Multipel skleros (MS) är en kronisk, neurologisk sjukdom som drabbar centrala nervsystemet (CNS). Sjukdomen är autoimmun där kroppens eget immunförsvar angriper nervvävnaden, framför allt myelin, och därefter uppstår en inflammatorisk reaktion. Nerverna i det centrala nervsystemet påverkar många olika funktioner och eftersom myelinet som omsluter nervfibrerna blir inflammerat på olika ställen, både i hjärnan och i ryggmärgen, uppkommer olika typer av symptom. Symptomen för MS kan skilja sig mycket från en individ till en annan, och från skov till skov. Om McDonald-kriterierna uppfylls, som bygger på att minst två separata CNS-lesioner skilda i rum och med minst en månads intervall påvisas utan en tydlig anledning, ställs diagnosen MS. Individer med MS producerar autoreaktiva T-celler som deltar i bildningen av inflammatoriska lesioner längs myelinskidan, men det finns även CNS-celler som gliaceller och astrocyter, som deltar i inflammationsprocessen. Vävnadstypen HLA-DR2 är förknippad med ökad risk att få MS. Den vanligaste och den milda formen av MS, skovvis förlöpande MS, bromsas med immunmodulerande läkemedel som interferon beta, fingolimod, m.fl. Syftet med det här litteraturarbetet var att utreda den kliniska effekten av fingolimod, den första orala behandlingen mot MS, på möjliga skador som förekommer på grund av MS. Studierna som granskades i detta arbete hämtades från Pubmed. Resultatet visar att fingolimod har klinisk effekt vid MS, men det behövs fler studier för att undersöka säkerhetsprofilen mer ingående. I en studie under två år reducerades den årliga skovfrekvensen från 0.4 till mindre än hälften av detta. I en annan två-årig studie, där fingolimod jämfördes med interferon-beta 1a, fanns det en signifikant större reduktion i den årliga skovfrekvensen i fingolimodgruppen (årlig skovfrekvens < 0,20) än i interferongruppen (årlig skovfrekvens 0,33). Majoriteten av patienterna (>70 %) förblev skovfria med fingolimodbehandlingen i båda studierna. Det fanns inga studier där fingolimod kombinerades med annan MS-behandling, vilket skulle vara intressant att se i framtiden. / Multiple sclerosis (MS) is a chronic neurological disease that affects the nerves within the central nervous system (CNS). The disease is autoimmune where the body's own immune system attacks nerve tissue, especially myelin, causing an inflammatory reaction. The nerves of the central nervous system serve many different functions and since the myelin surrounding the nerve fibers becomes inflamed at different places, both in the brain and the spinal cord, a variety of symptoms result. Symptoms of MS can differ greatly from one individual to another, as well as from relapse to relapse. If the McDonald criteria, which are based on at least two separate CNS lesions separated in space and at least one month apart demonstrated without a clear reason, are fulfilled a diagnosis of MS is set. Individuals with MS produce autoreactive T cells involved in the formation of inflammatory lesions along the myelin sheath, but CNS cells, glial cells and astrocytes, also participate in the inflammatory process. Tissue type HLA-DR2 is associated with increased risk of developing MS. The most common and mild form of MS, relapsing-remitting MS, can be slowed down with immunomodulatory drugs such as interferon beta, fingolimod, etc. The aim of this literature study was to investigate the clinical efficacy of fingolimod, the first oral therapy for MS, on the possible damage that occurs due to MS. The studies examined in this work were taken from Pubmed. The results demonstrate that fingolimod has clinical efficacy in MS, but more studies are needed to investigate the safety profile in more detail. In a study over two years the annualized relapse rate was reduced from 0.4 to less than half of this. In another two-year study in which fingolimod was compared with interferon-beta 1a, there was a significantly greater reduction in the annualized relapse rate in the fingolimod group (annualized relapse rate <0.20) than in interferon group (annualized relapse rate 0.33). The majority of patients (>70 %) remained relapse-free with fingolimod treatment in both studies. There were no studies that combined fingolimod with other MS treatments, which would be interesting to see in the future.

Phenotypic screening of drug molecules relies on the generation of a specific response; however the means by which this is elicited often remains unknown. Affinity chromatography is a valuable tool in the discovery of drug binding partners and may even allow the elucidation of the wider interactome of the initial drug target. The introduction of easily cleavable linkers and affinity-independent elution protocols to affinity chromatography is of current interest, since they render the technique much more adaptable with respect to the characterisation of biologically active species of interest. This thesis details the application of a novel azobenzene linker developed by the Hulme group for use in affinity-independent chromatography. The first chapter reviews recent developments in affinity chromatography and describes the synthesis of an affinity linker toolbox with both affinity-dependent and affinity-independent linkers. These linkers are functionalised with an azide moiety for use in CuAAC coupling to alkynyl derivatives of bioactive small molecules and have been modified to include photoreactive groups giving a series of linkers for use in the identification of less abundant, or low affinity, proteins. The first drug investigated, anisomycin (ANS), is a small molecule which was initially introduced as an antibiotic drug (Flagecidin). At nanomolar concentrations ANS has been shown to affect the mitogen activated protein kinase (MAPK) pathways; downstream effects of these pathways are thought to play a role in a range of pathological disorders such as Alzheimer’s disease, cancer and spinal muscular atrophy (SMA). ANS is thus a candidate for drug repurposing. Although the downstream effects of MAPK/SAPK pathway activation induced by anisomycin are well-documented, the cellular target has yet to be revealed. Previous work by the Hulme group has shown that the N-propargyl anisomycin derivative (I) retains the biological activity of the lead compound ANS. Thus to evaluate the cellular protein targets, N-propargyl ANS (I) was coupled onto the linker toolbox to create an ANS affinity probe library as described in chapter 2. The second drug investigated, fingolimod, was introduced as an immunomodulating drug (Glienya) for the treatment of multiple sclerosis (MS). This small molecule has also been shown to have anti-cancer properties in a range of cancer cell lines; however the precise mechanism by which this is effected is unknown. Literature precedent shows that terminal modification of fingolimod generates analogues which still retain biological activity. Thus a novel fingolimod alkyne derivative (II) was synthesised and used to create an affinity probe library as described in chapter 3. Chapter 4 describes affinity pull-down experiments conducted with the aim of finding the protein target(s) of ANS and fingolimod, using the affinity probe libraries generated in chapters 2 and 3. This chapter concludes with a discussion of the implications of these findings and directions for future study.

Pancreatic ductal adenocarcinoma is an extremely lethal cancer that is difficult to treat. A better understanding of the biology of pancreatic ductal cancer will help to develop targeted therapies that may improve clinical outcomes. Recently, the lipid signaling molecule sphingosine-1-phosphate (S1P) has emerged as a driver of malignant behavior in many types of cancer. Its role in pancreatic cancer remains unknown. Pancreatic cancer cells express high levels of the S1P receptor known as S1PR1, which is the receptor most important for mediating growth and migration through S1P signaling. In addition, the subcellular expression of the sphingosine kinases is altered in pancreatic cancer cells, which may contribute to their malignant behavior. Exogenous S1P increases pancreatic cancer cell migration, while inhibition of S1P signaling decreases the metabolic activity of pancreatic cancer cells as well as their ability to invade and migrate. Taken together, these results demonstrate the importance of S1P signaling in maintaining malignant behavior in pancreatic cancer cells. In addition, inhibition of S1P signaling represents a potential therapeutic target in pancreatic ductal cancer.

Multipel skleros (MS) är en kronisk immunologisk sjukdom som slår på centrala nervsystemet (CNS) och kan leda till minskad neurologisk funktion. I Sverige finns 17 500 personer som fått en MS-diagnos. Den vanligaste åldern för insjuknande är 20-40 år och sjukdomen är dubbelt så vanlig bland kvinnor som bland män. Om patienterna inte får behandling finns risk för kraftiga funktionsnedsättningar. Syftet med detta arbete var att undersöka effekten och säkerheten med det perorala läkemedlet fingolimod jämfört med intravenös behandling med natalizumab vid MS. Arbetet utformades som en litteraturstudie där sökningar gjordes i PubMed genom Linnéuniversitetets bibliotek. De sökord som användes var "multiple sclerosis AND drug therapy AND fingolimod" samt "multiple sclerosis AND drug therapy AND natalizumab". Sökningarna ledde till att 7 studier granskades. Studierna visade att både fingolimod och natalizumab ger bättre effekt än placebo och de har dessutom bra säkerhet. Det var dock inga stora skillnader mellan de två substanserna. Vad gällde risken för progredierande funktionsnedsättning och risken för allvarliga biverkningar tycktes fingolimod vara något mera fördelaktigt, medan natalizumab hade en liten fördel vad gällde antalet gadoliniumförstärkta lesioner. Då man i några av fingolimodstudierna inte använde sig av intention-to-treat (ITT) och dubbelblindning kan läkemedlets effekter i dessa studier framstå som bättre än vad de är och de små förbättringar i effekter som sågs i resultaten blir osäkra. För att säkert kunna säga om det är någon skillnad i effekt och säkerhet mellan preparaten vore det önskvärt med nya dubbelblindade långtidsstudier av fingolimod där ITT använts vid analysen. Det vore också intressant med en randomiserad klinisk prövning som jämför fingolimod med natalizumab. De resultat som framförallt bör studeras är skovfrekvens, risk för ökande funktionsnedsättning och risk för allvarliga biverkningar, då dessa parametrar borde vara av störst värde för patienterna.

Objectives: Adherence to multiple sclerosis (MS) treatment is essential to optimize the likelihood of full treatment effect. This prospective, observational, single-center cohort study investigated adherence to fingolimod over the 2 years following treatment initiation. Two facets of adherence - implementation and persistence - were examined and compared between new and experienced users of disease-modifying treatments (DMTs). Materials and methods: Implementation rates were based on the proportion of days covered and calculated as percentages per half-yearly visits and over 2 years, captured through refill data, pill count, and self-report. Nonadherence was defined as taking less than 85.8% of prescribed pills. Implementation rates were classified as nonadherent (< 85.8%), suboptimally adherent (>= 85.8% but. 96.2%), and optimally adherent (>= 96.2%), including perfectly adherent (100%). Persistence, ie, time until discontinuation, was analyzed by Kaplan-Meier analysis. Reasons for discontinuation were recorded. Results: The cohort included 98 patients with relapsing MS, all of whom received a dedicated education session about their medication. Of these 80% were women, 31.6% had fingolimod as first DMT, and 68.4% had switched from other DMTs. The mean implementation rate over 2 years was 98.6% (IQR(1-3) 98.51%-98.7%) and did not change significantly over time; 89% of measurements were in the optimally adherent category, 45.6% in the perfectly adherent category. There was one single occurrence of nonadherence. New users of DMTs were 1.29 times more likely to be adherent than experienced users (OR 1.29, 95% CI 1.11-1.51; P < 0.001), but not more persistent. Nineteen of 98 patients discontinued fingolimod. Conclusion: The very high implementation rates displayed in this sample of MS patients suggest that facilitation by health care professionals in preserving adherence behavior may be sufficient for the majority of patients. Targeted interventions should focus on patients who are nonadherent or who stop treatment without intention to reinitiate.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12871号 / 論医博第2087号 / 新制||医||1006(附属図書館) / 31589 / 北海道大学大学院医学研究科臨床医学コース / (主査)教授 三森 経世, 教授 長澤 丘司, 教授 河野 憲二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die 2009 erstmals entdeckte Spezies C. auris erlangte binnen kürzester Zeit zunehmend weltweite Aufmerksamkeit. Vor allem die Tendenz der Multiresistenzentwicklung und das rasche Auslösen von nosokomialen Infektionen erschweren den Umgang und die Therapie von C. auris Infektionen im Vergleich zu anderen Candida Spezien. Diese Dissertationsarbeit umfasst eine systematische Resistenzanalyse der im NRZMyk vorhandenen Stammsammlung aus C. auris und C. parapsilosis Isolaten, um Aufschluss über den Wirkmechanismus von Amphotericin B in Hefepilzen zu erlangen. Anhand der zunächst durchgeführten Amphotericin B-Resistenztestungen kristallisierten sich CAU37 und CAU43 mit MHK-Werten bis zu 12 µg/ml als stark Amphotericin B-resistente Isolate heraus. Die Analyse der Sequenzierungsergebnisse zeigte bei beiden Stämmen eine Mutation im ERG4 Gen an Position 576, welche nicht eindeutig als alleinige Ursache für die verminderte Amphotericin B-Empfindlichkeit festgelegt werden konnte. Dennoch wurde im Rahmen eines Survival Assays bei beiden Amphotericin B-resistenten Isolaten anfänglich eine konzentrationsabhängige Aktivität gegenüber Amphotericin B festgestellt, bevor ein Nachwachsen der Kulturen beobachtet wurde. Somit wurde die Vermutung aufgestellt, dass lediglich ein Teil der aufgebrachten Candida-Zellen abgetötet wird und dies in einer Vermehrung der überlebenden Zellen resultiert. Des Weiteren konnte im Rahmen von Resistenztestungen mit dem Sphingolipidinhibitor Myriocin nachgewiesen werden, dass vor allem in Amphotericin B-resistenten Isolaten eine deutliche Wirkungsverstärkung des Polyens hervorgerufen wird. Diese Sensitivitätssteigerung ist allgemein bei allen C. auris Isolaten zu beobachten, fällt bei resistenten Stämmen jedoch deutlich stärker aus. Hierdurch kam die Annahme auf, dass Amphotericin B-Resistenzen auch in möglichen Veränderungen des Sphingolipid-Haushaltes begründet sein könnten. Darüber hinaus scheint Myriocin keinen Einfluss auf Fluconazol-resistente oder FKS-mutierte Echinocandin-resistente C. auris Stämme zu haben. Das ebenfalls untersuchte und von Myriocin abgeleitete Medikament Fingolimod hatte jedoch ebenfalls keinen wirkungsverstärkenden Effekt. Allerdings reagierte ein Großteil der C. auris Isolate (57,6 %) sensitiv gegenüber dem neusten medizinisch bekannten Triazol Isavuconazol und es konnte erstmalig ein ECV-Wert von 0,03125 µg/ml festgelegt werden. Ein valider Vergleich von C. auris zu C. parapsilosis war aufgrund der mangelnden Anzahl an C. parapsilosis Isolaten jedoch nicht möglich / The species C. auris, which was first discovered in 2009, quickly attracted worldwide attention. In particular, the development of multidrug resistance and the rapid onset of nosocomial infections complicate the management and treatment of C. auris infections compared to other Candida species. This dissertation comprises a systematic resistance analysis of the strain collection available at the NRZMyk from C. auris and C. parapsilosis isolates in order to shed light on the mechanism of action of amphotericin B in yeast fungi. CAU37 and CAU43 ermerged as highly amphotericin B-resistant isolates in the initially performed amphotericin B resistance tests, with MIC values up to 12 µg/ml. Sequencing results showed a mutation in the ERG4 gene at position 576 in both strains, which can`t be clearly identified as the main cause of the reduced susceptibility to amphotericin B. Nevertheless both amphotericin B-resistant isolates initially showed a concentration dependent activity against amphotericin B, followed by a regrowth of the cultures. The hypothesis is, that only some of the applied Candida cells are killed, resulting in a proliferation of the surviving cells. Furthermore the resistance tests with the sphingolipid inhibitor Myriocin in combination with amphotericin B showed that sublethal myriocin concentrations increased the C. auris susceptibility to amphotericin B. This increase in sensitivity is generally observed in all C. auris isolates, but is significantly stronger in resistant strains. This leeds to the assumption that amphotericin B resistance can also be due to possible changes in the sphingolipid balance. Furthermore, myriocin does not appear to have any influence on fluconazole-resistant or FKS-mutated echinocandin-resistant C. auris strains. Fingolimod, a drug also investigated and derived from Myriocin, doesn`t have any enhancing effect either. However the majority of C. auris isolates (57.6 %) reacted sensitively to the latest medically known triazole isavuconazole and for the first time an ECV value of of 0.03125 µg/ml could be determined. A valid comparison of C. auris to C. parapsilosis was not possible due to the lack of C. parapsilosis isolates.

La polyradiculonévrite inflammatoire démyélinisante chronique (PIDC) est une pathologie neurologique auto-immune du système nerveux périphérique dont la physiopathologie est actuellement mal connue, pour laquelle les options thérapeutiques sont peu nombreuses et dont il n’existe pas de modèle animal fiable. Le premier objectif de ce travail était de valider et de caractériser un modèle animal de PIDC par immunisation de rat Lewis avec le peptide P0(180-199) palmitoylé. Les animaux ont développé une pathologie chronique ou à rechute qui a pu être caractérisée aux plans clinique, histologique, électrophysiologique et immunologique. Les résultats sont en faveur d’un modèle fiable et reproductible mimant bien la PIDC humaine. Le deuxième objectif de ce travail était de tester, sur notre modèle, le fingolimod, modulateur des récepteurs à la sphingosine 1-phosphate, comme potentiel traitement de la pathologie. Le fingolimod a permis, dans notre modèle, de diminuer la sévérité et la chronicité de la maladie, d’améliorer les paramètres électrophysiologiques, de diminuer l’infiltration par les cellules inflammatoires et les anomalies immunologiques. / Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune pathology of the peripheral nervous system whose pathophysiology is currently poorly understood, for which there are few therapeutic options and no reliable animal model. The first aim of this work was to validate and characterize an animal model of CIDP by immunization of rat Lewis with the palmitoylated peptide P0(180-199). The animals developed a chronic or relapsing pathology that could be characterized clinically, histologically, electrophysiologically and immunologically. The results are in favor of a reliable and reproducible model that mimics the human CIDP. The second aim of this work was to test, on our model, the fingolimod, sphingosine 1-phosphate receptor modulator, as potential treatment of the pathology. In our model, fingolimod has reduced the severity and the chronicity of the disease, improved electrophysiological parameters, reduced infiltration by inflammatory cells and recue immunological abnormalities.

Les neuropathies à anticorps anti-MAG sont secondaires à une gammapathie monoclonale IgM dirigée contre la MAG des gaines de myéline des nerfs périphériques. Le traitement est celui de l’hémopathie sous‐jacente. Même si les thérapeutiques sont de plus en plus efficaces, les hémopathies restent le plus souvent incurables. Le rituximab est couramment utilisé dans le traitement des neuropathies à anticorps anti‐MAG, mais son efficacité n’a pas pu être clairement démontrée dans deux études contrôlées. Le FTY720 ou fingolimod est un sphingolipide, analogue de la sphingosine, qui inhibe les récepteurs de la sphingosine-1-phosphate (S1P). Il est utilisé comme immunosuppresseur dans la Sclérose en Plaques. Des études ont également rapporté un effet cytotoxique du FTY720 dans des hémopathies sans toutefois clairement expliquer son mécanisme d’action. L’objectif de ce travail est d’élucider les mécanismes moléculaires de l’effet cytotoxique du FTY720 dans un modèle d’hémopathie B, la leucémie lymphoïde chronique (LLC). Des cellules leucémiques primaires de LLC et une lignée cellulaire MEC1 ont été utilisées comme modèle expérimental in vitro. Le FTY720, comme la sphingosine, entraîne une cytotoxicité dose‐dépendante dans la LLC. Cet effet, médié par la forme non phosphorylée de FTY720, est indépendant des récepteurs au S1P. Le FTY720 induit l’expression de marqueurs d’apoptose: exposition de la phosphaJdylsérine, clivage de PARP et de caspase 3. Cependant sa toxicité apparaît indépendante des caspases. La lipidation accrue de LC3 et la formation d’autophagolysosomes indiquent que le FTY720 augmente également le flux autophagique. Cependant, des inhibiteurs de l’autophagie ne permettent pas de bloquer la mort cellulaire induite par le FTY720, suggérant que l’autophagie a ici un rôle protecteur vis à vis de la toxicité du FTY720. Plusieurs éléments permettent de conclure que le FTY720 est responsable d’une nécrose cellulaire : aspect morphologique de nécrose en microscopie électronique, perméabilisation membranaire précoce avec relocalisation cytoplasmique de HMGB1, libération extracellulaire de LDH, perméabilisation de la membrane lysosomale associée à une activation des cathepsines. Au niveau moléculaire, l’action du FTY720 n’est pas bloquée par la nécrostatine 1, indiquant que la nécrose induite par le FTY720 est indépendante de RIPK1 (receptor interacJng protein 1), une kinase clef des voies extrinsèques de nécrose cellulaire programmée. Par contre, nos travaux ont établi l’implication de DRP1 (dynamin related protein), une enzyme régulatrice de la fission mitochondriale, dans le processus de nécrose induite par le FTY720. En plus d’une relocalisation précoce de DRP1 à la mitochondrie accompagnée d’une augmentation de sa phosphorylation sur des sites régulateurs de son activité, nos expériences montrent que la suppression de son expression par interférence à ARN dans les cellules leucémiques réduit fortement la mort cellulaire induite par le FTY720. Le FTY720 est donc responsable dans la LLC d’une nécrose cellulaire programmée dépendante de DRP1. Nos résultats illustrent l’implication des sphingolipides dans la régulation de la survie cellulaire et dans les voies de nécrose programmée. Le FTY720 a un mode d’action original différent de l’apoptose induite par les chimiothérapies classiques. Le FTY720 pourrait donc être une alternative thérapeutique dans les néoplasies B résistantes aux chimiothérapies usuelles et dans certaines manifestations auto‐immunes des hémopathies comme les neuropathies à anticorps anti‐MAG. / Fingolimod (FTY720) is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of haematological malignancies. Previous studies have indicated a role for FTY720 in inducing autophagy and caspase-independent cell death in cancer cells through incompletely characterized molecular mechanisms. Our study thus aims at a beeer understanding of the way of action of FTY720. In chronic lymphocytic leukaemia (CLL) cells, FTY720 induced cell death with typical features of apoptosis, including phosphatidylserine exposure and caspase-3 activation, and features of autophagy, including LC3 conversion, autophagolysosome formation and lysosomal cathepsins activation. However, neither caspase nor autophagy blockade prevented the cytotoxic effect of FTY720, suggesting another mechanism of cell death. Using electron and fluorescence microscopy, flow cytometry and biochemical analyses, we found that FTY720 treatment increased a fraction of annexin V-/7-AAD+ cells both in primary and transformed leukemic cells and induced morphological changes representative of necrosis, including oncosis, mitochondrial and plasma membrane alteration. FTY720 treatment resulted in increased plasma membrane permeability as shown by the extracellular translocation of the nuclear high mobility group box 1 (HMGB1) protein and by the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH). Interestingly, cell death induced by FTY720 was not prevented by pharmacological inhibition of RIPK1 and PP2A. In contrast, FTY720--‐induced necrosis was accompanied by an early relocation to the mitochondria of Dynamin Related Protein 1, DRP1. Importantly, FTY720 stimulation led to ma tior changes in the phosphorylation of serine residues associated with the mitochondrial fission activity of DRP1. Finally, siRNA--‐mediated knockdown of DRP1 significantly reduced necrotic cell death induced by FTY720. In this study, we thus demonstrate that in leukemic cells the cytotoxic effect of the immunosuppressive drug Fingolimod involves a DRP1--‐dependent regulated necrosis. These observations are important in line of the future development of Fingolimod as a new therapeutic agent in haematological malignancies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22336号 / 医博第4577号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 渡邉 大, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM