Browsing by Title

Lead molecule search is the first part of drug design. This process can be done using computerized docking of ligands into target proteins. Usually this requires expensive software and powerful computer systems specifically made for the process. There are however some programs that are available for free and can be run on home computers. The purpose of this Master's Thesis was to see how these free software can be used for the task of docking and also to create a method or a guideline for such work. Protein kinase C (PKC), a popular target for drug design, was chosen as a target of inhibitor design. PKC is part of a larger family of serine/threonine kinases and formed of 10 isoforms all with different effects on cellular functions. The large amount of related kinases and the similarities in their sequences make finding selective inhibitors a difficult process. Homology models of all PKC isoforms in three known conformations solved by x-ray diffraction (pdb: 1XJD, 2I0E and 3A8W) were created using Modeller. Into these models a set of possible ligands from the free database of molecules ZINC was docked using Autodock Vina utilizing a script created for docking multiple ligands into multiple targets. The dockings resulted in some interesting results. Six molecules were recognized as possible lead molecules for further research. None of these molecules had any patents or previous results of protein kinase inhibition connected to them. The most interesting result was the finding that coluracetam, a nootropic drug of the racetam family, might be a protein kinase inhibitor. Racetams are usually considered drugs that lead to PKC activation. It has been proposed that inhibitors may prolong the lifetime of kinases in the cells leading to increased activity in the long term. In our opinion coluracetam might prove to be a good tool for studying the complex way kinase activity is modulated. The methods and scripts used in this work will be released for free use.

In the United States pharmacists have prescribed medicines and managed patient's drug therapy since the 1970s, and in the United Kingdom pharmacists have been authorization to prescribe medications since 2003. The discussion about the right of Masters of Science in Pharmacy will be renewed prescriptions during the last decade in Finland but few Finnish studies have been published from the subject. In the document Medicines Policy 2020 published by Ministry of Social Affairs and Health states that by prescribing should be used cost-effective modes of operation. The knowledge about pharmacist prescribing benefits and costs, and also prescribing practice in Finland, is needed to evaluate the cost-effectiveness of pharmacists' authorization to prescribe and to support the decision-making concerning pharmacist prescribing. The aim of this master's thesis is to gather all existing knowledge about the economic and other effects of pharmacist prescribing using a systematic literature review method. The aim of theoretical part of this master's thesis is to explain the Finnish prescribing, the participation of pharmacists in drug therapy management in Finland and internationally pharmacist prescribing. The empirical part of this master's thesis is also to assess the quality of the studies of pharmacist prescribing benefits and costs using quality assessment checklists. In addition, this thesis describes the principles of the cost and benefit analyses, economic evaluations and systematic literature reviews. As a result of the literature search were found 1825 references. Based on the inclusion and exclusion criteria, 17 studies were selected to include in the systematic review. Of these studies three were economic assessments, 8 randomized controlled trials and 6 observational studies. The quality of these studies was assessed using four quality assessment checklists. On the basis of a systematic literature review pharmacist prescribing has been studied in the treatment of type 2 diabetes, hypertension, dyslipidemia, anticoagulation, chronic pain, emergency contraception and minor ailments and renewal of long-term medicines. Pharmacists reduced blood pressure by providing follow-up care with prescribing compared with the usual care, but not compared with the case management, which does not include prescribing. In addition, the follow-up care was to improve the treatment results of type II diabetes. The results obtained in the care of dyslipidemia were partly unclear. In the clinic follow-up care with prescribing could be reduced LDL-cholesterol, but not the risk of cardiovascular disease compared with the control group. In the pharmacy follow-up care had no effect on the treatment of patients with LDL-cholesterol compared with the control group. In addition, pharmacist prescribing improved how well patients stayed within INR target range. Pharmacist medication review with pharmacist prescribing achieved in the care of chronic pain patients differed few from the results of pharmacist medication review with feedback for a general practitioner. Pharmacist prescribing could reduce errors in inpatient medication compared with usual care. Much uncertainty is connected to the results of the study. The limited amount of studies, heterogeneity of the studies and methodological quality make the evaluation of real effects more difficult. The included studies of pharmacist prescribing were so heterogeneous. In addition treated disease, assessed benefits and scope of working environment were varied in included studies. Pharmacist prescribing was often studied as part of other care or pharmaceutical service, such as chronic disease management or medication review. The quality assessment of the included studies revealed several sources of bias. The available research information is the insufficient reliable evaluation of economic and other effects of pharmacist prescribing and the need for the further research is big.

There are dozens of bromotyrosine alkaloids extracted from the marine sponge Pseudoceratina purpurea. Bromotyrosines have for example antibacterial, antiviral and antitumor activity in vitro and therefore bromotyrosines are potential drug lead molecules. By far, bromotyrosines have been extracted from different marine sponges of the Verongida order for the use in biological activity research. Collecting marine sponges by scuba diving is neither fast nor ecological and therefore finding an effective synthesis strategy is vital so that the research could continue. One new bromotyrosine, purpurealidin I, was found from the marine sponge Pseudoceratina purpurea in the four year (2010-2014) Marex-project. The aim of this thesis was to synthesize the compound purpurealidin I and its derivatives. Synthesized compounds were tested against hepatitis C and chikungunya viruses. It is important to find new potent drug molecules, because approximately 350 000-500 000 people die from hepatitis C and there is no curative medication for the chikungunya. Purpurealidin I is synthesized from tyrosine and tyramine parts, which will be put together to form an amide bond in the final step of the synthesis. The synthesis of purpurealidin I was not completed during the Master's thesis. However there were two purpurealidin I derivatives and four purpurealidin I tyramine part derivatives that were successfully synthesized. One of the compounds is purpurealidin E, which can be extracted from the sponge Pseudoceratina purpurea. The t-Boc derivative of purpurealidin E was examined against hepatitis C and chikungunya and the compound showed moderate activity against hepatitis C virus, but it wasn't active against chikungunya virus. The original plan to synthesize purpurealidin I is possible, although some reactions and purification of crude products need to be optimized in order to get better yields. For the future research derivatives of the t-Boc derivative of purpurealidin E should be synthesized and studied against hepatitis C virus.

Pyrazoles are five-membered nitrogen containing heterocycles, whose derivatives can be widely used in medicinal chemistry. One of the most common ways to produce pyrazoles is 1,3-dipolar cycloaddition, where the dipole containing heteroatom is reacting with a dipolarophile, and forming a cycloadduct. Among others, mesoionic sydnones have been used as dipoles in 1,3-dipolar cycloadditions of pyrazoles. During the last decades solid phase methods, where either a dipole or dipolariphile is being temporarily bound to solid support, have been exploited in 1,3-dipolar synthesis. With the aid of solid phase methods, the synthesis can be controlled chemically by protecting groups that react easily. Also the isolation and purification can be made easier by using these techniques. The 1,3-dipolar solid phase methods can be combined with microwave techniques, to make the synthesis shorter and more effective. The goal of this work was to synthesize new N-unsubstituted pyrazoles, starting from sydnones bound to solid support and alkynes, with use of 1,3-dipolar cycloaddition reaction and to purify and analyze prepared compounds. The aim was also to develop a new 1,3-dipolar solid phase method so, that the end products could be cleaved easily in a traceless manner, and that there would be minor need for purification. There was also an aim to make the last step of the synthesis faster and easier with microwave reactor, and by using this method to standardize the conditions used in the cycloaddition-dehydration reaction step. The AMEBA-resin was chosen to be the solid support in this synthesis. Its traceless cleavage by trifluoroaceitic acid made possible not only the formation of the desired structure, but also effortless purification of the end product. The amino group of pyrazole was protected by the solid support during the N-nitrosation, so that after the traceless cleavage of the resin, N-unsubstituted pyrazoles were obtained. By changing the amino acids used in this synthesis, it was possible to alter the structure of the synthesized pyrazoles, and to create four structurally new pyrazoles. Microwave method used during the last step of synthesis for heating shortened the reaction step significantly, and also the yields of end products were better compared to conventional heating. During this work a functioning and developable 1,3-dipolar solid phase method was created, that can be utilized to synthesize pyrazoles and other compound of similar nature also the in future.

Pharmaceutical industry is supervised by several competent authorities. These authorities all over the world inspect manufacturers in order to make sure they comply with the Good Manufacturing Practice (GMP) guidelines and produce quality products. If non-compliance with the guidelines is detected, the authorities can revoke manufacturing licenses and deny access of the products. Recent trend in pharmaceutical industry is that the Active Pharmaceutical Ingredient (API) manufacturing is concentrated in few factories. If this kind of manufacturer is declared non-compliant and is therefore unable to supply an API, it can lead to drug shortages. This research aimed to find out what kind of quality problems occur in API manufacturing. Because of the concentration trend, it is important to understand what kind of problems the manufacturers do struggle with to prevent any risk for shortages. This research aimed also to determine how much the quality problems in API manufacturing can impact on drug shortages. Also, the number and location of these non-compliance cases were investigated. The chosen time frame was 2016-2018. Several databases were used as information sources in this research. These databases are maintained by the authorities in the U.S. and Europe and they contain information about the inspections and the GMP deficiencies they have found during these inspections. With the information collected from the databases, an inductive content analysis was conducted to determine the reasons for non-compliance with GMP in API manufacturing. Other information (e.g. locations, names of APIs) was also collected from the databases and analysed to answer the rest of the research questions. Results show that the biggest problem areas in API manufacturing were data integrity and analytical testing. Other problems relating to documentation occurred also. The amount of these cases was quite stable, and the relative proportion declined during the time period. Comparison between the list of APIs and drug shortage databases showed that even over 30% of the non-compliant APIs were later in shortage. The effect was greater in Finland than in the U.S. Therefore, it was concluded that the most significant GMP deficiencies in API manufacturing were poor data integrity and inappropriate analytical testing procedures. Secondly, the number of non-compliance cases in API manufacturing has not increased during this time, but these problems may have had an impact on drug availability problems.

Native nanofibrillated cellulose is wood-derived, animal-free biocompatible biomaterial which has proved the suitability of nanoscale cellulose fiber based hydrogels for 3D cell culturing and wound healing applications. The problem of freeze-drying nanofibrillated cellulose hydrogel (NFCh) has been the aggregation of the hydrophilic fibrils of the NFC during freeze-drying, which leads deformed freeze-dried cake and unsuccessful reconstitution of the sample. Molecular Dynamic (MD) simulations have been earlier applied in formulation design of NFCh for freeze-drying successfully by screening excipients based on their attraction to the surface of NFCh. The weakness of MD simulations is it can only model the fresh formulation system intend to freeze-dry, but not the actual freeze-drying process and the effect of it and the excipients to the material. To evaluate the protecting properties of excipients and therefore the accuracy of the MD simulations detailed information about changes in the physical state and molecular orientation of the formulation before and after freeze-drying is needed. Non-invasive and label-free Raman spectroscopy can be used to determine vibrational modes of molecules to investigate changes in molecular orientation of the material. The aim of this study was to investigate the possible molecular changes induced by freeze-drying of NFCh-based formulations utilizing Raman spectroscopy and evaluate the connection of the results to MD simulations. NFCh with different excipients was freeze-dried and physicochemical properties, rheology and Raman signal were measured before and after freeze-drying and compared to the literature of MD simulations. The principal component analysis (PCA) was done to the Raman spectra and differences evaluated. The spectra of all formulations differed before and after freeze-drying, and more detailed analysis was done to two most potential 0.8% NFCh based formulations, lactose 300 mM and lactose 250 mM + glycine 50 mM. They had great attraction to NFCh in MD simulations and very similar rheological properties before and after freeze-drying and reconstitution. The spectra of different state of both formulations different on areas between 400 - 500 cm-1 and 850 - 900 cm-1 based on PCA analysis contributing the mutarotation of lactose during freeze-drying and reconstitution. Freeze-drying and the absence of water molecules in NFCh formulation favor different ratios of β and α anomers than the fresh hydrated state which could be detected utilizing Raman spectroscopy. Therefore, Raman spectroscopy was confirmed to be a sensitive option to assess subtle changes in molecular orientation in fresh, freeze-dried, and reconstituted NFCh-based formulations, resulting in a detail knowledge of the molecular behavior of excipients which could be applied in MD simulations and design of better freeze-drying formulations in future.

Adverse drug events (ADE) are a major problem which deteriorates the quality of drug therapy. They cause significant morbidity and mortality each year. ADEs are often caused by incompatible drug combinations, drug-drug interactions (DDIs). Interprosessional collaboration between health care professionals is important in improving medication safety and preventig drug interactions. The aim of this study was to investigate the most common clinically significant drug-drug interactions in outpatient care and the role of pharmacist in preventing them. The study material was an interaction data which was collected in Helsinki University Pharmacy during August 2015. DDIs and the action needed by presecribers or pharmacists to handle them were collected. Only clinically significant interactions of the SFINX interaction database i.e. D- and C-interactions were recorded. The most common D-interactions (interactions to be avoided) were fluoroquinolones or tetracyclines combined with metal ions (calcium, iron, magnesium, aluminium) (14.7 % of D-interactions) and codeine or tramadol combined with CYP2D6 enzyme inhibiting antidepressants (12.6 %). C-interactions concerned most commonly interactions between antihypertensive drugs and NSAIDs (26.2 % of C-interactions). 59.6 % of D-interactions were interactions that might result in adverse drug reactions and 40.4 % were interactions that might result in therapeutic failure. For C-interactions numbers were 49.4 % and 50.6 %, respectively. Only a few interactions (1.6 %) led to contact with the prescriber from the pharmacy, and more often (1.8 %) the pharmacist advised the patient to contact the prescriber. 32.6 % of the interactions led to pharmacist's advice. The most typical interactions which can be prevented by pharmacist's advice were chelation interactions which can be prevented by taking drugs many hours apart from each other. 59.7 % of the interactions produced no action in pharmacy. Those concerned situations where the prescriber had planned the treatment and weighed up the benefits and risks of the medication, or interactions where the drugs had been in contemporary use for a long time, and thus the pharmacist assumed that the prescriber had planned the treatment. Pharmacists should intervene in drug-drug interactions easier. To avoid unnecessary calls, communication between prescribers and community pharmacies should be developed. Pharmacists' role in preventing DDIs could be improved for example by education and by updating the operations models in collaboration with other health care. Safe and efficient drug treatment should be ensured with interprofessional collaboration, and the responsibility should not be shifted to the patient alone.

Reverse-phase protein microarray, RPMA, is a novel and promising technology for proteomic profiling. The low sample consumption, high-throughput format, high sensitivity and good precision make RPMA attractive tool for clinical use. In RPMA, cellular lysates obtained from various sources (e.g. clinical samples, cell lines) are arrayed onto a substratum as a small spots such that an array is comprised of hundreds to thousands of different samples. The array is incubated with a capture molecule (e.g. antibody) that is validated to recognize the analyte of interest. Signal is created by labelled secondary antibody and the signal is detected by colorimetry, chemiluminescence or fluorescence methods. The literature part introduces the RPMA technology and its applications. RPMA have been utilized in versatile applications for example in cell signal pathway profiling, drug discovery and discovery and validation of biomarkers. In the future, it is hoped to allow individual therapy regimes and the evaluation of treatment efficacy. The aim of the experimental part was to culture various cell lines and prepare lysates for RPMA. The lysates were prepared of ARPE-19, HepG2, Hepa-RG, SKOV-3-ip1, SKOV-3, Caco-2, hCMEC/D3, HCE and D-407 cell cultures. The lysates were stored in -80 °C for subsequent use in RPMAs. The purpose was to optimize the method and based on the optimization studies, to print one RPMA. Cell lysates were arrayed onto nitrocellulose coated glass slide using Nano-Plotter (Gesim)-device which allows automated sample printing. β-actin and α-tubulin proteins were assessed from the samples. To create the signal, fluorescence dye was used, and detected at the visible wavelengths. Based on this study, more optimization is required. The detection method used in the RPMA was not optimal, but the experiment showed promising potential. By taking into account the development issues, the method performance can be significantly improved. Of these issues, perhaps the most important is to use infrared region for the signal detection instead of visible wavelengths.

River blindness (onchocerciasis) is a human helminth disease that is caused by Onchocerca volvulus filaria. It is endemic in tropical areas in Africa and Latin America. About 37 million people are infected. River blindness manifests in cutaneous and eye symptoms that are caused by the youngest forms (microfilariae). Against river blindness there has been mass drug treatments with ivermectin which has an effect on microfilariae. There is a need for a drug that kills adults or sterilizes females. A vaccine would be even better. Antibiotics are a new treatment because O. volvulus has Wolbachia bacterium as an obligate symbiont. Doxycycline kills at least 60 per cent of adults and sterilizes females. But the course lasts many weeks. A promising compound is emodepside that has a new mechanism of action for an anthelmintic drug. Numerous compounds has been examined to get drugs for filarial diseases. Some of them inhibit enzymes with which filariae evade human immune defence. Others disturb moulting that takes place four times. A good target for a drug is essential for the parasite but absent from mammals. Betulin is a triterpene that is abundant in birch bark. Betulin and many of its derivatives are pharmacologically active compounds that are examined particularly as cancer and HIV drugs. The research group of medicinal chemistry in University of Helsinki has synthesized and examined many derivatives. Some of them are promising for example against Leishmania protozoans, Chlamydia pneumoniae bacterium and alphaviruses. That is why those compounds should be tested against other causes of diseases like filariae. Both Wolbachia and C. pneumoniae have the same lipid biosynthesis pathway that is essential for both. Heterocycloadducts between betulin and nitrogen heterocycle were synthesized whose alcohols were oxidized to carbonyls. In both Leishmania donovani and L. braziliensis examinations the most effective compound was heterocycloadduct of formylbetulin. Although the compounds have not been examined against filariae, in future it would be worth an effort to synthesize derivatives that has nitrogen instead of carbonyls because the compound effective on C. pneumoniae is dioxime.

Breast cancer is the most common cancer in women worldwide and the number of new events is on the increase. Like many other serious diseases, breast cancer reduces patient’s health related quality of life (HRQoL) and breast cancer treatment burdens our society. Examination of breast cancer patient’s HRQoL makes it possible to calculate how effective breast cancer treatments are. Nevertheless, only cost-effectiveness analysis would further help us allocate the resources of our society in the best way possible. The aim of this study was to produce research about breast cancer treatment’s effects on patient’s HRQoL and to compare generic 15D- and EQ-5D-5L-instruments. The results can be used in the future research and the study might be useful, when it’s time to develop international protocol for measuring HRQoL. The study population included 152 breast cancer patients who were treated in HUCH and whose HRQoL were measured by 15D-, EQ-5D-5L- and VAS-instruments. All measurements were done twice, first before the treatments and then six months after the beginning of the treatments. 89 (58.6 %) patients answered both 15D-questionnaires and 81 (53.3 %) patients answered to both EQ-5D-questionnaires. 57 (37.5 %) patients didn’t respond to any questionnaire. Only some background information was available of this population. The average HRQoL for breast cancer patients’ was 0.92 before the treatments and 0.88 six months after the beginning of the treatments when measured by 15D. The same average HRQoL was 0.86 before the treatments and 0.80 six months after the beginning of the treatments when measured by EQ-5D-5L. During six months’ period, patients HRQoL reduced (-0.04) when it was measured by 15D and (-0.06) when it was measured by EQ-5D. The changes of HRQoL were clinically important (The minimum important change, MIC > ± 0,015) when measured by 15D. HRQoL reduced more with patients who received a mastectomy than with patients, who received a breast conserving surgery according to both instruments. According to the results, the chosen instrument has an effect of breast cancer patients’ HRQoL. It means that the chosen instrument also has an effect of treatment’s effectiveness. 15D offers higher HRQoL values, but EQ-5D offers a greater change in patient’s HRQoL. HRQoL was measured by two different generic instruments in two different times, which was assumed to be the strength of this study. The new 5L-version of EQ-5D-instrument was also used. This is possibly the first time, when 5L is used in this type of study.