Enzyme-catalyzed reactions are important to regulate steroidogenesis and nuclear receptor activation. The present investigation examines the role of steroid metabolism catalyzed by CYP7B1 for regulation of hormone receptor activation and the effects of vitamin D on enzymatic regulation of steroidogenesis. The study reports data indicating that CYP7B1 can regulate estrogenic signaling by converting estrogens into inactive or less active metabolites. Similar results were obtained for CYP7B1-mediated metabolism of some androgen receptor ligands, indicating that CYP7B1 can be involved also in the regulation of androgenic signaling. CYP7B1 substrates and metabolites were found to exert androgenic effects in a cell line-specific manner. Furthermore, cell line differences were observed in the expression pattern for androgen receptor comodulators. This thesis reports that 1α,25-dihydroxyvitamin D3 alters the gene expression and enzyme activity of CYP21A2 and CYP17A1 leading to suppressed production of aldosterone, dehydroepiandrosterone and androstenedione in adrenocortical cells. These are novel findings on vitamin D action. A mechanism is reported for the vitamin D-mediated regulation of the CYP21A2 gene. Data indicate that vitamin D receptor interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF) are key comodulators in this novel vitamin D receptor (VDR)-mediated mechanism. Furthermore, the results indicate that altered expression levels of VDIR and WSTF can shift the suppressing effect of vitamin D to a stimulatory effect. Also, epigenetic components were found to be involved in the effects of vitamin D on CYP21A2 transcriptional rate. In addition, a functional vitamin D response element was identified in the CYP21A2 promoter. This study also reports that 1α,25-dihydroxyvitamin D3 affects sex hormone production in a tissue-specific way. Gene expression and enzyme activity of aromatase were found to be downregulated in cells derived from breast, but not in cells derived from prostate and adrenal cortex. The production of estradiol and dihydrotestosterone was altered in a tissue-selective manner following vitamin D treatment. These findings are of importance for the discussion on vitamin D as a potential anti-breast cancer agent.

The introduction of artemisinin-based combination therapy (ACT) substantially reduced malaria-related mortality and morbidity during the past decade. Despite the widespread use of ACT, there is still a considerable knowledge gap with regards to safety, efficacy and pharmacokinetic properties of these drugs, particularly in vulnerable populations like children and pregnant women. In addition, there is growing evidence of widespread artemisinin-resistance across the Greater Mekong Subregion. Expedited delivery of novel antimalarial drugs with different mechanisms of action to the clinical setting is still far off; therefore, it is crucial to improve the use of existing antimalarial drugs for optimal outcome in order to prolong their therapeutic life span. This thesis focuses on utilizing pharmacometric tools to support this effort for malaria prevention and treatment. An extensive simulation framework was used to explore alternative malaria chemopreventive dosing regimens of a commonly used ACT, dihydroartemisinin-piperaquine. Different monthly and weekly dosing regimens were evaluated and this allowed an understanding of the interplay between adherence, loading dose and malaria incidence. A weekly dosing regimen substantially improved the prevention effect and was less impacted by poor adherence. This is also expected to reduce selection pressure for development of resistance to piperaquine. Population pharmacokinetics-pharmacodynamic models were developed for artesunate and the active metabolite dihydroartemisinin, effect on parasite clearance, in patients with artemisinin-resistant and -sensitive malaria infections in Southeast Asia. The modeling identified an association between parasite density and drug bioavailability. It predicted the presence of high levels of artemisinin resistant infection among patients in Cambodia and its spread into Myanmar. A nomogram to identify patients with artemisinin resistant infections was developed. Furthermore, the model was used to demonstrate the need for extended treatment duration to treat patients with artemisinin resistant infections. A population pharmacokinetic model developed from data on pregnant women in East Africa allowed further understanding of artemether-lumefantrine exposure in pregnant populations. It also suggested that the lumefantrine exposure in this population is not compromised. In summary, the results presented in this thesis demonstrate the value of pharmacometric approaches for improving antimalarial drug treatment and prevention. This ultimately contributes to overcoming the prevailing challenges to malaria control.

Amyloid PET imaging with [11C]PIB enabled detection of Aβ for the first time in vivo. However, [11C]PIB is a small molecule that binds only the insoluble Aβ plaque. Rather, the soluble Aβ aggregates are considered the cause of Alzheimer’s disease (AD). As such, a more sensitive and specific PET tracer is needed for tracking longitudinal AD pathology. Soluble Aβ aggregates likely interact with the metabotropic glutamate receptor 5 (mGluR5) to cause neurotoxic effects. However, with [11C]ABP688 PET we were unable to detect aberrant mGluR5 binding in AD mouse models, although we find elevated mGluR5 protein levels with immunoblotting. Antibodies are highly specific large molecules that can bind specifically to soluble Aβ aggregates, thus they can be a good marker for AD pathology. Unfortunately, due to their large size they cannot cross the blood-brain barrier (BBB). However, it is possible to shuttle antibodies into the brain by taking advantage of endogenous transporter systems on the BBB. By creating bispecific antibodies binding both to soluble Aβ aggregates and to the transferrin receptor (BBB target), we successfully transported the antibody into the brain and could visually detect soluble Aβ aggregates with PET. Recombinant expression further improved and optimized antibody design, creating smaller bispecific antibody-based constructs that had better pharmacokinetic properties allowing for earlier PET scanning (1 day instead of 3), and more sensitive signal. Lastly, using TCO-tetrazine click chemistry, we indirectly labeled our antibodies with fluorine-18, and could successfully perform PET already 11 h post-injection with a fluorine-18 labeled antibody.

Solvents play a crucial role in the Active Pharmaceutical Ingredient (API) manufacturing and are used in large quantities. Most of the industries incinerate the waste solvents or send it to waste management companies for destruction to avoid waste handling and cross-contamination. It is not a cost effective method and also hazardous to the environment. This study has been performed at AstraZeneca’s API manufacturing plant at Sodertalje, Sweden. In order to find a solution, a solvent recovery system is modeled and simulated using ASPEN plus and ASPEN batch modeler. The waste streams were selected based on the quantity and cost of the solvents present in them. The solvent mixture in the first waste stream was toluene-methanol in which toluene was the key-solvent whereas in the second waste stream, isooctane-ethyl acetate was the solvent mixture in which isooctane was the key-solvent. The solvents in the waste stream were making an azeotrope and hence it was difficult to separate them using conventional distillation techniques. Liquid-Liquid Extraction with water as a solvent followed by batch distillation was used for the first waste stream and Pressure Swing Distillation was used for the second waste stream. The design was optimized based on cost analysis and was successful to deliver 96.1% toluene recovery with 99.5% purity and 83.6% isooctane recovery with 99% purity. The purity of the solvents was decided based on the quality conventions used at AstraZeneca so that it can be recovered and recycled in the same system. The results were favorable with a benefit of €335,000 per year and preventing nearly one ton per year carbon dioxide emissions to the environment. A theoretical study for the recovery system of toluene-methanol mixture was performed. The proposed design was an integration of pervaporation to the batch distillation. A blend of polyurethane / poly(dimethylsiloxane) (PU / PDMS) membrane was selected for the separation of methanol and toluene mixture. The results of preliminary calculations show 91.4% toluene recovery and 72% methanol recovery with desired purity.

This work investigates the fluid flow in a cylindrical millilitre vial stirred by a magnetic stirred bar using Computational Fluid Dynamic (CFD). Stirred millilitre vials are used to study nucleation phenomena and crystallization as an outline of literature study of nucleation and crystallization phenomena and the role of stirring in this process. The baffle free vial was meshed with around 500,000 cells. To simulate the stirring a rotary frame and moving walls were used. Stirring speeds were between 100 and 1000 rpm where considered, correspondently to a stirrer Reynolds number between 260 and 2600. For stirring speeds bellow 500 rpm, simulations by both the both laminar flow model and the k-ε model where run, while above 500 rpm only k-ε was used. Results of the two models were very similar indicative the adequacy of k-ε to simulate the flow even at low Reynolds. The flow shows expected circulation pattern with upwards pumping close to side walls and downwards pumping in the centre of cylindrical vial. At 1000 rpm circulation patterns expands up to the top of the vial while at 300 rpm and lower the upper half of the vial is poorly mixed. The average turbulent energy of the flow is very low comparing with the squared stirrer tip speed and the power number decrees with Reynolds number, indicating that the flow is not fully turbulent.

För att motverka att innehållet i en behållare läcker ut krävs det att förslutningen är tät. För att förhindra läckage används någon form av tätning. På kapsyler i plast integreras ofta tätande element direkt i kapsylen i form av läppar eller kanter som pressar mot flaskan.Studien berör den inbyggda tätningen på en ”push-on”-kapsyl i Polypropencopolymer. Kapsylen tillverkas av Emballator Växjöplast AB och kallas DLM (dropless medicine) då den i de flesta fall monteras på glas- eller plastflaska som innehåller medicin. Kapsylens underdel pressas på flaskan permanent och den tvådelade kapsylen fungerar genom att en separat överdel skruvas på underdelen.Studien innehåller produktutvecklingssteg för att ta fram en uppdaterad konstruktion som ska klara av de problem som flaskans profil medför. Studien är upplagd som en DRM-studie och försöker svara på vad som påverkar tätningsförmågan hos en kapsyl och hur de tätande elementen kan konstrueras för att neutralisera flaskans form. Problemen uppstår främst när kapsylen är monterad på glasflaskan och roteras, då flaskan i vissa fall har en något oval profil. Det är när flaskan roteras som tätningen inte anpassar sig tillräckligt fort för att bli tät igen. Genom arbetets gång har man insett att i stället för att forma tätningen så att den följer behållarens form fortare, och riskera plastisk deformation på grund av relaxation hos materialet, väljer man att placera ytterligare en tätning på toppen. Ytan på toppen av flaskan utnyttjas, eftersom det är en ledig yta på både kapsyl och flaska,för att tätningarna ska komplettera varandra. Det görs för att kunna ha kvar den nuvarande tätningen och endast addera en topptätning som är mindre känslig för ovalitet hos flaskan. Studiens resultat bör ses som underlag för framtagning av prototyper och testning, då det inte går att garantera konstruktionens duglighet utan fortsatt testning / To prevent the contents of a container from leakage the closure of the container mustbe sealed. Some kind of seal is used to prevent leakage. The sealing part of plastic caps is usually integrated in the design of the cap in form of lips or edges that areputting pressure against the bottle.The study refers to the integrated seal on a “push-on” cap that is made of Polypropylene copolymer. The cap is manufactured by Emballator Växjöplast AB and is called DLM (dropless medicine) since it is mounted to a glass or plastic bottle containing medicine in most cases. The bottom part of the cap is pressed onto the bottle permanently and the two-part design works through a separate top part that is screwed onto the bottom part.The study contains product development steps to create an updated design that manages the problems caused by the profile of the bottle. The study is laid out as a DRM-study that is trying to answer what is affecting a cap´s ability to seal and how the sealing elements can be designed to neutralize the shape of the bottle. The problems are appearing mostly when the cap is mounted to the glass bottle and is rotated, since the bottle in some cases has an oval profile. It is when the bottle is rotated that the seal is not adapting fast enough to become sealed again.Through the process of the study, it has been realized that it is better to place a second seal on top of the bottle instead of designing a seal that follows the shape of the container. This is done to reduce the risk of plastic deformation due to relaxation of the material. The surface at the top of the bottle is utilized, since it is an unoccupied space at both the cap and the bottle, to have the seals complete each other. This is done to be able to keep the current seal and just add a top-seal that is less sensitive for ovality of the bottle. The results of the study should be seen as a basis for production of prototypes and testing since it not possible to guarantee the capability of the design without continued testing.

Paracetamol är ett vanligt använt läkemedel med analgesisk och antipyretisk effekt. Läkemedlet finns tillgängligt i ett flertal beredningsformer och doseringsstyrkor för användning både receptfritt och i sjukhusvården. Syftet med detta projekt var att undersöka påverkan av alternativ, off-label, dosering av paracetamol i pediatrisk vård, med hjälp av fysiologiskt baserad farmakokinetisk (PBPK) modellering. Modellen utvecklades först för en vuxen population genom integrering av in vitro, in vivo och in silico data för paracetamol. Efter detta extrapolerades concentrationskurvor till en pediatrisk population med hjälp av ontogeni-information. Modellen validerades i både vuxna och barn, och var tillförlitlig för både peroral och intravenös dosering. Efter valideringen utfördes simuleringar för nio olika åldersgrupper baserat på rekommenderade doseringsprotokoll i Sverige. Simuleringarna visade att perorala tablettdoseringen var jämförbar med formulering i lösningsform, med snarlika maximumkoncentrationer och area-under-kurvan (AUC) för exponering. Hastigheten av magtömning influerade maximumkoncentrationer men inte AUC. Ytterligare testades modellens förmåga att prediktera plasmakoncentrationer i blodet efter överdosering med paracetamol. Dessa prediktioner fungerade bättre när läkemedelsmetaboliserande enzymer lämnades oförändrade, eller ökade något i aktivitet. Slutligen, den utvecklade PBPK-modellen kan användas för att säkert undersöka olika doseringsprotokoll och för design av pediatriska kliniska studier. / Paracetamol, a widely used analgesic and antipyretic drug, can be found in various formulations and doses for both home and hospital use. The aim of this study was to investigate the impact of off-label dosing of paracetamol in pediatric clinical practice using physiologically based pharmacokinetic (PBPK) modeling. The model was initially developed for adults by integrating relevant in vitro, in vivo and in silico data of paracetamol, after which the model was extrapolated for pediatrics by adding ontogeny information. The model was successfully validated in both adult and pediatric populations, and it showed accuracy for both oral and intravenous administration routes. After validation, simulations were conducted across nine different age groups following the recommended doses in Sweden. These simulations showed that tablet dose is comparable to solution dosing, resulting in nearly identical maximum concentrations and area under the curve (AUC) values. Furthermore, it was observed that gastric emptying time, which reflects the fed state of individuals, significantly influences the maximum concentration, with longer gastric emptying times resulting in lower and delayed peak concentrations. However, the gastric emptying time had no effect on the AUC values. Lastly, the model’s performance on overdose data was evaluated, and it turned out that it performs better when liver enzymes were not affected, or they were only slightly elevated. Finally, the developed PBPK model can be further used for safe and effective way of exploring dose banding and designing clinical trials in pediatrics.

<p>This thesis presents novel approaches for structural investigation of glucuronides using chemical derivatization in combination with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSⁿ).</p><p>Today, LC-ESI-MSⁿ is the dominant technique for quantitative as well as qualitative analyses of metabolites, due to its high sensitivity and selectivity. However, for compounds without an easily ionizable group, e.g., steroids, the sensitivity is limited. In the work presented in this thesis, a derivatization procedure forming a basic oxime significantly increased the detection sensitivity for the altrenogest glucuronide. </p><p>Furthermore, in structural evaluations of glucuronides, the limitation of LC-MSⁿ becomes evident due to the initial neutral loss of 176 u, i.e. monodehydrated glucuronic acid, which often makes it impossible to elucidate the structures of the conjugates. To solve this problem, the main part of the work described in this thesis was devoted to chemical derivatization as a means of facilitating the determination of the site of conjugation. </p><p>For the first time, the isomeric estriol glucuronides were evaluated using a combination of three reagents 2-chloro-1-methylpyridinium iodide (CMPI), 1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide (EDC), and 2-picolylamine (PA). Interestingly, the derivatization gave a selective fragmentation pattern leading to differentiation of the isomers. </p><p>Another derivatization reagent, 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC), was also tested for the first time in structural investigations. The isomeric glucuronides of morphine, formoterol, and hydroxypropranolol were evaluated. They can all be conjugated in aliphatic as well as aromatic positions. DMISC was proven to be useful in two ways. Firstly, the morphine and formoterol glucuronides that contained a free phenol could be differentiated from those that were conjugated in the aromatic position based on different reactivity. Secondly, for the aromatic <i>O</i>-glucuronide of 4’-hydroxypropranolol, DMISC was proven to react with the amine. This product gave a different fragmentation pattern compared to the corresponding derivative of the aliphatic glucuronide. </p>

Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences. This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities. The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies. The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.