Mastergradsoppgaver i farmasi

Advanced delivery system for skin and burns therapy : mupirocin as an antibacterial model drug

Berg, Ole Aleksander — Thu, 19 May 2011 22:00:00 GMT

Berg, Ole Aleksander
Trauma to the skin in the form of severe wound, particularly burns, can facilitate colonization of potentially life threatening bacterial infections. To prevent infections of the wounded area, antimicrobial agents are recommended as standard treatment. Topical administration of antimicrobial agents, such as mupirocin, can provide local therapy, while avoiding the risks of systemic administration. Mupirocin-in-liposomes-in hydrogels were proposed as advanced delivery system for this purpose. Up to now, no liposomal mupirocin for topical administration has been reported. Chitosan was selected as hydrogel matrix due to its biodegradability and in-built antimicrobial and wound healing potentials. Phosphatidylcholine liposomes containing mupirocin, namely non-sonicated and sonicated liposomes, were characterized for vesicle size and size distributions. Non-sonicated vesicles entrapped in average 74 and sonicated 49 % of mupirocin calcium, respectively. Sonication reduced the original vesicle size from around 1 micron down to 135 nm. Liposomes (10 %, w/w) were incorporated in chitosan hydrogels and liposomal hydrogels evaluated for their textural properties. Hydrogels were found to exhibit satisfactory adhesiveness and cohesiveness, with corresponding stability profile. Microbiological assessment confirmed antibacterial properties of liposomally entrapped mupirocin incorporated in hydrogels. In vitro and ex vivo (on pig skin) drug release profiles of various formulations containing mupirocin were performed on Franz diffusion cells. Liposomal hydrogels were compared with marketed mupirocin product, Bactroban® cream. The release studies showed that liposomal size affects the release of the incorporated drug. Liposomal hydrogels were shown to provide sustained release of incorporated mupirocin. In conclusion, liposomal hydrogels developed for mupirocin offer the potential to increase retention time and provide sustained release of a drug, which are important parameters for improved treatment of wounds, including burns.

Isolation and characterization of wax esters from Calanus finmarchicus

Andersen, Toril — Tue, 15 Jun 2010 22:00:00 GMT

Andersen, Toril
Calanus finmarchicus is a vast resource that until recently have been exploited to a minimal degree only. In the last few years harvesting and processing methods have been developed. There is a large market and great interest in products derived from marine oils. Most of the products today are derived from organisms at a high trophic level, i.e. products derived from cod liver oil. One disadvantage with these products is high levels of fat-soluble pollutants that require extensive purification before the products can be released to the market. As C. finmarchicus exists on the first trophic levels it is not polluted and is therefore very interesting as a substitution for cod liver oil, which is the main source for ω-3 fatty acids. In addition to this studies have shown that the oil extracted from the animal, Calanus oil, have beneficial properties and can reduce the risk for coronary heart disease, cancer and inflammation and this could be attributed to the contents of wax esters, high levels of a natural antioxidant, Astaxantine, and high levels of phytosterols. The aim of this thesis was to isolate and characterize the wax esters of the oil, and elucidate their structure. The wax esters were first separated from the Calanus oil with silica SPE and then transesterified to FAME and FAL. The two structures types were separated into two fractions with APS SPE. The FAME were analyzed as their picolinyl esters in GC-MS for structure elucidation and the FAL as their nicotinate esters and TMS ethers. The fractions from various SPE columns were tested by use of TLC and/or GC-MS to confirm that they contained the actual compounds. The FAME found were in a range of 14 to 22 carbon chain length, and there were saturated, monounsaturated and polyunsaturated FAME present. The PUFA were 18:4 (n-5, 7, 9, 11) and 22:5 (n-5, 7, 9, 11, 13). The FAL identified ranged from 16 to 22 carbon atoms in chain length, and were mostly monounsaturated, but one saturated compound was found. The next step would now be to analyze the wax esters in LC-MS to find the complete structure using the pieces of structure found in the FAME and FAL, as there are limited numbers of combinations of the FAME and FAL in a wax ester.

Structure/activity relationship of alpha-conotoxins targeting GABAB receptor

Køller, Katrine Sperstad — Wed, 15 Jun 2011 22:00:00 GMT

Køller, Katrine Sperstad
Conotoxins are small disulfide rich peptides derived from the venom of Cone snails. They target different receptors in the nervous system with high selectivity and potency making them valuable as drug leads or drug themselves. One group of conotoxins, α-conotoxins have been shown to have potential as treatment for neuropathic pain.

Design, synthesis and structural studies of relaxin receptor modulators

Elvheim, Iselin Sørstad — Tue, 31 May 2011 22:00:00 GMT

Elvheim, Iselin Sørstad
The relaxin family of peptides consists of seven structurally related peptides, with a wide variety of biological functions, ranging from involvement in reproductive processes to functions as neuroendocrine modulators. Because of the complex, two-chained structure of the relaxins, and their lack of specificity for the various relaxin family receptors (RXFPs), design of simpler, more selective analogues is important for further investigation of their biological functions and as potential drug leads. The aim of this project was to introduce a helical structure around the receptor-binding region of single-chain relaxin analogues. This was approached by utilising helix capping sequences on truncated relaxin B-chains and by grafting of residues important for binding onto a stable peptide scaffold. Helix capping enhanced the helical properties compared to previous single- chain analogues, but was unable to introduce a sufficiently stable helix. Despite the increased helical tendencies, no high-affinity analogues were found. As a result, the importance of Arg8 was investigated, and we have demonstrated that this residue might be more involved in binding than previously thought. We successfully synthesised a relaxin-3/chlorotoxin analogue, which appeared to fold correctly. However, the yield following oxidation was poor, and no bioactivity data or structural data confirming the correct fold was obtained within the time limits of the project. A secondary aim was to probe for favourable mutations around the receptor- binding region by synthesising a combinational library. We were able to successfully synthesise a library of peptides with mutations in one position by inserting a mixture of amino acids at this coupling step in the solid phase peptide synthesis (SPPS) procedure. Although no significant improvement in binding was seen for the analogues generated, important methodological advances were made that will be used to scan different positions for new contact points with the receptor.

Analyser av DKK3 som mulig målgen for Pax6

Mobasheri, Mina — Sun, 05 Jun 2011 22:00:00 GMT

Mobasheri, Mina
Pax6 og Pax6(5a) er to isoformer av den evolusjonært konserverte transkripsjonsfaktoren som blir uttrykt i blant annet hjernen, øyne, nese og pankreas hos virveldyr under embryoutvikling. Uttrykket av Pax6 vil vedvare hos voksne individer, blant annet i øyne, hjernen og pankreas. Den er viktig for vedlikehold av stamceller, men har også en stor betydning for celle differensiering i mange vev. Aniridia hos mennesker og small eye i mus er blant de sykdommene som forekommer pga. mutasjoner i ett Pax6-gen. Dersom det forekommer mutasjoner i begge Pax6 genene vil fosteret ved fødsel ikke ha øyne, deler av hjernen vil være manglende eller underutviklet og nasale strukturer vil også mangle. Derfor dør fosteret kort tid etter fødsel. Pax6 er også forbundet med kreftformer slik som glioblastoma. De to isoformene (Pax6 og Pax6(5a)) har forskjellige DNA bindings egenskaper, men blir uttrykt i de fleste celletyper samtidig, slik som i øyne. For å få en oversikt over hvilke målgener de to isoformene regulerer, ble det i forkant av dette prosjektet brukt genekspresjons microarray og en del av resultatene ble bekreftet ved hjelp av qPCR metoder. Det ble oppdaget et lite, men signifikant antall gener som ble regulert av Pax6 og/eller Pax6 i stabil transfekterte 3T3 fibroblaster. Noen av disse er felles målgen for begge isoformene. Men disse blir regulert i forskjellig grad av Pax6 og Pax6(5a). Et målgen som viste seg å bli regulert av både Pax6 og Pax6(5a) var Dickopf3(DKK3). DKK3 er et medlem av Dickkopf (DKK) familien som er antagonister av Wnt – signalveien. I disse undersøkelsene viste det seg at DKK3 er 18 fold oppregulert av Pax6(5a) i 3T3-fibroblaster fra mus, og 3 fold oppregulert av Pax6. Mulige målseter for Pax6 og Pax6(5a) i promoteren til både mus og menneske DKK3 genet har også blitt identifisert ved bruk av bioinformatikk. I denne oppgaven ble DKK3 promoteren fra mus og menneske klonet og ko–transfektert sammen med plasmider som kodet for Pax6 og Pax6(5a). Vi kunne tydelig se at mDKK3 promoteren ble positivt påvirket ved reporter gen assay analyser. Det ble også jort forsøk på å klone sebrafisk cDNA’ et som koder for DKK3, med tanke på å lage en probe for sebrafisk in situ hybridiseringer. Videre ble det utført transient transfeksjon av plasmider som koder for Pax6 og Pax6(5a) inn i 3T3-kontroll cellene for å se om korttids – uttrykket av Pax6 og Pax6(5a) gir samme resultat som de stabilt transfekterte cellelinjene med tanke på DKK3’ gen uttrykket. Protein uttrykket av Pax6 og Pax6(5a) ble bekreftet, men endringer i DKK3 RNA uttrykk kunne ikke identifiseres ved bruk av qPCR.

Evaluation of antiviral prophylaxis of cytomegalovirus in patients receiving liver, kidney or pancreas transplantation

Dyrhaug, Sara Ann — Thu, 19 May 2011 22:00:00 GMT

Dyrhaug, Sara Ann
Abstract Background– All solid organ transplanted patients are treated with immuno suppression medications to keep the immune system from rejecting the transplanted organ. Unfortunately the suppressed immune system makes the body more exposed for cancer and opportunistic infections. Among the most important viruses that causes one of these opportunistic infections is cytomegalovirus (CMV). A local project showed that not all transplanted patients on CMV prophylaxis get the right dose according to creatinine clearance (CrCl), and that the patients who had not been prescribed the recommended dose according to CrCl had a higher incident of CMV disease than those adjusted as recommended. The Quality improvement team for the transplant unit therefore suggested that a project could further investigate these issues. Aim and objectives – The aim of this project was to critically review and evaluate the processes in the prescribing and administration of valganciclovir for cytomegalovirus prophylaxis in liver, kidney or pancreas transplantation. Methods – Semi-structured one-to-one interviews with 2 nurses and 6 prescribers were conducted to establish current practice in prescribing, administration and monitoring of valganciclovir. A review of the local protocols at the transplant unit was conducted. To assess the harm a database analysis on the incident reporting system and a retrospective review of clinical records of 4 patients was undertaken. A questionnaire was developed for staff to self-assess the risk of harm of the whole process of CMV prophylaxis treatment. Pharmaceutical care issues relevant to CMV prophylaxis were recorded prospectively by clinical pharmacists over a two month period. Results – The semi-structured interviews with prescribers indicated that: 1) Prescribers often fail to recognise that the valganciclovir dose should be adjusted with changing CrCl. 2) That the laboratory test results taken at the clinic do not come back until the evening and are therefore not available at the time of prescribing, 3) There are gaps of knowledge, especially in the junior doctors. Reviews of local protocols suggested a need for update of the protocols and inclusion of detailed dosing guidance. The one incident reported in the database in terms of valganciclovir involved a missed dose. Case note reviews of four patients identified that dose adjustments are appropriate in 2 cases, 1 case was borderline and the other was a complex case but was judged to be appropriate. The questionnaire identified that there was agreement among healthcare professionals that there is a risk of errors that might lead to harm associated with all stages of valganciclovir use. Two clinical pharmacists recommended adjustment of valganciclovir 12 times in 7 patients in a time period of approximately two months. Conclusion – the outcomes from the interviews and pharmaceutical care issues analysis confirm the previous observation that the dose of valganciclovir is not always adjusted according to CrCl. Recommendations for improvement are to ensure modified guidelines are implemented to ensure all prescribers are aware of need for dose adjustment. Further work can be undertaken to measure the benefit after implement of the recommendations to assess improvement.

Karakterisering av modifiserte antistoffer innen radioimmunoterapi

Tobiassen, Tonje — Thu, 19 May 2011 22:00:00 GMT

Tobiassen, Tonje
Den prekliniske forskningen i farmasiselskapet Algeta har fokus på utvikling av nye behandlingsmetoder mot kreft ved bruk av målrettet alfa-terapi. I denne problemstillingen inngår bruk av modifiserte antistoffer merket med alfa-emitteren Thorium-227. Modifiseringen innebærer at en kjelator konjugeres til aminosyrene til antistoffet. Dette vil resultere i en heterogen fordeling av antall kjelatorer pr. antistoff . Denne fordelingen og det støkiometriske forholdet mellom kjelator og antistoff må karakteriseres i forbindelse med prekliniske forsøk. Masteroppgaven omhandler utvikling av en metode for å kunne karakterisere de modifiserte antistoffene. Målet med oppgaven er å utvikle en metode som skal kunne brukes til å bestemme kjelator-til-antistoff ratio (Drug to Antibody Ratio, DAR) for ulike syntesebatcher med varierende støkiometriske forhold. Delere av metoden bør kunne automatiseres og presisjonen bør være bedre enn 10%. I denne sammenhengen ble det utviklet en LC/MS basert metode for å måle konjugering av kjelatoren DOTA-NCS til antistoffet Trastuzumab ved bruk av gelkromatografi-massespektrometri. Metoden er basert på at konjugatene deglykosyleres med enzymet PNGase F og reduseres til heavy chain og light chain. DAR-verdien ble bestemt for både intakt -og redusert konjugat basert på nominelle støkiometrier fra 1:1 til 15:1. Modifiserte biologiske makromolekyler gir ofte opphav til svært komplekse massespektre på grunn av makromolekylenes størrelse samt ladningsfordelingen som følge av elektrosprayionisering. Hovedutfordringen i arbeidet har vært å utvikle en metode som gir massespektre med en kvalitet som gjør det mulig å bestemme DAR-verdier etter dataprosessering. Kvaliteten av råspekterne ble funnet å være en funksjon av signalstyrke og kompleksitet. Kompleksiteten av massespekterne til de reduserte konjugatene er mindre sammenlignet med massespekterne av intakte konjugater, noe som bidrar til sikrere bestemmelse av DAR-verdier for konjugater med nominell støkiometri over 5:1. I gelkromatografi-metoden som ble utviklet ble det brukt en kolonne med kolonnedimensjon på 2.0 millimeter indre diameter. Denne kolonnen ga ca. 5.9 ganger større signal for samme injiserte prøvemengde enn ved bruk av tradisjonell kolonnedimensjon på 4.6 millimeter indre diameter. Denne metodeforbedringen sammen med optimalisering av deglykosyleringsprosedyren og ionekildeparametere gjorde det mulig å bestemme DAR-verdier for DOTA-Trastuzumab konjugatene. Presisjonen ble gjennom partiell validering funnet til å være bedre enn 7% for alle støkiometriske forhold. Det ble vurdert at metoden er egnet til å måle antall DOTA konjugert til Trastuzumab med en nominell støkiometri fra 1:1 til 15:1 i henhold til målsettingen. Analyse av syntesebatcher med ulik støkiometri viste en tilnærmet lineær sammenheng mellom nominell DAR og funnet DAR med et reaksjonsutbytte på gjennomsnittlig 54% for intakte konjugater og 52% for reduserte konjugater (for 4 ulike støkiometrisk forhold). Dette reaksjonsutbyttet er i samme størrelsesorden som tidligere beskrevet i litteraturen. Det ble innledningsvis forsøkt utviklet omvendt fase HPLC-MS og UPLC-MS metoder, men disse ga ikke råspektre med god nok kvalitet til å kunne bestemme antall kjelatorer pr. antistoff. Alternative metoder beskrevet i litteraturen innebærer blandt annet transkjelatering fra Pb (II) arsenazo, radiometrisk titrering med Kobolt-57 eller merking av ligander med Karbon-14. Sammenlignet med disse alternativene er den utviklede LC/MS metoden relativt lite tidkrevende, ikke-radioaktiv og mer generell. I tidlig utviklingsfase innen radioimmunoterapi er det verdifullt å benytte karakteriseringsmetoder som er mer generelle fordi det åpner for mulighet til å bestemme DAR-verdier for konjugater med andre kjelatorer og eventuelt andre antistoffer.

A study of the prescribing and administration of immuno-suppressant medication in patients receiving liver, kidney and pancreas transplantation

Patel, Kinjal Tulsi — Thu, 19 May 2011 22:00:00 GMT

Patel, Kinjal Tulsi
Introduction There is little data on immuno-suppressant administration and prescribing to transplant patients. It was considered a high risk area because errors in prescribing and administration of immuno-suppressants can potentially have serious consequences like graft loss, side effects and even death. The reality was however that the lack of data meant that no one knew whether this was an area for improvement or not. The need for data collection was recognised and the aim of this study was to develop and validate a tool to inform the analysis of the patient journey (Failure Mode Effect Analysis) and identify opportunities for quality improvement of immunosuppressant medication use. (Time did not allow for the FMEA to be conducted). Methods One-to-one semi structured interviews were conducted with clinical staff (2 pharmacists, 2 nurses and 6 doctors) to explore their perceptions of high risk areas. A case study was done to define the patient journey and identify potential areas where the patient might be at risk of harm. Analysis of database of incident reports (from 2010) was conducted. Lastly, analysis of pharmaceutical care issues identified by clinical pharmacists (2 pharmacists) was done. Results Some of the areas identified by staff from interviews were; need for consistent education to patient by all healthcare professions, need for education of staff, communication with primary healthcare professions with regard to risk associated with immuno-suppressants, teamwork amongst the staff on the ward and documentation of interventions. A patient journey detailed where and when high risk processes could occur. The patient journey identified the following areas as high risk: nurses being busy, interrupted or not giving appropriate education. Patients being non-compliant in medications and follow-up meetings, doctors not having clear handwriting, doctors not writing the formulation of immuno-suppressant etc. Database analysis confirmed that Datix® was not a well used reporting system and incidents were mainly in the immuno-suppressant administration category. The incidents reported emphasised the need to follow safe use of medicines policy. Pharmaceutical care issues were not well documented and there were no consistent interventions to confirm particular high risk areas. Discussion The richest data came from interviews and highlighted actions that could be used to reduce risk of harm from immuno-suppressive drug therapy. The data collected can be used to generate an FMEA for agreement and use by a multidisciplinary team.

Lipidklasser og fettsyresammensetning i brunpølse (Cucumaria frondosa)

Nicolaisen, Jan-Roger Sandstad — Thu, 19 May 2011 22:00:00 GMT

Nicolaisen, Jan-Roger Sandstad
Different species of the sea cucumber are a well utilized resource in eastern parts of Asia, but not much used in western parts of the world. The annual harvest of sea cucumbers in Asian waters has been estimated to be over 100,000 tons. This has led to overfishing and decreasing catches. As a result, a demand for new fishing areas has occurred. Different species of sea cucumbers are observed in significant amounts along the Norwegian coastline and in other parts at the North Atlantic Ocean. Occurrence and demand indicates that sea cucumbers may become a new export product from Norway. One challenge is to develop good and effective harvesting methods for the sea cucumber (Cucumaria frondosa). For the most it is considered as a bycatch in context with more commercial fishery. The value of seafood can to a certain degree be determined by the kind of lipids and fatty acid that are present in the actual organism. Long chained omega-3 fatty acids are of special interest. The aim with the project was therefore to determine the lipid content and to analyze lipids from the sea cucumber with the use of thin layer chromatography and gas chromatography. The lipids were extracted and analyzed with the production of fatty acid methyl esters for further analysis with gas chromatography. The fatty acids from Cucumaria frondosa were identified by comparing observed retention times with retention times of known fatty acid methyl ester standards. The extracted oil was separated in the different lipid classes with the use of solid phase extraction. The fatty acid composition from each fraction was further analyzed by producing fatty acid methyl esters for gas chromatography. The analysis showed that the content of total fat was on average ~3.5 % based on freeze-dried material, and the content of long chained omega-3 fatty acids in Cucumaria frondosa was relatively high compared to the total fatty acid composition. Of the polyunsaturated fatty acids, in the total amount, EPA 20:5 n-3 (33 %) was the most abundant fatty acid. The relative amounts of the different fatty acids were different in the different lipid classes. EPA was now present with an amount of 70 % in the fraction consisting of polar lipids and only 11 % in neutral lipids. The most dominant lipid classes were polar lipids including phospholipids (~ 40 %) and free fatty acids (~ 20 %).

Investigation and optimization of liposome formulation for use as drug carrier for the anticancer agent Camptothecin

Naderkhani, Elenaz — Wed, 08 Jun 2011 22:00:00 GMT

Naderkhani, Elenaz
In this thesis, the method development and investigation of different liposomal formulations to incorporate and retain Camptothecin (CPT) is described. CPT is a potent anticancer drug that has shown to be active against a broad spectrum of cancers. However, due to its challenging physicochemical properties, like poor water solubility, severe toxic effects to normal tissues and instability, its clinical development has been limited for nearly 40 years. A strategy to overcome CPT’s challenging properties is to use liposome-based carrier system. By taking advantage of this carrier system, we may solubilise CPT in the phospholipid bilayer of liposomes, protect it from blood proteins and achieve a selective drug accumulation in tumor tissues or tumor-associated cells by enhanced permeability and retention effect (EPR). A good liposome formulation of clinical utility must fulfil two important criteria. The liposomal drug carrier must incorporate CPT in the liposomal bilayer in a relevant therapeutic concentration and be able to retain the drug within the liposome to make it bioavailable at the target site after i.v. administration. The focus of this thesis was to study different liposomal formulations and their ability to incorporate and retain CPT. Screening of eight different liposome formulations with respect to association with CPT was performed. The 1,2-di-oleyl-3 trimethyl-ammonium-propane (DOTAP) containing formulations showed superior incorporation capacity, giving an CPT incorporation of 250 µg/130 µmoles lipid. The DOTAP containing formulations exhibited as well a trend toward higher retention ability in serum compared to the other formulations. Although they showed better retention ability, only 25 % of the drug was associated with the liposomes, which is far from being optimal. One of the important criteria mentioned above for liposomes as drug delivery systems is their ability to remain stable in blood circulation for prolonged time in order to reach the specific target and to avoid rapid clearance by RES after i.v. injection. To achieve this, PEG decoration on the liposome surface can be employed. We chose to PEGylate DOTAP formulations in order to get a better understanding of this system. PEGylation lead, as expected, to increased stability of the liposomes, however a reduced incorporation capacity was observed. The presence of 1 % and 10 % PEG gave better retention and slower leakage from the liposomes. We conclude that DOTAP inclusion in our liposomes increased the incorporation of CPT into the lipid bilayer, that liposomal retention in our current formulations must be improved, and while PEGylation is necessary in order to prevent rapid in vivo clearance, the inclusion of PEG reduces incorporation, and therefore further studies are needed in order to improve incorporation of CPT in PEGylated liposomes.