| 1. |
- Aboye, Teshome Leta, 1975-
(författare)
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Engineering of the Ultra-stable Cystine Knot Framework of Microproteins : Design, Chemical Synthesis and Structural Studies
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations. Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used. Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.
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| 2. |
- Al Haj, Mahmoud
(författare)
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Effects of Dehydration and Blockade of the Renin-Angiotensin System in the One-humped Camel (Camelus dromedarius)
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The one-humped or the dromedarian camel is a pseudo-ruminant mammal, well adapted to the hot and dry climates of the desert. Its ability to withstand torrid heat and extreme desiccation is of paramount importance to its survival. The studies presented in this thesis were designed to investigate and document the effect of dehydration in the presence or absence of angiotensin II (Ang II) AT1 receptor blocker (losartan) on blood constituents, electrolytes, hormones, neurotransmitters as well as liver and kidney enzymes in a subset of dehydrated camels and to compare them with hydrated camels. Additionally, we studied the response of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) and revealed for the first time the cardiac storage form of BNP in the camel heart. Dehydration induced significant increments in packed cell volume (PCV), white blood cells (WBC), gamma glutamyl-transferase (GGT), serum sodium, creatinine and urea levels, and a doubling in plasma cortisol and arginine vasopressin (AVP) levels. At the same time dehydration caused significant decrease in body weights, plasma insulin like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3), and a 50% decrement in ANP and BNP levels. Moreover, dehydration with and without losartan resulted in significant changes in stress hormones and anti-oxidants in plasma, liver and kidney homogenates. Losartan on one hand enhanced the effect of dehydration resulting in significant increases in sodium, creatinine and urea levels. In addition losartan raised the binding affinity of Ang II AT2 receptors in the small intestine with 8-fold and with 16-fold for liver AT1 receptors, indicating that Ang II AT1 and AT2 receptor binding sites were present in camel's small intestine while only AT1 receptor binding sites were found in the camel liver. One the other hand losartan resulted in significant decrease in body weights impaired the rise in anti-diuretic hormone and reduced aldosterone level. Finally, we showed that the proBNP is the storage form of BNP in the camel heart.
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| 3. |
- Andersson, Hanna, 1979-
(författare)
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Design and Synthesis of Angiotensin IV Peptidomimetics Targeting the Insulin-Regulated Aminopeptidase (IRAP)
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Peptidomimetics derived from the bioactive hexapeptide angiotensin IV (Ang IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6) have been designed and synthesized. These peptidomimetics are aimed at inhibiting the insulin-regulated amino peptidase (IRAP), also known as the AT4 receptor. This membrane-bound zinc-metallopeptidase is currently under investigation regarding its potential as a target for cognitive enhancers. The work presented herein was based on stepwise replacement of the amino acid residues in Ang IV by natural and unnatural amino acids, non-peptidic building blocks, and also on the introduction of conformational constraints. Initially, we focused on the introduction of secondary structure mimetics and backbone mimetics. The C-terminal tripeptide His-Pro-Phe was successfully replaced by a γ-turn mimetic scaffold, 2-(aminomethyl)phenylacetic acid (AMPA), which was coupled via an amide bond to the carboxyl terminus of Val-Tyr-Ile. Substitution of Val-Tyr-Ile, Val-Tyr, Tyr-Ile and Tyr, respectively, by 4-hydroxydiphenylmethane scaffolds comprising a 1,3,5-substituted benzene ring as a central moiety unfortunately rendered peptidomimetics that were less potent than Ang IV. The subsequent approach involved the introduction of conformational constraints into Val-Tyr-Ile-AMPA by replacing Val and Ile by amino acid residues appropriate for disulfide cyclization or ring-closing metathesis. Chemically diverse structures encompassing an N-terminal 13- or 14-membered macrocyclic tripeptide and a C-terminal non-peptidic moiety were developed. Tyr2 and AMPA were modified to acquire further knowledge about the structure-activity relationships and, in addition, to improve the metabolic stability and reduce the polarity. Several of the compounds displayed a high capacity to inhibit IRAP and exhibited Ki values in the low nanomolar range. Hence, the new compounds were more than ten times more potent than the parent peptide Ang IV. Enhanced selectivity over the closely related aminopeptidase N (AP-N) was achieved, as well as improved stability against proteolysis by metallopeptidases present in the assays. However, additional investigations are required to elucidate the bioactive conformation(s) of the relatively flexible N-terminal macrocycles. The compounds presented in this thesis have provided important information on structure-activity relationships regarding the interaction of Ang IV-related pseudopeptides and peptidomimetics with IRAP. The best compounds in the series constitute important starting points for further discovery of Ang IV peptidomimetics suitable as tools in the investigation of IRAP and other potential targets for Ang IV. The literature presents strong support for the hypothesis that drug-like IRAP inhibitors would serve as a new type of future cognitive enhancers with potential use in the treatment of cognitive disorders, e.g. Alzheimer’s disease.
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| 4. |
- Andersson, Helén, 1982-
(författare)
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Experimental Studies of Endocrine Disrupting Compounds in Vascular Cells and Tissues
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epidemiological evidence suggest that exposure to endocrine disrupting compounds (EDCs) is a risk factor for diseases that involves the cardiovascular system but we know little about the mechanisms whereby these compounds can cause injury in the vasculature. The aim of this thesis was to characterize the effects and mechanisms of some EDCs in vascular cells and highly vascularized tissues. Elevated exposure to environmental EDCs is associated with an increased risk for cardiovascular diseases. In vitro studies demonstrated that the environmental EDCs, 1-nitropyrene, PCB126 and bisphenol A, caused distinct changes in primary human endothelial cells. 1‑Nitropyrene induced cell stress and DNA damage, PCB126 caused changes that indicate endothelial dysfunction and vasoconstriction, and BPA induced changes that indicate angiogenesis and vasoconstriction. Further studies demonstrated that long-term exposure of rats to BPA induced changes in rat cardiac tissues in vivo similar to those observed in human endothelial cells in vitro. The type of cellular alterations that were demonstrated is known to play to play a role in cardiovascular disease in humans. These findings suggest that environmental EDCs can cause damage to the human endothelium that may contribute to the development of cardiovascular disease. The beneficial effects of the pharmaceutical EDC tamoxifen in breast cancer treatment are compromised by an increased risk for bleedings, hyperplasia, and cancer in the endometrium. Ex vivo studies identified the glandular and surface epithelia as potential target sites for tamoxifen adduct formation and tamoxifen-induced cell stress the human endometrium. No signs of tamoxifen-induced changes were detected in the blood vessels. The results suggest that bioactivation of tamoxifen and subsequent cell injury in endometrial epithelial cells may play a role for tamoxifen’s side effects in the endometrium. Taken together, this thesis provide evidence that may help understanding how exposure to EDCs can increase the risk for diseases in that involves the cardiovascular system.
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| 5. |
- Andersson, Martin, 1978-
(författare)
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Phase Phenomena in Polymer Networks : Empirical Studies on the Influence of Hydrophobicity, Charge Density and Crosslinks on Macroion-Induced Phase Transitions in Polyelectrolyte Gels
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The thesis concerns polyelectrolyte gels in contact with oppositely charged proteins and surfactant micelles, and includes of four papers (I-IV). In paper I confocal Raman spectroscopy was introduced as a method to trace micelles and investigate the structure of gel-surfactant complexes, in phase separated gel spheres. In paper II, the binding of surfactants to microspheres (~50-100 µm) was investigated by means of a micromanipulator-assisted microscopy method. The two surfactants were found to display qualitative difference respect to degree of swelling, surfactant distribution in the gels, and the difference is discussed in terms of absence/presence of hydrophobic attraction to the polyelectrolyte gel network. Kinetics of volume change in gels were analyzed. Aggregation numbers of micelles in polystyrenesulfonate (PSS) solutions, obtained from fluorescence quenching measurements, are presented. In paper III, phase behaviour, protein assembly and diffusion, was studied in PSS gel microspheres. Interpretation of results was aided by measurements of osmotic swelling of individual gel networks, and by combining the results with studies of protein diffusion in macroscopic (cm-sized) gel spheres. Complexes formed were further analyzed with small angle x-ray spectroscopy. In paper IV phase behaviour of mixed ionic/nonionic surfactant micelles is investigated in cm-sized gel spheres. The coexistence of three phases, the formation of dense shells in the bulk of the gels and other phenomena are described for the first time, and the results are presented along with discussion on the charge-density of spherical micelles and of network induced hysteresis effects in gels. The composition and microstructure of phases are investigated by confocal Raman spectroscopy and small-angle x-ray scattering respectively. The results are interpreted with aid of highly detailed theoretical model calculations.
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| 6. |
- Axelsson, Linda, 1977-
(författare)
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Development of HIV-1 Protease Inhibitors and Palladium-Catalyzed Synthesis of Aryl Ketones and N-Allylbenzamides
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of palladium-catalyzed reactions to introduce new carbon-carbon bonds is a fundamental synthetic strategy that has been widely embraced due to its high chemo- and regioselectivity and functional group tolerance. In this context, Pd(0)-catalyzed aminocarbonylations using Mo(CO)6 instead of toxic and gaseous CO and with allylamine as the nucleophile were investigated. The aminocarbonylated product dominated over the Mizoroki-Heck product, and (hetero)aryl iodides, bromides and chlorides gave N-allylbenzamides in good yields.In this thesis improvements to an existing protocol for the Pd(II)-catalyzed synthesis of aryl ketones from five benzoic acids and a variety of nitriles are also presented. Addition of TFA improved the yields and employing THF as solvent enabled the use of solid nitriles, and the aryl ketones were isolated in good yields.The pandemic of HIV infection is one of the greatest public health issues of our time and approximately 35.3 million people worldwide are living with HIV. There are currently many drugs on the market targeting various parts of the viral reproduction cycle, but the problems of resistance warrant the search for new drugs. HIV-1 protease makes the virus mature into infectious particles. In this thesis a new type of HIV-1 protease inhibitor (PI) is presented, based on two of the PIs on the market, atazanavir and indinavir, but it has a tertiary alcohol, as well as a two-carbon tether between the quaternary carbon and the hydrazide β-nitrogen. A total of 25 new inhibitors were designed, synthesized and biologically evaluated, the best compound had an EC50 value of 3 nM.Based on this series a project aimed at synthesizing macrocycles spanning the P1-P3 area was initiated. Macrocycles often tend to have an improved affinity and metabolic profile compared to their linear analogs. Introduction of a handle in the para position of the P1 benzyl group proved difficult, despite efforts to synthesize intermediates containing either a bromo-, hydroxy-, methoxy-, silyl-group protected hydroxy- or an alkyne-group. The lactone intermediate was abandoned in favor of an alternative synthetic route and initial studies were found to be promising. This new approach requires further investigation before the target macrocycles can be synthesized.
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| 7. |
- Barclay, Victoria K.H.
(författare)
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Development of LC-MS/MS Methods for the Analysis of Chiral and Achiral Pharmaceuticals and Metabolites in Aqueous Environmental Matrices
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the development of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the trace analysis of active pharmaceutical ingredients (APIs) and their metabolites in aqueous environmental matrices. The research was focused on the development of chiral LC-MS/MS methods for the analysis of fluoxetine and metoprolol, as well as their chiral metabolites in environmental water samples. A method was also developed for the achiral compounds, diazepam and nordiazepam. The LC-MS/MS methods were validated by the use of the isotope-labeled compounds. As these isotope-labeled compounds were not found in the wastewater samples, the validation could be assessed at trace level concentrations in the actual matrices in which the analytes were detected. The analytes were extracted from the water samples using solid phase extraction methods. Different types of solid phase extraction sorbents were evaluated. Fluoxetine and norfluoxetine were extracted through the use of a mixed mode polymeric based extraction sorbent. A hydrophilic and lipophilic balanced sorbent was employed for the simultaneous extraction of metoprolol and its metabolites, the base α-hydroxymetoprolol and the acidic metabolite deaminated metoprolol. Moreover, silica based C18 extraction discs were applied for the sample preparation of diazepam and nordiazepam. The chromatographic separations were conducted in reversed phase LC with MS compatible mobile phases. The enantiomers of fluoxetine and norfluoxetine were simultaneously separated using the chiral stationary phase (CSP), α1-acid glycoprotein (AGP). The Chiral AGP column was also applied for the separation of the enantiomers of deaminated metoprolol. For the simultaneous separation of the metoprolol enantiomers and the four stereoisomers of α-hydroxymetoprolol, the cellobiohydrolase (CBH) protein based CSP was used. An octadecyl silica based LC column was applied for the separation of diazepam and nordiazepam. The analytes were detected by the use of tandem quadrupole mass spectrometry operating in selective reactive monitoring mode. High resolution MS, employing a quadrupole time-of-flight (QqTOF) mass analyzer, was utilized for the identification of an unknown compound in wastewater samples. The APIs and their metabolites, as well as their respective enantiomers, were quantified in raw and treated wastewater from Uppsala, Sweden along with surface water from the River Fyris in Uppsala.
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| 8. |
- Baverel, Paul
(författare)
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Development and Evaluation of Nonparametric Mixed Effects Models
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A nonparametric population approach is now accessible to a more comprehensive network of modelers given its recent implementation into the popular NONMEM application, previously limited in scope by standard parametric approaches for the analysis of pharmacokinetic and pharmacodynamic data. The aim of this thesis was to assess the relative merits and downsides of nonparametric models in a nonlinear mixed effects framework in comparison with a set of parametric models developed in NONMEM based on real datasets and when applied to simple experimental settings, and to develop new diagnostic tools adapted to nonparametric models. Nonparametric models as implemented in NONMEM VI showed better overall simulation properties and predictive performance than standard parametric models, with significantly less bias and imprecision in outcomes of numerical predictive check (NPC) from 25 real data designs. This evaluation was carried on by a simulation study comparing the relative predictive performance of nonparametric and parametric models across three different validation procedures assessed by NPC. The usefulness of a nonparametric estimation step in diagnosing distributional assumption of parameters was then demonstrated through the development and the application of two bootstrapping techniques aiming to estimate imprecision of nonparametric parameter distributions. Finally, a novel covariate modeling approach intended for nonparametric models was developed with good statistical properties for identification of predictive covariates. In conclusion, by relaxing the classical normality assumption in the distribution of model parameters and given the set of diagnostic tools developed, the nonparametric approach in NONMEM constitutes an attractive alternative to the routinely used parametric approach and an improvement for efficient data analysis.
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| 9. |
- Bergstrand, Martin, 1977-
(författare)
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Application of Mixed-Effect Modeling to Improve Mechanistic Understanding and Predictability of Oral Absorption
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Several sophisticated techniques to study in vivo GI transit and regional absorption of pharmaceuticals are available and increasingly used. Examples of such methods are Magnetic Marker Monitoring (MMM) and local drug administration with remotely operated capsules. Another approach is the paracetamol and sulfapyridine double marker method which utilizes observed plasma concentrations of the two substances as markers for GI transit. Common for all of these methods is that they generate multiple types of observations e.g. tablet GI position, drug release and plasma concentrations of one or more substances. This thesis is based on the hypothesis that application of mechanistic nonlinear mixed-effect models could facilitate a better understanding of the interrelationship between such variables and result improved predictions of the processes involved in oral absorption. Mechanistic modeling approaches have been developed for application to data from MMM studies, paracetamol and sulfapyridine double marker studies and for linking in vitro and in vivo drug release. Models for integrating information about tablet GI transit, in vivo drug release and drug plasma concentrations measured in MMM studies was outlined and utilized to describe drug release and absorption properties along the GI tract for felodipine and the investigational drug AZD0837. A mechanistic link between in vitro and in vivo drug release was established by estimation of the mechanical stress in different regions of the GI tract in a unit equivalent to rotation speed in the in vitro experimental setup. The effect of atropine and erythromycin on gastric emptying and small intestinal transit was characterized with a semi-mechanistic model applied to double marker studies in fed and fasting dogs. The work with modeling of in vivo drug absorption has highlighted the need for, and led to, further development of mixed-effect modeling methodology with respect to model diagnostics and the handling of censored observations.
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| 10. |
- Björnsson, Marcus, 1973-
(författare)
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Pharmacometric Models in Anesthesia and Analgesia
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Modeling is a valuable tool in drug development, to support decision making, improving study design, and aid in regulatory approval and labeling. This thesis describes the development of pharmacometric models for drugs used in anesthesia and analgesia.Models describing the effects on anesthetic depth, measured by the bispectral index (BIS), for a commonly used anesthetic, propofol, and for a novel anesthetic, AZD3043, were developed. The propofol model consisted of two effect-site compartments, and could describe the effects of propofol when the rate of infusion is changed during treatment. AZD3043 had a high clearance and a low volume of distribution, leading to a short half-life. The distribution to the effect site was fast, and together with the short plasma half-life leading to a fast onset and offset of effects. It was also shown that BIS after AZD3043 treatment is related to the probability of unconsciousness similar to propofol.In analgesia studies dropout due to lack of efficacy is common. This dropout is not at random and needs to be taken into consideration in order to avoid bias. A model was developed describing the PK, pain intensity and dropout hazard for placebo, naproxen and a novel analgesic compound, naproxcinod, after removal of a wisdom tooth. The model provides an opportunity to describe the effects of other doses or formulations. Visual predictive checks created by simultaneous simulations of PI and dropout provided a good way of assessing the goodness of fit when there is informative dropout.The performance of non-linear mixed effects models in the presence of informative dropout, with and without including models that describe such informative dropout was investigated by simulations and re-estimations. When a dropout model was not included there was in general more bias. The bias increased with decreasing number of observations per subject, increasing placebo effect and increasing dropout rate. Bias was relatively unaffected by the number of subjects in the study. The bias had, in general, little effect on simulations of the underlying efficacy score, but a dropout model would still be needed in order to make realistic simulations.
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