Currently L-DOPA is the drug most commonly used for the treatment of Parkinson’s disease (PD). However, the long-term use of L-DOPA is associated with the development of motor fluctuations and dyskinesias. Treatment mainly addresses the dopaminergic features of the disease and leaves its progressive course unaffected. An optimal treatment would be a combination of both motor and non-motor symptom relief with neuroprotective properties. Two drug targets have attracted the attention for PD treatment, namely monoamine oxidase B (MAOB) and adenosine A2A receptors. MAO-B inhibitors enhance the elevation of dopamine levels after L-DOPA treatment, improve motor functions and may also possess neuroprotective properties. The antagonistic interaction between A2A and dopamine receptors in the striatopallidal pathway, which modulates motor behaviour, has also become a potential strategy for PD treatment. Blockade of the A2A receptor exerts both anti-symptomatic and neuroprotective activities and offer benefit for motor symptoms and motor complications. This thesis seeks to synthesize novel drug treatments for PD by exploring both MAO-B inhibitors and adenosine A2A receptor antagonists and to assess the prospects for drug modification to increase activity. MAO-B inhibitors - Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5- sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC50 values in the nanomolar range. The most potent inhibitor, 5- (benzylsulfanyl)phthalimide, exhibits an IC50 value of 0.0045 μM for the inhibition of MAO-B with a 427–fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy. It has recently been reported that nitrile containing compounds frequently act as potent MAO-B inhibitors. In an attempt to identify additional potent and selective inhibitors of MAO-B and to contribute to the known structure-activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC50 values in the nanomolar range. In general, the sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding sulfanylbenzonitrile homologues. Among the compounds evaluated, 4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC50 = 0.025 μM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson’s disease. Adenosine A2A receptor antagonism - Most adenosine A2A receptor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substituted heterocyclic compounds. In an attempt to discover high affinity A2A receptor antagonists for PD and to further explore the structure-activity relationships of A2A antagonism by the xanthine class of compounds, this study examines the A2A antagonistic properties of series of (E)-8-styrylxanthine, 8-(phenoxymethyl)xanthine and 8-(3- phenylpropyl)xanthine derivatives. The results document that among these series, the (E)-8- styrylxanthines are the most potent antagonists with the most potent homologue, (E)-1,3-dietyl- 7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a Ki value of 11.9 nM. This compound was also effective in reversing haloperidol-induced catalepsy in rats. The importance of substitution at C8 with the styryl moiety was demonstrated by the finding that none of the 8- (phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines exhibited high binding affinities for the A2A receptor. It was also concluded that (E)-8-styrylxanthines are potent A2A antagonists with particularly the 1,3-dietyl-7-methylxanthine substitution pattern being most appropriate for high affinity binding. Conclusion - The results of these studies have established that all of the sulfanylphthalimides, sulfanylphthalonitriles and sulfanylbenzonitriles examined display significant MAO-B inhibitory properties in vitro with IC50 values in the low μM to nM range. Good A2A receptor affinity was demonstrated by the xanthines containing a styryl moiety, while the phenoxymethyl and phenylpropyl xanthines exhibited poor activity. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Currently L-DOPA is the drug most commonly used for the treatment of Parkinson’s disease (PD). However, the long-term use of L-DOPA is associated with the development of motor fluctuations and dyskinesias. Treatment mainly addresses the dopaminergic features of the disease and leaves its progressive course unaffected. An optimal treatment would be a combination of both motor and non-motor symptom relief with neuroprotective properties. Two drug targets have attracted the attention for PD treatment, namely monoamine oxidase B (MAOB) and adenosine A2A receptors. MAO-B inhibitors enhance the elevation of dopamine levels after L-DOPA treatment, improve motor functions and may also possess neuroprotective properties. The antagonistic interaction between A2A and dopamine receptors in the striatopallidal pathway, which modulates motor behaviour, has also become a potential strategy for PD treatment. Blockade of the A2A receptor exerts both anti-symptomatic and neuroprotective activities and offer benefit for motor symptoms and motor complications. This thesis seeks to synthesize novel drug treatments for PD by exploring both MAO-B inhibitors and adenosine A2A receptor antagonists and to assess the prospects for drug modification to increase activity. MAO-B inhibitors - Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5- sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC50 values in the nanomolar range. The most potent inhibitor, 5- (benzylsulfanyl)phthalimide, exhibits an IC50 value of 0.0045 μM for the inhibition of MAO-B with a 427–fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy. It has recently been reported that nitrile containing compounds frequently act as potent MAO-B inhibitors. In an attempt to identify additional potent and selective inhibitors of MAO-B and to contribute to the known structure-activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC50 values in the nanomolar range. In general, the sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding sulfanylbenzonitrile homologues. Among the compounds evaluated, 4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC50 = 0.025 μM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson’s disease. Adenosine A2A receptor antagonism - Most adenosine A2A receptor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substituted heterocyclic compounds. In an attempt to discover high affinity A2A receptor antagonists for PD and to further explore the structure-activity relationships of A2A antagonism by the xanthine class of compounds, this study examines the A2A antagonistic properties of series of (E)-8-styrylxanthine, 8-(phenoxymethyl)xanthine and 8-(3- phenylpropyl)xanthine derivatives. The results document that among these series, the (E)-8- styrylxanthines are the most potent antagonists with the most potent homologue, (E)-1,3-dietyl- 7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a Ki value of 11.9 nM. This compound was also effective in reversing haloperidol-induced catalepsy in rats. The importance of substitution at C8 with the styryl moiety was demonstrated by the finding that none of the 8- (phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines exhibited high binding affinities for the A2A receptor. It was also concluded that (E)-8-styrylxanthines are potent A2A antagonists with particularly the 1,3-dietyl-7-methylxanthine substitution pattern being most appropriate for high affinity binding. Conclusion - The results of these studies have established that all of the sulfanylphthalimides, sulfanylphthalonitriles and sulfanylbenzonitriles examined display significant MAO-B inhibitory properties in vitro with IC50 values in the low μM to nM range. Good A2A receptor affinity was demonstrated by the xanthines containing a styryl moiety, while the phenoxymethyl and phenylpropyl xanthines exhibited poor activity. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Background: Vitamin-K antagonists (VKA) are a class of oral anticoagulant medications used to prevent blood clots. The anticoagulant intensity of VKAs is measured with a blood test known as the International Normalized Ratio (INR). Traditionally, international guidelines have recommended INR tests every 4 weeks for all patients. However, adherence to these guidelines has never been investigated in real world settings. The objectives of this study were to describe adherence to INR testing in Saskatchewan among patients receiving VKA medications, and to identify predictors of optimal adherence. Methods: This was a retrospective cohort study of VKA users in Saskatchewan captured in the administrative data between 2003 and 2010. Physician claims for anticoagulation monitoring were used as a proxy for INR testing. Adherence to INR testing was measured using the Continuous, Multiple-Interval Measure of Medication Gaps (CMG). Individuals were considered adherent if adherence by the CMG was at least 80%. Hierarchical (random effects) logistic regression models were developed to identify important predictors of optimal INR monitoring. Individual physician identification was considered a random effect in these models. The dependent variable was the achievement of optimal adherence, defined as ≥80% adherence to the 4-week test interval. Results: Among 17,388 VKA users, 42% resided in rural areas and virtually all (99%) were monitored by a general practitioner. During a median follow-up of 514 days, 50% of patients exhibited at least 74% adherence to INR testing if a 4-week interval was used as the reference standard. However, the estimated median adherence increased dramatically to 98% when the benchmark for optimal testing was lengthened to every 12 weeks. The most prominent risk factors for poor adherence to INR monitoring appeared to be rural residence (rural vs. urban OR 0.55, 95% CI 0.47-0.64 among subjects age ≥75 years) and duration of VKA therapy (≥731 vs. 35-90 days OR 0.04, 95% CI 0.03-0.05). Discussion: Adherence to INR testing appeared to be acceptable for most VKA-treated patients in Saskatchewan. However, this data indicated that adherence might be more problematic in the subgroup of rural residents. Possible explanations include reduced access to testing facilities or the shortage of physicians in rural areas. Further research is required to understand if poor access is the underlying cause of non-adherence to INR testing in the rural population.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Stuttgart, Univ., Diss., 2003.

Regensburg, Univ., Diss., 2002.

INTRODUCTION: For many years, stress and anxiety disorders have taken a heavy toll on the American population. Affecting approximately 40 million individuals over the age of 18, the discovery of treatment options is very important. Ever since the 1950s, a wide variety of compounds have been discovered and proven to have antagonistic properties for such disorders. For the last three decades, however, researchers have focused on a specific peptide that was discovered in 1981 by Dr. Wylie Vale and his colleagues at the Salk Institute in San Diego, California, corticotropin releasing factor (CRF). CRF is a 41 amino acid peptide that has been shown to play a very important role in an organism's endocrine response to stress through the activation of the hypothalamic–pituitary–adrenal (HPA) axis. Ever since its discovery, the identification and characterization of the CRF receptors and family members have allowed for the development of novel peptide and non–peptide antagonists. Unfortunately, these compounds have been unsuccessful in the progression to later stage clinical trials that could lead to promising therapeutics. There are two receptor subtypes for this family of peptides known as CRFR1 and CRFR2. While there have been many compounds identified that can block CRFR1, currently, there are no known selective non–peptide antagonists for the CRFR2 subtype. As the two receptor subtypes share 70% sequence identity, close observation of the functional properties of antagonist ligands for CRFR1 may lead to the development of such ligands for CRFR2. METHODS: In our current study, we focused on two residues in transmembrane domains (TMD) 3 (His199) and 5 (Met276) of CRFR1 that have proven to be important for the function of the highly selective small molecule antagonist antalarmin. In order to further prove the importance of these sites, we have mutated the two corresponding amino acids in CRFR2β to those of CRFR1: V215H in TMD 3 and V292M in TMD 5. In addition, we mutated a third amino acid residue, M293I, in order to avoid the positioning of two adjacent methionine amino acids. With this mutant construct, CRE–luciferase and cyclic AMP radioimmunoassay methodologies were used to observe the function of antalarmin on CRFR1, the mutant and wild type CRFR2β. The accumulation of cAMP was measured intracellularly following stimulation by the CRF receptor peptide agonists sauvagine, isolated from frog, and urocortin 1, isolated from rat. RESULTS: For the initial CRE–luciferase functional assay, we used the CRF receptor agonist sauvagine on our mutant CRFR2β to indirectly measure the accumulation of intracellular cAMP through the enzyme luciferase. In the presence or absence of the antagonist antalarmin, there were no significant changes on the function of the mutant CRFR2β. On the other hand, when directly measuring the accumulation of intracellular cAMP via radioimmunoassay, antalarmin successfully showed a functional inhibitory effect on the mutant CRFR2β receptor. As expected, Ucn1 stimulation of CRFR1 in the presence of antalarmin indicated a decrease in the EC50 for the peptide agonist, and thus an inhibitory effect by antalarmin. Compared to CRFR1, we observed a similar effect for Ucn1 stimulation of the mutant CRFR2β receptor in the presence of antalarmin. While the presence or absence of antalarmin did not have a significant inhibitory effect on the wild type CRFR2β, it can be concluded that the mutant CRFR2β receptor possessed similar properties to the CRFR1 receptor with respect to antalarmin antagonist activity. CONCLUSION: In our study, we were able to further support the importance of the two amino acid residues in TMD 3 and 5 of CRFR1 for the function of small molecule antagonists. In addition, we were able to show that antalarmin, a small molecule antagonist known to be highly selective for CRFR1, can have a functional inhibitory effect on the mutant CRFR2β. The progressive study of these discrete differences between the two CRF receptor subtypes may enable the discovery of novel selective non–peptide CRFR2β receptor antagonists.

Syftet med studien är att jämföra framställningen av skräck i originalversionen och nyinspelningen av skräckfilmen IT. Med en semiotisk innehållsanalys analyserar vi framställningen av antagonisten och effekter i valda scener från båda filmerna. Materialet som studeras är fem nyckelscener från respektive version. Studiens syfte är att jämföra hur framställningen av skräck har förändrats i filmerna samt få en djupare förståelse hur synen på skräck i dagens samhälle kan utgöra en faktor i förändringen. För att komma åt denna struktur används teorier angående ideologiteori, representationsteori och semiotisk teori. Metoder vi använder är semiotik som metod och analyser av effekterna i film. Med en semiotisk jämförelse av de valda scenerna och framställningen av antagonisten kan vi besvara studiens frågeställningar. Analysen har genomförts med en semiotisk denotation av handlingen följt av en djupare jämförande analys. Studiens resultat visar att det finns både likheter och även stora skillnader mellan de valda scenerna. Eftersom det är en sådan stor skillnad på miljön och ljussättningen mellan alla nyckelscener så får man en känsla av att det krävs mer för att bli rädd idag än vad det var på 1990-talet när allt var soligt och fina miljöer. Den äldre versionens monster skulle kunna associeras med stereotypiska monster samtidigt som den senare versionens monster var mer främmande och nyskapade. Antagonisten skiljer sig drastiskt från versionerna och visar tydligt att det krävs mer effekter och övernaturliga fenomen för att bli räddare idag.

Post-ischemia depression (PID) is a condition that affects approximately 30% of survivors from stroke or cardiac arrest and has an important impact on patients’ quality of life. Previous studies support important roles of the endocannabinoid (eCB) system in depression and brain ischemia. This study attempts to link all three variables together by investigating the role and mechanism of eCB signaling in the development of PID. A global ischemia + hypotension model was used to induce a PID phenotype in CD1 mice. Three ischemic time frames were tested, and even though all three could induce significant cell death in the CA1 region of the hippocampus, only the 15-minute time point led to an increased immobility time on the forced swimming test (FST). The main goal of this study was to investigate the effect of a cannabinoid type-I receptor (CB1R) antagonist/inverse agonist, AM281, on the development of two depressive symptoms: anhedonia, measured with the sucrose preference test (SPT), and behavioral despair, measured with the FST. AM281 administration was able to significantly reduce the symptoms of anhedonia and behavioural despair. Subsequently, the mechanism behind this antidepressant-like effect was investigated. Administration of bicuculine with AM281 did not significantly affect the antidepressant effect on the FST, therefore suggesting that AM281 does not act on GABAergic synapses. A similar protocol was adopted with NVP-AM077, where its administration combined with AM281 was able to block the effect of AM281, thus confirming the importance of glutamatergic synapses for the antidepressant effect of AM281. Furthermore, the administration of a TAT-GLUR2 peptide did not significantly affect the effect of AM281, implying that the astroglial cell-mediated LTD (long-term depression) at glutamatergic synapses is not involved in the antidepressant effects of AM281. Finally, a bilateral intra-BLA (basolateral nucleus of the amygdala) administration of AM281 was able to reduce the immobility time on the FST. In conclusion, these results highlight the important contribution of BLA glutamatergic synapses to the antidepressant-like effect conferred by AM281.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22042号 / 医博第4527号 / 新制||医||1039(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 佐藤 俊哉, 教授 湊谷 謙司, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM