Browsing by master's degree program "Master 's Programme in Pharmacy"

The problems caused by hypromellose in sterile filtration of ophthalmic products in the pharmaceutical industry were investigated. The research project was performed at NextPharma Oy's ophthalmics manufacturing facility in Tampere during the autumn of 2020. Hypromellose is an excipient commonly used in ophthalmic products as a viscosity enhancer to prolong the contact time of the preparation on the eye surface. In the ophthalmics compounding process, hypromellose is first dispersed by slowly sprinkling it into a hot solution and thoroughly mixing, after which the solution is cooled to room temperature. During cooling, the hypromellose dissolves and gels, increasing the viscosity of the solution. Incomplete dispersion or dissolution of hypromellose during the manufacturing process can slow down the filtration rate or even clog the filter completely due to undissolved hypromellose polymer material. Hypromellose is an industrially produced cellulose derivative that often contains some amounts of unreacted cellulose and other sparingly soluble polymer particles as impurities, which can also cause problems in filtration processes. Sterile filtration is a commonly used sterilization method for ophthalmic products, in which the prepared bulk solution is filtered through a 0.1 to 0.2 µm pore size filter membrane into a sterile receiving vessel. Due to the very small pore size, sterile filters are easily clogged if the solution contains poorly dissolved material. The purpose of this work was to collect additional information on the possible causes of clogging caused by hypromellose and to determine whether the filterability of a solution containing hypromellose can be improved by optimizing the manufacturing process parameters. The design of experiments was prepared, creating a two-level full-factorial test matrix without replicates and with three centre points. Four different process parameters were used (mixing time, mixing speed, dispersion temperature, and cooling temperature). Minimum and maximum levels for the parameters were obtained in the initial tests, after which the test solutions were prepared and filtered in a randomized order according to the test matrix. The aim of the screening was to find out which parameters were affecting the filterability and what would be their optimal combination that would maximize the filtration rate and the yield of filtration. Finally, the optimized parameters were used to test different batches of hypromellose, comparing the results to previous filtration tests. Additionally, an alternative hypromellose dispersion method was tested to minimize the amount of insoluble material remained during the dispersion and cooling steps. Of the parameters tested, mixing speed was the least significant, while cooling temperature had the most effect on the filtration results. The solutions with lower cooling temperature had better filtration results, which may be due to reduced aggregation of hypromellose due to increased hydration of the polymer chains. The temperature behaviour of hypromellose solutions could be an interesting subject for further investigation. Longer mixing times and higher dispersion temperatures produced slightly better filtration results on average, but the differences were not statistically significant. Most challenging in the study was controlling the temperature and mixing of the solutions, and the retention of insoluble hypromellose material at the walls of the compounding vessel. The alternative dispersion method gave promising preliminary results, but the method still requires further testing. It would be important to also find the root cause of the filter clogging mechanism e.g., by further analysing the clogged filter membrane. The study provided additional useful information of the behaviour of hypromellose solutions in solution preparations and during sterile filtration, which has been helpful in solving production problems.

Solids most commonly come in two broad forms: crystalline or amorphous. Crystalline solids have a regular, organized long-range structure of atoms and crystals, and are characterized by having a distinct shape, specific volume, and melting point. They can also have multiple polymorphs. On the other hand, amorphous solids do not usually have a regular long-range atomic and crystal structure and their molecules are more easily separated, which makes them more soluble in their surroundings compared to crystalline solids. However, despite this, short-range order can also occur. To improve the solubility of crystalline solids, co-amorphous systems can be created by mixing together two or more chemically different compounds in a way that they don't form a regular crystalline structure, but rather an irregular, amorphous one. Co-amorphous systems can be analyzed qualitatively or quantitatively. Qualitative analysis is often the main focus when studying amorphous matter, as it can be difficult to accurately quantify these materials using techniques based on crystal structures. Additionally, many amorphous systems are made up of complex mixtures of polymers with different chemical and physical properties. This study aimed to determine the most effective method for obtaining quantitative information about the co-amorphization of indomethacin and tryptophan. Three analytical techniques were used for this purpose: differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and Raman spectroscopy. The co-amorphous system was created by mixing together α-indomethacin and tryptophan, γ-indomethacin and tryptophan, and amorphous indomethacin and tryptophan. This study showed that DSC, XRPD, and Raman spectroscopy are effective in providing quantitative information about crystallinity and crystal size. These techniques were able to accurately detect and characterize discrete residual crystals, and were able to measure and quantify the amount of these substances. Even though these methods may not be able to detect nanoscale structures with precision, they still provided valuable information about the crystalline and amorphous nature of the samples studied. Additionally, the fact that similar quantitative results were obtained using different analysis methods further supports the reliability of these techniques. Of all the techniques discussed, Raman spectroscopy was able to identify even small residual crystals, resulting in the highest calculated crystallinity percentage.

Biologisten lääkkeiden tarjonta ja käyttö ovat lisääntyneet voimakkaasti viimeisen vuosikymmenen aikana. Biologiset lääkkeet voivat parantaa reumasairauksien ja useiden muiden pitkäaikaissairauksien hoitotuloksia. Biologiset lääkkeet ovat yleensä perinteisiä pienimolekyylisiä lääkkeitä kalliimpia, ja siksi biologisen alkuperäislääkkeen kanssa kliinisesti samanarvoiseksi kehitettyjen edullisempien biosimilaarien käyttöä pyritään edistämään osana rationaalista lääkehoitoa. Potilaan näkemyksillä ja niiden huomioimisella on hoitoon sitoutumisen ja hoidon tulosten kannalta suuri merkitys. Potilaiden näkemyksistä ja kokemuksista biologisista lääkkeistä ja niiden vaihdosta on melko vähän tutkimuksia. Tutkimuksen tavoitteena oli tutkia reumapotilaiden tietämystä ja näkemyksiä biologisista lääkkeistä ja biosimilaareista sekä kokemuksia niiden ei-lääketieteellisestä lääkevaihdosta. Lisäksi tutkittiin reumapotilaiden uskomuksia omasta lääkityksestään, halukkuutta osallistua lääkitykseen liittyvään päätöksentekoon lääkärin kanssa ja biologisten reumalääkkeiden käyttäjien lääketiedon lähteitä. Tutkimus toteutettiin sähköisenä kyselynä Yliopiston Apteekin kanta-asiakkaille ja kertomalla siitä Reumaliiton ja IBD- ja muut suolistosairaudet ry:n viestinnässä 18.–30.1.2021. Kohderyhmänä olivat aikuiset avoterveydenhuollon reuma-, IBD- ja muut suolistosairaus-, sekä ihopsoriasispotilaat, jotka käyttivät adalimumabin tai etanerseptin alkuperäistä biologista lääkettä (BA) tai biosimilaaria (BS) tai ainoastaan perinteisiä pienimolekyylisiä lääkeitä (PL). Tässä tutkimuksessa tarkasteltiin vastauksia 260 reumapotilaalta, joista biologisia lääkkeitä oli käyttänyt 75 (BA-käyttäjiä 35, BS-käyttäjiä 40) ja perinteisiä lääkkeitä 185. Ei-lääketieteellisen lääkevaihdon kokeneita oli 17. Tutkimuksen teoreettisena viitekehyksenä käytettiin terveysuskomusmallia. Ensisijaiset lopputulosmuuttujat olivat faktorianalyysillä muodostettuja summamuuttujia. Erot lääkekäyttäjäryhmien (BA, BS, PL) välillä ja taustamuuttujien vaikutukset testattiin tilastollisilla analyyseillä. Vastanneista potilaista 94 % tunnisti biologisen lääkkeen käsitteen, mutta biosimilaari-käsite tunnettiin huonommin (34 %). Suurin osa potilaista (73–78 %) luotti biosimilaarien olevan ominaisuuksiltaan samankaltaisia alkuperäisvalmisteiden kanssa. Lääkärin tekemään biologisten lääkkeiden vaihtoon luotettiin enemmän (89 %) kuin mahdollisesti apteekissa tehtävään vaihtoon (40 %). Tutkimuksessa todetut kokemukset biologisten lääkkeiden vaihdosta biosimilaariin olivat pääosin positiivisia. Tutkimuksen reumapotilailla oli hyvä tietämys biologisista lääkkeistä, mutta he tunsivat huonommin biosimilaarin käsitteen ja mitä biosimilaarit ovat. Heillä oli yleisesti ottaen luottavainen näkemys siitä, että biosimilaari on ominaisuuksiltaan alkuperäisvalmistetta vastaava lääke. Biologisten ei-lääketieteelliseen vaihtoon suhtauduttiin melko myönteisesti, mutta lääkärin toteuttamaan lääkevaihtoon suhtauduttiin luottavaisemmin kuin mahdollisesti apteekeissa tehtävään vaihtoon. Lisää tutkimustietoa tarvitaan muun muassa potilaiden biosimilaareihin kohdistuvan epävarmuuden syistä.

Poorly water soluble active pharmaceutical ingredients cause problems for the drug development. Solid state modification offers one approach to overcome the issue. In this study, co-amorphous systems and co-crystals were prepared with indomethacin at molar ratio of 1:1 using nicotinamide as a co-former. Co-amorphous systems were prepared by two different preparation methods: melting the physical mixture and then quench cooling it with liquid nitrogen and dry milling with a ball mill. Co-crystals were prepared by liquid-assisted ball milling. After that, the properties, dissolution, and physical stability of the formed formulations were investigated and compared. The characterisation methods were differential scanning calorimetry, X-ray powder diffraction, Fourier-transform infrared spectroscopy, polarised light microscope and scanning electron microscope. In addition, the solubility and physical stability of the formulations were investigated. Co-amorphous systems were successfully prepared by quench cooling the melt and co-crystals by liquid-assisted ball milling. Dry milling did not induce the formation of co-amorphous systems. In the intrinsic dissolution test, both the co-amorphous system and co-crystal enhanced the dissolution of crystalline indomethacin. When examining the dissolution rate with the paddle apparatus, it was observed that the co-crystal had the highest dissolution rate among both powder and tablet samples. The co-amorphous powder sample floated on the surface of dissolution medium which impeded the dissolution of indomethacin. However, co-amorphous tablet sample showed a higher dissolution rate than crystalline indomethacin. Stability testing (25 °C, 18 %RH) showed that the co-amorphous system recrystallised into a co-crystal after two weeks of storage, while the co-crystal was found to stay stable the whole study period.

During co-processing, magnesium stearate can induce surface coating on carrier particles in powders which contain at least one other component in addition to magnesium stearate. Magnesium stearate is sometimes added to powder mixtures as it is known to have beneficial effects on powder characteristics, such as physical and chemical stability, and flowability. In order to fully optimize and control the coating/mixing process, it is necessary to be able to characterize the quality of surface coating. Various methods can be used in determining the coating of powder particles. They can roughly be divided into two different categories: direct and indirect methods. For example, spectrophotometric instruments, which are used to visually express the element distribution on particle surface, are considered direct methods. Indirect methods include methods in which coating parameters are inferred using other properties such as water sorption and powder flowability. Principally direct methods have been used in previous studies to determine the quality of coating. Therefore, the area of interest was especially to study indirect methods and compare them to results obtained using direct methods. Having knowledge of the suitability of indirect methods would be interesting as they might have many benefits compared to direct methods, such as quicker analysis speed and cost-efficiency. The aim of the study was to examine the suitability of direct and indirect methods in studying the surface of powders containing magnesium stearate and active pharmaceutical ingredients (APIs) or lactose, more closely how magnesium stearate was placed on carrier particles as well as the uniformity and the thickness of coating layer. The used methods were selected using literature and own consideration while taking the available equipment into account. The powders containing API (d50 < 10 μm) or lactose (d50 > 80 μm) with magnesium stearate had substantially differing characteristics and thus behaved differently. Therefore, there were differences in the suitability of analytical methods in determining surface of powders. Powders containing lactose and magnesium stearate were able to be examined using direct methods (SEM-EDS and ToF-SIMS) and several indirect methods. Samples with API and magnesium stearate were able to be studied with fewer methods. Validation of the suitability of these methods need more research. However, according to the results from this study, it is probable that surface characterization of studied co-particles can be achieved with direct, but also with indirect methods.

There are certain characteristics in children’s medication process, such as weight or body surface area-based drug dosing and off-label use of medications, that expose children to medication errors. Small children especially are prone to physical injuries resulting from medication errors. High-alert medications bear a heightened risk of causing significant, even life-threatening harm to a patient when used in error. The aim of this study was to promote children's medication safety by identifying medication errors and contributing factors to errors associated with the use of high-alert medications in pediatric medication process in a hospital environment. The data of this retrospective register study consisted of voluntary medication error reports (HaiPro) made in the pediatric and adolescent units at Helsinki university hospital (HUS). ISMP's (Institute for Safe Medication Practices) list of high-alert medications in acute care settings was used to limit the data. The data was analyzed by using both quantitative and qualitative methods. The aim of the quantitative analysis was to report the frequencies (n) and proportions (%) of high-alert medications and routes of administration and the aim of the qualitative analysis was to identify the types of medication errors and contributing factors in the data. ISMP’s high-alert medications accounted for approximately one-fifth (19.7%) of all medication error reports made in pediatric and adolescent units in 2018–2020. Twelve medications and intravenous route covered approximately 65.0% of all high-alert medications and routes of administration mentioned in the data. Medication errors were mostly identified in medication administration stage (43.3%) and administration errors were often preceded by prescribing errors. Dosing errors (20.5%) and documenting errors (16.8%) were the most common medication error types in the data. Errors associated with dosing and infusion rate were most often involved in severe medication errors. The most frequently identified contributing factors in the data were associated with the work situation and conditions, documenting and information transfer or medications. More detailed risk analysis considering high-alert medications and the intravenous medication process and targeting preventive barriers to identified risk areas are recommended in pediatric and adolescent units in the future. Barriers should be planned to cover the entire medication process. Among different types of medication errors, multiple dosing errors and errors during the programming of infusion rate require special attention in the future.

Inhibition of the cytochrome P450 enzymes is one of the most significant factors causing drug-drug-interactions, and thus one of the most important objects of study at preclinical drug development. CYP-inhibition can be either reversible or irreversible. Although different inhibition mechanisms are well known, their evaluation in vitro is still challenging. Thus, the development of more accurate and efficient in vitro methods is important and as a continuous target of interest. Immobilized enzyme microreactors (IMER) have presumably several advantages over traditional in vitro methods and have been presented as a promising tool for drug metabolism studies in vitro. The purpose of this work was to evaluate the suitability of a novel flow-through based immobilized enzyme microreactor in determining the CYP enzyme kinetic parameters. The developed immobilization protocol is based on attaching biotinylated human liver microsomes to a thiolene-based microreactor coated with Streptavidin. To validate the developed method, the activity of the CYP2C9 enzyme was assessed using the recommended model reaction by authorities, that is 4-hydroxylation of diclofenac. The enzyme kinetic parameters i.e., enzyme affinity (Km) and activity (Vmax), determined with the developed IMER were comparable to the values previously published in the literature and determined in static in vitro conditions. In addition, the inhibition of CYP2C9 enzyme by four model inhibitors (fluconazole, nicardipine, sulfaphenazole and miconazole), was examined by determining the IC50 (half-maximal inhibitory constant) values for each compound and by monitoring the reversibility of the CYP2C9 enzyme for 90 minutes after the inhibitor was removed from the feed solution. The IC50 values determined with the developed method for all inhibitors were well in line with previous publications, showing fluconazole (IC50 22 µM) to be the weakest inhibitor of CYP2C9 enzyme and the other examined inhibitors caused more potent inhibition (IC50 for sulfaphenazole 1.3 µM; IC50 for miconazole 1.3 µM; IC50 for nicardipine 0.67-1.1 µM). The reversibility of the CYP2C9 enzyme was examined by removing the inhibitor from the feed solution and monitoring the recovery of the enzyme activity via diclofenac 4-hydroxylation. Based on the results obtained with developed IMER, the inhibition of fluconazole and sulfaphenazole was reversible and thus well in line with previous studies. In contrast, on account of data obtained with IMER, inhibition by miconazole and nicardipine was not reversible, although these compounds have previously been reported to be reversible CYP2C9 inhibitors in vitro, which may be due to the strong aggregation tendency of these compounds. The study shows that the developed flow-through based IMER is well suited for studying inhibition of CYP enzymes However, to utilize the developed technology in CYP enzyme inhibition research, it’s applicability in determining enzyme inhibition should still be evaluated with more comprehensively with several CYP isoenzymes.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with the neuropathological hallmark of intraneuronal inclusions called Lewy bodies (LB). Accumulation of α-synuclein (α-syn) and cellular components into LBs coincides with degeneration of dopaminergic neurons in the midbrain, substantia nigra. Degeneration of dopaminergic neurons eventually leads to motor dysfunctions. Currently, the treatments for PD are symptomatic. For this reason, new disease-modifying treatments are needed to slow down or prevent the disease progression. Neurotrophic factors (NTFs) have been an interest of research for a couple of decades because of their neuroprotective properties. The main aim of this study was to investigate if brain-derived neurotrophic factor (BDNF) reduces pre-formed fibril (PFF) induced aggregation of α-syn in dopaminergic neurons. PFF-model was used to mimic the accumulation of LBs in neurons, as PFFs induce aggregation of endogenous α-syn in neurons. Additionally, the dose dependence of BDNF was tested. The secondary objective was to investigate the interaction of tropomyosin receptor kinase B (TrkB) signaling pathway and α-syn aggregation using TrkB agonists and antagonists. The cultured dopaminergic neurons isolated from the midbrain of mouse embryos were treated with PFFs on the day in vitro (DIV) 8. BDNF or control treatments were added either 1 hour after the PFF-treatment or on DIV 12. Neurons were fixed on DIV 15 and fluorescent immunohistochemistry was performed. After the detection of fluorescence with automated, high-content imaging, image analysis was done for quantifying dopaminergic neurons, and dopaminergic neurons positive for LB-like aggregates by using unbiased image analysis CellProfilerTM software. Both BDNF and positive control glial cell line-derived neurotrophic factor (GDNF) significantly reduced LB-like aggregates in dopaminergic neurons at both timepoints. GDNF was more effective at both timepoints than BDNF. Both tested doses of BDNF lowered the number of LB-like aggregates, but a more robust effect was seen with the higher dose. The highest tested dose for the TrkB agonists was toxic to the cultured dopaminergic neurons, whereas the lower doses did not affect either the survival or the number of LB-like aggregates. BDNF promoted the survival of the dopaminergic neurons despite the survival-reducing adverse effect of TrkB antagonist K252a. This study provided new information on the effects of exogenously added BDNF on PFF-model with primary neuronal culture. Research on the underlying mechanisms of α-syn aggregation and the protective effects of NTFs can forward the development of new therapies against PD.

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.

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.