Ziel der vorliegenden Promotionsarbeit war die Untersuchung von Sepsis-assoziierten Veränderungen des Arachidonsäure (AA)-Metabolismus und die Identifikation differentiell regulierter AA-Metabolite mit Prüfung ihres diagnostischen Potentials bei Patienten mit Sepsis unter Anwendung eines in-vitro Lipopolysaccharid (LPS) Vollblutaktivierungs-Modells. In Zellüberständen von nicht-aktiviertem und LPS-aktiviertem Heparinblut (25 Sepsis- Patienten, 15 Gesunde) wurden AA-Metabolite mittels Flüssigkeitschromatographie-Tandem- Massenspektrometrie analysiert. In einer unabhängigen Kohorte (10 Sepsis-Patienten, 3 Gesunde) wurden nach RNA-Isolation aus Zellmaterial zusätzlich Target-Gene des AAMetabolismus (Cyclooxygenase (COX)-2 und mikrosomale Prostaglandin-E-Synthase (mPGES)-1 mittels quantitativer Reverse Transkriptase-Polymerase Kettenreaktion (RT-PCR) untersucht. Es konnte eine differentielle Freisetzung von AA, AA-Analoga und der COX-assoziierten Metabolite Prostaglandin (PG) E2, 11-Hydroxyeicosatetraensäure (HETE) und Thromboxan (TX) B2 zwischen Patienten und gesunden Kontrollpersonen gezeigt werden. Sepsis-Patienten wiesen dabei gegenüber Gesunden eine deutlich reduzierte Freisetzung von AA und den COXassoziierten Metaboliten 11-HETE und PGE2 auf. Das Ausmaß der reduzierten Mediatorenfreisetzung bei Sepsis-Patienten war mit der Schwere der Erkrankungssymptomatik und dem klinischen Outcome assoziiert. Auf Genexpressionsebene zeigte sich eine reduzierte Induzierbarkeit der COX-2 mRNA-Expression bei Sepsis-Patienten gegenüber Gesunden, jedoch eine erhaltene Induzierbarkeit auf der Ebene der mPGES-1.:I Bibliographische Beschreibung………………………………………………………2 II Abkürzungen……………………………..……………………………………………4 III Einleitung 1. Epidemiologie und Definition der Sepsis……………………………………………..…6 2. Pathophysiologie der Sepsis……………………………………………………………..7 3. Stellenwert und Limitationen labordiagnostischer Marker bei Sepsis…………………..8 4. Eicosanoide und Sepsis…………………………………………………………………10 5. In-vitro LPS Vollblutaktivierungs-Modell für die Prüfung der Eicosanoidantwort auf Genexpressions- und Mediatorenebene……………..................12 6. Bestimmung von Eicosanoiden mittels LC-MS/MS........................................................16 7. Zielstellung der Arbeit……………………………………………………………….....18 IV Publikation…………………………………………………………….......................19 V Zusammenfassung der Arbeit………………………………………………………29 VI Literatur……………………………………………………………………………...32 VII Erklärung über die eigenständige Abfassung der Arbeit.………..…………….....35 VIII Lebenslauf………………………………………………………………………........36 IX Spezifizierung des wissenschaftlichen Beitrages zur Publikation...........................38 X Danksagung………………………………………………………………………......39

In forensic toxicology, opioids are frequently associated with drug abuse or drug-related death cases. An optimal method for use in the identification and quantification of opioids in a complex blood matrix is of paramount importance. Along with the ability to identify and quantitate opioids, this method should be accurate, sensitive, and selective. The application of sample pre-treatment and solid-phase extraction are common to purify and concentrate the target analytes before analyzing with liquid chromatography-tandem mass spectrometry. The purpose of this study was to compare the performance of two standard operating procedures, adopted by the Massachusetts State Police Crime Laboratory Toxicology and the Biomedical Forensic Sciences– Toxicology Laboratory at Boston University School of Medicine, for detecting opioids in blood. A total of eight drugs were analyzed: 6-monoacetylmorphine, codeine, fentanyl, hydrocodone, morphine, norhydrocodone, oxycodone, and oxymorphone. Comparison was performed using the parameters studied as part of method validation, including calibration model, bias, precision, carryover, interferences, ionization suppression/enhancement, and recovery. The results indicated that the method from Massachusetts State Police provided a better performance with between-run precision, interferences from matrix and other commonly encountered drugs, matrix effect at high concentration (250 ng/mL) and matrix recovery. Meanwhile, the method from Biomedical Forensic Sciences showed less bias, within-run precision, and matrix effect at low concentrations. Carryover and internal standard interference were comparable in both standard operating procedures. The calibration models were adjusted by altering the selection of regression model for improved quantification method performance. The volume of solvents, sample matrix, as well as time, were taken into consideration in accessing the overall performance of identification and quantitation. Both procedures were comparable yet the one from Massachusetts State Police was more beneficial in identifying the target analytes with greater sensitivity and selectivity and the one from Biomedical Forensic Sciences was more economical and efficient.

Drogpartiklar i utandningsluft är ett nytt forskningsområde med stora möjligheter att utvecklas för sjukdomsdiagnostik. En stor fördel är att provtagning kan utföras med större enkelhet och att proceduren kan upplevas som mindre integritetskränkande än till exempel blod- och urinprov. Det övergripande syftet med det här examensarbetet var att undersöka egenskaperna hos en apparat för provtagning vid namn SensAbues, som är framtagen för att samla in partiklar i utandningsluft för drogtestning. En del av syftet var att undersöka den variation som existerar i resultaten från tidigare forskning kring droganalys via utandningsprov. Examensarbetet utfördes utifrån litteraturstudier samt sex experiment. Utifrån teori och resultat ges rekommendationer på förändringar och förbättringar på provtagaren. Experiment I-III var relaterade till SensAbues egenskaper. De egenskaper som undersöktes var provtagarens flödesmotstånd (experiment I), hur partiklarna fördelar sig över provtagaren (experiment II) samt partikelstatistik gällande utandade partiklar samt provtagarens insamlingseffektivt (experiment III). I experiment IV-VI undersöktes anledningen till tidigare resultats variation, där det framförallt var tre hypoteser som låg som grund för experimentuppställningarna: 1) Att ökat flödesmotstånd skulle stimulera partikelbilandet och därmed öka koncentrationen av utandade drogpartiklar (experiment IV). 2) Att en speciell andningsmanöver skulle stimulera partikelbilandet och därmed öka koncentrationen av utandade drogpartiklar (experiment V). 3) Att partiklar från munhålan, som till exempel saliv, kontaminerar filtret vilket är anledningen till variationen i tidigare resultat (experiment VI). Resultaten från experiment I visar att det mesta flödesmotståndet sitter i provtagarens munstycke. I experiment II undersöktes spridningen av metadonpartiklar över provtagarens olika delar. Resultatet visar att det förekommer stor spridning och det verkar som att endast en liten del, cirka 3 % av metadonpartiklarna som andas ut fastnar i filtret, det vill säga den del som analyseras i laboratoriet. I experiment II går det inte att dra några slutsatser om provtagarens effektivitet, det vill säga hur stor andel av utandade metadonpartiklar som fångas upp av provtagaren, detta undersöks i experiment III. I experiment III undersöks provtagarens effektivitet och resultaten visar att provtagaren fångar upp cirka 99 % av utandade partiklar (alla utandade partiklar, ej drogpartiklar) Resultaten i experiment IV visade att ökat flödesmotstånd inte verkar ha någon betydelse för partikelkoncentrationen (generellt utandade partiklar, ej drogpartiklar). Resultaten i experiment V visade inte på att den speciella andningsmanövern bidrar till ökad koncentration av utandade drogpartiklar jämfört med vanlig provtagning. Det mest troliga skälet till tidigare forsknings varierande resultat är att partiklar från munhålan kontaminerar filtret, vilket visas i resultatet från experiment VI. Utifrån resultaten i experiment I-VI ges rekommendationer för utveckling av provtagaren. / Analysis of drug particles in exhaled air is a new research area with great potential for medical diagnoses. A major advantage is that sampling can be performed much easier and that the procedure may be perceived as less intrusive than, for example, blood or urine samples. The overall aim of this thesis work was to investigate some of the properties of a sampler named SensAbues, which is designed to collect particles in exhaled air. Another part of the purpose was to examine the variation in the results of previous research related to drugs through expiration. The work was based on literature studies and six separate experiments. Experiments I-III was related to SensAbues properties. The properties examined were the sampler's flow resistance (experiment I), how the particles are distributed over the sampler (experiment II) and particle statistics regarding exhaled particles and the sampler's collection effectiveness (experiment III). In experiments IV-VI previous research results variety was examined based on primarily three hypotheses: 1) Increased resistance would stimulate the amount of exhaled particles and thereby increase the concentration (experiment IV). 2) That a special breathing maneuver would stimulate the amount of exhaled particles and thereby increase the concentration (experiment V). 3) That particles from the oral cavity, such as saliva, would contaminate the filter and explain the variation in previous research results (experiment VI). The results from experiment I show that most of the flow resistance is located in the sampler's nozzle. In experiment II it shows that there is a great distribution of methadone particles over the sampler's different parts. Only a small part, about 3% of methadone particles exhaled trapped in the filter, which is the part that is analyzed in the laboratory. In experiment II, it is not possible to draw any conclusions about the sampler's efficiency, this examined in experiment III. In experiment III the sampler's effectiveness is examined and the results show that the sampler captures approximately 99% of exhaled particles (all exhaled particles, not drug particles). Results in experiment IV showed that the increased flow resistance does not seem to have any bearing on the particle concentration (generally exhaled particles, not drug particles). The results of experiment V did not show that the particular breathing maneuver contributes to increased concentration of exhaled drug particles compared with normal breathing. The most likely reasons for the variation in previous research are that particles from the mouth contaminate the filter, as shown in the results of experiment VI. Experiments I-VI leads to recommendations for further development of the sampler.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18209号 / 薬博第799号 / 新制||薬||237(附属図書館) / 31067 / 京都大学大学院薬学研究科創薬科学専攻 / (主査)教授 石濱 泰, 教授 松﨑 勝巳, 教授 加藤 博章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Kritiskt sjuka patienter med infektioner är en börda för sjukvården och 70 % av alla patienter på intensivvårdsavdelningar är ordinerade antibiotika. Antibiotika binder till proteiner i blodet, men enbart den icke-proteinbundna (fria) fraktionen kan diffundera över kapillära membran och binda till receptorer. Standardproteinbindningsgrad för olika antibiotika har utvecklats från studier på friska frivilliga och doseringen av läkemedlen är anpassade därefter. Den totala koncentrationen av antibiotika i patienters blod är vanligen representativ för den farmakologiska effekten. Dock kan vissa sjukdomar påverka proteinbindningsgraden vilket resulterar i en större eller mindre mängd fria antibiotika i blodcirkulationen. Det här kan i sin tur resultera i toxicitet eller otillräcklig effekt av läkemedlet. Syftet med det här projektet var att utveckla en analytisk metod för att bestämma den fria koncentrationen av Beta-Lactam antibiotikan cefotaxim, flukloxacillin, kloxacillin och piperacillin i plasma. En metod utvecklades med ultrafiltrering för extraktion av den fria fraktionen och högupplösande vätskekromatografi och tandem masspektrometri, UHPLC-MS/MS, för kvantifiering av analyterna. Metoden validerades delvis enligt den Europeiska Läkemedelsmyndighetens riktlinjer för bioanalytisk metodvalidering. / Infections in critically ill patients are a problem for the healthcare system and at any one time, 70 % of all intensive care unit (ICU) patients are treated with antibiotics. Antibiotics bind to proteins in the blood, but only unbound drug can diffuse over capillary membranes and bind to the targeted receptor. Standard protein binding percentages for antibiotics have been developed from studies on healthy volunteers and dosing regimens for patients are adapted accordingly. The determination of the total concentration of antibiotics in patients’ blood samples is, based on the standard percentages, ordinarily representative for the pharmacological effect of the antibiotic. However, certain conditions that are common in critically ill patients can alter protein binding percentages, resulting in a larger or smaller unbound fraction. This in turn can result in toxicity or therapeutic failure. The aim of this project was to develop an analytical method for the determination of the unbound concentration of the Beta-Lactam antibiotics cefotaxime, flucloxacillin, cloxacillin and piperacillin in plasma. A method was successfully developed using ultrafiltration for the extraction of unbound analytes and ultra high performance liquid chromatography tandem mass spectrometry, UHPLC-MS/MS, for their quantification. The method was partly validated according to the European Medicines Agency’s guidelines on bioanalytical method validation.

TRPC6 (transient receptor potential cation channels; canonical subfamily C, member 6) is widespread localized in mammalian tissues like kidney and lung and associated with progressive proteinuria and pathophysiological pulmonary alterations, e.g., reperfusion edema or lung fibrosis. However, the understanding of TRPC6 channelopathies is still at the beginning stages. Recently, by chemical diversification of (+)-larixol originating from Larix decidua resin traditionally used for inhalation, its methylcarbamate congener, named SH045, was obtained and identified in functional assays as a highly potent, subtype-selective inhibitor of TRPC6. To pave the way for use of SH045 in animal disease models, this study aimed at developing a capable bioanalytical method and to provide exploratory pharmacokinetic data for this promising derivative. According to international guidelines, a robust and selective LC-MS/MS method based on MRM detection in positive ion mode was established and validated for quantification of SH045 in mice plasma, whereby linearity and accuracy were demonstrated for the range of 2–1600 ng/mL. Applying this method, the plasma concentration time course of SH045 following single intraperitoneal administration (20 mg/kg body weight) revealed a short half-life of 1.3 h. However, the pharmacological profile of SH045 is promising, as five hours after administration, plasma levels still remained sufficiently higher than published low nanomolar IC50 values. Summarizing, the LC-MS/MS method and exploratory pharmacokinetic data provide essential prerequisites for experimental pharmacological TRPC6 modulation and translational treatment of TRPC6 channelopathies.

Abstract : The major objective of our study is to investigate DNA damage induced by soft X-rays (1.5 keV) and low-energy electrons (˂ 30 eV) using a novel irradiation system created by Prof. Sanche’s group. Thin films of double-stranded DNA are deposited on either glass and tantalum substrates and irradiated under standard temperature and pressure surrounded by a N[subscript 2] environment. Base release (cytosine, thymine, adenine and guanine) and base modifications (8-oxo-7,8-dihydro -2’-deoxyguanosine, 5-hydroxymethyl-2’-deoxyuridine, 5-formyl-2’-deoxyuridine, 5,6-dihydrothymidine and 5,6-dihydro-2’-deoxy uridine) are analyzed and quantified by LC-MS/MS. Our results reveal larger damage yields in the sample deposited on tantalum than those on glass. This can be explained by an enhancement of damage due to low-energy electrons, which are emitted from the metal substrate. From a comparison of the yield of products, base release is the major type of damage especially for purine bases, which are 3-fold greater than base modifications. A proposed pathway leading to base release involves the formation of a transient negative ion (TNI) followed by dissociative electron attachment (DEA) at the N-g lycosidic bond. On the other hand, base modification products consist of two major types of chemical modifications, which include thymine methyl oxidation products that likely arises from DEA from the methyl group of thymine, and 5,6-dihydropyrimidine that can involve the initial addition of electrons, H atoms, or hydride ions to the 5,6-pyrimidine double bond. / Résumé: L'objectif majeur de ce projet étude est d'étudier les lésions d'ADN induites par les rayons X mous (1,5 keV) et des électrons de faible énergie (˂ 30 eV) à partir d'un nouveau système d'irradiation créé par le groupe du Pr. Sanche. De minces couches d'ADN double brin sont déposées soit sur du verre ou sur les substrats de tantale. Celles-ci sont irradiées sous une température et pression environnante, mais dans une atmosphère de N[indice inférieur 2]. Les bases relâchées (cytosine, la thymine, l'adénine et la guanine) et les produits de modification de base (8-oxo-7,8-dihydro-2'-désoxyguanosine, 5-hydroxyméthyl-2'-désoxyuridine, 5-formyl-2'-désoxyuridine, 5,6-dihydrothymine et 5,6-dihydrouridine) sont analysés et quantifiés par LC-MS/MS. Nos résultats révèlent un plus grand rendement de dommages dans les échantillons déposés sur le tantale que celles sur le verre. Cette différence peut être expliquée par l’interaction des électrons de faible énergie qui sont photo émis des substrats métalliques. D'après les résultats obtenus, la libération de bases est un produit majeur en comparaison avec la modification de bases. Ceci provient, en particulier, surtout des purines qui libèrent la base a un niveau trois fois plus grand que la modification de la base. Une voie proposée, conduisant à la libération de base, implique la formation d'ions négatifs transitoires (TNI), suivie par l'attachement d'électrons dissociatifs (DEA) à la liaison N-glycosidique. En outre, les produits de modification de base sont composés en deux grands types de modifications chimiques. L’un des produits est l’oxydation du groupe méthyle de la thymine, qui probablement consiste de en d'hydrure (-H[indice supérieur -]) par l'intermédiaire de DEA. Alors que l’autre modification chimique est la formation de 5,6-dihydropyrimidine qui implique l'addition d'hydrure à la double liaison du 5,6-pyrimidine.

<p>Disease-specific compounds (biomarkers) are analyzed in clinical laboratories to assist with diagnosing diseases. This thesis describes development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS) based tests for diagnosing a diverse group of endocrine and metabolic diseases. The analytical methods used on-line and off-line sample extraction and analytical derivatization as means of enhancing the analytical sensitivity, specificity and clinical utility. All developed methods were extensively validated and reference intervals for the biomarker concentrations were established in blood samples of healthy adults and children. Advantages of the LC-MS/MS as an analytical technique include possibility of simultaneous measurement of multiple analytes and ability of confirming their identity. In this thesis we proposed and evaluated approaches for the assessment of the specificity of analysis in the methods that use tandem mass spectrometry detection. To enhance throughput of the LC-MS/MS tests for the biomarkers that have endogenous or exogenous isomers an approach was developed for quantitation of isomers from unresolved chromatographic peaks. Using methods developed in this thesis we performed a study of the steroidogenesis in ovarian follicles of healthy women and women with polycystic ovary syndrome (PCOS). Obtained data on the steroid concentrations and associations between the steroid metabolites in the pathway would be helpful for better understanding of the ovarian pathophysiology. Potential biomarkers of PCOS were identified in the thesis; further studies will be necessary to confirm their clinical utility.</p>

Les dernières décennies ont été marquées par une augmentation du nombre des cas de cancers, ce qui a subséquemment conduit à une augmentation dans la consommation des agents de chimiothérapie. La toxicité et le caractère cancérogène de ces molécules justifient l’intérêt crucial porté à leur égard. Quelques études ont fait l’objet de détection et de quantification des agents de chimiothérapie dans des matrices environnementales. Dans ce projet, une méthode utilisant la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) précédée d’une extraction sur phase solide (SPE) automatisée ou en ligne a été développée pour la détection et la quantification d’un groupe de six agents de chimiothérapie. Parmi ceux-ci figurent les plus utilisés au Québec (gemcitabine, méthotrexate, cyclophosphamide, ifosfamide, irinotécan, épirubicine) et présentant des propriétés physico-chimiques et des structures chimiques différentes. La méthode développée a été validée dans une matrice réelle représentant l’affluent d’une station d’épuration dans la région de Montréal. Deux des six composés cytotoxiques étudiés en l’occurrence (cyclophosphamide et méthotrexate) ont été détectés dans huit échantillons sur les neuf qui ont été recensés, essentiellement au niveau de l’affluent et l’effluent de quelques stations d’épuration de la région de Montréal. Les résultats des analyses effectuées sur les échantillons réels ont montré qu’il n’y avait pas de différence significative dans la concentration entre l’affluent et l’effluent, et donc que les systèmes d’épuration semblent inefficaces pour la dégradation de ces molécules. / The last few decades have been marked by an increase in the number of cancer cases, which subsequently led to an increase in the consumption of chemotherapeutic agents. The toxicity and the carcinogenicity of these molecules justify the increased interest. Few studies have been conducted to detect and quantify chemotherapeutic agents in environmental matrices. In this project, a method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) preceded by an online solid-phase extraction (SPE) has been developed for the detection and quantification of a group of six chemotherapeutic agents, which are among the most commonly used in Quebec (gemcitabine, methotrexate, cyclophosphamide, ifosfamide, irinotecan, epirubicin) and having different physico-chemical properties and different chemical structures. The developed method was validated in a real water matrix representing the influent of a sewage treatment plant in the Montreal area. Two of the six studied cytotoxic agents (cyclophosphamide and methotrexate) were detected in eight samples of the nine taken mainly at the influent and effluent of some treatment plants in the Montreal area. The results of the analysis of real samples showed that there was no significant difference in concentration between the influent and effluent. This also demonstrates the inadequacy of the current wastewater treatment approaches to remove those compounds.

Mass spectrometry is being increasingly used in biomarker research mainly due to its ability to achieve high selectivity coupled with high sensitivity. This dissertation focuses on quantitative mass spectrometric investigations of two protein biomarkers i.e. serum thymidine kinase 1 (TK1) and human osteopontin (OPN). First part of this research was focused on developing a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for measuring the activity of TK1 in serum by monitoring the conversion of a TK1 specific exogenous substrate, 3’-deoxy-3’-fluorothymidine (FLT), to its mono-phosphorylated form 3’-deoxy-3’-fluorothymidine monophosphate (FLT-MP). A method to quantify FLT-MP on LC-MS/MS was developed and validated. The method was linear over the range of 2.5-2000 ng/mL with a mean correlation coefficient of 0.9935. Using the developed method, serum TK1 activity was measured in serum from hepatocellular carcinoma (HCC) patients and age-matched controls under standardized conditions. A sub-population of the HCC patient samples showed an almost 20-fold enhanced TK1 activity compared to the controls. A method was developed and validated for quantifying human osteopontin from plasma using immunoaffinity isolations coupled with microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide ‘GDSVVYGLR’ which is unique to hOPN was identified and used as a signature peptide for this method. The method was validated over a range of 25-600 ng/mL. The performance of the method was compliant with USFDA validation guidance. In addition, a stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF’ were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. In the digestion variability studies, the use of extended SIL peptide as internal standard limited the total variability within ±30%. Alternatively, when SIL peptide was used as internal standard the variability ranged from -67.4% to +50.6 %. The applicability of the validated method was demonstrated by analyzing plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. More than 9-fold increase in the mean plasma hOPN concentration was seen in 30% of the breast cancer patient samples (n=10) in comparison to the healthy volunteer samples. In a proof of concept investigation, a stable isotope labeled signature peptide was evaluated as an internal standard to compensate for immunocapture variability during quantification of human osteopontin (hOPN) by immunoaffinity coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well. The immunocapture variability ranged from -80.9 % to +77.0 % when the IS was added after immunocapture and from -37.5% to +20.3% when the IS was added before immunocapture. The lower variability demonstrates the ability of SIL-IS peptide to compensate for variation during immunocapture.