonisten sairauksien yleistymisen ja väestön ikääntymisen takia. Pitkäaikaisten sairauksien hoidossa lääkehoitojen rationaalisuus korostuu, mutta WHO:n arvioiden mukaan noin puolet lääkkeiden määräämisestä, toimittamisesta, käytöstä ja myynnistä toteutuu epärationaalisesti. Tämä lisää terveydenhuollon ammattilaisten vastuuta lääkehoidon vaikutusten seurannassa ja potilaan hoitoon sitouttamisessa myös reseptien uudistamisessa. Reseptien uudistamiskäytäntöjä on kuitenkin tutkittu vähän niin Suomessa kuin maailmanlaajuisesti. Tässä pro gradu -tutkielmassa tavoitteena oli tarkastella nykyisiä reseptien uudistamiskäytäntöjä perusterveydenhuollon lääkäreiden näkökulmasta. Tavoitteena oli tarkastella, minkälaiset tekijät vaikuttavat lääkäreiden työskentelyyn sekä potilaan lääkehoidon kokonaisuuden hallintaan ja turvallisuuteen reseptien uudistamistilanteissa. Lisäksi kartoitettiin lääkäreiden ratkaisuehdotuksia uudistamiskäytäntöjen kehittämiseksi. Tutkimus toteutettiin laadullisena monimenetelmätutkimuksena Kirkkonummen terveysasemilla. Tutkimuksessa hyödynnettiin triangulaatiota ja tutkimusaineisto koostui reseptien uudistamistilanteiden varjostuksesta sekä kahdesta lääkäreiden ryhmähaastattelusta. Tutkimukseen osallistui yhteensä 12 lääkäriä, joista viisi osallistui varjostusvaiheeseen ja seitsemän haastatteluvaiheeseen. Aineisto kerättiin huhti-heinäkuun 2019 aikana. Tutkimuksen teoreettisena viitekehyksenä oli inhimillisen erehdyksen teoriaan perustuva järjestelmälähtöinen näkökulma. Tutkimusaineisto analysoitiin aineistolähtöisellä sisällönanalyysillä, jossa varjostus- ja haastatteluaineistosta etsittiin tutkimuksen tavoitteiden kannalta merkittäviä ilmaisuja. Reseptien uudistaminen on lääkäreiden näkökulmasta monivaiheinen prosessi. Prosessiin vaikuttivat useat uudistamista helpottavat ja vaikeuttavat järjestelmä-, potilas- ja lääkelähtöiset tekijät. Lääkärit tunnistivat ongelmakohtia uudistamisprosessin jokaisesta vaiheesta. Lääkäreiden mukaan etenkin tietojärjestelmien epäkäytännölliset ominaisuudet ja tekniset ongelmat sekä ajantasaisten lääkitystietojen ja tiedonkulun puutteet olivat uudistamistilanteissa ongelmallisia ja tekivät uudistamisesta työlästä. Myös kiire ja uudistettavien reseptien suuri määrä vaikeuttivat uudistamista. Ongelmien takia lääkärit kokivat, ettei lääkehoitojen seurantaa voitu tehdä uudistamistilanteessa perusteellisesti. Lääkäreiden ehdotuksia uudistamisprosessin kehittämiseen olivat uudistamisen parempi koordinointi, tietojärjestelmien ja tiedonvälityksen kehittäminen sekä moniammatillisen yhteistyön ja potilaan osallistamisen lisääminen.
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(2009)Stakes ja Lääkehoidon kehittämiskeskus määrittelevät lääkitysturvallisuuden lääkkeiden käyttöön liittyväksi turvallisuudeksi, joka voi vaarantua missä tahansa lääkehoidon vaiheessa. Yksi lääkitysturvallisuuteen vaikuttava lääkehoidon osa-alue on lääkeneuvonta. Koska lääkkeitä käytetään ammattilaisilta saatujen ohjeiden perusteella, on lääkeneuvonnan kehittäminen tärkeää. Lääketietouden lisäämisen ohella lääkeneuvonnalla voidaan vaikuttaa lääkkeiden käyttäjien asenteisiin. Lapsen lääkitykseen kohdistuva lääkeneuvonta on haasteellista, sillä huomio täytyy kiinnittää sekä lapseen että vanhempiin. Tutkimuksessa selvitettiin sairaalasta kotiutettavan lapsipotilaan lääkeneuvonnan sisältöä ja kehitystarpeita sekä lääkeneuvonnan toteuttamiseen vaikuttavia asioita. Lisäksi tutkimuksen avulla selvitettiin vanhemmille kotiutuksen jälkeen ongelmallisia lääkehoitoon liittyviä asioita. Tutkimusaineistona käytettiin HUS:n Lastenklinikalla työskentelevien sairaanhoitajien teemahaastatteluja. Toisena aineistona käytettiin vanhempien osastolle soittamia, lasten kotiutuksen jälkeistä lääkehoitoa koskevia puheluita. Aineiston keräys tapahtui hoitajien avulla heidän kirjatessaan tarkoitusta varten muodostettuun lomakkeeseen ylös ne lääkehoitoa koskevat kysymykset, joita vanhemmat soittavat osastoille kotiutuksen jälkeen. Keräykseen osallistuivat HUS:n infektio-osaston, lastentautien osaston ja munuais- ja elinsiirto-osaston hoitajat. Syyskuun 2009 aikana suoritettuihin teemahaastatteluihin osallistui 10 infektio-osaston sairaanhoitajaa. Kuten aiemmissakin tutkimuksissa on todettu, sairaanhoitajilla on suuri vastuu sairaalassa annettavasta lääkeneuvonnasta. Tutkimustulosten mukaan sairaanhoitajat pyrkivät kattavaan lääkeneuvontaan. He painottavat erityisesti perheen pärjäämistä kotona havainnollistamalla antamaansa neuvontaa kirjallisen materiaalin ja antotekniikkaan liittyvän opetuksen avulla, sekä kiinnittämällä huomiota perheen asenteisiin lääkehoitoa kohtaan. Esteinä lääkeneuvonnan toteuttamiselle koettiin sekä vanhempien että hoitajien kiire, joidenkin vanhempien vääränlainen asenne, hoitajien ja vanhempien kielimuurista johtuvat kommunikaatio-ongelmat, neuvontatilan rauhattomuus sekä hoitajille suunnattujen yhteisten ohjeistuksien puute. Lääkeneuvontaan liittyviksi kehityskohteiksi nousivat sekä vanhemmille että hoitajille suunnatut kirjalliset ohjeet, lääkkeisiin liittyvä lisäkoulutus, neuvonnan ajoitus, neuvontatila ja osastofarmaseutin osallistuminen neuvontaan. Kahden kuukauden tutkimusjakson aikana loka-marraskuussa 2009 kirjattiin osastoilla yhteensä 26 kotiutetun potilaan lääkehoitoa koskenutta puhelua. Infektio-osastolle tulleet yhteydenotot (n=7) koskivat pääasiassa lääkkeen annosteluaikoja ja kuurin tarkkaa kestoa. Yksi puhelu koski haittavaikutuksen ilmaantumista ja yksi antotekniikkaa. Lastentautien osastolle tulleista puheluista (n=11) kymmenen koski insuliinien annostusten muuttamista verensokeriarvojen korjaamiseksi. Munuais- ja elinsiirto-osastolle vanhemmat soittivat yleensä kysyäkseen annosteluun liittyviä erityisasioita, kuten annostelua oksennuksen, väärän lääkkeenantoajan ja yliannoksen jälkeen.Munuais- ja elinsiirto-osastolla puheluita kirjattiin yhteensä kahdeksan. Tutkimuksen mukaan hoitajien antama lääkeneuvonta on perusteellista. Lääkehoitoon liittyviä asioita jää kuitenkin vanhemmille epäselväksi, vaikka huomiota kiinnitetään niihin asioihin, joissa vanhemmat tarvitsevat eniten tukea. Osastoille kehitetyn lääkeneuvontamallin tarkoitus on auttaa hoitajia lääkeneuvonnan toteutuksessa, mutta huomionarvoista olisi myös osastofarmaseutin mahdollisuus osallistua neuvontatilanteeseen. Jatkotutkimuksissa olisi hyvä selvittää, millä tavalla neuvontamallia hyödynnetään neuvonnan toteuttamisessa. Erityisesti lapsille suunnattua lääkeneuvontaa on kehitettävä.
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(2013)Hospital pharmacies and drug centers are responsible for pharmaceutical services for inpatient care in the public health care in Finland. Each of the 20 hospital districts have a central hospital pharmacy. Every hospital district is a member of one of the five regional hospital groups (called erva-alue). In each regional hospital group the area's university hospital is responsible for the specialized hospital care. Most of the regional hospital groups cooperate in drug purchasing. The drug purchasing policies need to be in line with the legislation regulating public sector's purchasing policies. Usually procurement and organizing a tender competition are coordinated by university hospitals. With centralization hospital pharmacies can get cost-benefits. This study deals with drug purchasing policies in hospitals and regional hospital group cooperation in Finland. The objective was to explore drug purchasing process in hospital pharmacies and related cooperation in regional groups. The study was carried out as a postal survey which was sent to the head pharmacists of all 20 hospital districts in spring 2012. The survey instrument was reviewed by selected experts and revised according to their comments before it was sent to the respondents. Most of the questions were open-ended enabling the respondents to reflect their opinions. The response rate was 90% (n=18). All respondents answered to the most of the questions. There were seven procurement groups. Most of the regional cooperation groups procured drugs together. Only Helsinki University Hospital's (HYKS) regional cooperation group did not procure and organize a tender competition together. Purchasing period was generally two years. Usually procurement was centralized to the university hospitals in the regional groups. The hospital pharmacies that had two years purchasing periods reasoned the duration of the period most commonly by cost savings. The pharmacies that had a three-year or longer period explained its length by drug safety. The areas also differed in the way they involved specialists in selecting pharmaceutical products and making final decisions. The expertise of the specialists involved varied widely. Some areas involved a very broad range of experts, while some others had few. The drugs were selected independently by or within groups. Procurement criteria varied a lot, but the main criterion in all the responses was price or total cost-effectiveness. The respondents reported that drug safety was considered in the procurement but its inclusion as a purchasing criterion was challenging. Few of the respondents reported having studied cost savings of using purchasing groups. However, cost savings were believed to be significant. Particularly, the respondents reported that workloads had decreased because of the cooperation in procurement. Some changes were reported to happen in the drug procurement processes of some purchasing groups. All these ideas concerning drug purchasing policies and cooperation are described in the research report. For example, some head pharmacists indicated their willingness to have national cooperation in establishing drug guidelines. Most of them were satisfied with the current cooperation and purchasing policies and were ready to continue and develop the cooperation. The study achieved its goal in exploring drug purchasing policies and cooperation between hospitals in Finland. The study can perform as a baseline evaluation for further studies in the field. It also provides useful information to those people working on drug procurement and purchasing policies.
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(2020)Salvinorin A is a dissociative hallucinogen found in the plant Salvia divinorum. Unlike other hallucinogens it is a selective kappa-opioid receptor antagonist with no affinity to serotonin receptor 5-HT-2A. Modern case studies suggest low, regularly used salvinorin A doses might have antidepressant properties. In animal studies salvinorin A causes both pro- and antidepressant behaviour. Other hallucinogens, such as classical psychedelics psilocybin and LSD, show great promise as rapid acting antidepressants in multiple clinical trials focusing on treatment resistant depression. The most well-known rapid acting antidepressant drug ketamine belongs to the same group of dissociative hallucinogens as Salvinorin A. The use of subanesthetic ketamine has become an integral part of treatment resistant patient care in Finnish healthcare. Ketamine as well as chronic treatment with traditional antidepressants induce plasticity via BDNF-TrkB signaling. The antidepressant mechanism of classical psychedelics is mostly unknown, but they have been shown to promote neuroplasticity by increasing the expression of immediate-early genes and spinogenesis in cortical neurons. The experimental part of this master’s thesis examines the acute effects of salvinorin A on the signaling pathways associated with antidepressant response in C57BL/6 mice. To better characterize the effects of salvinorin A an open field test of 100 min was carried out in addition to phosphorylation studies. Single high dose (5-10 mg/kg) of salvinorin A causes a robust reduction in locomotor activity almost immediately after i.p. administration in mice. However it does not affect the phosphorylation of proteins associated with antidepressant response, nor does it affect BDNF m:RNA expression in mouse prefrontal cortex. According to previous studies, the therapeutic effects of salvinorin A might be present only at low doses or in regular microdosing.
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(2023)Silmätipat ovat silmälääkkeiden yleisin annostelumuoto, mutta silmään imeytyvän lääkeaineen osuus jää pieneksi. Esimerkiksi glaukoomaa sairastaa maailmanlaajuisesti noin 80 miljoonaa potilasta, mutta glaukoomalääkkeiden hyötyosuus etukammiossa on vain 1–7 %. Päällimmäiset syyt heikkoon hyötyosuuteen ovat lääkeaineen nopea poistuminen silmän pinnalta mm. systeemiverenkiertoon sekä sarveiskalvon heikko läpäisevyys. Tästä syystä lääkeaineen permeaatio sarveiskalvon läpi on ollut tutkimuksen kiinnostuksen kohteena. Lääkeaine voi imeytyä silmän pinnalta etukammioon sarveiskalvon läpi passiivisella diffuusiolla ja aktiivisesti transporttereiden välityksellä. On vielä pitkälti epäselvää, että mitä transporttereita löytyy aktiivisena ihmisen sarveiskalvosta ja että kuinka paljon eri lääkeaineet hyödyntävät transporttereita imeytyessään. Tämän tutkimuksen tarkoituksena oli kartoittaa transportterien aktiivisuutta ihmisen sarveiskalvon epiteelisolulinjassa (HCE) sekä kanin eristetyssä sarveiskalvossa. Tutkimuksessa käytettiin radioleimattuja lääkeaineita, joita käytetään silmälääkkeenä (kliinisesti tai tutkimusvaiheessa) ja joiden tiedetään olevan eräiden transporttereiden substraatteja ja/tai inhibiittoreita. HCE-soluilla tehtiin in vitro aika-lineaarisuus- ja inhibitiosolunottokokeita ja kanin eristetyllä sarveiskalvolla permeabiliteettikokeita ex vivo. Tulosten mukaan kaikilla lääkeaineilla näyttäisi olevan aktiivista kuljetusta HCE-soluissa, mutta aktiivisen kuljetuksen osuus ja että mitkä transportterit ovat vastuussa kuljetuksesta, on epäselvää. Kiinnostava tulos oli, että inhibiittoreista MK-571 inhiboi metotreksaatin solunottoa HCE-soluissa sekä permeabiliteettia kanin sarveiskalvon läpi apikaali-basolateraalisuunnassa. Tulokset viittaavat influksitransportterien inhibitioon, mutta tarkempia johtopäätöksiä on hankala tehdä. Silmän ja sarveiskalvon transportteritutkimus on vielä alkutekijöissä ja lisää tutkimustietoa aiheesta tarvitaan.
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(2011)QSPR (Quantitive structure property relationship) describes relationship between descriptors and biological activity. Therefore, QSPR models are useful tools in drug discovery. The literature review summarizes existing corneal, intestinal and blood brain barrier permeability models. The most common descriptors are hydrophobicity, polar surface and H-bonding capability. Also, the size of molecule may have influence on permeability even though the results are sometimes contradictory. Descriptors might have limiting values such as those presented in Lipinski's ‖rule of five‖. Drug candidate should not have 'rule of 5' values outside of the useful range, otherwise the per oral absorption of the compound may be compromised. In the literature review the transporter activity in cornea, intestine and blood brain barrier is described. Currently, many QSPR-models have been developed to predict interactions of drug candidates with transporters. The purpose of experimental part was to build in silico -model of corneal passive permeability for early ocular drug discovery. QSPR-model was built using permeability data and molecular descriptors of 54 molecules. Corneal permeability coefficients in rabbits were obtained from the literature. Octanol-water partitition coefficient at pH 7,4 (logD) and the total number of hydrogen bonds were the descriptors in the final model. The final equation was log(permeability coefficient) = -3,96791 - 0,177842*Htotal + 0,311963*logD(pH7,4). For this model R2 was 0,77 ja Q2 was 0,75. The model was evaluated using an external data set of 15 compounds and by pharmacokinetic modeling. Predicted permeability coefficients were used to simulate the aqueous humour concentrations of sevent compounds at steady-state. In addition corneal absorption coefficient (Kc) was simulated for 13 compounds and these values were compared to predicted permeability. The predicted permeability coefficients correlated well with experimental permeability coefficients. In addition aqueous humour concentrations can be simulated in steady state using predicted (QSPR) permeability coefficients. The final QSPR-model may be used in ocular drug discovery and development.
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(2018)During and after myocardial infarction, millions to a billion cells die off. Scar tissue formed by fibroblasts replaces the injured myocardium during recovery. While the newly formed tissue is durable and prevents rupture of the heart, it doesn´t contribute to pump function. Depending on the extent of cardiomyocyte loss, the remaining functional myocardium get strained. Adult mammalian heart has inadequate capacity to regenerate after such injury. In case of sustained substantial increase in workload, the compensatory mechanisms turn into pathological processes including excessive fibrosis and myocyte apoptosis. The progressive decline of hearts contractile function results in heart failure (HF). Current drug treatments for managing HF aim to prevent progression of the disease and relieve symptoms. ACE inhibitors, beta blockers and diuretics are effective along with healthy lifestyle. No practical treatments are available to restore cardiac function yet. Human myocardium normally regenerates, but only 1% or less of myocytes get replaced yearly. Heart’s resident stem/progenitor cells (CPCs) likely play a role in the turnover. The aim of this study was to develop a screening method to identify small molecules that possibly promote differentiation of cardiac progenitor cells to cardiomyocytes. Cell population differentiated from mouse embryonic stem cells (mESCs) was used as a model for CPCs. Directed differentiation protocol of mESCs used here promotes commitment to cells of cardiac mesoderm, part of which will further differentiate to cardiac progenitors. The resulting population at day 6 is heterogenous but many of these cells are progenitors that turn into cardiomyocytes (CMs) by day 8. 10 000 cells per well are plated on 384 well plates at day 5. Test compounds are added at day 6 and removed day 8 for effect in progenitors and day 7-9 for effect in early cardiomyocytes. 0,1% DMSO is used as vehicle and Wnt pathway inhibitor XAV939 as positive control. The effects are quantified with plate reader on day 9. E14 derived mESC reporter line was used. Myl2v-eGFP + SMyHC3-RFP double reporter line allows the specific identification of ventricular CMs with green fluorescence and atrial CMs with red fluorescence. Plate reader measures the total fluorescence of the wells at 485/520nm on day 9, which is used as a readout for ventricular CMs. The fluorescence intensity depends on the amount of GFP+ cells but also on the level of Myl2v expression. Atrial CMs could be quantified similarly but the population doesn´t contain enough RFP+ cells. The assay was shown to reliably point out ‘hits’ that have a strong effect. Any compounds that only produce a moderate effect could be a false negative, however. The effect on cardiac progenitors could likely be increased by simply adding the compounds earlier on day 5. Variability of key reagents causes the main technical troubles through unpredictably affecting cytokine concentrations which decreases the amount of cardiac progenitors. Partially similar screening assays are being used by the big pharma where they cryopreserve progenitors in bulk for later use, thus simplifying and speeding up their method. Same approach could be adopted.
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(2013)There is currently no vaccine or specific treatment available for diseases caused by alphaviruses. Marine organisms have attracted interest for the past decades as unexplored sources of new pharmaceuticals and marine-derived substances might provide novel new lead molecules also for antivirals. The aim of the study was to identify marine-derived replication inhibitors acting against Chikungunya virus. Chikungunya virus is an arthropod-borne virus that belongs to the Alphavirus genus and causes a disease characterized by febrile illness and persistent arthralgia. Several epidemic disease outbreaks have occurred in recent years. A sample library of 373 marine-derived extracts, isolated compounds and synthetic molecules was screened for antiviral activity by using a genetically modified mammalian cell line. The cell line expresses viral replication proteins together with fluorescent and luminescent proteins as detection markers. Secondary evaluation including determination of cytotoxicity and dose-response was performed for samples active in the primary screening phase. Based on the primary screening results, 32 samples (8.6% of the total screened library) were found active against Chikungunya virus replication. The active samples were extracts and isolated compounds; none were synthetic molecules. False positives were excluded based on secondary assay results and finally nine non-cytotoxic samples with dose-dependent inhibitory activity against Chikungunya virus replication were identified. The used screening method is a safe and suitable choice for preliminary identification of Chikungunya virus replication inhibitors. Assays taking use of infectious viruses or other virus types are nevertheless needed for future studies to get more detailed information on action of active samples. The identified samples with antiviral activity should additionally be further studied with regards to isolation of active components, sustainable collection or cultivation and possible synthetic production and optimization.
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(2015)The properties of liquid and gas flows in microscale systems differ from those in macroscale; microfluidics is a field of science in which these properties are investigated and utilized for the development of microscale systems. Acoustofluidics is a branch of microfluidics focusing on the movement (acoustophoresis) or localization (acoustic trapping) of particles in microchannels using ultrasound. In this work, the suitability of a new miniaturized method for the screening of cell-drug interactions was investigated. In the method, the cells were acoustically trapped within a glass capillary, enabling liquid movement (generated with a syringe pump) in the capillary while the trapped cell cluster remains stationary. In this manner, the trapping of cells, their incubation with a drug solution, rinsing, and the elution could be done using the same capillary. The sample preparation was done using a miniaturized solid phase extraction technique (integrated selective enrichment target, ISET), and the analysis was done with matrix assisted laser desorption/ionization mass spectrometry (MALDI MS). The drug compounds investigated were selective serotonin reuptake inhibitors (SSRI). The research was conducted in five phases. In the first phase, a suitable solid phase extraction method for the drug compounds was investigated. In the second phase, the performance of the acoustic trap was investigated by acoustically trapping polystyrene beads and Coulter counting them. In the third phase, the method was modelled by conducting drug binding studies using cation exchange beads instead of cells. In the fourth phase, the drug binding studies were conducted by investigating the binding of drug compounds to human platelets and yeast cells. Platelets were chosen due to the expression of serotonin transporter, the molecular target of SSRI drugs, on their cell membranes. Also a cell membrane preparation containing serotonin transporter was used for the binding studies. In addition, memory effects occurring in the method were investigated. In the fifth phase, comparative drug binding studies without acoustic trapping were conducted. The suitability of the method for the screening of cell-drug interactions could not be thoroughly substantiated, but further research and method development are required. The reason for this was the inadequate sensitivity of the method, because of which large drug concentrations had to be used. This lead to the increased occurrence of memory effects.
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(2013)Alzheimer's disease is a neurodegenerative brain disease and it is the leading cause of dementia worldwide. However, there are not any medical treatments available to slow down or cure the disease. The typical microscopic changes in Alzheimer patients' brain are extracellular amyloid deposits and intracellular neurofibrillary tangles. Serine/threonine kinases are protein kinases that take part in the regulation of cellular functions. At least protein kinase C (PKC), glycogen synthase kinase 3 (GSK-3), cyclin-dependent kinase 5 (CDK5) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) are involved in the pathogenesis of Alzheimer's disease. There are currently molecules in development that either activate or inhibit these protein kinases in order to stop the progression of the disease. PKC is an interesting kinase considering this project. It has been shown that PKC activation prevents the formation of amyloid deposits and protects neurons from premature death. This could slow down or prevent the progression of the disease. The purpose of this study was to investigate the effects of dialkyl 5-(hydroxy-methyl)isophthalates (HMI-1a3 and HMI-1b11) on SH-SY5Y-neuroblastoma cell proliferation and morphology with live cell imaging and to Alzheimer's disease-related Wnt, ERK1/2 and PKC signaling pathways with Western blotting. The main purpose was to evaluate the potential of the compounds for further in vitro and in vivo experiments. According to the results of this study both isophthalates, HMI-1a3 and HMI-1b11, had good binding affinities to PKCα and PKCδ. Both of them caused a dramatic increase in ERK1/2 phosphorylation which may be due to PKC activation and may thus suggest a PKC-dependent mechanism of action. However, the possible PKC activation did not cause downregulation of the PKC-isoforms α, β and δ. In addition, both HMI-1a3 and HMI-1b11 inhibited SH-SY5Y cell proliferation. HMI-1a3 was cytotoxic at 20 µM, while HMI-1b11 did not cause any cell death. Both compounds also induced neurite outgrowth. In addition, HMI-1a3 increased the amount of β-catenin. That could indicate the activation of Wnt-signaling, which is inhibited in Alzheimer's disease. Both of the compounds have potential for further studies because of the good binding to PKC and the beneficial effects on neurite outgrowth and Wnt signaling.
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(2012)Literature review: Cognitive deficits of schizophrenia include disturbances in executive functions, working memory, attention and information processing. Improved understanding of the neurobiology of these deficits depends on the availability of reliable and carefully validated animal models, which can assist the development of novel pharmacotherapies. The glutamate hypothesis of schizophrenia arises from observations that substances which block a type of glutamate receptor known as N-methyl-D-aspartate-receptor (NMDAR) induces schizophrenia-like condition. The evidence strongly supports the use of NMDAR antagonists to model schizophrenia in animals. In this literature review various cognitive animal models of schizophrenia are presented. Also heterogeneity in the effects of NMDAR antagonists, at the cognitive level, following single-dose or long-term exposure is reviewed and discussed. Experimental part: Attentional set shifting task (ASST) is a cognitive animal model, which models animal's cognitive flexibility or ability to shift attentional set. The ASST has been modified for use with mice. Validation of the test in mice is still inadequate. The main purpose of this study was to investigate whether ASST is suitable for an outbred ICR mouse strain. The current study failed to demonstrate the suitability of ICR mice in this test. Though results did prove that ICR mice are capable of performing the technical requirements of the test. The pharmacological focus of this study was to investigate in mice how a subchronically administrated NMDAR antagonist dizocilpine (MK-801) (0.03-0.1 mg/kg, 10-14 d, i.p.) influences the ability to shift attentional set. With our experimental design we could not measure the ability to shift attentional set thus we cannot conclude whether or not MK-801 influenced this cognitive domain. Results did reveal that MK-801, as administrated above, did not alter the motivation or motor functions in ICR mice. According to literature and this current study it is obvious that more research is needed to clear ASST suitability for mice. Future studies should focus to investigate how the components of the experimental arrangement in ASST affect the test performance.
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(2022)OATP1B1 is an influx transporter that is predominantly expressed in the liver, and it mediates the uptake of many clinically important endogenous compounds and drugs from portal vein blood into hepatocytes. OATP1B1-mediated uptake affects the rate of hepatic elimination of substrate drugs, directly affecting their plasma concentrations. Some naturally occurring single nucleotide variants (SNVs) in the SLCO1B1 gene encoding OATP1B1 can alter the transport function of the transporter resulting in alterations in pharmacokinetics, efficiency and toxicity of substrate drugs. The aim of this master´s thesis was to examine the effect of four naturally occurring SNVs of the SLCO1B1 gene on transport activity, expression, and localization of the OATP1B1 transporter in vitro. SNVs 170G>A (R57Q), 388A>G (N130D), 452A>G (N151S) and 758G>A (R253Q) were created using site-directed mutagenesis in the SLCO1B1 gene presenting in the pENTR221 plasmid. Recombinante baculoviruses were produced in Sf9 cells using the Bac-to-Bac® Baculovirus Expression System and used to transduce HEK293 cells for the overexpression of OATP1B1 wild type and variant proteins. An uptake assay was used to study the transport activity of the OATP1B1 variants in HEK293 cells. Western blotting was used to study the expression of OATP1B1 proteins in membrane vesicles. Immunofluorescence staining was used to determine the localization of OATP1B1 wild type and variants in HEK293 cells. Transport activity of the OATP1B1 variants R57Q and R253Q was significantly decreased compared to wild type. In contrast, transport activity of the N130D ja N151S variants was not significantly altered. The reasons for the changes in transport activity could not be reliably estimated due to the failure to measure the expression levels of OATP1B1 proteins by Western blotting. However, immunofluorescence microscopy revealed that the localization and expression of the all the studied OATP1B1 in baculovirus transduced HEK293 cells were comparable to the wild type. The results of this master´s thesis indicate that SNVs 170G>A and 758G>A can impair the transport activity and substrate uptake functions of OATP1B1 in vitro. Additional in vitro studies of transport activity, expression and localization of the variants R57Q and R253Q will be required to confirm these results. In the future, the R57Q and R253Q variants should be also studied for their possible clinical significance in pharmacokinetics and pharmacodynamics of substrate drugs, as SNVs 170G>A and 758G>A may increase the exposure and the risk for adverse effects of OATP1B1 substrate drugs.
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(2013)Cellulose has already been used as an industrial raw material for over a century. However, during recent years the nanostructural features of the naturally occurring biopolymer have been fully investigated and characterized through different processing methods as nanofibrillar cellulose (NFC). This has led to a rapid development of novel cellulose based nanoscale materials and advancements in the field of composite materials. NFC offers interesting specific properties that differ from many other natural and synthetic polymers, such as self-renewable raw materials, semi-crystalline morphology, broad chemical modification capacity, biocompatibility and biodegradability. Biocompatibility and the biomimetic aspects of NFC have enabled the fabrication of nanoporous membranes and scaffolds that can function as medical devices (e.g. tissue engineering, wound healing, novel active implants). In this study, the properties of plant-derived NFC, as potential injectable drug releasing hydrogel "implants" were investigated. Three different sized candidate molecules were selected (123I-NaI, 123I-β-CIT and 99mTc-HSA, from small to large respectively) and investigated with the use of a small animal SPECT/CT molecular imaging device. Study compounds were mixed with the NFC biomaterial and injected into the pelvic region of mice. Drug release was observed for a period of 24 hours and the results were compared to saline/study compound control injections. In addition, 99mTc labeled NFC hydrogels were prepared for dual label tracing to observe the hydrogel positioning during the SPECT/CT acquisitions. For the smaller compounds (123I-NaI, 123I-β-CIT), no differences were found in the drug release or absorption in between the NFC biomaterial and saline injections. However, a clear difference was found with the large compound (99mTc-HSA). In the NFC hydrogel, the rate of release was slower and the distribution of 99mTc-HSA was more concentrated around the area of injection. In addition, the NFC hydrogel did not migrate from, or disintegrate, at the site of injection, suggesting a robust enough structural integrity to withstand normal movement and activity. In conclusion, the labeling of NFC was found to be a reliable and simple method. NFC hydrogels have the potential use as drug releasing medical devices with larger compounds. NFC matrix did not have any controlled release effect on the studied small molecules. Therefore further studies are required for more specific conclusions.
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(2018)Flowability is an important powder character and, despite decades of research, there are still issues in finding a suitable measurement method. Common challenges are sample size and methodology’s suitability for cohesive powders due to their ability to form vault structures. Powder flowability properties depend strongly on particle features such as size and shape. As particles are in contact with other particles and materials, they receive electric charge and form bonds. In addition to these variables, the gravity and shear stress affect the powder. A combination of all these determine the powder properties such as flowability. Besides the particle properties, process and preservation conditions and especially humidity affects the powder properties significantly. Hence, the powder’s flow behavior varies in different conditions. There are several measurement devices available but none of them is able to yield intrinsic values. Reliability of the measurements presents another challenge as the measured values cannot be directly compared with published literature. Moreover, the flow measurement of cohesive powders is either impossible or extremely difficult with the devices currently available and the sample size needs to be sufficient. Hence, there is a need for new devices, which measure powder flow easily in small-scale. Small sample size is important especially when developing new, expensive drugs since their properties need to be explored in order to develop a new formulation. The aim of the empirical study was to develop a device, which measures particularly the flowability of cohesive powders in small-scale. A ground for the study was a device developed at University of Helsinki, which measures powder flowability by utilizing horizontal movement. In addition, the device breaks the problematic vault formation of cohesive powders by jolts. In the study a cuvette, which utilizes the horizontal movement and measures the powder flow, was developed. Flowability tests were run with five powders – Acetaminophen, Pharmatose 80M, Pharmatose 200M, Emcompress®, Avicel PH-101, Avicel PH-102, Avicel PH-200 and Maize Starch. The results were promising and the device was capable of classifying the powders by their flowabilities but more research is still needed.
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(2023)Poorly water soluble active pharmaceutical ingredients cause problems for the drug development. Solid state modification offers one approach to overcome the issue. In this study, co-amorphous systems and co-crystals were prepared with indomethacin at molar ratio of 1:1 using nicotinamide as a co-former. Co-amorphous systems were prepared by two different preparation methods: melting the physical mixture and then quench cooling it with liquid nitrogen and dry milling with a ball mill. Co-crystals were prepared by liquid-assisted ball milling. After that, the properties, dissolution, and physical stability of the formed formulations were investigated and compared. The characterisation methods were differential scanning calorimetry, X-ray powder diffraction, Fourier-transform infrared spectroscopy, polarised light microscope and scanning electron microscope. In addition, the solubility and physical stability of the formulations were investigated. Co-amorphous systems were successfully prepared by quench cooling the melt and co-crystals by liquid-assisted ball milling. Dry milling did not induce the formation of co-amorphous systems. In the intrinsic dissolution test, both the co-amorphous system and co-crystal enhanced the dissolution of crystalline indomethacin. When examining the dissolution rate with the paddle apparatus, it was observed that the co-crystal had the highest dissolution rate among both powder and tablet samples. The co-amorphous powder sample floated on the surface of dissolution medium which impeded the dissolution of indomethacin. However, co-amorphous tablet sample showed a higher dissolution rate than crystalline indomethacin. Stability testing (25 °C, 18 %RH) showed that the co-amorphous system recrystallised into a co-crystal after two weeks of storage, while the co-crystal was found to stay stable the whole study period.
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(2011)Use of natural xanthine derivates in medicine is complicated with their physical properties. Theobromine is poorly soluble while theophylline is highly sensitive to hydration. The aim of this study was to improve bioavailability of xanthines by co-crystallization, theophylline was also cocrystallized with carboxylic acids (capric, citric, glutaric, malenic, malonic, oxalic, stearic, succinic) and HPMC. Co-crystallization was performed by slow evaporation and ball milling. Physical stability was checked by wet granulation and water sorption methods, solubility was measured by intrinsic tablet dissolution. Theobromine formed co-crystal with other xanthines and theophylline interacted with all acids except stearic and HPMC, the latter showed alternative interactions based on hydrogen bonding. Hydration resistance was good in theophylline:succinic acid co-crystal and excellent in complexes containing capric, stearic acids and HPMC. Theophylline:HPMC showed improved solubility. The reported approach can promote use of xanthines and can be recommended for other compounds with similar problems.
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(2019)Background and objectives: Cells secrete extracellular vesicles (EV) and it has been found that cells communicate via EVs. EVs are liposome-like vesicles. Membrane is consisting of a lipid bilayer and hydrophilic moiety is inside the vesicle. It has been found that EVs carry e.g. nucleic acids, lipids and proteins. The aim of this master thesis was to determine whether EVs can transport non-coding RNA (siRNA) into the central nervous system through the blood-brain barrier. In the literature review, investigated methods which has been used to load siRNA into the EVs and how EVs are transported through the blood-brain barrier. The aim of the experimental part was to produce and isolate EVs and to load FAM-labeled dsDNA and siRNA into EVs by physical methods such as sonication and electroporation. Fluorescence measurements were taken to demonstrate FAM-labeled DNA loading into EVs and the functionality of the siRNA-loaded EVs was measured by measuring the expression level of the gapdh gene. Methods: Extracellular vesicles were produced in ARPE-19 and PC-3 cells. EVs were isolated from the cell culture medium by two-step differential centrifugation (DC) and further purified by gradient centrifugation (GC) by using the OptiPrep™-reagent. OptiPrep™-reagent was purified by Amicon 10kDa filtration tubes. The average particle size and size distribution of the isolated EVs were determined by NTA analysis, protein concentration was measured by colorimetric BCA method and EVs were characterized by Western blot method using HSP70 and CD9 antibodies. EVs were loaded with 21 bp length FAM-labeled dsDNA or siRNA by sonication or electroporation. Free nucleic acid and OptiPrep™-reagent were purified from EVs by the size-exclusion chromatography with Sephacryl (S-300) column. Loading efficient of the EVs were studied by measuring the fluorescence (ex 485 nm, em 520 nm) and qPCR method was used to demonstrate the functionality of the siRNA loaded EVs. In qPCR, the expression level of the gapdh gene was measured in dividing ARPE-19 cells. Results: DC and GC purified ARPE-19 and PC-3 EVs had an average particle size of about 140 nm and were successfully characterized by Western blot method. PC-3 EVs were produced in the bioreactor and the yields were enough for loading experiments. ARPE-19 cells produced only small amounts of EVs in culture flasks. The size-exclusion chromatography was a good method to purification free nucleic acids from EVs. The sonication method did not cause EVs to be degradation under the conditions used. Based on fluorescence measurement, FAM-labeled dsDNA could not be loaded into EVs. The functionality of siRNA-loaded EVs could not be demonstrated in ARPE-19 cell experiments. After electroporation large number of EVs were lost and this method of loading siRNA into EVs did not proved to be suitable. Conclusions: ARPE-19 EVs must be produced in the bioreactor to produce enough EVs for loading experiments. The EV purification protocol should be further optimized since the recovery-% of EVs were low after several purification steps. The size-exclusion chromatography is suitable for the purification of the free siRNA from EVs, but the chromatography method needs further optimization and miniaturization. Loaded EVs should be produced by aseptically or alternatively sterilized prior to ARPE-19 cell assay. Physical loading method, such as sonication, can be scaled to larger scale. Sonication method should be optimized e.g. by experimenting with higher temperatures and longer sonication times. The probe sonicator should be tested instead of the water bath sonicator. According to the literature review, the use of extracellular vesicles as carriers for biomolecule delivery into the central nervous system seems to be promising.
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(2015)Parkinson's disease is a progressive neurodegenerative movement disorder which is characterized by the death of dopaminergic neurons in the substantia nigra. In addition, other neuropathological features of the disease are intracytoplasmic protein inclusions as well as oxidative and ER stress. Currently there is no cure for Parkinson's disease so there is a need for novel therapies which could stop the disease progression. Because neurotrophic factors can promote the survival of neurons they might be beneficial for the treatment of Parkinson's disease. Cerebral dopamine neurotrophic factor (CDNF) has proven to be neuroprotective and neurorestorative in rodent models of Parkinson's disease. However, the development of new therapies requires relevant disease models. The defects of the current models of Parkinson's disease increases the need for better and more descriptive disease models. The literature review of this thesis presents an overview of ER stress and oxidative stress. Their role in Parkinson's disease 6-OHDA, MPTP, α-synuclein and rotenone models is also reviewed. The experimental part consists of three studies. The aim of the first study was to establish a preformed α-synuclein fibril mouse model of Parkinson's disease. The development of the lesion was studied by testing the motoric skills with balance beam, rotarod, wire hanger and cylinder test. In addition, TH and α-synuclein immunostainings from striatum and substantia nigra sections was performed. In the second study the effect of CDNF on mice behaviour and TH- and α-synuclein-immunoreactivity in the α-synuclein fibril mouse model was examined. The same motoric behaviour tests as in the first study were used. The purpose of the third experimental part was to investigate the effect of CDNF and GDNF on ER stress proteins in 6-OHDA rat model of Parkinson's disease. The levels of ER stress markers GRP78 and phosphorylated eIF2α were analyzed by Western Blot. The results of the studies were promising. In further studies the effect of α-synuclein fibrils on mouse behaviour and TH- and α-synuclein-immunoreactivity could be studied for longer time. The effect of CDNF on α-synuclein aggregation could also be studied further. The expression levels of other ER stress markers could be investigated so it would clarify the effect of CDNF and GDNF on ER stress.
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