A prognostic factor is a marker or a feature that can be used to estimate the risk of recurrence of disease, metastatic spread and clinical outcome. Despite intensive search for more sophisticated markers in renal cell carcinoma (RCC), few have added prognostic information to earlier described factors like stage of disease, nuclear grade, tumour type, and in metastatic disease; performance status, anaemia, hypercalcaemia and increased erythrocyte sedimentation. In the dominating tumour type, clear cell renal RCC (cRCC), hypoxia is common, leading to an up-regulation of hypoxia inducible factor (HIF). The majority of cRCC have a mutation in the von Hippel Lindau gene (VHL-gene), which regulates HIF and in turn leads to up-regulation of a number of target genes for potential growth factors. The aim of the study was to evaluate the possible prognostic information of a few factors associated to pVHL/HIF, anemia and/or hypercalcaemia in RCC; erythropoietin (EPO) and it´s receptor (EPO-R), carbonic anhydrase IX (CA IX), parathyroid hormone-related protein (PTHrP) and osteopontin (OPN). Patients diagnosed with RCC between 1982-2007 were included in the studies. The tumour tissue expressions of EPO, EPO-R and PTHrP were assessed using immunohistochemistry. Serum/plasma levels of EPO, CA IX, PTHrP and OPN were also analyzed using immunometric methods. Our study demonstrated that the expression of EPO and EPO-R were related, and the expressions differed significantly between RCC types. The serum EPO levels did not associate to the tumour expression of EPO or EPO-R, indicating that circulating EPO derives from other sources than tumour cells. Erythropietin receptor expression was more frequent in advanced stages of disease, but neither EPO, nor EPO-R, were independent prognostic factors for survival. Serum CA IX levels were higher in cRCC compared to papillary RCC (pRCC). In cRCC, the CA IX serum levels correlated positively to TNM stage, but serum CA IX did not add independent prognostic information. Parathyroid hormone-related protein is a cause of hypercalcaemia in malignancy, and we observed that circulating PTHrP related to hypercalcaemia in RCC. The tumour expression of PTHrP associated positively to serum PTHrP, but not to serum calcium. We found an association between PTHrP and OPN in plasma, and both plasma PTHrP and OPN were positively associated to TNM stage.  Neither serum/plasma PTHrP nor tumour expression of PTHrP were independent prognostic factors for survival. The serum OPN levels were higher in pRCC but no impact on survival was observed in this RCC type. In contrast, plasma/serum OPN was an independent prognostic factor for disease-specific survival in cRCC. Our results support a role for these factors in RCC. The expressions vary between tumour types, which can be explained by different gene aberrations. Some of the factors have a close relation to para-malignant symptoms like hypercalcaemia. Most of the factors correlate positively to TNM-stage, reflecting a relation to advanced disease. Although expression of EPO, EPO-R, PTHrP and CA IX did not add independent prognostic information, the results might contribute to greater understanding of important mechanisms and associations in RCC. Osteopontin is a strong independent prognostic factor in cRCC, and should be further evaluated as a tool in the clinic when treating RCC patients.

Carbonic anhydrases (CAs, EC 4.2.1.1) are a class of metalloenzymes that catalyze the hydration of CO2 under physiologic conditions and are involved in many physiological and pathological processes. Modulation of CA activity, particularly CA inhibition is exploited pharmacologically for the treatment of many diseases such as cancer, glaucoma, edemas, mountain sickness. CA activation has been less frequently investigated till recently. Genetic deficiencies of several CA isozymes are reported in the literature and reflect the important role of carbonic anhydrases in human physiology and homeostasis. Activation of CA isozymes in brain have been correlated recently with spatial learning and memory. Based on these premises, activators of CA isozymes have the potential to alleviate mild dementias and to act as potential nootropic agents. In chapter 3, continuing our long-term interests towards the development of potent and selective CAAs, we carried out X-ray crystallographic studies with a small series of pyridinium histamine derivatives, previously developed as CAAs by our group. This study revealed important insights into the binding of this class of activators into the active site of CA II isozyme. A potent pyridinium histamine CAA 25i was successfully crystallized with CA II isozyme and was found to bind into the hydrophobic region of the active site, with two binding conformations being observed. This is one of the very few X-ray crystal structures of a CAA available. Based on the findings of this X-ray crystallographic study and building on our previously developed ethylene bis-imidazole CAAs, we advanced a novel series of lipophilic bis-imidazoles. Enzymatic assays carried out on purified human CA isozymes revealed several low nanomolar potent activators against various brain-relevant CA isozymes. Bis-imidazole 30e was found to be a nanomolar potent activator for CA IV, CA VA and CA IX. Due to their conjugated structure, these CAAs were also fluorescent and therefore were fully characterized in terms of photophysical properties, with several representatives proving to display very good fluorophores. The very good activation profile against several different CA isozymes, along with excellent fluorescence properties recommend these compounds as great molecular tools for elucidation of role of CA isozymes in brain physiology, as well as towards improvement of memory and learning. Focusing on inhibition of CA isozymes, it must be stressed that over the last decade a clear connection had been established between the expression of CA IX and CA XII and cancer. Since cancer is the second most common cause of death in the world, we explored the possibility to kill cancer cells via inhibition of different CA isozymes present in cancer cells. The membrane bound carbonic anhydrase IX (CA IX) isozyme represents a particularly interesting anticancer target as it is significantly overexpressed in many solid tumors as compared to normal tissues. In malign tissues this CA isozyme was found to play important role in pH homeostasis and promotes tumor cell survival, progression and metastasis. Thus, CA IX represents a potential biomarker and an appealing therapeutic target for the detection and treatment of cancer. CA IX can be targeted either through the development of small or large molecular weight, potent, and selective inhibitors or through the development of CA IX targeted drug delivery systems for selective delivery of potent chemotherapeutic agents. Building on these premises, in this dissertation, we also revealed our continuing efforts towards the development of potent and selective CA IX inhibitors along with their translation into the development of CA IX targeted drug delivery systems. In chapter 4, we designed a series of small molecular weight (MW) ureido 1,3,4-thiadiazole sulfonamide derivatives employing the “tail approach”, through the decoration of established sulfonamide CA inhibitor warheads with different tail moieties via ureido linker. The generated CAIs were tested against tumor associated CA IX and CA XII isozymes and off-target cytosolic isozymes CA I and CA II, and were revealed to be moderate to highly selective and nanomolar, even sub-nanomolar, potent CA IX inhibitors. Several potent pan-inhibitors were also identified in this section. We assessed these CAIs for their in vitro cell killing ability using MDA-MB 231 breast cancer cell line expressing CA IX and CA XII. The most efficient CAI proved to be ureido-1,3,4-thiadiazole-2-sulfonamide 69, which showed subnanomolar potency against purified human CA IX and CA XII isozymes, with good selectivity against CA I and CA II, and consistent, statistically significant cancer cell killing. In Chapter 5, continuing our efforts towards the development of potent and selective CA IX inhibitors, we designed, synthesized, characterized and evaluated a new series of PEGylated 1,3,4-thiadiazole-2-sulfonamide CAIs, bearing different PEG backbone length. We increased the PEG size from 1K to 20K, in order to better understand the impact of the PEG linker length on the in vitro cell killing ability against CA IX expressing cancer cell lines and also against a CA IX negative cell line. In vitro cell viability assays revealed the optimum PEG linker length for this type of bifunctional bis-sulfonamide CAIs in killing the tumor cells. The most efficient PEGylated CAI was found to bis-sulfonamide DTP1K 91, which showed consistent and significant cancer cell killing at concentrations of 10−100 μM across different CA IX and CA XII expressing cancer cell lines. DTP1K 91 did not affect the cell viability of CA IX negative NCI-H23 tumor cells, thus revealing a CA IX mediated cell killing for these inhibitors. In chapter 6, we decided to further explore the possibility of using CA IX as a targeting epitome for the development of a gold nanoparticle-based drug delivery system. We translated the oligoEG- and PEGylated CAI conjugates into efficient targeting ligands for gold nanoparticle decoration along with chemotherapeutic agent doxorubicin (Dox), in a novel multi-ligand gold nanoplatform designed to selectively release the drug intracellularly, in order to enhance the selective tumor drug uptake and tumor killing. We were successful in developing compatible CAI- and Dox- ligands for efficient dual functionalization of gold nanoparticles. Our optimized, CA IX targeted gold nanoplatform was found to be very efficient towards killing HT-29 tumor cells especially under hypoxic conditions, reducing the hypoxia-induced chemoresistance, thus confirmed the potentiating role of CA IX as a targeting epitome. / Pharmaceutical Sciences

Background: Angiogenesis is recognised as a critical step in tumour progression. The angiogenic switch is activated by various trigger signals, such as hypoxia, low pH, and genetic mutations. Renal cell carcinoma (RCC) is often an aggressive tumour, and advanced disease has limited treatment options and bad prognosis. This study was focused on markers of angiogenesis in RCC: endoglin (CD105) and CD31 assessing microvessel density (MVD), and carbonic anhydrase (CA) IX and hypoxia-inducible factor (HIF)-2α expressed at hypoxia. Upregulation of HIF is also associated with inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene, which is common in conventional/clear cell (c)RCC. Method: A tumour bank containing 308 tumours from patients operated 1982-2003 was used. The tumours were well characterised regarding tumour type, TNM stage, nuclear grade, tumour size, and patient survival. The tumours were prepared in tissue microarrays and fresh frozen in whole sections. To analyse the expression of endoglin, CD31, CA IX, and HIF-2α mRNA, immunohistochemistry and real-time PCR were used. Results: There was a higher endoglin expression in cRCC than in papillary (p)RCC and chromophobe (ch)RCC, and a higher CD31 expression in cRCC than in pRCC. MVD correlated inversely to TNM stage and nuclear grade in cRCC. There was also an inverse correlation between tumour diameter and CD31 expression in cRCCs. Patients with cRCC with high MVD had a more favourable prognosis than patients with lower MVD. Endoglin and CD31 were not independent prognostic factors. The CA IX expression was higher in cRCC than in pRCC and chRCC. Patients with cRCC expressing low CA IX had a significantly less favourable prognosis compared with those with higher expression. CA IX is an independent prognostic factor. There was a higher HIF-2α mRNA expression in cRCC than in pRCC and chRCC. In cRCC, there was a significant inverse correlation between HIF-2α mRNA expression, and TNM stage and nuclear grade. There was also an inverse correlation between HIF-2α mRNA expression and tumour size among patients with cRCC. HIF-2α was not an independent prognostic factor. Conclusion: In these studies, the factors related to hypoxia and vascularity were all inversely correlated to tumour aggressiveness in cRCC. MVD, CA IX, and HIF-2α expression were also higher in cRCC than in pRCC and chRCC. The relationship between angiogenesis, vascularity, and hypoxia is ambiguous. A line of reasoning including mutations increasing angiogenesis in advanced disease may also be applied to RCC. Measurements of individual angiogenic factors seem to provide prognostic information, and can potentially be combined in patient monitoring and treatment.