Faculty of Pharmacy

 

Recent Submissions

  • Parviainen, Heli (Helsingin yliopisto, 2020)
    Statins are a commonly used group of drugs that reduce the cholesterol levels in blood and have been shown to reduce cardiovascular morbidity and mortality. However, a considerable percentage of patients experience adverse effects during statin treatment. Statin adverse effects have been associated with genetic polymorphisms and drug-drug interactions that affect the elimination and active transport of these drugs. A more comprehensive knowledge of statin metabolism may be a step towards better management of statin treatments. Statin metabolism both in vivo and in vitro has been subject of study for years. In vitro incubation conditions may considerably affect the observed clearance, and results obtained with different methods or in different laboratories may not be directly comparable to each other. No single in vitro study on a wide panel of statins has previously been conducted. Six statins and some of their metabolites, fourteen compounds in total, were included in the study. The intrinsic clearance (CLint) of these molecules was investigated in vitro on human liver microsomes (HLM) and a panel of eleven cytochrome P450 (CYP) enzymes recombinantly expressed in E. coli. Observed CLint values for each compound in HLM and for each compound-CYP pair with observed depletion were calculated. The percentual contributions of each CYP enzyme to the metabolism of the compounds was calculated. The results obtained with recombinant CYP enzymes (rcCYP) were complemented with studies on HLM with specific chemical inhibitors of CYP enzymes. In this study the metabolism of statin lactones seemed to be faster than the metabolism of the corresponding statin acids. Atorvastatin lactone, 2-hydroxy atorvastatin lactone, 4-hydroxy atorvastatin lactone and simvastatin were extensively metabolized. Atorvastatin, 2-hydroxy atorvastatin, 3R,5S-fluvastatin, 3S,5R-fluvastatin, pitavastatin lactone and simvastatin acid showed intermediate metabolism. 4-hydroxy atorvastatin, pitavastatin, pravastatin and rosuvastatin rates of metabolism were below quantification limit. CYP3A4 had a major role in the metabolism of atorvastatin and its metabolites, simvastatin and simvastatin acid. CYP3A4 also had activity towards pitavastatin lactone. CYP2C9 had a high activity towards both 3R,5S-fluvastatin and 3S,5R-fluvastatin. CYP2D6 may play a part in the metabolism of pitavastatin lactone. CYP2C8 may have some activity towards simvastatin and simvastatin acid. The data is mostly in agreement with previous in vitro and in vivo studies regarding both the metabolism rate of statins and the contributions by different CYP enzymes to the metabolism of statins. Due to the screening nature of the study and some methodological constraints, these data should be considered as preliminary and require confirmation in further studies.
  • Juntunen, Maiju (Helsingin yliopisto, 2020)
    Cancer immunotherapy refers to therapy strategies that utilise the mechanisms of the immune system to treat cancer patients. The benefits of the approach include the possibility for specific targeting and utilisation of the host immune system. The treatment methods include cancer vaccines, oncolytic viruses (OVs), cell-based immunotherapies and antibodies. The interplay between the cancer and the immune system has been observed crucial for the progress of the cancer and the success of immunotherapies. An immune inflamed tumour microenvironment has been observed beneficial for the success of several therapy methods. Many immunotherapy methods rely on detecting tumour specific antigens that are used to guide the therapy agent to the target site. This strategy poses challenges when considering tumour immune evasion mechanisms, which can cause downregulation of target antigens, and heterogeneity of tumour cells and patients. OVs have the advantage of not requiring predetermined target structures to exert their effect to the tumour cells. They cause direct tumour cell lysis and induce immune responses, and may be modified to express additional genes, including immunostimulatory agents. However, virus-related immunosuppressive mechanisms and a rapid viral clearance may limit their effects. A Western Reserve (WR) Vaccinia virus (VACV) is a highly oncolytic virus strain but the virus has been observed to suppress the function of the cyclic guanosine monophosphate adenosine monophosphate synthase – stimulator of interferon genes (cGAS STING) innate immune pathway which has been shown to have a significant role in anti-tumour immune responses. The aim of this study was to create a WR VACV encoding a dominantly active (D A) STING and to determine whether the virus is capable of activating the cGAS STING pathway. The effects were compared to a corresponding virus vvdd tdTomato that does not have the STING encoding gene. The pathogenicity of viruses was controlled by a double deletion of the thymidine kinase and vaccinia growth factor genes which restricts the virus replication to tumour cells. Transgene fragments were cloned from template plasmids by polymerase chain reactions (PCRs) and joined together in a Gibson Assembly (GA) reaction to form a STING-P2A-eGFP gene insert. The insert was attached to a shuttle vector pSC65-tdTomato by restriction enzyme digestion, ligation and transformation in Escherichia coli. The correct transgene plasmid construct was verified by Sanger sequencing and PCRs. The transgene was inserted to a modified WR VACV vvdd-tdTomato-hDAI by a homologous recombination. The newly created VVdd STING-P2A-eGFP virus was purified by plaque purification. The STING protein expression was studied by an immunocytochemistry (ICC) assay. The immune signalling pathway activation was examined by testing nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation in RAW-Blue cells and dendritic cell activation and maturation in JAWS II cells. The cell viability after iinfection was studied with four cell lines; A549, B16-F10, HEK293 and MB49. The D-A STING expressing virus was produced successfully. The ICC experiment verified the capability of the VVdd STING-P2A eGFP to produce the STING protein in the infected cells. The preliminary findings indicate that the VVdd STING-P2A-eGFP virus activates the NF-κB signalling in the RAW-Blue cells and that the activation is dependent on the STING expression. The activation level is relative to the infection concentration at MOI range 0,001 to 0,1. The findings suggest that the VVdd-STING-eGFP virus can induce innate immune signalling via the STING pathway. The reference virus did not activate the signalling. The in vitro experiments also indicated that the STING virus may induce DC activation and maturation. We observed a trend of CD86 and CD40 expression upregulation on the JAWS II DCs. The effects to the cell viability were inconclusive. More studies should be conducted to verify the results. The effects of the virus should be studied in more advanced cancer models that take into account the complexity of the immune system. These preliminary results indicate the that the VVdd-STING-P2A-eGFP virus could stimulate the immune signalling through the STING pathway.
  • Eriksson, Veronica (Helsingin yliopisto, 2020)
    Migraine was ranked as the second largest cause of disability in 2016 in the Global Burden of Disease (GBD) study. People with migraine have a greater disability and a lower health-related quality of life than those of the general population. Many migraine patients experience functional and emotional impairment due to their disease. Migraine can limit their daily activities and impact their private, professional and social life. Migraine affects the patient also in between the attacks and can impact their education, career and their family and loved ones. Comorbid diseases and failed treatment lines add to the burden of migraine. Furthermore, migraine also imposes an economic burden. Stigma is described as the hidden burden of disease. Chronic migraine patients have been found to have higher stigma than episodic migraine patients. Even though migraine is one of the most common disabling headache disorders, it is still both under-recognised, under-diagnosed and under-treated. The objectives of this study were to determine the extent of the burden and the stigma of migraine in adult Finnish migraine patients. This study aimed to produce comprehensive and current information about migraine and its severity in Finland, highlighting the burden it poses on the migraine patients as well as on society. Migraine is most prevalent among the working aged population, which increases the societal burden of the disease. This study was conducted as a cross-sectional electronic survey amongst adult Finnish migraine patients. The participants were contacted through the Finnish Migraine Patient Advocacy Group. The questionnaire consisted of the already existing and validated Migraine Disability Assessment (MIDAS) Questionnaire and of measures developed by the author. The final data consisted of 608 responses. Of all respondents with 8 or more headache days a month, over 90% were categorised in the severe disability group (MIDAS grade IV), thus having similar disability to those with 15 or more headache days a month (i.e. respondents with probable chronic migraine). The proportion of respondents with severe disability (MIDAS grade IV) was greater in the present study (65.0%) than in a study conducted in Finland in 2000 (47%), indicating that migraine disability in Finland might have become more severe during the past two decades. The mean level of headache pain in the present study was 6.2 (on a scale of 0-10) and pain was the aspect that most respondents viewed as the worst aspect of migraine. This highlights the importance of proper pain management in migraine care. Many of the respondents were also at risk for medication overuse, which highlights the importance of monitoring medication use and informing the patients about possible risks. Stress was reported as the most common migraine trigger, and reducing stress at the workplace was also reported as the most important way of how migraine could better be managed at the workplace. Almost half (44.4%) of all respondents felt stigmatised due to their migraine. Reasons for this stigma and suggested solutions on how to reduce/manage the stigma were quite similar. The ignorance of others was the most reported reason for their migraine stigma, and increasing awareness and correct information about migraine was the most reported way of reducing the stigma. Many of the respondents had faced, due to their migraine, belittlement at work, from family and friend and from healthcare professionals. Facing belittlement from healthcare professionals was reported to have happened often by 11.5% and sometimes by 34.7% of all respondents. Of all respondents, 55.6% worried often and 29.8% worried sometimes about the onset of the next migraine attack. The majority of the respondents had severe disability based on their MIDAS grades. Many other aspect of the burden were reported as well, inculding stigma, reported by almost half of the respondents. Further and future studies need to be conducted to get an even better understanding of the burden and stigma of migraine experienced by adult Finnish migraine patients. This includes further and more intricate quantitative and qualitative analyses of the data from this study, as´well as studies with new perspectives based on the results found in this study.
  • Eriksson, Veronica (Helsingin yliopisto, 2020)
    Migrän rankades som den näst största orsaken till funktionshinder år 2016 i Global Burden of Disease (GBD) -studien. Människor med migrän har större funktionshinder och en lägre hälsorelaterad livskvalitet än befolkningen i allmänhet. Många migränpatienter upplever funktions- och känslomässig nedsättning på grund av sin sjukdom. Migrän kan begränsa deras dagliga aktiviteter och påverka deras privata, yrkesmässiga och sociala liv. Migrän påverkar patienten också mellan attackerna och kan påverka deras utbildning, karriär, familj samt nära och kära. Komorbida sjukdomar och misslyckade behandlingslinjer ökar migränbördan. Utöver dessa så medför migrän även en ekonomisk börda. Stigma beskrivs som den dolda sjukdomsbördan. Kroniska migränpatienter har visat sig ha högre stigma än episodiska migränpatienter. Även om migrän är en av de vanligaste funktionshindrande huvudvärkstörningarna, är den fortfarande både underigenkänd, underdiagnostiserad och underbehandlad. Syftet med denna studie var att fastställa omfattningen av migränbördan och migränstigman hos vuxna finländska migränpatienter. Studien siktade på att producera omfattande och aktuell information om migrän och dess svårighetsgrad i Finland, med betoning på den börda det medför för migränpatienterna och samhället. Migrän är vanligast bland befolkningen i arbetsför ålder, vilket ökar den samhälleliga bördan av sjukdomen. Denna studie genomfördes som en tvärsnittsundersökning med hjälp av en elektronisk enkät bland vuxna finländska migränpatienter. Deltagarna kontaktades via Migränföreningen i Finland. Frågeformuläret bestod av den redan existerande och validerade MIDAS-enkäten och av frågor som utvecklats av författaren. De slutliga datan bestod av 608 svar. Av alla respondenter med 8 eller fler huvudvärksdagar i månaden kategoriserades över 90% i gruppen för svår funktionsnedsättning (MIDAS grad IV) och hade därmed liknande funktionshinder som de med 15 eller fler huvudvärksdagar i månaden (dvs.respondenter med trolig kronisk migrän). Andelen respondenter med svår funktionsnedsättning (MIDAS grad IV) var större i denna studie (65,0%) än i en studie som genomfördes i Finland år 2000 (47%), vilket tyder på att funktionshinder på grund av migrän i Finland kan ha blivit svårare under de senaste två decennier. Den genomsnittliga nivån av huvudvärkssmärta i den aktuella studien var 6,2 (på en skala från 0-10) och smärta var den aspekt som de flesta respondenter betraktade som den värsta aspekten av migrän. Detta understryker vikten av korrekt smärtlindring vid migränvård. Många av respondenterna löpte också risk för överanvändning av läkemedel vilket betonar vikten av att övervaka läkemedelsanvändningen och informera patienterna om möjliga risker. Stress rapporterades som den vanligaste migränutlösaren, och att minska på stress på arbetsplatsen rapporterades också som det viktigaste sättet för hur migrän bättre kunde hanteras på arbetsplatsen. Nästan hälften (44,4%) av alla respondenter kände sig stigmatiserade på grund av migrän. Anledningarna till denna stigma och de föreslagna lösningar på hur man kan minska/hantera stigman var ganska lika. Okunnigheten hos andra var den mest rapporterade orsaken till deras migränstigma, och ökad kännedom och korrekt information om migrän var det mest rapporterade sättet att minska stigmatiseringen. Många av de tillfrågade hade, på grund av sin migrän, utsatts för nedvärdering på jobbet, från familj och vänner och från hälsovårdpersonal. Utsättning för nedvärdering från hälsovårdpersonal rapporterades ha hänt ofta av 11,5% och ibland av 34,7% av alla respondenter. Av alla respondenter oroade sig 55,6% ofta och 29,8% oroade sig ibland för början av nästa migränattack. Majoriteten av respondenterna hade svår migränrelaterad funktionsnedsättning baserat på deras MIDAS-grad. Många andra aspekter av bördan rapporterades också, inklusive stigma, vilket rapporterades av nästan hälften av respondenterna. Ytterligare och framtida studier måste genomföras för att få en ännu bättre förståelse för migränens börda och stigma hos vuxna finländska migränpatienter. Detta inkluderar ytterligare och mer invecklade kvantitativa och kvalitativa analyser av datan från denna studie, liksom studier med nya perspektiv baserade på resultaten av denna studie.
  • Räntilä, Sanna (Helsingfors universitet, 2010)
    The purpose of this study was to describe cat owners' problems that relate to cat medication, especially from the drug formulation point of view. Oral, topical, eye and ear administration routes were included into study. There are few compliance and palatability studies made for cats and dogs in Finland and abroad, but this kind of descriptive study relating different drug formulation has never been done before. This study was carried out as Internet survey questionnaire study and it was addressed to cat owners who visited in academic veterinary hospital for small animals and those municipal and private veterinary offices that were randomized into the study. Additionally, the survey study was addressed to cat owners who had medicated their cats during January-March 2010. Those cat owners were contacted through Internet discussion sites. In the veterinary offices the office staff selected the proper candidates for the study and distributed invitations to participate. For distributing invitations the main criteria was that the cat owner received veterinary medication prescription or got directions for using some medication in cat. 59 answers were received in the study and 84 % of all formulation were administered via oral route. The products were antimicrobial and paracite medicines, cardiovascular and anti-inflammatory medicines. Based on the study results most of the problems were related to oral and ear administration routes. Cat showed low compliance and unwillingness to take pills and capsules because of the unpleasant smell, taste and mouth feel of the product. Tablet and capsule form medicines caused problems to the owners, because it was often necessary to adjust the dose by splitting and cutting half the tablet. This made it difficult for owners to follow given medication instructions. The consistency of liquid medicine forms was described sticky and package material thick and stiff. Because of these factors cat owners had difficulties to evaluate the amount of drops to administer to cats ear or eye and the amount remaining in the medicine bottle. According to the study results there is a need for palatable and easily administered medicines that will be taken readily by cats. It should also be possible to adjust to dose as described. The survey questionnaire is a convenient study method for descriptive purposes and it should be carefully considered what kind of sampling method to use and how to carry out the sampling in practice.
  • Nordström, Sabina (Helsingfors universitet, 2010)
    Parkinson's disease is a chronic progressive neurodegenerative disorder, characterized by muscle rigidity, hypokinesia, tremors and bradykinesia. The cause of symptoms in Parkinson's disease is loss of dopaminergic nerve cells in the substantia nigra, which attenuates the nigrostriatal dopaminergic signaltransmission. Oxidative stress, mitochondrial dysfunction, protein misfolding and aggregation, inflammation, excitotoxicity, apoptosis and other routes for cell death, and loss of neurotrophic factors have shown to be mechanisms in the pathogenesis of Parkinson's disease. Microglia might have a double role in the pathogenesis of Parkinson's disease. Microglia stimulated by Į- synuclein does not only produce toxic factors such as certain cytokines and reactive oxygen and nitrogen species, which contribute to the neuronal cell death but also produce anti-inflammatory cytokines and neurotrophic factors, which can be neuroprotective. Deeper knowledge of the mechanisms underlying Parkinson's disease is needed for developing restorative medicines. Three different neurotrophic factor families are known to be important in the research of Parkinson's disease. The GDNF-family consists of glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN). The neurotrophin-family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophins NT3 and NT4/5. The most recently discovered family is the MANF-family, which consists of mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF). In Parkinson's disease the neurotrophic factors could stop, slow or ideally even reverse the neurodegeneration in the dopaminergic system and decrease the functional decline of the neurons. Research has already shown that GDNF has both a neurorestorative and neuroprotective effect in animal models of Parkinson's disease. Clinical trials have however shown controversial results. The challenge with neurotrophic factors can be the administration to the brain through the blood-brain-barrier, sideeffects because of receptor binding in other organs or sites of the body and low diffusionrate. Research of both MANF and CDNF has shown promising neurorestorative and -protective results in vivo. Local diffusion of MANF has been shown to be better than of GDNF. In this Master's thesis research was done on whether MANF and CDNF have a neurorestorative effect on the dopaminergic nerve cells in mixed primary culture in vitro after 6-OHDA exposure. The aim of the study was to receive information about whether MANF and CDNF are as effective as GDNF at repairing celldamages caused by 6-OHDA in vitro in this experimental model. GDNF was used as a posivite control in this study. The results from this study suggest that MANF might have a neurorestorative effect, but this effect is much smaller than with the neurotrophic factor GDNF. The results show no neurorestorative effect with CDNF. Neither the dopamine uptake nor the tyrosine hydroxylase staining showed statistical significance.
  • Lipsanen, Tuomas (Helsingfors universitet, 2010)
    Medication review is relatively new intervention in Finland that potentially promotes safe and rational use of medicines. During the recent years, the effectiveness of different kind of medication review procedures has been evaluated in many countries. The results considering the cost-effectiveness have varied and conclusions have been difficult to make. Because of the limited resources, the funders and decisionmakers need evidence-based effectiveness data to get the best possible value for the money spent in health care. The aim of the study was to gather, analyze and summarize the published data of costeffectiveness of the medication review by means of a systematic review. The aim was also to represent the medication review as a concept, procedure and study objective. This study was conducted as an analysis of study methods applied in published studies on cost-effectiveness of medication review procedures. In total, 947 references were found using a systematic literature search covering three electronic databases (Medline, CRD and IPA). Most of the references were excluded based on titles and abstracts, and 85 full-text articles were evaluated. After the duplicates were removed, 11 articles met the requested inclusion criteria and were entered to the study. There was a lot of variation between selected articles. In five articles the description of the medication review was not detailed or the intervention was not equal to the expected content level. Also the outcomes measures used in the studies varied. Most studies measured the use of medicines or the number of drug-related problems in different ways. Quality of life was applied as an outcome measure only in five studies and none of the studies were able to show statistically significant differences between intervention and control groups. Mortality was measured in four studies. In most studies the definition and calculation of the costs was limited and inadequate for proper economic evaluation. It was also common that only the direct drug costs from patient's perspective were calculated. The cost of pharmacist's working hours was taken into account in five studies. The incremental analysis was performed only in one study which also got high quality scores compared to all other studies. On the whole the economic evaluations in the selected studies were of low quality and performed in simple a way.
  • Aaltonen, Kalle (Helsingfors universitet, 2010)
    Rheumatoid arthritis is an inflammatory autoimmune disease with prevalence of 0,8 per cent of Finnish people. Rheumatoid arthritis may lead to immobility and premature death. Treatment of Rheumatoid Arthritis includes disease modifying anti-rheumatic drugs and surgery. TNF-blockers are efficacious new drugs, which halt the progression of joint destruction caused by inflammation. The first TNF-blocker to receive permission of the national agency for medicines was Infliximab in 1999. Since then Infliximab has been followed by Etanercept, Adalimumab, Golimumab and Certolizumab. TNF-blockers have been found to be more efficacious than placebo in both clinical trials and register studies. In addition they are considered to be safe enough for clinical use despite the increased risk for tuberculosis and certain cancers. The number of patients annually treated with TNF-blockers in Finland increased threefold between 2004 and 2008. In 2008 the medication costs per patient were 11 669€ for Etanercept and 13 074€ for adalimumab. Systematic literature review is a study, which searches, identifies and combines individual studies. Usually Systematic reviews include a meta-analysis, which uses statistical methods to combine the results of the studies. Meta-analysis aims for increasing power and generalisibility of the studies and reducing the potential bias in individual studies. In order not to introduce bias by itself the systematic review must be done following the methods approved by the scientific community. In addition the process must be documented in detail. Following a predefined search strategy the systematic literature search found 5308 references. After a process involving the evaluation of the patients, intervention, control, outcomes, study design and the risk of bias 27 studies were selected to be included in the systematic review and meta-analysis. Of the included studies, nine had adalimumab, six had etanercept, five had infliximab, four had golimumab and three certolizumab as intervention. TNF-blocker was used either alone or in combination with methotrexate whereas control was either placebo or methotrexate. Altogether, there were 11 533 patients in the intervention group and 9027 in the control group. The results of the meta-analysis indicate reveal that the patients treated with TNF-blockers are twice as likely to reach a 20 % increase on ACR criteria compared to control patients. The likelihood to reach improvements of 50 and 70 % was 3 and 3.5 times higher, respectively. There were no statistically significant differences in efficacy between individual TNFblockers. Increasing the dosage of a TNF-blocker did not increase efficacy. However, combination of TNF-blocker and methotrexate was superior to monotreatment of TNF-blocker without increasing the likelihood of discontinuation of treatment. There were no statistically significant differences between the efficacy of TNF-blocker monotherapy and methotrexate. Adalimumab, infliximab and certolizumab lead more often to treatment discontinuation compared to etanercept and golimumab, which do not differ from control. This systematic review probably found all studies that investigated the efficacy of TNF-blockers in a randomized controlled trial. Study selection and evaluation were based on widely accepted methods. This study has two weaknesses. Firstly, literature search and study selection and evaluation were done only by a single researcher. Secondly, unpublished studies and study results were not actively obtained outside electronic databases.
  • Turku, Ainoleena (Helsingfors universitet, 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.
  • Bahadori, Tadjmohammad (Helsingfors universitet, 2010)
    This master's thesis explored the activities of interpreters used by immigrants in Finnish health care. The main aim was to find out the actual roles of interpreters in working life and how these roles compare and contrast with the roles defined in interpreters` professional code of practice. Additionally, this study explored: what are the most important roles and competencies in the interpreters work from their own perspective and how they perceive their impartiality and proficiency. The interpreters` professional code of practice, Forsander`s (1996) study on interpreters roles and Jalbert`s (1998) classification of interpreter roles were used as a theoretical background of this study. Structured interviews were conducted among interpreters (n=32) working in metropolitan Helsinki. The interpreters were recruited from one interpretation center employing altogether 60 interpreters. The interview guide was based on a previous literature and included questions on interpreters work: their professional code of practice, roles, skills and competencies needed in working life. Interpreters perceived that the role of oral translator, cultural brokers and social role of the various expert institutions were the most important roles in their work. The least important roles were: the role of witnesses, counselor, and an additional source of information. The interpreters reported that they need special support and training in their work. This training should be organized conjointly both with native Finns and other cultures representatives. A shared understanding between different cultures can also be in focus in interpreter services. Similarly with previous studies, language and communication difficulties were found as a major challenge in Finnish health care. Interpreters highlighted that they customers are commonly dissatisfied with the health care due to a continuous rush and lack of time, and attitudes of health care personnel. Immigrants were satisfied with maternity clinic services and high level of technology applied in Finnish healthcare. Interpreters also mentioned that healthcare professionals` cultural skills and experiences are varying: the more they have experience of immigrants, the easier is communication. Interpreters perceived that the majority of healthcare professionals were positive to immigrants and were interested in developing their own cultural competencies. The roles reported by interpreters were in line with the roles defined in interpreters` professional code of practice. Additionally, the characteristics of a competent interpreter as mentioned in the code of practice were also perceived as important among interpreters. This research highlighted the need for cultural education among health care professionals.
  • Huynh, Thi Le Hang (Helsingfors universitet, 2010)
    In the written part of my master -thesis I discuss about GDNF signalling and more specifically how the changes in the GDNF/GFRα1/Ret signaling affect the nigrostriatal dopaminergic neurons in different mutant mice. In the animal models of Parkinson's disease the neuroprotective and neurorestorative effects of exogenous GDNF are very clear which raises hope for use of GDNF in treatment of Parkinson's disease. In intact animals GDNF stimulates the function of nigrostriatal dopaminergic system. Revealing the role of GDNF/GFRα1/Ret signaling in development, maintenance and protection of nigrostriatal dopaminergic system will certainly help in search for treatment of neurodegeneration in Parkinson's disease. In knockout mouse models GDNF/GFRα1/Ret signaling is not crucial for prenatal nigrostriatal dopaminergic neuron development, but it has been shown that it plays an important role in the early postnatal development. Also, it was shown that reduced GDNF/GFRα1/Ret signaling compromises nigrotriatal dopaminergic system in heterozygous GDNF/GFRα1/Ret knockout mice. However the physiological roles of endogenous GDNF and its signalling in the nigrostriatal dopaminergic neurons are not very well understood. In the experimental part of my master -thesis I studied how reduced endogenous GDNF signaling affects the dopaminergic system after 6-OHDA induced neurotoxicity in the conventional heterozygous GDNF mice. Besides that I examined the effects of elevated endogenous GDNF on dopaminergic system of 7 days old so-called GDNF hypermorphs mice. The effects of reduced endogenous GFRα1 levels on dopaminergic system of 20 days old GFRα1 hypomorphs have also been studied. The obtained date showed that mice with the reduced levels of endogenous GDNF are not more susceptible to the 6-OHDA induced neurotoxicity than the wild type littermates. Elevated endogenous GDNF levels did not affect early postnatal development of the nigrostriatal dopaminergic system in GDNF hypermorphs mice as revealed by normal intensity of TH staining in striatum and normal number of TH-positive cells in the substantia nigra pars compacta. Reduced levels of endogenous GFRα1 levels did not affect monoamine levels in the striatum of GFRα1 hypomorph mice.
  • Widell, Kim (Helsingfors universitet, 2010)
    The human tissue kallikreins (KLKs) form a family of 15 closely related serine proteases (KLK1-15). KLK3 is better known as prostate specific antigen (PSA) and it is highly prostate-specific. Kallikreins are attracting increased attention due to their role as biomarkers for screening, diagnosis, and monitoring of various cancers. Although PSA is a very useful marker for prostate cancer in the blood, the expression level of PSA is higher in normal prostatic epithelium than in tumour tissue, and it is further reduced in poorly differentiated tumours. It has been postulated that PSA activators (stimulating compounds) could be beneficial for patients with prostate cancer. The development of peptides as clinically useful drugs is greatly limited by their poor in vivo stability and low bioavailability. As the problems in using peptides as drugs are mainly arising from the peptide backbone, the focus for synthesizing peptide mimicking compounds is on the peptide backbone and how to replace it. The aim of this work was to replace the central disulfide bridge in the most potent PSA activating peptide C-4 by a hydrocarbon linker that had been previously synthesized in the research group. Two strategies for synthesis of the pseudopeptides were applied: a) synthesis of all peptide bonds on solid support by tailoring the reactions for this particular peptide, and b) synthesis of a monocyclic pseudopeptide in solution that could be incorporated directly into the standard protocol of solid phase peptide synthesis. The first strategy (a) proved to be tedious and would have required a lot of optimization to be successful. The cleavage conditions of the orthogonal protecting groups were not directly compatible with synthesis on solid support. The second strategy (b) also proved to be tedious, epimerization at the histidine residue was very prone in the solution phase even with standard peptide coupling reagents. However, the possibility to monitor all steps of the synthesis and to purify intermediate products made this synthetic route more attractive for this type of pseudopetide. The work in this master thesis resulted in a useful strategy to synthesise the desired pseudopeptides.
  • Juuti, Hanne (Helsingfors universitet, 2010)
    The blood-brain barrier protects brain from xenobiotics that are in blood. Different in vivo and in vitro methods have been developed for studying blood brain barrier and those can be found in the literature. There are only few computational models pharmacokinetics of compounds in the brain. In this study permeability factors, which were measured in vitro or in vivo, were collected from literature. Additionally two different pharmacokinetic computer models of blood-brain barrier were described. One of which is called microdialysis model and the other efflux model. Microdialysis model is a very simple two compartmental model, the compartments being the blood and the brain. Five substances were simulated according to the values measured in vivo in rat. The model did not correlate well with the in vivo results, because of the simplicity of the model as the model missed the compartment of brain tissue and the kinetics of transporters. Efflux model has three compartments, blood, blood brain barrier endothelial cells and brain. The model was used to study the impact of the of efflux transporter at the luminal barrier of endothelial cells and passive permeability to the steady-state concentration of a compound in the brain extracellular fluid with theoretical simulations. The relation between free drug concentrations in blood and brain extracellular fluid (Kp,uu) was studied. The impact of Michaelis-Menten kinetics of efflux transporter to the concentration of compound was shown in the results. The efflux model is suitable for theoretical simulations. It is possible to add new active transporters. With theoretical simulations the results from in vitro and in vivo studies can be combined and the different factors can be studied in one simulation.
  • Vanhanen, Jenni (Helsingfors universitet, 2010)
    Histamine is an important neurotransmitter in peripheral as well as in central nervous system. Histaminergic neurons modulate various functions such as sleep-wake cycle, energy metabolism, memory and pain. In addition the brain histaminergic system has been shown to play a role in reinforcement, addiction and addiction related behaviors. After finding the H3 receptor in 1980s it was realised how essential the neuronal histamine is in modulating several central nervous system (CNS) disorders. H3 receptor modulates the synthesis and release of histamine. Furthermore it modulates the release of various other neurotransmitters, such as serotonin, noradrenalin, dopamine, glutamate, γ-aminobutyric acid (GABA) and acethylcoline. The H3 receptor is predominantly expressed in the brain and therefore it is an attractive target for various CNS indications. For more than a decade H3 receptor has gained the interest of many pharmaceutical companies. Several H3 receptor ligands, mainly antagonists or inverse agonists, have been assessed in preclinical as well as in clinical studies. So far there are not enough clinical data on the safety and efficacy of H3 receptor ligands, but there is a strong possibility that H3 receptor antagonists will be used in the treatment of various important disorders, including narcolepsy, schizophrenia and cognitive disorders. Earlier in our research group it was shown that H3 receptor ligands play a major role in ethanol related behaviors. These observations were confirmed in the practical part of this Master's thesis. H3 receptor modulates ethanol stimulation as well as ethanol reinforcement. Both H3 receptor antagonists, ciproxifan and JNJ-10181457 were able to inhibit ethanol-evoked conditioned place preference (CPP). This means they were able to inhibit ethanol reward and reinforcement. Ciproxifan also increased ethanol stimulation. Immepip on the other hand did not alter ethanol-evoked CPP, but it totally inhibited the stimulation of locomotor activity by ethanol. The dopaminergic system regulates both reward and motor functions. The postsynaptic H3 receptors have been shown to be able to heteromerize with both dopamine D1- and D2- receptors in striatum. The formed heteromers modulate dopaminergic neurotransmission in vitro, which may lead to alterations in behavior in vivo. It is therefore possible that the responses we have seen on a behavioral level in this Master's thesis project are due to interactions between histaminergic and dopaminergic systems in striatal areas. The H3 receptor is an interesting target in the drug development of various CNS disorders. The responses seen in this Master's thesis project also indicate that the blockade of H3 receptor inhibit ethanol reward and reinforcement. In conclusion, these findings indicate that H3 receptor antagonists could possibly have therapeutic potential in treating ethanol addiction.
  • Saarelainen, Taija (Helsingfors universitet, 2010)
    Steroid hormones are involved in many physiological functions such as stress response and the maintenance of salt-water balance and pregnancy. Concentrations of steroids in the body fluids are generally very low (below ng/ml). Steroid hormones are metabolically associated and changes in mutual concentration levels of different steroids may signify a disease. Methods that allow the measurement of various steroids simultaneously are of great importance in investigating the role of steroid metabolism for example in formation of cancer. The aim of this work was to develop a sensitive and selective method for simultaneous quantification of 16 steroids in plasma. Nano liguid chromatography-microchip electrospray ionization-tandem mass spectrometry (nanoLC-µESI-MS/MS) was used in order to achieve good sensitivity. C18 enrichment column and separation column, and an electrospray tip were integrated onto the chip that was used in this work. Mass spectrometric parameters were optimized by using a MS calibration and diagnostic chip. It was noticed that the structure of steroids plays an important role on how the compound behave in electrospray ionization. Steroids with 4,5-ene-3-one-structure had much lower limits of detection than steroids without conjugated double bonds (0,075-0,5 ng/ml and 5-25 ng/ml respectively). The chosen sample pretreatment method to extract the steroids from plasma did not work properly, because it was able to extract only a third of the compound's real concentration. Analysis of some compounds was also difficult because of the background noise coming from plasma. The method development was therefore decided to continue with eight steroids that were well detectable and had 4,5-ene-3-one-structure. The limits of detection were 0,075-0,5 ng/ml in biological matrix for these compounds. Eight knock out and seven wild type mouse plasma samples were analyzed using the validated method. The method was able to quantify aldosterone, corticosterone and androstenedione. Developed method did not meet all the aims of this work. Derivatizated compounds, different equipment or totally new method should be used in order to accomplish the aims.
  • Hannula, Mirva (Helsingfors universitet, 2010)
    Prolyl oligopeptidase (POP, prolyl endopeptidase, EC 3.4.21.26) is a serine-type peptidase (family S9 of clan SC) hydrolyzing peptides shorter than 30 amino acids. POP has been found in various mammalian and bacterial sources and it is widely distributed throughout different organisms. In human and rat, POP enzyme activity has been detected in most tissues, with the highest activity found mostly in the brain. POP has gained scientific interest as being involved in the hydrolyzis of many bioactive peptides connected with learning and memory functions, and also with neurodegenerative disorders. In drug or lesion induced amnesia models and in aged rodents, POP inhibitors have been able to revert memory loss. POP may have a fuction in IP3 signaling and it may be a possible target of mood stabilizing substances. POP may also have a role in protein trafficking, sorting and secretion. The role of POP during ontogeny has not yet been resolved. POP enzyme activity and expression have shown fluctuation during development. Specially high enzyme activities have been measured in the brain during early development. Reduced neuronal proliferation and differentation in presence of POP inhibitor have been reported. Nuclear POP has been observed in proliferating peripheral tissues and in cell cultures at the early stage of development. Also, POP coding mRNA is abundantly expressed during brain ontogeny and the highest levels of expression are associated with proliferative germinal matrices. This observation indicates a special role for POP in the regulation of neurogenesis during development. For the experimental part, the study was undertaken to investigate the expression and distribution of POP protein and enzymatic activity of POP in developing rat brain (from embryonic day 14 to post natal day 7) using immunohistochemistry, POP enzyme activity measurements and western blot-analysis. The aim was also to find in vivo confirmation of the nuclear colocalization of POP during early brain ontogeny. For immunohistochemistry, cryosections from the brains of the fetuses/rats were made and stained using specific antibody for POP and fluorescent markers for POP and nuclei. The enzyme activity assay was based on the fluorescence of 7- amino-4-methylcoumarin (AMC) generated from the fluorogenic substrate succinyl-glycyl-prolyl-7-amino-4-methylcoumarin (Suc-Gly-Pro-AMC) by POP. The amounts of POP protein and the specifity of POP antibody in rat embryos was confirmed by western blot analysis. We observed that enzymatic activity of POP is highest at embryonic day 18 while the protein amounts reach their peak at birth. POP was widely present throughout the developmental stages from embryonic day 14 to parturition day, although the POP-immunoreactivity varied abundantly. At embryonic days 14 and 18 notably amounts of POP was distributed at proliferative germinal zones. Furthermore, POP was located in the nucleus early in the development but is transferred to cytosol before birth. At P0 and P7 the POP-immunoreactivity was also widely observed, but the amount of POP was notably reduced at P7. POP was present in cytosol and in intercellular space, but no nuclear POP was observed. These findings support the idea of POP being involved in specific brain functions, such as neuronal proliferation and differentation. Our results in vivo confirm the previous cell culture results supporting the role of POP in neurogenesis. Moreover, an inconsistency of POP protein amounts and enzymatic activity late in the development suggests a strong regulation of POP activity and a possible non-hydrolytic role at that stage.
  • Kinnari, Päivi (Helsingfors universitet, 2010)
    Most new drug molecules discovered today suffer from poor bioavailability. Poor oral bioavailability results mainly from poor dissolution properties of hydrophobic drug molecules, because the drug dissolution is often the rate-limiting event of the drug's absorption through the intestinal wall into the systemic circulation. During the last few years, the use of mesoporous silica and silicon particles as oral drug delivery vehicles has been widely studied, and there have been promising results of their suitability to enhance the physicochemical properties of poorly soluble drug molecules. Mesoporous silica and silicon particles can be used to enhance the solubility and dissolution rate of a drug by incorporating the drug inside the pores, which are only a few times larger than the drug molecules, and thus, breaking the crystalline structure into a disordered, amorphous form with better dissolution properties. Also, the high surface area of the mesoporous particles improves the dissolution rate of the incorporated drug. In addition, the mesoporous materials can also enhance the permeability of large, hydrophilic drug substances across biological barriers. T he loading process of drugs into silica and silicon mesopores is mainly based on the adsorption of drug molecules from a loading solution into the silica or silicon pore walls. There are several factors that affect the loading process: the surface area, the pore size, the total pore volume, the pore geometry and surface chemistry of the mesoporous material, as well as the chemical nature of the drugs and the solvents. Furthermore, both the pore and the surface structure of the particles also affect the drug release kinetics. In this study, the loading of itraconazole into mesoporous silica (Syloid AL-1 and Syloid 244) and silicon (TOPSi and TCPSi) microparticles was studied, as well as the release of itraconazole from the microparticles and its stability after loading. Itraconazole was selected for this study because of its highly hydrophobic and poorly soluble nature. Different mesoporous materials with different surface structures, pore volumes and surface areas were selected in order to evaluate the structural effect of the particles on the loading degree and dissolution behaviour of the drug using different loading parameters. The loaded particles were characterized with various analytical methods, and the drug release from the particles was assessed by in vitro dissolution tests. The results showed that the loaded drug was apparently in amorphous form after loading, and that the loading process did not alter the chemical structure of the silica or silicon surface. Both the mesoporous silica and silicon microparticles enhanced the solubility and dissolution rate of itraconazole. Moreover, the physicochemical properties of the particles and the loading procedure were shown to have an effect on the drug loading efficiency and drug release kinetics. Finally, the mesoporous silicon particles loaded with itraconazole were found to be unstable under stressed conditions (at 38 qC and 70 % relative humidity).
  • Backman, Nina (Helsingfors universitet, 2011)
    Screening of drugs of abuse has to combine sensitivity, selectivity and repeatability. The conventional screening methods include immunoassay screening followed by a more sensitive confirmation method. The aim of the study was to develop a simple, yet sensitive sample preparation method for screening of benzodiazepines and amphetamine derivatives in urine samples with silicon micropillar array electrospray ionization chip (µPESI) coupled to mass spectrometric analysis. Another aim was to evaluate the suitability of µPESI in biological sample analysis. Ideally, the developed method would provide an alternative to immunoassay screening method in forensic urine analysis. The sample preparation methods were separately optimized for benzodiazepines and amphetamine derivatives. Methods used included solid- phase extraction with Oasis HLB cartridge and C18-phase containing ZipTip®-pipette tip, liquid-liquid extraction, and dilution and filtering without prior extraction. Optimization focused, however, on ZipTip®-extraction. The compounds were spiked in blank urine to their cut-off levels, 200 ng/ml for benzodiazepines and 300 ng/ml for amphetamine derivatives. For benzodiazepines, every extraction phase was optimized. The sample pH was adjusted to 5, the ZipTip® phase was conditioned with acetonitrile and washed with a mixture of water (pH 5) and acetonitrile (10 % v/v) and the sample was eluted with a mixture of acetonitrile, formic acid and water (95:1:4 v/v/v). For amphetamine derivatives, pH values of sample and solvents were optimized. The sample pH was adjusted to 10, the ZipTip® phase was conditioned with a mixture of water and ammoniumbicarbonate (pH 10, 1:1 v/v), washed with a mixture of water and acetonitrile (1:5 v/v) and the sample was eluted with methanol. The optimized methods were tested with authentic urine samples obtained from Yhtyneet Medix Laboratories and compared to the results of quantitative GC/MS analysis. Benzodiazepine samples were hydrolyzed prior to extraction to improve recovery. All samples were measured with Q-TOF Micro apparatus and hydrolyzed benzodiazepine samples additionally with microTOF apparatus in Yhtyneet Medix Laboratories. Based on the results the developed method needs more optimization to function properly. The main problems were lack of reproducibility and poor sample ionization. Manual sample preparation and adding to the chip sample introduction spot increased variation. Authentic benzodiazepine samples gave false negative and authentic amphetamine derivative samples false positive results. False negatives may be due to the lack of sensitivity and false positives due to the contamination of sample cone, chips or solvents.
  • Tyyskä, Miia (Helsingfors universitet, 2009)
    Diabeetikoiden määrä lisääntyy jatkuvasti. Samalla hoitokulut ovat kasvaneet merkittävästi. Paras tapa hillitä kustannusten kasvua on hoitaa diabetesta mahdollisimman hyvin. Näin voidaan ehkäistä myös diabetekseen liittyvien oheissairauksien syntyä. Diabeteksen hoidossa on tärkeää kiinnittää huomiota hoidon jatkuvuuteen ja potilaan hoitoon sitoutumiseen. Apteekin henkilökunnan asema on noussut yhä keskeisemmäksi diabeetikon hoitoon sitoutumisen edistäjänä. Tämän pro gradu -tutkielman tavoitteena oli selvittää, mikä on apteekin farmaseuttisen henkilökunnan rooli diabetespotilaan hoitoon sitouttamisessa, elämäntapamuutosten toteuttamisessa ja niiden pysyvyyden varmistamisessa. Asiaa tarkasteltiin voimaantumisen teorian näkökulmasta. Tarkoituksena oli selvittää, miten voimaantuminen yksilössä tapahtuu, miten sitä voidaan apteekkineuvonnalla edistää ja mikä on apteekin rooli ulkopuolisena voimaannuttajana. Tämän tutkimuksen aineisto on kerätty Mäntyharjun Havu apteekissa ja se on osa laajempaa tutkimusta, jonka päätavoitteena on kehittää ja testata apteekkeihin soveltuva yksilökeskeinen toimintamalli tyypin 2 diabeteksen hoidon tukemiseen. Toimintamalli perustuu säännöllisiin neuvontatapaamisiin apteekissa. Pro gradu -tutkielmaan analysoitavaksi valittiin tutkimusjoukosta (n=19) ne, joilla tapahtui apteekkiohjelman aikana eniten positiivisia muutoksia yksilötasolla sekä elämäntapamittareilla mitatuissa arvoissa että kliinisissä parametreissa (n=4). Kvaliatiivisessa analyysissä käytettiin sekä deduktiivista että induktiivista lähestymistapaa. Vaikka diabeetikoilla oli tietoa sairaudesta ja elämäntapojen merkityksestä, niin käytännön tasolla jokainen henkilö kaipasi hoitoon ja erityisesti muutosten toteuttamiseen tukea ulkopuoliselta taholta. Apteekin rooli ulkopuolisena voimaannuttajana koettiin erityisen keskeiseksi. Tapaamiset loivat oikeanlaisen ympäristön ja ilmapiirin elämäntapamuutosten toteuttamiseen ja voimaantumisprosessin etenemiseen. Voimaantuminen ruokavaliomuutoksiin oli koko intervention aikana melko nousujohteinen prosessi. Sen sijaan voimaantuminen liikunnalliseen elämäntapaan oli aaltoilevaa. Apteekkitapaamiset sosiaalisena tapahtumana paransivat asiakkaan hoitoon sitoutumista. Asiakas koki, että häntä kohdellaan yksilönä kokonaisvaltaisesti. Voimaantuakseen yksilö tarvitsi aikaa. Vuoden mittaisen intervention aikana voitiin saavuttaa pysyviä muutoksia elämäntapoihin, mikäli yksilöllä itsellään oli halu ja motivaatio sitoutua tukiohjelmaan. Tämä tutkimus osoitti, että tämänkaltaista apteekkiohjelmaa tarvitaan. Nykyisessä kiireyhteiskunnassa ihmiset arvostavat, jos jollakin on aikaa paneutua yksilöön itseensä ja hänen sairautensa hoitoon kokonaisvaltaisesti.
  • Saarikko, Elina (Helsingfors universitet, 2010)
    Biopharmaceutical Classification System (BCS) is a scientific framework for classifying drug substances based on their aqueous solubility and intestinal permeability. When combined with dissolution of the drug product, the BCS takes into account three major factors that govern the rate and extent of drug absorption. For a BCS biowaiver, the in vitro dissolution study may be used as a surrogate for in vivo bioequivalence studies. Currently, BCS I drugs are accepted as biowaiver candidates by EMEA, FDA and WHO. EMEA and WHO also accept class III drugs in some conditions. The main difficulty in classifying drugs according to BCS is the determination of permeability. Biopharmaceutics Drug Distribution Classification System (BDDCS) was introduced to provide a surrogate for permeability. If the major route of elimination is metabolism, then the drug exhibites high permeability. There are two parts in this master thesis. BCS and BDDCS are discussed and evaluated in the literature part. The focus is in the BCS III drugs. The purpose of the experimental part is to evaluate BCS III drug, hydrochlorothiazide as a biowaiver candidate. Solubility of the drug substance and dissolution of the drug product was determined. Aim of the permeability studies with Caco-2 cells were to study if hydrochlorothiazide permeates by passive diffusion across the monolayer. Importance of paracellular diffusion was evaluated by opening tight junctions with EDTA. Influence of dissolution rate was evaluated by theoretical simulation. According to the results of this study, hydrochlorothiazide has good solubility in aqueous buffer. It has been reported to diffuse passively across the epithelial cells but in this study permeability increased when concentration decreased. This may be due to active transport. Hydrochlorothiazide diffuses partially through the tight junctions. Dissolution of the hydrochlrothiazide tablet was very rapid. Drug eliminates almost entirely by metabolism, it is also BDDCS class III drug. EMEA and WHO accept BCS III drugs as biowaiver candidate if dissolution rate is very rapid. According to this, hydrochlorothiazide could be suggested as a biowaiver candidate. There are also other issues to be considered, for example excipients used in tablets. Since hydrochlorothiazide has been discovered to be absorbed in the upper part of the small intestine, the influence of excipients is especially important. This possible influence should be evaluated before the final decision of biowaiver.

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