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  • Hämäläinen, Noora (2021)
    Mini-tablets are 1-3 mm in diameter and administered as a single tablet or as a multi-particulate formulation. Mini-tablets are an attractive alternative for conventional solid dosage forms due to the ease of administration and the possibility for combination and individualised drug therapy. In mini-tablet production, good flowability of the formulation is critical as minor variations in die filling can lead to significant changes in mini-tablet weights. In addition, to reduce weight variation, the particle size should not exceed 1/3rd of the die diameter. This study aimed to determine the influence of the granule size on mini-tablet weight variability and content uniformity. The feasibility of direct compression, as well as high-shear wet granulated and roller-compacted formulations, were evaluated. From the nine final formulations manufactured, particle size distribution, Hausner ratio, Carr’s index, angle of repose and flowability were determined. The mini-tablets were made on a rotary tablet press using single punches of 3 mm in diameter. Content uniformity and weight variation of the mini-tablets were determined. The direct compression formulation had the smallest particle size, and the roller-compacted formulation milled through a 1.0 mm and 1.25 mm square screen had the largest particle size. Surprisingly, the RC 0.8 mm grater screen formulation had a very wide particle size distribution and is classified as a very fine blend. The wide particle size distribution might result from a high fill ratio during the milling of the roller-compacted ribbons. The four different high-shear wet granule formulations had a very similar particle size distribution. According to the Hausner ratio and Carr’s index values, the flow properties of the formulations varied between fair and very poor, while according to the angle of repose, the flow properties were between excellent and poor. However, all nine formulations were used to make mini-tablets with acceptable uniformity of mass, mini-tablets were within ± 8 % of the target weight, and none exceeded the 10 % limit set by Ph. Eur. The weight variation is small, as indicated by the low RSD of 1.0-2.9 %. The differences in the weight variation may be attributed to segregation due to particle or granule size and density. This is further supported by the fact that no force feeder or vacuum was utilised in the rotary tablet press, possibly causing re-circulation of the formulation and shearing forces. In addition, the fill level of the feeder might have varied between the nine formulations and affect the weight variation in a way that is not recognised in this study. Only direct compression formulation was within the limits of uniformity of content of single-dose preparations set by Ph. Eur. In the final formulations, the amount of paracetamol was in the HSWG 0.8 mm round screen 98.5 % and in the RC 1.0 mm square screen 97.7 %. These results suggest that the formulations contained an adequate amount of paracetamol, which does not explain why the mini-tablets made from high-shear wet granules did not meet the content uniformity criteria. Furthermore, the weight variation might not entirely explain why high-shear wet granulated formulations performed so poorly in the content uniformity analysis. In summary, that direct compression is a feasible manufacturing method for mini-tablets of 3 mm in diameter. However, further studies are needed on the content uniformity of mini-tablets made using high-shear wet granulated and roller-compacted formulations as these did not meet the content uniformity criterion. In particular, the content uniformity of the mini-tablets made from the high-shear wet granulated formulations was not acceptable, and the reason for this was not identified.
  • Sundberg, Enikö (2024)
    Viimeaikaisten tutkimusten perusteella suun sairauksilla on havaittu olevan vakavia systeemisiä vaikutuksia sekä vaikutuksia yleisterveyteen ja hyvinvointiin. Antiseptisia valmisteita käytetään tulehduksellisten suun sairauksien hoidossa, mutta niillä on todettu riittämätöntä tehoa ja paikallisia sivuvaikutuksia. Antimikrobisia aineita käytetään vakavampien suun tulehdusten hoitoon, mutta ne lisäävät antibioottiresistenssin riskiä ennaltaehkäisevässä ja toistuvassa käytössä. Kaksoisvalohoito vähentää mikrobilääkkeiden ja antiseptisten aineiden tarvetta ja siten haitallisia sivuvaikutuksia. Varsinkin ennaltaehkäisevänä hoitona kaksoisvalon uskotaan aiheuttavan vähemmän haittaa, mutta olevan silti riittävän tehokas estämään tulehduksia. Fotodynaamista hoitoa (PDT) on tutkittu mm. parodontiitin, peri-implantiitin ja suun punajäkälän hoidossa. Satunnaistettu, yhdessä keskuksessa toteutettava kliininen lääketutkimus suunniteltiin ja toteutettiin määrittämään säännöllisesti käytetyn antibakteerisen lääkinnällisen laitteen, Lumoral®-kaksoisvalohoidon, tehoa ja turvallisuutta plakin hallinnassa ja ienterveydessä julkisessa terveydenhuollossa. 40 molempaa sukupuolta edustavaa, 37–77-vuotiasta koehenkilöä, joilla oli diagnosoitu parodontiitin vaihe II tai korkeampi, jotka harjaavat hampaitaan päivittäin ja käyttävät hammaslankaa tai hammasharjaa, satunnaistettiin 1:1 kontrolli- ja tutkimusryhmään. Kaikilta koehenkilöiltä kerättiin aMMP-8 ja mikrobiologiset näytteet ja kliiniset parametrit, kuten PPD, BOP, VPI, sekä lähtötilanteessa että tutkimuksen lopetuskäynnillä. Tutkimusryhmä sai aPD-hoitoa päivittäin noin 5 viikon ajan. aPD-hoito suoritettiin CE-merkityllä Lumorinse®-suuvedellä yhdessä CE-merkityn Lumoral®-valoaplikaattorin kanssa. Suuvettä purskuteltiin minuutin ajan, jonka jälkeen annosteltiin 10 minuutin ajan samanaikaisesti 405 nm aBL ja 810 nm lähi-infrapuna (NIR) LED-valoa, kokonaissäteilyaltistuksen ollessa 40 J/cm2. Seurantakäynnit suoritettiin keskimäärin 7 viikon kuluttua lähtötilanteen arvioinnista, jolloin tutkimusryhmässä oli keskimäärin 8 päivän hoitotauko. Tutkimustulokset viittaavat Lumoral® aPDT -hoidon käytön vähentävän ienverenvuotoa, auttaen hoitamaan tulehduksellisia suusairauksia, kuten parodontiittia, peri-implantiittia ja ientulehdusta. Suurempi hyöty on osoitettu tupakoimattomille henkilöille. Tutkimustulokset viittaavat tupakoimattomien henkilöiden, joilla on diagnosoitu vaiheen II tai III parodontiitti, voivan hyötyä Lumoral® hoidosta arvioitaessa syviä ientaskuja. Tutkimus osoittaa potilaiden, joilla on vaiheen II tai sitä korkeampi parodontiitti ja joilla on jo kohtalaisen hyvät kotihammashoitorutiinit, olevan motivoituneita näkemään ylimääräistä vaivaa hoitonsa parantamiseksi. Lumoral®-hoidon tehokkuuden arviointia syvien ientaskujen paranemisessa varten tarvitaan lisätutkimuksia. Lumoral®-hoidon vaikutusta hammasplakkiin, parodontiitin vaiheeseen ja mikrobiologiseen taakkaan tulee varmistaa lisätutkimuksin. Tupakkatuotteiden vaikutus Lumoral®-hoidon tehoon tulee vahvistaa. Optimaalista käyttötiheyttä ja Lumoral®-hoidon kestoa tulee tutkia lisää. Lumoral®-hoidon hyöty potilailla, joilla on vaikea vaiheen IV ja asteen C parodontiitti, tulee varmistaa kohdennetuin tutkimuksin.
  • Ignatius, Adele (2021)
    Misfolding and aggregation of alpha-synuclein (α-syn) protein, leading to dysfunctional proteins and toxic protein aggregates, are seen as major factors in the pathogenesis of Parkinson’s disease (PD). Direct protein-protein interactions (PPI) between α-syn and a serine endopeptidase, prolyl oligopeptidase (PREP), have been shown to increase α-syn aggregation. Small molecular PREP inhibitors, in turn, have been shown to reduce the ɑ-syn aggregation process both in vitro and in vivo. Inhibition of PREP has been shown to have dual effects on ɑ-syn aggregation: first of all, blocking PREP mediated seeding and secondly, inducing the clearance of ɑ-syn aggregates via increased autophagy. Thus, PREP inhibitors should be further studied as a potential treatment for PD and other synucleinopathies. In this study, we evaluated the effect of two different PREP inhibitors, 4-phenylbutanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047) and HUP-115 in a virus vector-based unilateral A53T-ɑ-syn overexpression mouse model. AAV-A53T-ɑ-syn injections used in this study caused a significant increase in oligomer-specific alpha-synuclein (ɑ-synO5) immunoreactivity and a mild dopaminergic neuron loss, together with mild motor deficits. Neither 2-week PREP inhibition with KYP-2047 or 4-week PREP inhibition with HUP-115 reduced ɑ-synO5 immunoreactivity or protected dopaminergic neurons in the substantia nigra (SN). Concordant to this, the treatments did not restore the slight behavioral deficit AAV-A53T-ɑ-syn injections caused in the cylinder test. In previous studies, PREP inhibition with KYP-2047 decreased ɑ-synO5 immunoreactivity, attenuated dopaminergic neuron loss and restored behavioral deficits in other α-syn overexpression mouse models. It is suggested that PREP inhibitors mainly have an effect on soluble ɑ-syn oligomers, rather than insoluble fibrils. In case A53T-ɑ-syn forms insoluble fibrils too rapidly in mice, overexpression of A53T-ɑ-syn might not be a suitable option when studying the effects of PREP inhibitors. Our results suggest that further characterization of this model in mice is much needed before drawing any conclusions about the effect of these PREP inhibitors.
  • Jaskari, Iida (2022)
    Multiple sclerosis is a progressive inflammatory disease of the central nervous system that affects young adults. The pathological hallmark of MS is the degradation and loss of oligodendrocytes resulting in demyelination. Damage to axons caused by demyelination severely impairs physical function. Currently there is no cure for MS, but current drugs aim to modify the course of the disease and relieve symptoms. However, they are unable to promote the repair of damaged myelin sheaths, and thus new therapies are needed. In this study, the effect of V-MANF on remyelination was investigated in two commonly used experimental toxin models. V-MANF is a modification of the endoplasmic reticulum located protein MANF, which has been found to have neuroprotective and regenerative properties. Additionally, MANF can regulate ER stress, which contributes to demyelination in MS. The effect of V-MANF on lysolecithin-induced demyelination was examined in organotypic cerebellar brain sections from C57B/6 mice. The study was conducted exceptionally using the brains of adult mice because they are a better model for neurodegenerative diseases. However, when analyzing the results, it was found that there was no demyelination in the tissue cultures, so the effect of V-MANF could not be analyzed. In the other study, C57B/6 mice were given dietary cuprizone for six weeks, followed by daily intranasal administration of either V-MANF or vehicle for seven days. Mice were subjected to behavioral experiments, in which a light/dark box test showed that V-MANFs had a potential anxiolytic effect in mice receiving cuprizone. No significant demyelination was observed by immunohistochemical analysis and therefore the effect of V-MANF on remyelination could not be assessed. However, the results of the study can be utilized in the design of further studies.
  • Turunen, Iida (2023)
    Metastatic prostate cancer is often fatal disease stage. Mechanism causing prostate cancer remains unknown, but possible mechanism relies on hormones. Testosterone may activate spontaneous cell division of oncogenes. Prostate cancer cells require androgen cell stimulation of AR to grow in early stages of prostate cancer, approximately 80-90% of prostate cancer cases are androgen dependent. 3bHSD1, encoded by HSD3B1, catalyzes the conversion of dehydroepiandrosterone to androstenedione and further to T and DHT. SNP (1245A to C) in HSD3B1 changes asparagine to threonine in position 367 resulting the enzyme accumulation and increased function. With androgen deprivation therapy castrate levels of testosterone are often achieved and it induces positive response in most PCa patients, but the polymorphism of 1245C is related with lower survival rate and higher probability for PCa to develop into CRPC. The aim of this study was to find out the effect of SNP in 3bHSD1 to androgen levels in patients treated with ADT. 32 patients were first genotyped based on SNP in the HSD3B1 gene (rs1047303) with 96.9 % success rate. 21 patients represented genotype AA, 9 AC and 1 patient CC. Other mutation in rs6203 was also detected. Genotyping was done by isolating DNA from blood samples and preparing it further for Sanger sequencing. Steroid analysis was performed by using LC/MS, using liquid-liquid extraction as sample preparation method. Altogether 21 steroids were analyzed from serum samples. Samples were taken every 3 months, during 33 months period for longest. The concentrations of T and DHT were reduced in AA genotype group after ADT as was expected to happen in all of the groups. In fact, the only significant changes were seen in AA genotype with for example the concentrations of previously mentioned T, DHEA and also A4. The changes in measured androgen levels cannot be generalized to concern especially the CC genotype, as there was only one patient homozygote with the mutation. Even though these results gave promising data of possible androgen synthesis pathways, a similar study must be rerun with larger patient data to be sure of the characteristics of different genotypes. Also, the effect of SNP in rs6203 remains still unknown.
  • Niittymäki, Erika (2021)
    Since the discovery of ketamine’s antidepressant response, numerous of studies have been observed it to alleviate depressive symptoms rapidly and effectively within hours. This is a significant advantage compared to traditional antidepressants, which take weeks to show treatment efficacy. Ketamine is a N- methyl-D-aspartate receptor (NMDA) antagonist and its underlying mechanism of is proposed to be in its ability to increase synaptic plasticity and this is ultimately believed to improve mood. On a molecular level, the antidepressant effects have been observed to be dependent on the activation of tropomyosin receptor kinase B (TrkB) signalling pathway. However, the antidepressant mechanism of ketamine remains still poorly understood as no new NMDA-antagonist or other rapid-acting antidepressants have been successfully developed for clinical use despite many years of effort. Therefore, some have proposed that the missing pieces of understanding its antidepressant effects might be linked to ketamine’s ability to modify sleep patterns and circadian-related molecules. Ketamine has especially been demonstrated to increase slow-wave activity during the following night of treatment and these changes have been shown to predict the clinical outcome in patients with major depressive disorder (MDD). Slow-wave activity is a low-frequency and high-amplitude wave seen in electroencephalography, which is highly expressed during the deepest stage of sleep, and this has been prominently found to be reduced in MDD patients. Even more intriguing, there are indications that ketamine might increase slow-wave activity also immediately after its administration. During this time, TrkB signalling is observed to became active. Following these molecular findings, we sought to investigate the link between the TrkB signalling pathway and two prominent processes occurring during slow-wave sleep. During slow-wave sleep processes such as (1) reduction of brain’s energy expenditure and (2) the activation of glymphatic system is known to occur. The glymphatic system is as lymphatic-perivascular network, which is responsible for clearing the brain from the metabolic waste. Thus, in this study, our objective was to investigate whether by causing an acute decline in adenosine-triphosphate (ATP) production or by stimulating the glymphatic network, we could activate the same plasticity-related pathways as ketamine is capable of activating in mice prefrontal cortex. The results of this study suggest that acute metabolic reduction can trigger pathways regarding synaptic plasticity. The metabolic inhibitor, 2-deoxy-D-glucose and mercaptoacetate (2DG+MA), was found to phosphorylate the TrkB receptor and its downstream signalling molecules GSK3β and p70S6K, while MAPK was dephosphorylated. These results correlate with the previous findings of ketamine’s effect after its administration. We also found a plasticity-related marker, MAP2, to be heavily phosphorylated by 2DG+MA, indicating 2DG+MA having a surprising role on neuroplasticity. These results are promising indication of understanding the rapid effects of ketamine and might even give important insight to developing novel antidepressants. However, these findings are only preliminary, and more research is needed to directly link antidepressant effects and energy metabolic inhibition together, as our study did not directly investigate antidepressants and depression-like behaviour in mice.
  • Snellman, Nana (2023)
    Chlamydia pneumoniae is an intracellular Gram-negative bacterium, that can cause respiratory infections. Infections are typically mild or asymptomatic, but it can also lead to more severe infections, for example, pneumonia. Severe infections might need antibiotic treatment. When the bacteria are exposed to stressful conditions, they can change to a chronic, persistent form. Amoxicillin and penicillin are known to transform bacterium into persistent forms. Antibiotics are not effective for persistent infection very often. Amoxicillin is the recommended treatment for pneumonia in Finland and worldwide, which is problematic from the perspective of C. pneumoniae. That is why there is a need for effective treatment for persistent C. pneumoniae infection. Probiotics and their by-products short chain fatty acids (SCFAs) are known to have beneficial effects on human health. Based on the current knowledge, SCFAs and other probiotic by-products are known to inhibit pathogen bacterial growth. Thus, SCFAs could have a potential effect on the treatment of C. pneumoniae infection. The aim of this work is to study the impact of SCFAs, acetate, propionate, and butyrate on C. pneumoniae infection and its antibiotic susceptibility. To study the impact of acetate, propionate and butyrate on C. pneumoniae infection and its antibiotic susceptibility, three different infection models were used: productive C. pneumoniae infection model with A549 cells, amoxicillin-induced persistent infection model with A549 cells, and persistent infection model with THP1 cells. Bacterial growth was followed with immunofluorescence and the number of the bacterial genome was studied with quantitative polymerase chain reaction (qPCR). The studied SCFAs did not have a significant impact on productive C. pneumoniae infection. With amoxicillin- induced persistent infection, the results were varying. For example, sodium acetate, and propionate showed some increase in bacterial growth on the first infection, but with sodium butyrate, there were not any impact. The only SCFA that decreased the bacterial growth in the persistent infection model with THP1 cells was sodium butyrate (200 μM). The same treatment also decreased the number of bacterial genomes with qPCR in the same infection model. In addition, the same condition increased the antibiotic susceptibility of persistent C. pneumoniae to azithromycin in THP1 cells. In conclusion, the studied SCFAs seemed to have more impact on C. pneumoniae infection with human immune cells compared to human lung epithelial cells. Based on this study, sodium butyrate could have positive impacts against persistent C. pneumoniae infection. Nevertheless, further studies of the impact of sodium butyrate on persistent C. pneumoniae infection are needed.
  • Savolainen, Heikki (2018)
    Tablet manufacturing requires both high-quality equipment and powder blend with high flowability and compactability and low segregation tendency. The process is complex and tablet formation process still remains not fully understood. Adequate powder flow is a necessity for the pharmaceutical manufacturing process, i.e., powder flowability and flow properties play a great role when designing manufacturing processes for solid dosage forms. As such, the powder characteristics need to be investigated. However, one property is seldom enough to predict the flowability of a powder in specific processes and different test methods need to be used to fully understand the tableting performance of a particular powder. It is crucial to know how the assessed properties reflect the manufacturing conditions. The need for test batches and the use of empirical testing still exists despite the numerous powder characterization tests available. The main aim of the study was to understand the influence of material properties, flow properties and segregation tendencies on both the processability of a formulation during tablet compression and the critical quality attributes, such as mass, tensile strength and dose uniformity of the final drug product. Additionally, testing of an in-line NIR method to observe the homogeneity of the powder inside the force feeder right before the compression step and transmission Raman as an at-line method for tablet content were also evaluated. A number of powder characterization tests were employed in order to fully understand the impact of the formulation on the process performance. Three formulations with different particle size of the active substance and mannitol were used throughout the study. Both the sifting segregation and fluidization segregation tests’ results predicted the formulations’ tabletability particularly well. Fluidization segregation test predicted the changing composition of the formulation throughout tableting whereas sifting segregation results showed the constantly fluctuating API concentration in the manufactured tablets. Moreover, the Raman results confirmed the tablets of variable content despite the offset caused by the different particle size of the raw materials used. The functionality of the NIR in the force feeder was tested successfully. The residence time distribution could be determined at a sufficient level to point out tablets of a bad quality from the batch on grounds of the NIR data. Results from the powder flow property tests were rather conflicting. Angle of repose, Carr’s index and volume flow rate gave the best characterizing results, whereas the mass flow rate, shear test with higher normal stress in pre-shear gave the worst results, considering the experienced flow character of the formulations. As stated above, different flow property tests may give conflicting result, and hence, it is crucial to know which results are the most relevant ones. Furthermore, the right settings for the test should be known to gain applicable results, best exemplified by the shear cell test.
  • Eriksson, Veronica (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.
  • Silfvast, Saga (2016)
    Heart failure is a major public health problem and a leading cause of mortality worldwide. The most common cause of heart failure is myocardial infarction. Following a myocardial infarction, a large number of cardiomyocytes die and cardiac muscle is replaced by fibrotic scar tissue. Since the adult heart has inadequate endogenous regenerative capacity, loss of muscle tissue often causes a progressive decrease in cardiac function eventually leading to heart failure. At the moment heart transplantation is the only curative treatment for heart failure, but the low number of donor hearts is limiting the use of this treatment option. As current drugs only slow down the progression of the disease, there is a great need for new regenerative treatments. Direct cardiac reprogramming is a new approach for generating cardiomyocytes for cardiac regeneration. Unlike pluripotent stem cell-based strategies, direct reprogramming enables conversion of a terminally differentiated cell type directly into another cell type without first producing a pluripotent intermediate. Due to their abundancy and role in the repair of myocardial injury, fibroblasts represent an attractive starting cell type for direct cardiac reprogramming. Fibroblasts have been directly reprogrammed to induced cardiomyocytes (iCMs) by overexpression of key cardiac transcription factors, microRNAs (miRNA) or by modulating specific signal transduction pathways with small-molecule compounds. Despite successful reports of direct reprogramming both in vitro and in vivo, the efficiency of direct reprogramming remains, however, too low for potential clinical applications. The aim of this M.Sc. thesis work was to establish direct reprogramming of mouse embryonic fibroblasts (MEFs) to iCMs by viral overexpression of cardiac transcription factors Hand2 (H), Nkx2.5 (N) Gata4 (G), Mef2c (M) and Tbx5 (T) and a small-molecule compound screening platform for identifying small-molecule compounds that could enhance the reprogramming efficiency and potentially replace cardiac transcription factors in direct cardiac reprogramming. In accordance with previous publications MEFs were successfully directly reprogrammed to iCMs using both HGMT and HNGMT cardiac transcription factor combinations. The screening platform was tested using the TGF-β inhibitor SB431542, which has recently been reported to increase the cardiac reprogramming efficiency. In line with previous publications, the reprogramming efficiency was significantly increased by treatment with SB431542. Initial tests with other small-molecule compounds did not have a positive effect on the reprogramming efficiency. The results of this M.Sc. thesis work verify previous publications and demonstrate a method for in vitro small-molecule compound screening, which can be used to identify compounds that increase the reprogramming efficiency in direct cardiac reprogramming. However, the results shown here are only preliminary and more replicates are needed in order to confirm the current results. Nonetheless, the results of this thesis work set a foundation for finding small-molecule compounds that in the future might be used to target direct cardiac reprogramming as a regenerative therapy for myocardial infarction and heart failure.
  • Kahma, Helinä (2014)
    Active transport processes in the basolateral (sinusoidal) membrane of hepatocytes have an important role in the hepatic clearance and overall disposition for several types of drugs. Organic anion transporting polypeptides (OATPs) expressed in the sinusoidal membrane have been shown to mediate the sodium-independent hepatic uptake of broad range of drugs and they have been associated with clinically relevant drug-drug interactions (DDIs) and genetic polymorphisms. The literature review focuses on sinusoidal OATP transporters and on the pharmacokinetic effects of OATP-mediated hepatic uptake. In addition, current methods to investigate the interactions between drugs and transporters are discussed, with the emphasis on methods applicable to study uptake transporters. The aim of the experimental part of the master's thesis was to determine if two clinically used drugs, entacapone and fluvastatin, are actively transported from blood into rat and human hepatocytes, and to assess the role of OATP transporters in the hepatic uptake of the drugs in comparison with known OATP substrates, estrone 3-sulfate (E3S) and taurocholic acid and broad OATP inhibitor rifamycin SV. The uptake kinetics of compounds of interest were determined in freshly isolated and cryopreserved rat hepatocytes and in cryopreserved human hepatocytes using the oil-spin method. Uptake clearances (CLuptake) via active uptake (CLactive) and passive diffusion (Pdiff) were calculated from the initial uptake data over a 1 - 200 µM and 1 - 50 µM concentration range for entacapone and fluvastatin, respectively. The half-maximal inhibitor concentration (IC50) of E3S uptake transport was determined for entacapone in a competitive uptake experiment over a 10 - 400 µM concentration range. Fluvastatin uptake showed active saturable transport kinetics in rat hepatocytes with a Km value of 6 µM, whereas entacapone uptake in rat hepatocytes was somewhat linear and did not inhibit E3S uptake at clinically significant concentrations, with an IC50 value of 240 µM. Significantly lower hepatic uptake of taurocholate and entacapone was observed between rat and human hepatocytes, indicating species differences in hepatic uptake processes, although cryopreservation may have had an effect on the noticed difference. The results suggest that murine Oatp transporters do not have a significant contribution to hepatic uptake of entacapone. However, this should be confirmed with future studies with more repetitions and a reliable quantification method.
  • Kouri, Riikka (2011)
    The p53-family consists of three transcription factors, p53, p73 and p63. The family members have similar but also individual functions connected to cell cycle regulation, development and tumorigenesis. p53 and p73 act mainly as tumor suppressors. During DNA damage caused by anticancer drugs or irradiation, p53 and p73 levels are upregulated in cancer cells leading to apoptosis and cell cycle arrest. p53 is mutated in almost 50 per cent of the cancers, causing the cancer cells unable to undergo cell death. Instead, p73 is rarely mutated in cancer cells and because of that could be more viable target for anticancer therapy. The network surrounding the regulation of p73 is extensive and has several potential targets for cancer therapy. One of the most studied is Itch ligase, the negative regulator of p73 levels. Gene therapy directed towards knockdown of Itch ligase is a potential approach but in need for more in vivo proof. p73 has two isoforms, transactivating TA-forms and dominant-negative ΔN-forms. The specific regulation of these isoforms could also offer a possible way for more effective cancer treatment. The literature work includes information of structures, isoforms, functions and possible therapeutic targets of p73. Also the main therapeutic approaches to date are introduced. The experimental part is based on transfection and cytotoxicity studies done e.g. in pancreatic cancer cells (Mia PaCa-2, PANC1, BxPc-3 and HPAC). The aim of the experimental work was to optimize the conditions for effective transfection with DAB16 dendrimer nanoparticles and to measure the cytotoxicity of plain dendrimers and DAB16-pDNA complexes. Also the protein levels of p73 and Itch ligase were measured by Western blotting. The work was done as a part of a bigger project, which was aiming to down regulate Itch ligase (negative regulator of p73) by siRNA/shRNA. Tranfection results were promising, showing good transfection efficacy with DAB16 N/P30 in pancreatic cancer cells (except in BxPc-3). Pancreatic cancer cells showed recovery in 3 days after they were exposed to plain dendrimer solution or to DAB16-pDNA. Measurement of protein levels by Western blotting was not optimal and the proposals for the improvement regarding e.g. the gels and the extracted protein amounts have been done.
  • Heiman, Johanna (2012)
    This work evaluated the use of roller compaction as granulation method for hydroxypropyl methylcellulose (HPMC) based hydrophilic extended release matrix tablets. Roller compaction is a dry granulation method where powder material is fed through a hopper between two counter-rotating rolls and pressed into a ribbon like compact. The compact is thereafter milled to obtain granules. Two full factorial experimental designs (DoE) were set up using two model active pharmaceutical ingredients (API). Paracetamol was chosen as a model for a highly soluble API that deforms mainly by fragmenting, whereas ibuprofen was used as a model for poorly soluble and plastically deforming API. The effect of process parameters, the roll pressure and the ratio between feeder screw speed and roll speed as well the effect of particle size of API and HPMC on the manufacturability and release robustness were investigated. Both compositions with medium drug load were successfully compacted into ribbon. Roller compaction increased the particle size and bulk density of the tablet mass. However, the methods used for evaluation of flow properties gave contradictive results on whether the flow properties were enhanced after dry granulation. The loss of compactibility after granulation was observed, as the tensile strength of tablets prepared of granules was in most cases lower than that of directly compacted powder. Exceptionally, two of the ibuprofen granulations showed compactibility similar to that of the initial powder blends. Increased roll pressure resulted in denser ribbon and narrower particle size distribution for granules. However, high roll pressure had a tendency to decrease the tablet tensile strength. This is most probably due to the particle size enlargement and work hardening phenomenon during the double compaction. The use of large particle size HPMC improved the permeability of the powder blend and the flow properties of the granulations. Tablet dissolution testing showed that the large sized HPMC particles were unable to percolate through the tablet and form a consistent network. Roller compaction helped to break down the large HPMC agglomerates and distribute them more evenly within the tablets. No significant difference in release profiles was observed for tablets prepared using granules roller compacted with different parameters.
  • Karhunen, Emilia (2018)
    Functional in vitro cultured human hepatocytes are needed in different applications in biomedical research. Treatment for liver diseases is usually liver transplantation, but due to the lack of healthy donors, cell therapy using hepatocytes is considered as a better option. Drug industry will also need representative liver models to test metabolic profiles of drug molecules. Primary human hepatocytes are studied in cell therapy and disease modelling, but they have also drawbacks. In vitro they do not proliferate efficiently, and they are short-lived. In vitro differentiated human pluripotent stem cells (hPSCs) to hepatic fate are an alternative for the primary human hepatocytes. Especially human induced pluripotent stem cells (hiPSCs) are widely studied because they are easily available, and they even make personalized therapy possible without problems with ethical issues related to the human embryonic stem cells (hESCs). Differentiation to hepatic fate includes several steps before mature functional hepatocyte-like cells are formed. Hepatocytes are derived from the definitive endoderm (DE) which is one of the germ layers formed in the gastrulation process. Efficient induction of hPSCs into DE lineage would be a good starting point for generating mature hepatocyte-like cells in further hepatic differentiation. Different protocols to differentiate hPSCs in vitro into DE have been published. In vitro cell culture systems should well represent the environment of the target tissue because signals from the environment guide the differentiation. Three-dimensional (3D) cell culture systems are widely studied, because they better mimic the in vivo microenvironment of cells than two-dimensional (2D) cell culture. The aim of the thesis was to study the efficacy of the 3D differentiation of hiPSCs into DE. Before starting the 3D differentiation, differentiation protocol was optimized and the effect of ROCK inhibitor Y-27632 was investigated. Differentiation medium was supplemented with Y-27632 during the whole 6 days differentiation, because survival of the cells and formation of the spheroids were improved, and gene expression studies of pluripotency markers and several DE markers did not show evident effect of Y-27632 on the gene expression of hiPSCs. The main objective in the studies was also to investigate possible differences between different 3D culture conditions on hiPSCs differentiation into DE. Also, the effect of the spheroid size on differentiation was examined. Two different hydrogels were used as a matrix material in the experiments: basement membrane extract (BME) and nanofibrillar cellulose (NFC) hydrogels. Suspension culture was used as a biomaterial-free 3D culture system. Experiments were performed with three spheroid sizes: 200 cells/spheroid, 500 cells/spheroid and 1000 cells/spheroid. Efficacy of differentiation to DE lineage was estimated by studying protein and mRNA expression of some of the DE markers (HNF3B, SOX17, CXCR4, CER1), pluripotency marker OCT4, mesendoderm marker Brachyury and hepatoblast marker HNF4A in the cells. Spheroids differentiated in suspension and NFC were analysed by flow cytometry to get the number of DE positive live cells and dead cells using CXCR4 and 7-AAD double staining. Besides flow cytometry, protein expression of some of the key markers were studied by immunofluorescent staining and further confocal imaging. Viability of the spheroids in BME hydrogel culture were investigated using live/dead staining followed by confocal imaging. BME hydrogel culture was left out from the further experiments due to the morphology of the spheroids and results from viability and protein expression studies. Spheroids in suspension started DE differentiation faster compared to NFC culture. Suspension and NFC cultures yielded high number of double positive cells in flow cytometry and bright fluorescence of other DE markers was seen in the confocal images. NFC hydrogel proved to be a promising 3D culture system by supporting the differentiation of hiPSCs. Flow cytometry results and gene expression studies propose that four days long 3D differentiation would be efficient to produce sufficient number of DE cells. Smaller spheroids showed higher number of DE positive cells than bigger spheroids on day 2 but gene expression studies showed difference in DE marker expression between size conditions rather in later days in differentiation and it was the opposite. Experiments showed signs of more efficient differentiation of the smaller sized spheroids in the beginning of differentiation. But further studies are needed to verify the obtained results and both draw conclusions about the possible differences between different 3D culture systems and explore the best size of the spheroid for hepatic differentiation. However, results obtained from the studies are useful for designing further experiments.
  • Ruutiainen, Henna (2022)
    In health care, the most patient safety incidents occur from medication errors, to which pediatric patients in particular are susceptible. According to James Reason's Theory of Human Error, errors inevitably occurs in an individual's actions, causing potential harm. The prescribing phase has been identified as a specific risk point in the pediatric medication-use process, and therefore defences must be established to prevent or stop errors before they reach the patient. Such system-centric barriers are, for example, electronic health record (EHR) systems that can include computerized physician order entry (CPOE) systems where e.g., medication orders and prescriptions can be made. Knowledge-based clinical decision support (CDS) tools such as dose range check or dose calculator can be integrated into the CPOE system to assist in the prescribing process. The objective of this systematic review was examine the effects of CPOE systems with CDS functions on preventing wrong dose errors in pediatric inpatient orders and outpatient prescriptions. This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 criteria and Synthesis Without Meta-analysis (SWiM) items as an extension to PRISMA criteria. The Joanna Briggs Institute’s (JBI) recommendations from JBI Manual for Evidence Synthesis on mixed methods was used as a guide to conduct this review. Additionally, Cochrane Handbook for Systematic Reviews of Interventions was utilized to conduct the synthesis examining the wrong dose error effectiveness. The study protocol according to the prior defined eligibility criteria was registered in PROSPERO. The literature search was implemented in four databases (MEDLINE Ovid, Scopus, Web of Science and EMB Reviews), reference lists and grey literature in January 2022. Two independent reviewers conducted the study selection and data extraction of the eligible studies using a Covidence software platform. Vote counting method was used to describe and analyze the quantitative findings of the studies exploring the characteristics of CPOE-CDS systems reducing wrong dose errors and regarding their effectiveness on error prevention. JBI’s critical appraisal tools and Grades of Recommendation, Assessment, Development and Evaluation (GRADE) approach were used to define the quality of the studies. A total of 18 studies met the inclusion criteria. The studies had been published in 2007–2021 and majority (13/18) considered only inpatient orders. Almost all (n=16) studies had customized or homegrown CPOE-CDS system and the most used CDS tools were dose range check (78%, 14/18), dose calculator (45%, 8/18) and dosing frequency check (45%, 8/18). When implementing new or customizing the used CPOE-CDS system usually alert functions were added (n=9) and in total alerts were present in 15 studies. Statistically significant reduction in wrong dose errors (overall, overdosing or underdosing errors) was reported in eight studies. None of the studies (n=18) found an overall increase of wrong dose errors. CPOE systems with CDS functions have a great potential to reduce wrong dose errors and promote pediatric medication safety. CPOE-CDS system customization for pediatric population, implementing CDS alerts and the use of dose range check tool seem to be most advantageous when aiming to prevent wrong dose errors. However, CPOE-CDS systems cannot prevent all wrong dose errors as human errors continue to occur and the implemented CPOE-CDS systems can pose new risks such as alert fatigue. Therefore, systematic actions are needed to optimize the safe use of CPOE-CDS systems in pediatrics. More studies are needed particularly on the effectiveness on wrong dose error prevention comparing basic and advanced CDS tools and the effects of different individual CDS functions on wrong dose errors.
  • Miinalainen, Annika (2022)
    OATP2B1 is a transmembrane transport protein expressed widely in the human body and transports both endogenous compounds and several drugs from outside the cell into the cytoplasm. The abundant expression of OATP2B1 in pharmacokinetically important tissues such as in the intestine, liver, and kidney suggests an important role in the drug absorption and elimination process, although research data on the clinical significance of OATP2B1 is still limited. Several drugs inhibit the function of OATP2B1, creating a risk for drug-drug interactions. OATP inhibition by some inhibitors is time-dependent, which may lead to more potent in vivo effects than expected. In this study, the time dependence of OATP2B1 inhibition by five different drugs was evaluated using OATP2B1-overexpressing HEK293 cells. IC50 values of inhibitors for OATP2B1-mediated uptake of DBF and E1S were determined with and without preincubation for 20 minutes. In addition, the in vivo interaction potential of the inhibitors in the intestine, liver, and other tissues was evaluated by utilizing the FDA and EMA guidelines. All five drugs showed effective and concentration-dependent OATP2B1 inhibition with IC50 values of 0.12– 8.82 µM. Furthermore, the inhibition of OATP2B1-mediated DBF uptake by ticagrelor and atorvastatin was time-dependent, while the effect of pre-incubation remains below the limit for the other inhibitors. The inhibitory effect of ticagrelor continued even after the inhibitor was removed from the inhibition buffer. All five inhibitors showed the potential to cause in vivo OATP2B1 inhibition in the intestine, which could result in decreased absorption of the co-administered substrate drug. About erlotinib, the risk of interaction also appeared in the liver, which could reduce the transfer of the substrate drug to the liver and thus lower its elimination rate. In this study, pre-incubation did not affect the in vivo interaction potential of the inhibitor drugs. The results indicate that drug-induced inhibition of OATP2B1 may be time-dependent and therefore can lead to interactions at lower concentrations than expected. For this reason, evaluating the time dependence would be appropriate when assessing transport protein-mediated interaction risk. The results of this study can be utilized in designing clinical interaction studies and in understanding the results.
  • Aalto, Hanna (2016)
    Atypical antipsychotics (AAPs) can be used to treat severe behavioural symptoms of dementia when certain conditions are fulfilled. They are not considered as primary treatment for these symptoms due to their possible serious side effects that are found to be more common in elderly dementia patients. Package leaflets (PLs) are one of the most important sources of medicine information for elderly patients. Evidence-based medicines information is the prerequisite for decision-making and success of pharmacotherapy. The aim of this study was to evaluate the usability and informational content of atypical antipsychotic PLs from the perspective of the elderly. Additionally, the content of medicines information for the elderly found in PLs was compared to similar medicines information targeted to health care professionals (HCPs). Medication Information Design Assessment Scale (MIDAS) was used to evaluate the usability of the most commonly used AAPs (olanzapine, quetiapine and risperidone) among the elderly in Finland. To evaluate the informational content of the PLs and summaries of product characteristics (SmPCs) all the references for the elderly were identified using certain keywords. The informational content concerning elderly from the PLs was compared to information targeted to HCPs in Beer's criteria, Current Care Guideline for memory disorders, Database of medication for the elderly, Martindale and SmPCs. The usability of the PLs in this study was found to be insufficient. The mean MIDAS-credit was 6,4 (n=61; range 5,0-8,0), the maximum credit being 13. Sufficient line spacing and limiting the length of line were among the poorly represented features in the PLs in this study. The occurrence of sufficient font-size varied. Good contrast, headings, usage of upper and lower case in text and bullet points were among the well-represented features. All the PLs included in the content-analysis (n=106) contained at least three references to the elderly. The way the information was presented and how well it stood out from the leaflet varied. The SmPCs contained useful information targeted to elderly that was not found in corresponding PLs. Actions need to be taken to improve the usability and content of product specific medicines information from the perspective of the elderly. Medicine authorities and the pharmaceutical industry have the authority to make these improvements possible. Scientific data and concrete tools are needed to facilitate the change.
  • Konttinen, Riikka (2017)
    Hepatitis C virus disease is transmitted through blood. Chronic hepatitis C causes liver damages such as liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. It is estimated that there are approximately 20 000 - 30 000 patients infected with hepatitis C virus in Finland. For many years pegylated interferon and ribavirin has been standard of care. However standard of care causes side effects and an adequate treatment response can't be achieved with it. There have been effective direct-acting antivirals available on market which are directed against structural proteins and enzymes of the virus from 2014 onward. These second generation direct-acting antivirals are effective, safe and well tolerated. The only disadvantage is the high price of these medicines which restricts them for severe liver damage patients. More information about cost-effectiveness of second generation direct-acting antivirals is needed to support the decision making. The aim of this master thesis is to describe current care, guidelines, and costs of hepatitis C in Finland. Thesis also describes the principles of economic evaluation and systematic literature review. The purpose of the thesis is to assess cost-effectiveness of second generation direct-acting antivirals versus standard of care in treating of hepatitis C by means of systematic literature review and evaluate the quality of cost-effectiveness analyses. Previously published studies were used to analyze the cost-effectiveness of second generation direct-acting antivirals. In total of 435 references were found through systematic literature search. In addition, two studies were found from the bibliographies of already included studies. Altogether 26 studies were included in the systematic review of which 25 were original studies and one was previously published systematic literature review. The most relevant data of the studies was gathered and analyzed. The quality of the studies was assessed by using three checklists. It is difficult to make conclusions about cost-effectiveness of second generation direct-acting antivirals based on previously published reviews because only one review was found through systematic literature search. The incremental cost-effectiveness ratio (ICER) of second generation direct-acting antivirals varied between dominance and 1 135 655 € /QALY compared to standard of care. When compared to another second generation direct-acting antiviral, ICER of second generation direct-acting antivirals varied between dominance and 65 281 € /QALY. It was also analyzed how stage of liver damage affects the incremental costeffectiveness of second generation direct-acting antivirals. The ICER of second generation direct-acting antivirals was between 299 € - 85 195 € /QALY when treating patients with cirrhosis. When treating non-cirrhotic patients, the ICER of second generation direct-acting antivirals was between 2182 € - 177 679 € /QALY.The connection between funder of the study and the ICER of second generation direct-acting antivirals was also analyzed. The ICER was 1717 € - 86 056 € /QALY in studies funded by pharmaceutical company. The ICER was 299 € - 1 135 655 € /QALY in studies funded by other party. Based on the results of the thesis second generation direct-acting antivirals might be cost-effective compared to current standard of care in treating hepatitis C. The cost-effectiveness ratio of second generation direct-acting antivirals is lower in cirrhotic patients than in non-cirrhotic patients. The incremental cost-effectiveness ratio is lower when pharmaceutical company funds a study. The quality of the cost-effectiveness analyses included in the thesis varied greatly which makes it difficult to draw conclusions and interpretate the results. This study has several strengths. First, literature search was conducted systematically and transparently. Second, quality of the reviewed studies included was assessed by care. Finally, reporting of the results is transparent and repeatable. The study has also some limitations. Selection of the reviewed studies, data extraction and quality assessment of the studies was conducted by one person which may increase the possibility of human error.
  • Hallila, Susanna (2013)
    There is a strong need for new in vitro methods in early drug development that predict in vivo conditions more reliably. One of the prerequisites for successful drug therapy is sufficient permeability. A drug needs to be transported through a cell membrane before it can have a pharmacological effect. Therefore, the drug-cell interactions are studied in the early stage of the drug development process. The literature review of this work covers the traditional in vitro and in silico methods of predicting the permeability of drugs across the intestinal membrane. The widely applied methods are reviewed briefly and the predictability of the methods is evaluated. Moreover, the surface plasmon resonance (SPR) technique is introduced. The principle of SPR and its applications for predicting intestinal permeability using lipid membranes resembling the intestinal membrane and for studying drug-cell interactions are discussed. The advantage of the SPR technique is that it is an optical method which allows real-time monitoring under a constant flow without labeling agents. The aim of the experimental part of this work was to evaluate the suitability of the SPR technique for cell-based studies to monitor drug-cell interactions in native cellular environments. Previously, the SPR technique has been almost merely used in routine biomolecular interaction analysis. Recently, the SPR technique has also been applied to cellbased assays but in those studies the reason for the SPR signal responses is generally poorly discussed. The objective of the experimental study was to evaluate and optimize different cell culturing approaches for living cell sensing for SPR, i.e. cells immobilized on the roof of the PDMS molded flow channel in the SPR instrument and cells immobilized directly on the SPR sensor surface. ARPE-19 cells were immobilized on the PDMS substrates but the challenge of imaging cell monolayers on PDMS molded SPR flow channels suggested that immobilizing the cells directly on the SPR sensor surface would be a more straightforward procedure. Hence, ARPE-19 and MDCKII cell culturing protocols were optimized for successful immobilization of confluent cell monolayers directly on the SPR sensor surface. However, ARPE-19 cells showed poor resistance against shear stress in the flow channel; whereas MDCKII cells showed much better resistance against shear stress in the flow channel. Therefore, only MDCKII cells immobilized on the SPR sensor surfaces were used for drug-cell interaction studies. After three days of culture MDCKII cells were exposed to test compounds in separate SPR measurements. The used test compounds were propranolol, D-mannitol, D-glucose and HSPC:Chol liposomes. During the SPR measurements, the changes in the SPR peak minimum angular position and SPR peak minimum intensity were recorded in real-time, and these were further used for analysis after the measurements. The results showed that clear differences in both SPR signals between propranolol and D-mannitol were observed when the cells were exposed to the test compounds. Propranolol diffuses effectively by the transcellular pathway into cells whereas D-mannitol uses the paracellular pathway. This indicates that the introduced SPR approach may be a potential in vitro method in order to provide real-time information on the permeability of drugs and possibly on cell uptake mechanisms of nanoparticles for a better mechanistic understanding of drug-cell interactions on a cellular level.
  • Lehtola, Minna (2018)
    Tramadol products for cats are not commercially available. Problems may occur when dividing a tablet registered for humans due to uneven distribution of active ingredient within a tablet and bitter taste of tramadol. Minitablets have multiple benefits, including small size, better uniformity of content, coatability and fast administration, in comparison to a divided conventional tablet. The purpose of this study was to develop minitablets which are possible to coat with a taste masking coating. Physical and chemical properties of tramadol hydrochloride, such as water solubility, temperature behavior and hygroscopicity were studied. Additionally, compatibility of tramadol hydrochloride with excipients was studied by a 3-month stability exam. The pre-tests of granulation were carried out by using lactose or ascorbic acid as an active ingredient to model tramadol hydrochloride. The granulation was performed with high shear granulator and tableting with a rotary tablet press. The only variable factor between the granulation batches was the amount of granulation fluid. The impact of the amount of granulation fluid to the tableting properties was examined by determining particle size distribution, Carr index and Hausner ratio. Uniformity of mass, uniformity of content, hardness, disintegration time and dissolution were examined. The study revealed that tramadol hydrochloride did not have incompatibilities with the examined excipients. Tramadol hydrochloride was not hygroscopic even though it was found out to be freely soluble in water. Tablets with adequate hardness were successfully compressed of both granulated masses and the direct compression mass. However, the direct compression mass had more undesirable properties regarding the processes. Most batches fulfilled the requirements set for uniformity of mass and uniformity of content. Although the purpose of this study was to develop a tablet for veterinary medicine, the results in this study may be utilized in developing a formulation for pediatric medicine.