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C-MANF:n farmakokineettiset ominaisuudet ja vaikutukset ALS:n geneettisessä SOD1-G93A-hiirimallissa (2021)Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons in brain and spinal cord. The degeneration of motor neurons leads to muscle atrophy and paralysis. Currently there is no cure for ALS. Available drugs for ALS can lengthen the survival time by a couple of months. Several factors involve the pathophysiology of ALS, such as endoplasmic reticulum stress and neuroinflammation. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a protein which has shown neuroprotective effects on animal models of Parkinson disease and brain ischemia. C-terminal fragment of MANF can cross the blood-brain barrier, allowing it to be administered subcutaneously instead of injected directly into the brain. The experimental part consists of two parts. The aim of the first part was to study the pharmacokinetic properties of next generation MANF (C-MANF). The aim of the second part was to elucidate the effect of twice a week administered subcutaneous injection of C-MANF in genetic SOD1-G93A mouse model and its neuroprotective effects by assessing protection of lumbar motor neurons. Pharmacokinetic properties of C-MANF were determined in wild type mice after a single subcutaneous injection of C-MANF at different time points by using indirect ELISA assay. The effects of C-MANF in SOD1-G93A mouse model were assessed by subcutaneous injection of either C-MANF or PBS twice a week and by monitoring clinical score and motor behavior of mice from 10 weeks of age to clinical endpoint. Hematoxylin eosin staining was used to study neuroprotective effects of C-MANF. C-MANF administered subcutaneously is absorbed into the blood circulation and the highest serum concentration of C-MANF is after 60 minutes of dosing. Subcutaneously injected C-MANF also crosses the blood-brain barrier and reach the brain in 120 minutes. C-MANF did not preserve motor function or ameliorated ALS symptoms in SOD1-G93A mouse model. In this study C-MANF did not increase the survival of SOD1-G93A mice. C-MANF did not significantly protect motor neurons from degeneration even though there was a slight trend between the groups. No beneficial effects were observed with C-MANF in SOD1-G93A mouse model and therefore the dose and frequency of administration of C-MANF were not optimal. Subcutaneously injected C-MANF provides a safer dosing option for neurodegenerative disorders.
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(2021)Huntington’s disease (HD) is a rare but devastating neurodegenerative disease, progressively culminating in severe brain atrophy and death. The disease is caused by an inherited mutation resulting in a CAG trinucleotide repeat expansion in the huntingtin gene, leading to the production of a neurotoxic protein, known as mutant huntingtin, with an abnormally long polyglutamine stretch. Even though the genetic background of HD is known, the cellular pathways affected in the disease are complex and not completely understood. Increasing evidence indicates that endoplasmic reticulum (ER) stress – a condition of disturbances in normal ER activity, leading to accumulation and aggregation of misfolded proteins in the ER lumen – is a central factor in the pathogenesis of HD and other neurodegenerative diseases. In the literature review of this thesis, known pathogenic cellular mechanisms of HD and how these cellular mechanisms are connected to ER stress, are discussed. Unpublished data from previous studies in our laboratory have indicated that the ER luminal protein canopy homolog 2 (CNPY2) could play a role in the regulation of neuronal survival, including the viability of mutant huntingtin expressing neurons. The aim of the experimental part of this study was to gain insight into a possible function of CNPY2 in HD, by examining the levels of the protein in neuronal models of HD under various conditions, such as ER stress, and by searching for potential interacting partners of CNPY2 amongst known ER stress regulators. The obtained results show that the levels of CNPY2 are increased in striatal neurons expressing mutant huntingtin, and that the secretion of CNPY2 is increased by these neurons, compared to control neurons expressing normal huntingtin. Further, we show that CNPY2 interacts with the major ER stress regulator binding immunoglobulin protein (BiP) in human neuroblastoma cells treated with the ER stress inducer tunicamycin, and that the intracellular levels of CNPY2 are altered by tunicamycin treatment. Together, these findings indicate that CNPY2 could be involved in the pathogenesis of HD. However, further research on the functions of CNPY2 and its role in ER stress regulation is required to understand the nature of this involvement.
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(2022)Viral promoters are an essential part of a normally functioning virus. Their main task is to drive the transcription of genes which govern hijacking of cell function and replication of viral particles. In addition to supporting normal function of a virus, they can be used to drive the transcription of transgenes which can be used in different therapies. In oncolytic therapies, transgenes can be used to prime the host system against neoplasms which has been shown to generate long term anti-tumour immunity. Human adenoviruses (Ad) are commonly used as a platform for oncolytic virotherapies. Human Ad’s replicate poorly in mouse tumour cell lines, yet some promoters, which are included in the viral constructs to drive the transcription of beneficial transgenes, are able to function. Currently it is unknown whether E3, the native promoter of adenovirus 5 of the E3 region, is capable of functioning in murine cell lines. In this thesis we study whether human cytomegalovirus promoter (CMV) and E3 differ in their efficacy to drive the transcription of the mOX40L and mCD40L transgenes. In the experimental part of this thesis, we compared the efficacies of two viral promoters, AdE3 and AdCVM, in transcribing mOX40Land mCD40L in vitro. Efficacy of transcription was assessed through immunofluorescence and flow cytometry in human and murine cell lines. Furthermore, the effects of promoters on viral infection, killing and replication were evaluated in burst assay and the colorimetric MTS proliferation assay. MTS and burst assay were conducted to confirm if viral infection, killing and replication occurs in human and murine cell lines. Both AdE3 and AdCMV were able to infect and kill human cell lines and cell viability decreased in correlation to the number of viral particles used. In murine cell lines, no decrease in cell viability was detected in the 4T1 cell line. In burst assay, viral replication was observed for both AdE3 and AdCMV in the human MDA-MB-436 cell line. In murine CT26 cell line, no replication was observed for AdE3 or AdCMV constructs. Immunofluorescence assay was performed to visualize transgene expression and localization. Results indicated that mOX40L was localized on cell surface while mCD40L was detected both outside and inside of the cytosolic compartment. Flow cytometry results revealed that both AdE3 and AdCMV constructs are capable of efficiently transcribing mOX40L in human cell lines. In the flow cytometry results for AdE3, two large cell populations with different fluorescence intensities were detected. AdCMV lacked this feature which is postulated to be due to higher lytic activity of the viral construct. In murine cell lines, HCMV could produce mOX40L, but production in murine cell lines was severely attenuated compared to human cell lines. mOX40L produced by the AdE3 construct did not differ from the baseline and was deemed incapable of producing mOX40L in murine cell lines. For the purpose of studying novel virotherapeutics the results of this thesis would indicate that human CMV can be used to drive expression of transgenes in murine cell lines. Despite this, it is preferable to use host specific viruses and promoter sequences for a better translation between mice and humans. Viruksen promoottorit ovat keskeisessä osassa toimintakykyisessä viruksessa. Virus promoottorin päätarkoituksena on geenien transkriptio, mitkä vastaavat solun keskeisten toimintojen kaappaamisesta ja virus partikkeleiden replikaatiosta. Näiden toimintojen lisäksi promoottoreita voidaan käyttää transgeenien transkriptiossa, mitä voidaan hyödyntää sairauksien hoidossa. Onkolyyttisissä terapioissa transgeenejä voidaan käyttää virittämään kehon immuunipuolustus taistelemaan kasvainkudosta vastaan. Ihmisen adenovirusta käytetään usein onkolyyttisten viroterapioiden alustana. Ihmisen adenovirus (Ad) replikoituu hyvin heikosti hiiren syöpäsoluissa, mutta osa adenovirukseen sisälletyistä eksogeenisistä promoottoreista, joita käytetään terapeuttisten transgeenien transkription ajamiseen, kykenee toimimaan ja tuottamaan haluttua proteiinia. Tällä hetkellä ei tiedetä, kykeneekö E3, joka on adenoviruksen E3 lokuksen promoottori, toimimaan hiiren solulinjoissa. Tässä tutkielmassa selvitämme ihmisen sytomegalovirus promoottorin (CMV) ja E3 eroa niiden tehossa ajaa mOX40L ja mCD40L transgeenien transkriptiota. Kokeellisessa osuudessa vertailimme kahden virus promoottorin, E3 ja CMV, eroa niiden tehossa ajaa mCD40L ja mOX40L transkriptiota in vitro. Transkription tehoa tutkittiin immunofluoresenssin ja virtaussytometrian avulla ihmisen ja hiiren syöpäsolulinjoissa. Tämän lisäksi promoottorien vaikutusta virus infektioon, replikaatioon ja kykyyn tappaa soluja arvioitiin burst kokeella ja kolorimetrisellä MTS menetelmällä. MTS ja burst kokeiden avulla varmistettiin AdE3 ja AdCMV virusten kyky infektoida, tappaa ja replikoitua ihmisen ja hiiren syöpäsolulinjoissa. Sekä Ad3 ja AdCMV todettiin kykenevän infektoimaan ja tappamaan ihmissyöpäsoluja ja solujen viabiliteetin lasku korreloi virus partikkeleiden määrän kanssa. Hiiren 4T1 syöpäsoluissa ei todettu solujen viabiliteetin laskevan. Burst kokeessa havaitsimme sekä AdE3 että AdCMV kykenevän replikoitumaan ihmisen MDA-MB-436 solulinjassa. Hiiren CT26 solulinjassa kummankaan viruksen ei havaittu kykenevän replikoitumaan. Immunofluoresenssi kokeessa visualisoimme transgeenien ilmentymisen ja paikantumisen. Tulokset osoittivat, että mOX40L paikantui solun pinnalle. mCD40L havaittiin puolestaan sekä solun ulkopuolella että sytosolissa. Virtaussytometria kokeen tulokset osoittivat, että sekä AdE3 ja AdCMV pystyivät tehokkaasti ilmentämään mOX40L ihmisen solulinjoissa. AdE3 virtausytometria tuloksissa löydettiin kaksi solupopulaatiota, joilla oli toisistaan poikkeavat fluoresenssi intensiteetit. Tätä ilmiötä ei havaittu AdCMV:lla infektoiduilla soluilla, mikä saattoi johtua korkeammasta lyyttisestä aktiivisuudesta. Hiirisolulinjoissa CMV kykeni ilmentämään mOX40L, mutta transkription teho oli selvästi alhaisempi verrattuna ihmissolulinjoihin. E3 promoottorin ilmentämä mOX40L ei eronnut kontrollista ja sen todettiin olevan kykenemätön tuottamaan mOX40L hiirisolulinjoissa. Tuloksemme osoittavat, että ihmisen CMV promoottori kykenee ilmentämään transgeenejä hiiren 4T1 ja CT26 solulinjoissa. On kuitenkin huomattava, että isäntälajille natiivien virusten ja promoottorien käyttö olisi tarkoituksenmukaisempaa tulosten käännettävyyden kannalta hiiristä ihmisiin.
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(2010)The aims of this work were (1) to compare the three dimensional structures of different S- adenosylmethionine (SAM)-dependent methyltrasferases and (2) to screen in silico a commercial library for potential methyltransferase inhibitors. In this work we decided to focus on DNA methyltransferase-like enzyme (DNMT2) and catechol-O-methyltransferase(COMT). There were two different parts in my work. The first part was to analyze the 3Dstructures of DNMT2 and COMT in relation with their amino acid sequences. The structures of DNMT2 and COMT were compared together by means of superimposition with Sybyl 8. The ligand binding properties were studied by manual and automatic docking of known inhibitors in order to understand the binding specificity of these methyltransferases. The softwares I used for docking were Autodock 4.2 and Gold 4.0. The sequence alignments and superimposition of the known crystal structures showed that the structures of DNMT2 and COMT share a similar fold. Furthermore the main similarities between the structures of these enzymes are in the co-enzyme binding sites. The only significant difference in the binding sites is the place of one tyrosine residue, which causes a slight change in the conformation of the bound co-enzyme. Unlike co- enzyme binding sites, the substrate binding sites of DNMT2 and COMT are different. There is indeed a bound magnesium ion in the substrate binding site of COMT but not in the substrate binding site of DNMT2. Because the substrate binding sites are more different than the co-enzyme binding sites, we decided to screen the potential active ligands only at the substrate binding sites. The second part of the work was virtual screening. I used a subset of 20.000 molecules of ChemBridge DIVER Set that can be purchased commercially. The softwares I used for library preparation were CONCORD and Balloon, from which Balloon created more reasonable 3D structures for the docking. I did two parallel screenings to the crystal structure of COMT (PDB code 3BWM) with docking program GOLD 4.0, which is the only program that can take account metal coordination. To DNMT2 I did two sets of screenings, one with GOLD 4.0 and another with Autodock 4.2. I used known COMT inhibitors as control in the COMT run and known DNA methyltransferase inhibitors as control in DNMT2 run. Before docking to the three dimensional structure of DNMT2, one loop near the substrate binding site had to be modeled. I used Swiss-Modeler and Modeller softwares for that. Docking to COMT was successful according to the rank of the known COMT inhibitors compared to the subset of the FIMM library that was screened. I created the hitlist of 60 compounds based on the scores of these compounds, pharmacophore search and visual examination. 30 of these compounds were purchased and are currently being tested. The results of the DNMT2 run were not as reliable as the results of COMT run mentioned before, since the DNMT2 run was unable to retrieve known inhibitors better than random. The reason for that can be the quality of the model of the missing loop or the chosen controls. Furthermore only one of the ten small molecules that we used as controls is proved to be DNMT2 inhibitor, the others are DNMT1 and DNMT3 inhibitors and while the binding sites of DNMT1, DNMT2 and DNMT3 are very similar, they are, however, not completely identical.
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Comparison of The U.S. and Finnish pharmaceutical markets and their impact on health care spending (2020)Theoretical framework: The consolidated pharmaceutical market is becoming increasingly global and the same international pharmaceutical companies operate around the world in different countries, responsible for drug development and production. The high costs of developing novel medicines and the motive for higher profits has led to elevating price level of pharmaceuticals and health care services. Finland and the U.S. offer two extremes at the pharmaceutical market. The pharmaceutical market field in Finland is very structural and rigid, and medicine prices are regulated by law. In the U.S. the prices are based on the laws of supply and demand and the prices differ by different states, retailers and insurance policies. A small-scale longitudal price comparison is also reviewed to showcase the effect of continuously rising medicine prices. Study objective: The idea of this study is to describe and compare pricing mechanisms of pharmaceuticals and price differences between two very different market structures and review how these might affect the cost-effectiveness of national health care spending. These divergences are also mirrored to survey recent global pharmaceutical market problems such as drug shortages, possibly due to less appealing markets of higher price regulation policies. Materials and methods: Price data were collected from national, official, open-source databases. National health care expenditure and comparison to GDP was collected from publications by the OECD. All monetary values have been presented in both currencies (EUR and USD) to present more comparable values. Results: When compared to other OECD-countries the U.S. spent distinctly the largest amount of funds on health care per capita. Finland’s national health care costs were thousand times minor in total spending and less than a half per capita when compared to those of the U.S. With lower expenditure Finland manages to offer access to public, government-funded health insurance program. Meanwhile the prices of prescription medicines in Finland have decreased significantly, the prices for have continuously elevated in the U.S. Conclusions: The outcome of this study is that free markets and a complex supply chain, compared to more regulated markets with more transparency, have higher overall price level in pharmaceuticals and health care services. Free markets and sufficient intellectual property rights are more enticing to pharmaceutical companies. They promote new innovations and developing of much-needed novel therapies to modern health problems, such as AIDS and the global threat of worsening situation of antibiotic resistance. More regulated markets may create problems such as drug shortages and are often considered complex and less appealing market systems due to high level of administrative work but conserve the cost-effectiveness of the use of public funds.
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(2020)Parenteral products are sterile products that are administered as injection, infusion or implantation. Administration of the contaminated parenteral product can cause severe consequences such as sepsis meningitis and even death. Most of the parenteral products used at the hospitals needs to be compounded (e.g. dissolved, diluted) before administration. Whenever possible, compounding should be done in biological safety cabinet using aseptic techniques. According to previous studies errors in aseptic techniques are quite common. Aim of this study was to compare three different environments as compounding area and their effect to the sterility of the compounded parenteral product. Based on the results of this study, changes to the protocols of the hospital could be made. Altogether 220 samples were compounded at two pediatric wards at HUS Helsinki University Hospital. Six volunteers (one pharmacist and five nurses) participated from both wards and each compounded 18 samples in three different environments (patient room, medicine room, biological safety cabinet). The samples were tested for the sterility by membrane filtration within 4 hours or after 24 hours of storage in the refrigerator. The investigator used an observation form to observe the compounding procedures. Environmental monitoring (settle plates) and monitoring of personnel (glove samples) were conducted. Almost all compounded samples (99%, n=213/215) were sterile. There were no significant differences in the contamination rate of the compounded samples between different environments. Five of the collected samples were excluded, because they were contaminated during the sterility test. According to observations, aseptic techniques were well followed. However, disinfection of the septum of the medicine bottle, hand hygiene and cleaning of the compounding area were observed to be deficiently completed. Even though there were lot of variation in the environmental and personnel monitoring the results were quite good. Results from the environmental monitoring were compared to the recommended limits of EU GMP for clean areas. One compounded sample was contaminated with Diezia maris and Corynebacterium mycetoides but the contaminants from the other contaminated sample could not be identified. Aseptic techniques were mainly well followed, however compounding should be done in the biological safety cabinet, since the environmental monitoring results show that the biological safety cabinet was only environment which was within the recommendation limits of the EU GMP for the compounding area of parenteral products. Protocols of the hospital could be changed, since there was no correlation between higher contamination rate of settle plates or compounded samples and not wearing mask and hair cover while compounding in the biological safety cabinet.
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(2012)The orexinergic system is a central regulator for sleep-wake rhythm and energy homeostasis. Dysfunction of the system is at least one of the reasons behind narcolepsy, in addition to which insomnia, obesity and certain cancers could be treated by targeting orexin receptors. The orexin system in human comprises two receptor subtypes, orexin receptor 1 and 2 (OX₁R and OX₂R respectively) as well as two cognate ligands, peptides orexin-A and -B. In this study the focus is on OX₁R and orexin-A. The aims of the study are (1) to propose a binding mode for orexin-A to OX₁R and (2) to understand the molecular interactions of OX₁R leading to receptor activation. I order to create 3D molecular models of OX₁R, a sequence alignment of the eight G proteincoupled receptors (GPCRs) that have been crystallized up to date was first generated by ClustalX and adjusted based on the superimposition by SYBYL-X. Structurally conserved regions were deduced from the alignment and used to add the orexin receptors. Five different models built with MODELLER were selected for their large binding cavity among a large pool of models. These models were constructed based on the chemokine receptor 4 (PDB Id:3ODU), as such and a modified version where TM3 was moved by 1 Å further from the center of the binding cavity, from the β₂-adrenoceptor (PDB Id: 2RH1) and from the adenosine receptor A2A (PDB Id: 2YD0), as such and with rotamer changes to few binding site residues. Orexin-A with straight conformation found by NMR (PDB Id:1WSO) was docked to these models using ZDOCK and RDOCK. In addition, an in-house docking protocol was implemented, but could not be validated. Docking poses were scored by purpose built knowledge based scoring function and clustered. High scoring clusters were then used to converge to three different binding modes. As a result, we suggest that the binding site of OX₁R consists of two hydrophobic walls, one from TM3 and TM5, the other from TM6 and TM7. Binding modes include a hydrogen bond network between the ligand and especially binding site residues Gln1263.32, Thr2235.46, Asn3186.55, Lys3216.58 and Tyr3117.43. Based on the binding modes, it is suggested that the OX₁R is activated by similar binding site contraction as β-adrenoceptors and adenosine A2A. The contraction in could result from the hydrogen bonds between ligand, Gln1263.32, Thr2235.46 and Asn3186.55. The hydrogen bonding of Thr2235.46 can also disrupt interactions between TM5 and TM3, an interaction which is identified as an important factor in keeping the receptor in the inactive state. The role of other ligand residues would be to direct ligand binding and keep the ligand in the helical conformation.
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(2023)Orphan medicinal products (OMPs) are pharmaceuticals, that are utilized in the treatment of rare diseases. Rare diseases are diseases with a prevalence of at most five individuals out of 10 000. Clinical trials with statistically robust clinical data are challenging to conduct with rare diseases, since patient populations are small and the amount of trial subjects enrolling into these trials are usually scarce. Rare diseases also represent a variety of different diseases with divergent properties (5000-8000 identified). This presents challenges in health technology assessment (HTA) when reimbursements for these treatments are assessed and decided, especially when these treatments are usually considerably expensive and burdening to national health care systems. The main objectives for this study and master´s thesis was to research via interviews with experienced professionals from pharmaceutical industry and officials as how to define, monitor and assess the clinical effectiveness of OMP treatments, how to enhance their market access, and how to develop the current conditional reimbursement system in Finland. The interviewees (n=12) all represented from their respective backgrounds and introduced opinions from their own occupational positions and frameworks based on their professional experience. The study was executed as a qualitative study utilizing semi-structured interviews with predetermined questions and themes between 6th of April 2023 and 8th of June 2023. The interviewees were initially contacted via email and phone by one of this thesis supervisors from a professional pool of individuals identified using purposive sampling. The interview transcriptions were examined and analyzed using content analysis, and they were coded and grouped into themes. When inquired, the most common opinions regarding how to define and monitor clinical effectiveness of OMP treatments, the most common answers emphasized individual clinical assessment, real world data (RWD) collection, consideration of symptom control and overall quality of life, economic effectiveness, and clinical expert assessment. Market access of OMPs could be enhanced with more flexible and bold applications for negotiations and agreements, and a need for uniform, predictable MEA procedures, parameters for treatment discontinuation, outcomes-based models, and earlier proactive start for initial negotiations on behalf of the payer (society). The existing conditional reimbursement system might be developed with outcomes-based models, increased dialogue and trust between companies and officials within the realm of negotiations, uniform and predictable MEA procedures, and already established MEA negotiation frameworks to fast tract OMP market access. Development of the existing conditional reimbursement scheme, as well as objective to enhance market access environment in Finland could be accomplished by novel, flexible, patient specific, holistic and bold systems with an emphasis on systematic collection of RWD. Uniform and predictable MEA procedures with predetermined negotiation frameworks could bring value through faster market access and valuable predictability for pharmaceutical companies in their operations. Rapid market access of OMPs could be beneficial via clinical effectiveness of the treatments, as well as through collecting valuable clinical data from the medicinal products.
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(2023)The most typical symptoms of dementia include impairment of cognitive brain functions, such as memory and thinking. Most common forms of dementia include Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, which is caused by degeneration of the frontotemporal lobe. Alzheimer's disease is the most common form of dementia, covering about 75% of all the cases. The pathophysiology of Alzheimer's disease includes beta-amyloid plaques and tau proteins, which accumulate in the brain, and which have been linked to damage to nerve pathways and the appearance of the typical symptoms of the disease. The disorder is progressive, but the exact cause remains unknown. However, old age (>65 years), the APOE-4 gene, lifestyle, and some comorbidities, such as cardiovascular diseases, are considered risk factors. Even though extensive research has been conducted, there is currently no curative treatment for Alzheimer's disease. Sleep disorders can be both a symptom of Alzheimer's disease and a risk factor for the onset of the disorder. Therefore, the mechanisms of sleep and circadian rhythm are connected to the pathophysiology of Alzheimer's disease, for example through the glymphatic system that cleans the brain mainly during deep sleep. Many drugs for Alzheimer's disease have a recommended time of administration. The dosing time can be very important issue in terms of the effectiveness of the drug. According to a recent study, sleep and circadian rhythm have not been considered in most studies on new rapid-acting antidepressants. Therefore, we carried out an analogous systematic literature review for Alzheimer's disease and dementia. The aim of this study was to find out whether sleep and circadian rhythm have been considered in the most cited preclinical and clinical drug research articles for Alzheimer's disease and dementia during the last decade (2010–2020). In addition, it was examined which drug groups the studied compounds belonged to, and what was the sex distribution of the test subjects in the studies. The number of subjects was also determined from clinical studies, and the animal species from preclinical studies. The research articles analysed in the study were collected with a systematic literature review of Scopus database. The study found that most studies did not include any consideration of sleep or circadian rhythm. Most of the investigated compounds were small molecules, followed by supplements and herbs, and rest classified as biological drugs. Most of the clinical trials were relatively small studies with less than a hundred subjects or hundreds of subjects. Among the 100 most cited clinical research articles, there were 14 reanalyses and observational studies that were not included in this analysis of subject numbers. In clinical studies, most of the test subjects were usually female, while preclinical studies used commonly male animals. To conduct more open and reliable science in the future, drug research should pay more attention to the subjects’ sleep patterns, the time of drug administration, and reporting on these issues in the articles, which is usually part of the requirements of scientific journals. This could potentially narrow the translational gap between preclinical and clinical research.
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(2023)Vascular endothelial growth factor C (VEGF-C) is the most studied of the growth factors that control the growth of lymphatic vessels (lymphangiogenesis) and belongs to the same VEGF family as VEGF-A, which controls the growth of blood vessels. The growth of blood vessels and lymphatic vessels is centrally related to the pathophysiology of several cancers that form solid tumours and wet macular degeneration. Unlike VEGF-A, VEGF-C is not currently (2023) a target molecule of any approved drugs, but in clinical trials in the indications mentioned above, combining a VEGF C inhibitor with VEGF-A inhibitors has provided better results than VEGF-A inhibitor monotherapy. The study's objective was converting a phage display library containing single-chain antibody variable fragments (scFvs) screened against VEGF-C into full IgG class antibodies. The scFvs had shown a binding affinity towards the human, mouse, or both VEGF-C variants. The DNA sequences of the best binders of the library had previously been cloned into pLK06H plasmids. The scFvs comprise the variable region of the light and heavy chain (VL and VH) but do not contain the constant regions of the antibody (CL and CHx). Using single-chain antibody fragments as drugs is limited because, in most indications, better stability of whole antibodies, lower immunogenicity, and a longer half-life enabling less frequent dosing is desirable. In addition, the Fc part of whole antibodies often mediates the drug effect, such as complement activation, and whole antibodies are also used as research tools. Secondly, the study aimed to investigate how changing the antibody format affects the binding affinity. To produce whole antibodies, original DNA sequences of pVitro-trastuzumab-IgGk1 plasmid encoding VH and VL regions were replaced with new VH and VL sequences from the phage display library. Several recombinant DNA technology methods were utilised, but the most crucial method was the commercially available NEBuilder HiFi DNA Assembly, which enabled the seamless joining of several DNA fragments into a recombinant DNA molecule in a single-tube reaction. The cloning workflow proved uncertain, as only one constructed antibody production plasmid was sufficiently amplified and expressed in bacterial and mammalian cell cultures. Suboptimal overlapping of DNA fragments and insufficient competence of the bacterial strain used in the transformation were probable bottlenecks. Therefore, as such, the method is not suitable for use on a large scale to convert single-chain antibody fragments into whole antibodies. Also, binding tests were not performed. However, the work done and the antibody production plasmid built is a good basis for further optimisation of the method. In the optimisation, attention should be paid, especially to the quality of the DNA primers and the competence of the bacterial cell line. Also, alternative cloning methods, such as restriction enzymes and ligases, could be used instead of the NEBuilder HiFi DNA Assembly.
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(2023)Adequate vaccine coverage and vaccine refusal have been prominently featured in the media during the COVID-19 pandemic. The decision-makers have considered adequate vaccine coverage important for maintaining the capacity of healthcare. The purpose of this study is to produce population-based research data about the factors affecting the willingness to take the COVID-19 vaccine. With the help of that information, it is possible to identify the factors that influence individual’s decision-making about whether to take the COVID-19 vaccine. The data of the study is the first Kansalaispulssi-data of the year 2022. Kansalaispulssi is a survey, made by the assignment of the State Council, which is used to research Finnish citizen’s views of the current topics. The research design is cross-sectional. The analysis of the study is done by crosstabulation using the Chi-square test. Age and financial situation of the person were related to the willingness to take the COVID-19 vaccine and the person's view of the role of the vaccine in preventing severe corona disease and getting rid of the COVID-19 pandemic. Age also influenced the person's view of the importance of the vaccine in preventing the COVID-19 disease. Gender, province, or educational level had no effect on a person's vaccine views in this study. Based on the research, Finnish citizens are very pro-vaccine. They also understand well the role of the vaccine in preventing a serious illness caused by the disease.
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(2022)Digitalization of health care and the corona pandemic have increased availability and use of online services provided by community pharmacies. In Finland, willingness to use online pharmacy services has been studied from population approach. Less is known about the user satisfaction with the core online pharmacy services such as dispensing and medication counseling services. This study aimed to investigate satisfaction with the University Pharmacy’s online services (ya.fi) from customers’ approach. Primarily, customer satisfaction with dispensing and medication counseling services was assessed. In addition, characteristics affecting customer satisfaction were analyzed. The conceptual framework of the study was Andersen's Model of Health Services Use. The data for this study was collected by a cross-sectional survey conducted in August 2020 among University Pharmacy’s online pharmacy customers who had made a purchase during the last three months. The survey instrument consisted mainly of structured Likert-scale questions, which were used to form two sum variables: satisfaction on online dispensing services (3 variables, Cronbach's alpha 0.803) and satisfaction on online counseling services (2 variables, Cronbach's alpha 0.883). Satisfaction on online dispensing services was studied through willingness to recommend and use the services in the future. Satisfaction on online counseling services was studied through a comparison of medication counseling on an online pharmacy and a conventional pharmacy. IBM SPSS (28) -software was used for statistical analysis consisting of bivariate (Kruskal-Wallis and Mann-Whitney U tests) and multivariate (generalized linear model) analyses to identify factors affecting satisfaction with dispensing and medication counseling services. Of 15 172 invitations sent to fill out the survey, 2555 eligible responses were received (16 %). Of the respondents, 92 % had concomitantly used the services of a conventional pharmacy. . The mean of satisfaction on online dispensing services on a scale from 1 to 5 (5 being the most positive option "completely agree") was 4.3 (SD 0.8). Similarly, the mean of satisfaction on online counseling services was 3.7 (SD 0.9). According to the multivariate analyses, significant characteristics affecting satisfaction on online dispensing services were age, form of living (alone/family with children/couple), purchase of prescription or OTC medicine, frequency of internet use and previous visits to a conventional pharmacy. Characteristics affecting satisfaction on online counseling services were education, purchase of prescription medicine, use of chat information service, frequency of internet use and previous visits to a conventional University Pharmacy outlet. The services on ya.fi online pharmacy rated most important by the respondents were services about medicine availabilities (in conventional University Pharmacy outlets and during a shortage) and information about medicines (prices, Kela reimbursements and information about customers' prescriptions). Customers were satisfied with online dispensing and counseling services. Online dispensing services received a higher satisfaction rate than online counseling services. Customers who had used the chat service and purchased a prescription medicine online were more likely to assess online counseling services to be equal or better than in a conventional pharmacy. Active use of internet and purchases of medicines online were factors connected to higher satisfaction with online dispensing services. Most online pharmacy customers had also visited conventional pharmacies. The results from this study can be utilized in the development of online and other pharmacy services.
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(2010)Cyclin dependent kinase 5 (Cdk5) is studied to take part in the migration neurons and development of brain. It is proven to participate also in the mediation of endothelial cell migration and angiogenesis. Angiogenesis is a crucial physiological mechanism in mediating wound healing and menstrual cycle among other functions. It is also important in some patophysiological processes like diabetic retinopathy and tumour outgrow. Tumour is shown to need its own vascularisation after reaching a size of 2-3 mm as a diameter in order to proceed growing. This makes Cdk5 a potential therapeutic target in regulating angiogenesis. In order to be activated, Cdk5 forms a complex together with its neuronal activator p35 or p39, or with their respective cleavage products p25 or p29. The mechanisms, how Cdk5 is activated in human ehdothelial cells has not been studied before. This master thesis is to evaluate the existence of Cdk5 activators p35 and p39 and their respective cleavage products in spreading human umbilical vein endothelial cells (HUVECs) to mimic the cell migration by freshly plating the cells. In our studies we performed western blot analysis and quantitative PCR analysis to investigate the expression of p35 and p25 in spreading HUVECs in different time points. We also performed an immunofluorescence assay to investigate the localisation of p35 and p25 in spreading HUVECs using confocal laser scanning microscopy (CLSM). The expression of p35 and p25 was also studied after growth factor stimulation (VEGF, FGF). The expression of the activator p39 in spreading HUVECs was studied using quantitative PCR method. Finally we investigated the interaction of Cdk5 with its activator p35 and its cleavage product p25 in spreading HUVECs using immunoprecipitation (IP). We were able to show in our studies that the activators p35 and p25 are expressed in HUVECs and that their expression is changing in spreading HUVECs in different time points. Additionally we were able to show, that p35 and p25 are partly localized in periphery in spreading cells. We were able to show that also the activator p39 is expressed in spreading HUVECs, but its relative amount was shown to be only a small portion of p35 in HUVECs. We were able to prove the interaction of Cdk5 with its activator p35 and p25 using immunoprecipitation, although the result could not be completely verified. Stimulation with growth factors showed no appreciable changes in the expression of p35 or p25. Based on the results, we can state that both the activator p35 and p39, and at least p25, the cleavage product p35, are expressed also in HUVECs. As they are neuronal activators of Cdk5 and Cdk5 has shown to participate in the mediation of angiogenesis and endothelial cell migration, the results amplify our hypothesis that also these activators might have a role in mediating endothelial cell migration and angiogenesis. Nevertheless to assure it, to specify the possible different roles of each activators and their interaction with Cdk5, further studies are needed.
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(2011)Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine's effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.
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(2012)Pharmacogenetics is the study of variations in DNA sequence as related to drug response, i.e. pharmacokinetics, drug efficacy and adverse effects. The literature review of the thesis covers pharmacogenetics of analgesics. The most studied genetic variations affecting the analgesics response are the 118A>G variant of µ-opioid receptor gene (OPRM1) and several variations in the genes coding for cytochrome (CYP) P450 enzymes. Also variations in the COMT gene and the ABCB1 gene coding for P-glycoprotein have been shown to modify the response to analgesics. Genetic polymorphism of CYP2D6, CYP3A4 and CYP3A5 enzymes was studied in the experimental part of the thesis. The aim of the study was to determine if the allele and haplotype frequencies of the CYP2D6, CYP3A4 and CYP3A5 gene variations are different between Finnish breast cancer patients and healthy volunteers. The results will be further used to explore whether the genetic polymorphism of these metabolic enzymes affects the response to a certain drug substance. The study population consisted of 996 Finnish breast cancer patients. Common genetic variants affecting the enzymatic activity of CYP2D6, CYP3A4 and CYP3A5 were studied. In addition to gene copy number, ten single nucleotide polymorphisms (SNP) of the CYP2D6 gene were genotyped. For CYP3A4 gene, genotyping was done for intron 6 SNP rs35599367 shown to decrease CYP3A4 gene expression. CYP3A5 SNP 6986A>G leading to splicing defect and premature STOP codon was also genotyped. Genotyping and copy number determination was done using PCR-based TaqMan® 5'-nuclease method. CYP2D6 haplotype analysis and phenotype predictions were derived based on genotype data. According to CYP2D6 enzyme activity individuals are commonly classified as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) or ultra-rapid metabolizers (UM). The frequencies of CYP2D6 phenotypic classes in our study population were the following: PM, 2.8%; IM 2.0 %; EM 87.7% and UM 7.6%. The haplotype and phenotype frequencies determined for breast cancer patients coincide with the values observed earlier for Finnish healthy volunteers. In our study population, the minor allele frequency (MAF) of the CYP3A4 rs35599367 SNP was 2.7% and the MAF of the CYP3A5 6986G>A SNP 7.6%. The MAF of CYP3A5 6986G>A SNP found in our study is in line with the previous findings for Finnish healthy volunteers. There are no previous publications on the frequency of CYP3A4 rs35599367 SNP in Finnish population. In conclusion, no differences were detected in the frequency of the studied CYP2D6 and CYP3A5 genetic variations between Finnish breast cancer patients and healthy volunteers. Frequency of CYP3A4 rs35599367 SNP in Finnish healthy volunteers should be determined in order to compare it with our findings in the population comprising of breast cancer patients. The results of this study can be further used to explore the effects of CYP2D6, CYP3A4 and CYP3A5 genetic polymorphism on drug response.
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(2020)Background: Alzheimer’s disease (AD) is a worldwide challenge for health care professionals and researchers. Every year, AD causes dementia for millions of patients. No preventive or curative medication is available despite continuous research. Amyloid-beta (Aβ) deposits in brain are one of the main pathological findings in AD. Accumulating Aβ peptides are thought to be the reason behind further disease progression. If the Aβ accumulation could be restricted or Aβ degradation increased their toxic effects would be prevented. Soluble oligomers and protofibrils are the most toxic species of Aβ. Most of the Aβ targeting drugs developed so far have not specifically targeted these toxic species. Neprilysin (NEP) is a major Aβ degrading enzyme that targets mostly the smallest species (monomers and dimers) of Aβ. Another common challenge for protein drugs has been passing the blood-brain barrier (BBB). Different strategies, such as utilising transferrin receptor (TfR) mediated transcytosis, have been studied for drug transport. For example, a rat anti-mouse TfR antibody, 8D3, or its fragments can be used for drug transportation. Objectives: To produce a recombinant protein, sNEP-scFv8D3, combining soluble NEP and single chain variable fragment of 8D3. Testing its ability to degrade different species and isoforms of Aβ in vitro and study in vivo brain uptake. Evaluate whether it is a promising model for future AD treatments. Methods: The recombinant protein was expressed in Expi293 cells and purified with affinity chromatography. The TfR binding was studied with TfR ELISA and enzymatic activity with MCA assay. Aβ ELISA was used for determining the Aβ degradation. Recombinant protein was compared to sNEP. In in vivo studies the brain uptake and blood half-life of radiolabeled sNEP-scFv8D3 of were studied on NLGF mice. Immunohistochemical analyses of brain cryo sections were done to evaluate the co-localisation of Aβ aggregates and sNEP-scFv8D3. Results and discussion: sNEP-scFv8D3 bound to TfR and showed similar enzymatic activity as sNEP. Both sNEP-scFv8D3 and sNEP were able to degrade monomeric Aβ-40 and Aβ-42 but no significant effect was seen on larger aggregates. In mice brain, sNEP-scFv8D3 was detected in same areas as Aβ aggregates. Compared to sNEP, our recombinant protein had better brain uptake. The blood half-life of sNEP-scFv8D3 was approximately 9.5 h and it was cleared fast from the brain. Already 6 h post injection, levels in the brain had dropped more than by half. Further studies are needed to determine whether sNEP-scFv8D3 is effectively transported across the BBB and if it can reduce brain Aβ levels in vivo. Conclusions: In the future, sNEP-scFv8D3 or its improved version could be used at the earliest stages of AD to prevent disease progression. Since sNEP-scFv8D3 degrades only small Aβ aggregates it could be combined with another drug targeting larger oligomers. Together they would decrease the total Aβ deposition in brain.
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(2016)Even though cancer treatment modalities have improved during last decades, there is still lack of specific, efficient and curative treatments especially in case of advanced and metastatic cancers. One relatively new approach is to use oncolytic adenoviruses, which selectively infect and kill cancerous cells leaving healthy cells unharmed. These viruses have shown to be effective especially when administered intratumorally and in combination with chemotherapeutics. However this approach has multiple challenges like rapid clearance by antibody neutralization in systemic administration. Another challenge is the cell entry of oncolytic adenovirus, which is mainly mediated by the Coxsackie-Adenovirus receptor and this receptor is downregulated in various cancer cells. Rapid clearance and reduced cell entry thus lead to decreased amount of oncolytic adenovirus in target cells and decreased efficacy. In order to overcome these limitations, this study explored the possibility to use cancer cell derived extracellular vesicles (EVs) as drug delivery system for oncolytic adenovirus. Since oncolytic adenoviruses have shown to be effective especially in combination with chemotherapeutics, the ability of EVs to deliver both oncolytic adenoviruses and chemotherapeutic drug paclitaxel was studied. The aims of this study were to i) study whether oncolytic adenoviruses could be encapsulated inside EVs (EV-virus complex) and load this complex with paclitaxel (EV-virus-PTX complex), ii) discover whether the surface charge or size distribution of EV-virus and EV-virus-PTX complexes differs from the control EVs and iii) study the infectivity/efficacy of EV-virus and EV-virus-PTX complex in comparison to noncapsulated adenovirus in vitro. Since this is a novel approach, the literature review focused on the characteristics, advantages and challenges of oncolytic adenoviruses and EVs. In order to determine whether cancerous cell are able to encapsulate oncolytic adenoviruses inside EVs, A549 lung cancer and PC-3 prostate cancer cells were infected with oncolytic adenovirus and the formed EVs were isolated form conditioned media using differential centrifugation. Paclitaxel was loaded into these EV-virus complexes with incubation. EV-virus complexes were imaged using transmission electron microscopy (TEM) (i). The characteristics of these EV-virus and EV-virus-paclitaxel complexes were studied by determining the surface charge by electrophoretic light scattering and the size distribution by nanoparticle tracking analysis (ii). In order to determine the infectivity/efficacy of these complexes in autologous use, three in vitro level assays were performed (cell viability, immunocytochemistry and transduction assay) (iii). In addition confocal microscopy was used to observe the localization of EV-virus complexes inside the cell. These studies pointed out that both cell lines were able to encapsulate oncolytic adenovirus inside EVs, which was observed by TEM. The size distribution of these EV-virus and EV-virus-PTX complexes may support this observation and the size was in range 50-500 nm. In addition the determined surface charge was shown to be similar in EV-virus and EV-virus-PTX- complexes when compared to control EVs derived from noninfected cells - however more specific assays in order to characterize the surface properties of EV-virus complexes are needed. As a main finding, these EV-virus and EV-virus-PTX complexes were shown to significantly increase the efficacy of oncolytic adenovirus in comparison to free oncolytic adenovirus, paclitaxel and paclitaxel+virus combination in all three in vitro assays. In addition localization of the EV-virus complex was seen with confocal microscopy imaging. These results indicate that EVs may enhance the delivery of oncolytic adenovirus into cancerous cells. Using EVs as a drug delivery system for both oncolytic adenovirus and chemotherapeutic drug paclitaxel was shown to increase the efficacy of oncolytic adenovirus in comparison to free virus. This characteristic could potentially enhance the targeting ability to cancerous cells and thus lead to decreased amount of side-effects of healthy tissues especially in case of chemotherapeutics. These promising results of this novel approach are however preliminary due to relatively low number of repetitions (n~3) and more research is needed especially in order to characterize, purify and concentrate the EV-virus complexes.
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(2011)Here, we demonstrate the application of desorption atmospheric pressure photoionization (DAPPI) as a screening method at the Criminal Laboratory of the Finnish National Bureau of Investigation for samples confiscated by the Finnish criminal police. DAPPI is a fast mass spectrometric technique to analysis compounds directly from the sample surface in ambient atmosphere. In DAPPI, the sample is thermally desorbed from the sample surface using hot solvent vapor, after which the analytes are ionised in the gas-phase by photon-initiated gas-phase reactions. DAPPI was applied to the direct analysis of confiscated drugs, anabolic steroids and explosives of various matrices without any sample preparation. Confiscated drug samples included e.g. tablets, powders, herbal mixtures, herbal products [Catha edulis (khat) leaves, opium, Cannabis sativa, Psilocybe mushrooms] and ampules and tablets containing anabolic steroids. Powders were sprinkled on a 2-sided tape on a microscope slide, after which the excess powder was shaken away from the tape surface. Liquid samples were analysed from a kitchen paper, after application of 1 Äl of oil from ampules. Other samples were analysed by simply placing them on the DAPPI sampling stage and by directing the solvent plume on the sample surface. DAPPI proved to be a fast and specific analysis technique to this type of forensic analysis. DAPPI does not require any sample preparation, which therefore is well suited for fast forensic analysis, especially for plant samples and oily anabolic steroids, which are considered very challenging with conventional methods. Contamination of the mass spectrometer could be avoided by adjustment of the distance of the sample from the mass spectrometer inlet. Memory effects or contamination of the MS instrument were not observed even after several weeks of DAPPI measurements. DAPPI was also used for trace detection of the explosives trinitrotoluene (TNT), nitroglycol (NK), nitroglycerine (NG), penitrit (PETN), cyclonite (RDX), octogen (HMX) and picric acid. These organic explosives are nitrated compounds, which are divided based on their chemical structure into nitroaromatics (TNT and picric acid), nitroamines (RDX and HMX) and nitrate esters (PETN, NG and NK). Explosive dilutions were analysed with DAPPI from a polymer surface [poly(methyl methacrylate), PMMA] after application and drying of 1 Äl of sample. Also forensic analysis of post-blast residues from different matrices were done. DAPPI was effective in the ionisation of nitroamines and nitrate esters as their adducts with anions such as nitrate, acetate, formate and acetate. TNT used to form negative molecular ions through electron capture and picric acid formed deprotonated molecules through proton transfer. A DAPPI-MS method was developed for all explosives but the identification of the very low concentration explosive traces from wild variety of matrices proved to be difficult.
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(2011)Lipids are fat soluble compounds that are derived from living tissues. Lipids have many important physiological functions. Developing methods for efficient lipid analysis is important since lipids can function as biomarkers in diseases. Additionally these methods can be used for the discovery of the biological processes of disease development. Lipids comprise of molecules with different polarity and structure. Several mass spectrometric ionization methods have been used in the analysis of lipids but they usually require sample preparation prior to the analysis. Desorption electrospray ionization-mass spectrometry (DESI-MS) and desorption photoionization-mass spectrometry (DAPPI-MS) are novel ionization methods that allow sample analysis straight from the matrix, such as tissue, usually without any sample preparation. DESI-MS has already been used in the analysis of different lipids, but DAPPI-MS has only been used in the analysis of steroids. The ionization of a range of lipid compounds (phospholipids, triglycerides, fat soluble vitamins, fatty acids, and steroids) by DAPPI-MS and DESI-MS was studied. Analysis conditions were optimized for all the different lipid classes with both DAPPI and DESI using standard samples. Some lipids were also analysed straight from pharmaceutical preparations. There were differences in the suitabilities of DAPPI-MS and DESI-MS for the ionization of different lipid classes. DAPPI-MS worked well for the ionization of nonpolar lipids like triglycerides, vitamins and fatty acids, but the phospholipids fragmented in the DAPPI-MS process and showed no molecular ion. Previous studies have shown that DESI-MS works well in the ionization of phospholipids, and this study showed that it works reasonably well for other lipid groups as well, with the exception of some of the nonpolar lipids. New knowledge was acquired especially about the suitability of DAPPI-MS for the analysis of different lipids. Based on the results it can be said that DAPPI-MS works equally well or better than DESI-MS in the ionization of most lipid classes. The DAPPI method should still be further developed so that phospholipids, which are very important lipids in human physiology, could be analysed by DAPPI-MS. As lipids were not analysed straight from a tissue sample, there are no conclusions about the suitability of DAPPI-MS for the analysis of lipids straight from tissue samples.
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(2010)The most important part in bioanalysis is the sample cleanup process which is usually the most laborious and time consuming part of the analysis and very susceptible to errors. A functional bioanalysis has to be quick, easily automated, sensitive, selective and stable. It also needs to be suitable for high throughput analysis. Desorption atmospheric pressure photoionization (DAPPI) is a novel direct desorption/ionization technique for mass spectrometry that enables direct analysis of solids from surfaces or liquid samples from a suitable sample plate often without any sample preparation. The suitability of DAPPI-MS for biological samples was investigated by measuring the limits of detection for selected opioids and benzodiazepines and screening them from authentic urine samples. Limits of detection were measured for standard solutions and spiked urine. Opioids and benzodiazepines were analyzed from post mortem urine samples with an optimized DAPPI-MS method. Post mortem urine samples were analyzed with and without sample preparation. Sample preparation improved the sensitivity of the method remarkably. About 50 % of the analytes were detected without sample preparation and almost 100 % after sample cleanup. It is however difficult to estimate the suitability of DAPPI-MS as a screening method because not all analyte concentrations of the urine samples were known. Therefore we cannot be certain weither the results obtained without sample preparation are caused by the suppression of the urine matrix or if the concentrations of the analytes are below the limits of detection. The reliability of the method can further be improved by investigating the metabolites of the analytes and improving the system towards automation. On grounds of this research DAPPI-MS should be used cautiously as a screening method for urine samples without sample preparation and with only high enough analyte concentrations. DAPPI-MS shows promise as a screening method for opioids and benzodiazepines from urine when the sample cleanup is used before the analysis.
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