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Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.

Purines as a class of compounds have been implicated in many biological systems, including as adenosine receptor antagonists. A method of synthesising 2,6,9-trisubstituted purines would be useful to produce small libraries of compounds for probing adenosine receptor selectivity. A library of trisubstituted purines has been achieved using a solid-phase methodology. The electronic properties of the substrate were found to result in difficulties with the loading of substrate onto the resin. Theoretical calculations provided the basis for mono-substitution in order to activate the substrate. This modified substrate has loaded onto the resin in reproducible and high yields. Amine and thiol, on-resin, C-2 substitution was shown to proceed at room temperature. This represents significantly milder conditions than are generally seen in the literature. This is due to the activating effect of the carbamate linker chosen on the pyrimidine ring. This also results in a faster reaction rate than is seen in the corresponding solution-phase reaction. This study showed that the electronic profile of the loaded substrate was responsible for the low alkylation on the carbamate nitrogen of loaded dichloro- or C-6 substituted chloropyrimidines. This reaction was modified by activating the pyrimidine ring via C-2 substitution and has been shown to go to completion with three different alkyl groups to give a clean product direct from resin cleavage. On-resin nitro reduction had been planned. The resin bound product would then be carried on to the next step of resin cleavage and cyclisation of the imidazole ring to give the final purine products. On resin reduction could not be achieved, however, cleavage of the compound from the resin and reduction in solution was found to be efficient as the cyclisation reagents could be included in this step without interfering with yield or purity of products and so this represents a clear improvement upon the planned synthesis. Efforts to fully characterise the library brought up issues of purine NMR. Extremely broad signals were observed in the proton spectra of many of the compounds making assignments difficult. Broad 13C NMR signals have also been observed. Restricted rotation about the substituent N-C bond is responsible for these problems. Crystal structure data has confirmed the double bond character of this bond with one of the substituted pyrimidines. High temperature NMR experiments have demonstrated how this can be overcome and the fine structure of the spectra observed. HMBC and COSY correlations have been used alongside the 1H and 13C spectra to allow full characterisation of the compounds wherever possible. Receptor homology models were created and updated for all four adenosine receptor subtypes. Known adenosine agonists and antagonists were created and minimised for use in docking experiments. Receptor docking experimental data is reported. Binding assays are being carried out by a third party and will be submitted for publication at a later date. A small library of 2,6,9-trisubstituted purines has been synthesised, exemplifying an efficient and robust method to achieve pure compounds for biological evaluation. A good level of diversity has been achieved at each combinatorial position (two substitutions and an N-alkylation). Final compounds have been isolated in good yields with a high level of purity.

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.

The high rate of relapse often seen in breast cancer patients has been suggested to be the result of a small subset of chemotherapy-resistant cancer stem cells (CSCs), believed to be responsible for initiating tumor formation. These CSCs possess the capability to self-renew and give rise to a hierarchy of cells which makes up the bulk of a tumor. Neurotransmitters have been suggested to influence CSC self-renewal and proliferation capabilities, and antagonists of neurotransmission pathways have been implicated as possible treatment methods for chemo-resistant tumors. Using nicotinic acetylcholine receptor (nAChR) antagonists in sphere-forming assays, we have identified a very promising candidate compound: MG624. We found this compound to have a high selectivity for sphere-forming cells over non-sphere-forming cells in vitro, in a dose-dependent relationship, across a panel of cell lines as well as in patient-derived xenograft cells. This was validated in two ex vivo assays, where tumor formation was significantly delayed in mice injected with MG624-treated HCC1954 cells at both the IC50 and IC90 of the compound, indicating that MG624 does indeed target functional BTICs. MG624 was also found to synergize with both taxotere and doxorubicin chemotherapies in vitro, and shrink tumors in NOD/SCID mice when combined with taxotere in vivo. MG624 in combination with taxotere was found to induce apoptosis, and prevent cells from entering into the M-phase of the cell cycle. Interestingly, MG624 was found to eliminate intratumoral fibroblasts in combination with taxotere, despite taxotere being found to recruit fibroblasts to the tumor site when used on its own. Most importantly, the combination of MG624 and taxotere was found to significantly delay tumor progression/relapse in mice, indicating that MG624 may be an excellent candidate compound to one day be combined with chemotherapy to provide durable remission to breast cancer patients. / Thesis / Master of Science (MSc)

Rats lesioned shortly after birth with 6-OHDA have been proposed to be a near-ideal model of severe Parkinson's disease, because of non-lethality of the procedure, near-total destruction of nigrostriatal dopaminergic fibers, and near-total dopamine (DA) denervation of striatum. There are scarce data that in Parkinson's disease, activity of the central histaminergic system is increased. Therefore, the aim of this study was to determine histamine content in the brain and the effect of histamine receptor antagonists on behavior of adult rats. At 3 days after birth, Wistar rats were pretreated with desipramine (20.0 mg/kg ip) 1 h before bilateral icv administration of the catecholaminergic neurotoxin 6-OHDA (67 μg base, on each side) or saline-ascorbic acid (0.1%) vehicle (control). At 8 weeks levels of DA and its metabolites l-3,4- dihydroxyphenylalanine (DOPAC) and homovanillic acid (HVA) were estimated in the striatum and frontal cortex by HPCL/ED technique. In the hypothalamus, hippocampus, frontal cortex, and medulla oblongata, the level of histamine was analyzed by immunoenzymatic method. Behavioral observations (locomotion, exploratory-, oral-, and stereotyped-activity) were additionally made on control and 6-OHDA neonatally lesioned rats. Effects of DA receptor agonists (SKF 38393, apomorphine) and histamine receptor antagonists (e.g., S(+)chlorpheniramine, H 1 ; cimetidine, H 2 ; thioperamide, H 3 agonist) were determined. We confirmed that 6-OHDA significantly reduced contents of DA and its metabolites in the brain in adulthood. Histamine content was significantly increased in the hypothalamus, hipocampus, and medulla oblongata. Moreover, in 6-OHDA-lesioned rats behavioral response was altered mainly by thioperamide (H 3 antagonist). These findings indicate that histamine and the central histaminergic system are altered in the brain of rats lesioned to model Parkinson's disease, and that histaminergic neurons exert a modulating role in Parkinsonian 6-OHDA-lesioned rats.

To determine whether noradrenergic nerves might have a modulatory role on the sensitivity or reactivity of histaminergic receptor systems in brain, behavioral effects of the respective histamine H1, H2 and H3 antagonists S(+)chlorpheniramine, cimetidine and thioperimide in control adult rats were compared to the effects in adult rats that had been lesioned as neonates with the noradrenergic neurotoxin DSP-4. On the 1st and 3rd days after birth rat pups were treated with either saline or DSP-4 (50 mg/kg sc), then returned to their home cages with the dam. At 8 weeks when rats were tested, S(+)chlorpheniramine (10 mg/kg ip) was found to increase locomotor activity in intact and DSP-4 lesioned rats, while cimetidine (5 mg/kg, ip) and thioperimide (5 mg/kg, ip) increased activity severalfold solely in the DSP-4 group. Exploratory activity, nociceptive activity, and irritability were little altered by the histamine antagonists, although oral activity was increased by thioperimide in intact and lesioned rats, and by cimetidine or S(+)chlorpheniramine in DSP-4 rats. High performance liquid chromatography with electrochemical detection was used to determine that DSP-4 produced a 90% reduction in frontal cortex, hippocampus and hypothalamus, with a 90% elevation of NE in cerebellum - reflecting reactive sprouting of noradrenergic fibers consequent to lesion of noradrenergic tracts projecting to proximal brain regions. These findings indicate that perinatal noradrenergic fiber lesioning in rat brain is associated with an altered behavioral spectrum by histamine H1, H2 and H3 receptor antagonists, thereby implicating histaminergic systems as modulators of noradrenergic systems in brain.

No / The 5-HT1A receptor has been extensively studied over the last two decades. There is a plethora of information describing its anatomical, physiological and biochemical roles in the brain. In addition, the development of selective pharmacological tools coupled with our understanding of psychiatric pathology has lead to multiple hypotheses for the therapeutic utility of 5- and in particular 5-HT1A receptor antagonists. Over the last decade it has been suggested that 5-HT1A receptor antagonists may have therapeutic utility in such diseases as depression, anxiety, drug and nicotine withdrawal as well as schizophrenia. However, a very compelling rationale has been developed for the therapeutic potential of 5-HT1A receptor antagonists in Alzheimer s disease and potentially other diseases with associated cognitive dysfunction. Receptor blockade by a 5-HT1A receptor antagonist appears to enhance activation and signaling through heterosynaptic neuronal circuits known to be involved in cognitive processes and, as such, represents a novel therapeutic approach to the treatment of cognitive deficits associated with Alzheimer s disease and potentially other disorders with underlying cognitive dysfunction.

Background: Pulmonary hypertension (PH) is a rare condition characterized by endothelial dysfunction and vascular remodelling, leading to increased pulmonary vascular resistance (PVR) and right ventricular heart failure. Endothelial dysfunction is associated with an imbalance between vasoconstrictor compounds, such as endothelin and thromboxane A2, and vasodilator compounds, such as prostacyclin and nitric oxide (NO). Asymmetric dimethylarginine (ADMA), a methyl derivate of L-arginine, inhibits synthesis of NO. Vardenafil, a phosphodiesterase type 5 inhibitor (PDE5-inhibitors), causes vasodilation through the NO/cGMP pathway. Aim: This thesis investigates the pharmacological effects and diagnostic utility of vardenafil in PH patients. In addition, to evaluate the change of L-arginine and dimethylarginines before and during PAHspecific therapy in PAH patients compared to patients with left ventricular heart failure (LVHF) and healthy subjects. Methods: The pharmacokinetics and hemodynamic effects of vardenafil were examined during right heart catheterization (RHC) in 16 PH patients and plasma concentrations were measured for up to nine hours after oral administration. In 20 PH patients, acute vasoreactivity test with vardenafil was performed during RHC. Hemodynamic responses were recorded, responders were defined and followed for up to seven years. Additionally, plasma ADMA, symmetric dimethylarginine (SDMA), L-arginine, L-citrulline and L-ornithine levels before and after PAH drug treatment were monitored in 21 PAH patients and compared to values measured in 14 LVHF patients and 27 healthy subjects. Results: Vardenafil concentrations increased rapidly to maximum plasma concentration (tmax 1h) and elimination half-life was 3.4 h. Patients co-medicated with bosentan had reduced vardenafil concentration. Significant acute hemodynamic responses were observed for mean pulmonary artery pressure (mPAP) (p<0.001), pulmonary vascular resistance (PVR) (p<0.001), cardiac output (CO) (p=0.015), cardiac index (CI) (p=0.010), systemic vascular resistance (SVR) (p<0.001) and PVR/SVR (p=0.002) and were related to plasma vardenafil concentrations. PAH patients had significantly higher ADMA and SDMA levels and significantly lower L-arginine levels and L-arginine/ADMA ratio compared with healthy subjects (p<0.001). L-arginine was also lower in PAH patients compared to patients with LVHF (p<0.05). WHO functional class and six minutes walking distance (6MWD) correlated to Larginine and L-arginine/ADMA ratio in PAH at baseline (p<0.05). At follow-up, patients on mono- or combinationtherapy with endothelin receptor antagonists (ERA) had lower ADMA levels than patients without ERA (p<0.05). In contrast, patients on PDE5-inhibitors had higher ADMA levels compared to patients without PDE5-inhibitors (p<0.05). Conclusion: Vardenafil is safe in acute vasoreactivity test in PH patients. Cardiopulmonary hemodynamic response was related to plasma drug concentrations. There was a high inter-individual variability of vardenafil pharmacokinetics and co-medication with bosentan caused a pharmacokinetic drug interaction. Baseline L-arginine and dimethylarginines levels were different in PAH patients compared to LVHF patients and healthy controls. PAH-specific treatment influenced L-arginine and dimethylarginines. Our data suggest that L-arginine might be useful for differentiating PAH from LVHF, and L-arginine/ADMA ratios were related to the severity of PAH and might be useful for follow-up evaluations of PAH patients.