Cancer remains one of the most dreaded diseases due to inevitable suffering and possible fatality. Only cardiac disease has caused more deaths than cancer. Present day cancer treatment involves radiation, surgery or chemotherapy. In chemotherapy, an anti-tumoral drug is used to treat the tumor either by killing or stalling the growth of the tumor cells. In certain types of cancer, for e.g. metastatic breast cancer, the first line of therapy is often chemotherapy. But the inability of current clinically approved drugs to selectively target tumor cells, ultimately results in side effects. To reduce these side effects, prodrug therapies have been developed. A prodrug is defined as a drug molecule inactivated by a temporary cap or carrier, subsequently removed by an external intra or extracellular stimulus. Several prodrug strategies such as ADEPT (Antibody–Directed Enzyme Prodrug Therapy) have been tested in clinical trials but have thus far met with limited success. In the wake of these limitations, development of photo-activatable prodrugs may be particularly desirable for minimizing the adverse side effects associated with current cancer chemotherapeutics. Photodynamic therapy (PDT) is a light dependent tumor treatment modality that has existed for many years. PDT involves a photosensitizer which is administered to the patient and later activated using the light of wavelengths between 650-800 nm. The activated photosensitizer creates singlet oxygen, which acts as cytotoxic agent to the tumor cells. But this approach has several drawbacks including slow uptake of the photosensitizer by the tumor cells and the dependence on molecular oxygen that is not always present at even moderate levels in the tumor tissues. To address these limitations of PDT, we developed a new prodrug concept called ‘Photocaged Permeability’ in our first project, and demonstrated drug delivery using this approach. The basis of this concept is that, by attaching a hydrophilic molecule to the drug via a photosensitive linker, the permeability of the drug could be restrained. But the drug could be released at the site of the tumor after irradiating with UV light. To achieve this goal, we designed and synthesized a photosensitive drug conjugate that was comprised of doxorubicin attached to a negatively charged, cell impermeable molecule, EDANS (5-((2-Aminoethyl) amino) naphthalein-1-sulfonic acid) via a photosensitive nitroveratryl linker. Later, we performed MTT (cell viability) assays using esophageal adenocarcinoma (JH-EsoAd1) cells to determine the efficiency of our drug conjugate to induce cell death. As expected our drug conjugate was able to induce cell death, but only in presence of light. But in the dark, the cells remained unaffected. Also, we did several control studies to substantiate the fact that the cell death was actually due to drug release but not due to light or other entities. Further, we performed FACS (Fluorescence Assisted Cell Sorting) and confocal assays to show that in dark, the drug conjugate did not permeate cells. But upon irradiation with UV light, the drug was released from the conjugate, permeated the cells and induced cell death. A weakness of the above mentioned approach is that the drug is “decaged” or photo-released from the conjugates only under UV light; which cannot be translated to physiological conditions. This is because the UV light cannot penetrate deeper than 5 mm into the human skin. As a result, tumor cells that are deeply embedded in the human body cannot be treated using these approaches. To address this problem, Near Infrared (NIR) light could be used as it penetrates deeper than UV. Recently, several groups have reported using Upconverting Nanoparticles (UCNP) for the purpose of drug activation. The basis of this phenomenon is that the incidence of NIR light on these particles initiates multi-photon processes, eventually emitting UV/VIS wavelengths. The advantage of the NIR is that it deeply penetrates into the human skin. In our latest project, we have designed a drug conjugate that would be attached to UCNPs. We envision that after grafting the drug conjugate onto the nanoparticles and irradiating it with NIR drug release will occur as a result of upconversion. The above two systems describes novel methodologies for controlled release of the drug. To further improve the efficacy of the drug action, we designed new photosensitive systems based on the concept of targeted drug delivery. Targeted drug delivery is a treatment methodology in which the modified chemotherapeutic drug with higher tumor affinity could be concentrated in the tumor tissues. In certain cases, the receptors of tumor cells are targeted for the purpose of therapy. Receptors are cell surface proteins that are expressed on their plasma membrane. A select few of them such as Folic Acid Receptor (FAR) and PSMA (Prostate Specific Membrane Antigen) are overexpressed in malignant cells. In our new designs, we attached folic acid and urea based (DUPA) ligand, which were previously reported to bind to FAR and PSMA receptors respectively. Cell studies are currently underway to determine the specificity of these drug conjugates in targeting tumor cells. Once we demonstrate the above drug delivery strategies in vitro and later in vivo, we will have established novel drug delivery systems that could potentially be applied towards chemotherapeutic treatment.

Glutamate carboxypeptidase II (GCPII) usually called prostate specific membrane antigen (PSMA) is membrane bound metallopeptidase expressed mainly in prostate carcinoma (PCa). Agents targeting GCPII suitable for both imaging and treatment of PCa are in development and they show promising results in advanced clinical trials. Some studies showed that GCPII may serve also as PCa blood serum marker, but this has not been validated due to the lack of methods suitable for accurate detection of GCPII in human blood. Moreover, GCPII is also expressed in brain, where it cleaves inhibitory N-acetyl-α-L- aspartyl-L-glutamate (NAAG) to release excitatory L-glutamate and GCPII inhibition has been shown to be neuroprotective in animal models of several neuropathies. Tight binding inhibitors of GCPII have been identified by rational design, but all have poor bioavailability and thus cannot be used in clinics. Identifying new scaffolds by 'brute force' screening methods is thus essential; however, no such method for GCPII has been developed so far. Glutamate carboxypeptidase III (GCPIII) is also expressed in brain and cleaves NAAG. It is thus an important protein for understanding of GCPII function as well as GCPII targeting in medicine. Here, we focused on development of novel methods for quantification of both...

Bakgrund: 68Gallium- Prostataspecifikt membranantigen – Positronemissionstomografi/Datortomografi (68Ga-PSMA-PET/DT) är en metod för att undersöka patienter med prostatacancer och eventuella metastaser. Denna undersökningsmetod har gjort att fler patienter kan få en diagnos i ett tidigt skede. Det saknas studier i patientupplevelser kring denna undersökning. Syfte: Syftet med studien är att beskriva patienters upplevelse av en 68Ga-PSMA-PET/DT-undersökning från remiss till utförd bildtagning.  Metod: En empirisk kvalitativ intervjustudie med semistrukturerade intervjufrågor genomfördes med 12 patienter. Resultatet analyserades med en induktiv kvalitativ innehållsanalys. Resultat: Resultatet visade att de flesta som intervjuades upplevde att de var välinformerade både muntligt från remitterande läkare, kallelse och den muntliga informationen som gavs under undersökningen. Majoriteten av patienterna upplevde inget obehag eller oro kring de olika momenten av undersökningen. Det framkom att muntlig information gällande risker med strålning till allmänheten inte givits till alla patienterna och studien fann att det fanns förbättringsbehov inom det området. Några upplevde att det var obehagligt att behöva ta diuretika, dricka vatten samt att ligga länge i PET/DT- kameran. Några av patienterna tyckte att undersökningen tog lång tid.  Slutsats: Studien visade att de flesta av patienterna kände sig välinformerade och väl bemötta. Studien fann däremot att det finns förbättringsområden vad gäller information efter undersökningen. Några patienter upplevde vissa förberedande moment som ansträngande, men en majoritet upplevde inga problem med undersökningen.

Background: Prostate cancer is the most prevalent form of cancer affecting men.In case of biochemical recurrence, positron emission tomography (PET) targeting prostate-specific membrane antigen (PSMA) on prostate tumor cells is primarily used, in combination with computed tomography (CT), for detection and localization of recurrence. Research for optimizing a PSMA-ligand with high affinity for tumor cells and minimal excretion to the urinary bladder is constantly ongoing, in order to allow better evaluation of the prostate and nearby regions. One such ligand is 18F-PSMA-1007, which was expected to be excreted in the urinary bladder at a rate of 5-10%. However, after switching from diagnostic to low-dose CT, the elimination of 18F-PSMA-1007 in the bladder was higher than expected. Purpose: To evaluate whether hydration during the accumulation period could affect the activity concentration in the bladder. Materials and Methods: The study involved analyzing PET-CT scans obtained from two prostate cancer patient groups who underwent 18F-PSMA-1007-PET with low-dose CT. The groups consisted of 20 participants each, with one group hydrating during the tracer’s accumulation time, while scans from the comparison group were obtained from a time point where patients did not receive instructions about water intake. The amount of radioactivity was measured by placing a standardized 3.00 cm Volume of Interest (VOI) on the bladder, which was then adjusted based on the individual size and shape of the patients' bladder. From the VOI, a standardized uptake value (SUV) was determined, which can be represented as either SUVmean or SUVmax. These values represent the average tracer concentration within a VOI and the highest concentration of the tracer in the urinary bladder, respectively. Results: SUV in the urinary bladder was lower for the hydrated group, where the SUVmean was 1,55 vs 4,5 (p=0,011) for the non-hydrated group. Similar values were obtained for SUVmax, 2,3 vs 6,65 (p< 0,003). Conclusion: This retrospective study suggests that water intake during the accumulation period leads to significantly lower activity concentration in the bladder among these patients, which benefits the detection of recurrences in adjacent areas.

The objective of this research was to develop and validate new macromolecular imaging agents to detect and characterize malignant tumours. Using well-defined, highly branched macromolecules called dendrimers as the structural scaffold, efficient functionalization of the periphery was demonstrated using “click” chemistry in order to prepare multivalent imaging probes. Furthermore, a transmetalation was demonstrated to displace chelated copper with technetium, enabling “click” reactions to be performed in the presence of the dipicolylamine (DPA), a ligand known to chelate many metals. The dendritic scaffold was functionalized with either hydrophobic or hydrophilic targeting vectors. The hydrophobic ligand, an acyloxymethyl ketone targeting the overexpression of cathepsin B exhibited poor in vitro affinity when coupled to either G1 or G2 dendrimers, despite the use of various linkers. A glu-urea-lys dipeptide, representing a hydrophilic prostate specific membrane antigen targeting vector, demonstrated excellent affinity in vitro. The lead compound, a G2 dendrimer bearing four PSMA targeting vectors attached via an alkyl spacer was further investigated in vitro and in vivo. Unfortunately, poor tumor uptake was observed and the compound was hypothesized to hydrolyze readily (<15min), based on the in vitro plasma stability data. To rectify the aforementioned problem, non neo-pentyl esters were replaced with either carbamate or ether linkages. In vitro plasma stability analysis of the analogous compounds demonstrated increased stability. In particular, the ether analogue was found to be most stable, with minimal degradation observed after 4 hours. / Thesis / Doctor of Philosophy (PhD)

PET/CT with prostate-specific membrane antigen (PSMA)-targeted tracers has been used in the diagnosis and staging of patients with clear cell renal cell carcinoma (ccRCC). For ccRCC primary tumors, PET parameters were shown to predict histologic grade and features. The aim of this study was to correlate PSMA PET/CT with histopathological findings in patients with metastatic recurrence of ccRCC. Patients with ccRCC who underwent PSMA-targeted PET/CT and subsequent histopathological evaluation of suspicious lesions were included. Specimens underwent immunohistochemical marking. Lesion diameter, volume and tracer uptake were correlated with the extent and intensity of molecular PSMA expression and with clinical findings. Twelve PET-positive lesions of nine patients were evaluated. Eleven ccRCC metastases and one prostate carcinoma were detected histopathologically. Molecular PSMA expression was detected in all lesions, which intensity and distribution did not correlate with PET parameters. PSMA-targeted PET/CT is a feasible tool for the evaluation of patients with ccRCC but cannot reliably predict histologic features of metastases. PSMA may also be expressed in malignant lesions other than ccRCC, leading to incidental detection of these tumors.

Currently, many patients with early-stage localized prostate cancer (PrCa) (D’Amico: low risk or low-intermediate risk) do not receive immediate therapy but are monitored within systematic AS programs. Prospective trials showed rates of stage reclassification and progression to the treatment of 20–40% over 2–5 years. However, in certain patients, PrCa progresses rapidly to an advanced stage that requires combined modality therapies, which carry increased risk for toxicity and poor outcomes. There is a need to identify biomarkers that can predict the risk for disease progression in this population. Research showed that dysregulation of metabolism is an important hallmark of cancer progression. Here, we pursued a pilot investigation of enzymes of de novo lipogenesis [ATP-citrate lyase (ACLY), Acetyl-CoA Carboxylase (ACC)], lipid oxidation [a-Methylacyl-CoA Racemase (AMACR)], glucose uptake [facilitative glucose transporter 1 (GLUT1)], and folate – glutamate metabolism (PSMA: prostate-specific membrane antigen) as potential biomarkers of PrCa progression in AS patients. With ethics approval from the Hamilton Integrated Research Ethics Board (HiREB), 40 AS patients were accrued prospectively from the Niagara Health System PrCa diagnostic program clinics and were asked to donate their biopsy tissue. 28 patients progressed on repeat biopsies at 12 or 24 months after initial diagnosis and were included in the “Progressed” group, and 12 did not who were included in the “Non-Progressed” group. Baseline diagnostic prostate core biopsy tissues of both groups were evaluated with H&E and immunohistochemistry (IHC) staining for ACLY, ACC, GLUT1, AMACR and PSMA expression (quantified by H-score). H-scores were evaluated in benign and malignant components (epithelial cells) and were compared between the two groups of patients. We observed statistically significant increased GLUT1 expression in malignant epithelial cells of the progressed group compared to the non-progressed group. Also, we found statistically significant increased PSMA expression in the benign epithelial cells of the progressed group compared to the non-progressed group. Further, our results demonstrated a statistically significant increase in ACLY and ACC expression in malignant epithelial cells compared to benign epithelial cells in the progressed group, while AMACR was detected solely in the malignant component. Overall, the results of this pilot study are consistent with the notion of induction of glycolytic metabolism, de novo lipogenesis and increased PSMA expression associated with the risk for PrCa progression. The levels of expression of PSMA within benign epithelial cells and GLUT1 within malignant epithelial cells may have value as predictive markers of risk for PrCa progression in AS patients. Future studies should investigate this concept systematically in larger AS cohorts. / Thesis / Master of Science (MSc) / Currently, many patients with localized prostate cancer do not receive immediate therapy and are monitored within systematic active surveillance (AS) programs. The main aim of AS management is to prevent overtreatment and treatment-related complications in patients who would otherwise have a good quality of life despite dealing with prostate cancer. However, many of these patients, especially those with low intermediate-risk prostate cancer have a significant risk for disease progression and metastasis. Additionally, there is a lack of promising tissue biomarkers to predict the risk for progression in AS patients at the time of initial diagnosis. Research showed that metabolism dysregulation is an essential hallmark of cancer progression, including prostate cancer. In this pilot study, we examined whether the expression of enzymes involved in lipid, glucose and protein metabolism could have value as biomarkers of risk for prostate cancer progression in patients managed with AS. The expression of five metabolic enzymes (ACLY, ACC, GLUT1, AMACR and PSMA) was examined in tumor and benign regions of diagnostic biopsies of the prostate obtained from men managed with AS. Our early results suggest that the expression of enzymes of protein (PSMA) and glucose (GLUT1) metabolism may have value as biomarkers of risk for prostate cancer progression and should be investigated further in systematic studies.

Introduction: Prostate-specific membrane antigen (PSMA)-based imaging and theranostics have played an important ole in the diagnosis, staging, and treatment of prostate cancer (PCa). We aimed to evaluate the acceptance and use of PSMA theranostics among German urologists.- Methods: An anonymous online questionnaire was sent via survio.com to the members of the German Society of Urology (DGU). - Results: Seventy-two percent of participants performed PSMA positron emission tomography (PET) imaging regularly in biochemically recurrent PCa. Overall, 61% of participants considered PSMA-radioligand therapy to be very useful or extremely useful. PSMA PET imaging in high-risk PCa is more often considered by urologists working in a university setting than in nonuniversity settings or medical practices (51% vs. 25%, p < 0.001). Most perform PSMA-radioligand therapy as an option after all approved systemic treatments for metastatic metastatic castration-resistant PCa (56%) or after cabazitaxel (14%). A total of 93.9% and 70.3% of respondents consider the lack of reimbursement by health insurance to be the main obstacle to using PSMA PET imaging or radioligand therapy, respectively. - Discussion/Conclusion: PSMA-based maging/theranostics are already widely applied but would find even more widespread use if reimbursement is clearly regulated by health insurance in Germany.

The aim of this thesis is to investigate the targeting potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as vectors for directed cell delivery of therapeutic or diagnostic compounds. Major capsid protein VP1 of MPyV is able to selfassemble into the noninfectious VLPs. Our main goal is to retarget these VLPs from its native receptor to the prostatic cancer cells by changing the receptor binding site in the surface-exposed loop of VP1. We introduced a peptide ligand CTITSKRTC, which binds prostate-specific membrane antigen (PSMA), by insertion or substitution into BC loop of VP1. These modifications did not change the stability of the particles and genetic substitution prevented the native receptor binding. PSMA-specific binding of modified VLPs was tested by pull-down assay and surface plasmon resonance. In order to further utilize these VLPs, we tested several approaches for preparation of VLPs as vehicles for compounds delivery into eukaryotic cells. Although the method for encapsidation of the DNA into the VLPs in cellular nuclear extracts, which mimic the in vivo conditions, did not enabled us to produce pseudocapsids, we successfully optimized procedure for dissassembly and reassembly of purified particles. This method will be use for encapsidation of molecules into the...

Submitted by PPG Medicina e Ci?ncias da Sa?de (medicina-pg@pucrs.br) on 2018-10-10T18:14:57Z No. of bitstreams: 1 Dissera??o CRIS rev_VFinal.pdf: 2998966 bytes, checksum: fef2ba937c2e3faa8a245d6da4b908ae (MD5) / Rejected by Caroline Xavier (caroline.xavier@pucrs.br), reason: Devolvido devido ? data de defesa cadastrada na publica??o (27/07/2018) estar diferente da data de defesa que consta na folha de aprova??o da banca (28/06/2018) no arquivo PDF. on 2018-10-16T17:23:19Z (GMT) / Submitted by PPG Medicina e Ci?ncias da Sa?de (medicina-pg@pucrs.br) on 2018-10-17T18:39:08Z No. of bitstreams: 1 Dissertac?a?o_Cristina_Sebastiao_Matushita rev_VFinal_corrigida.pdf: 2373644 bytes, checksum: 8a866e5c464e92b65ce80b925e027963 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-10-19T11:20:18Z (GMT) No. of bitstreams: 1 Dissertac?a?o_Cristina_Sebastiao_Matushita rev_VFinal_corrigida.pdf: 2373644 bytes, checksum: 8a866e5c464e92b65ce80b925e027963 (MD5) / Made available in DSpace on 2018-10-19T11:26:52Z (GMT). No. of bitstreams: 1 Dissertac?a?o_Cristina_Sebastiao_Matushita rev_VFinal_corrigida.pdf: 2373644 bytes, checksum: 8a866e5c464e92b65ce80b925e027963 (MD5) Previous issue date: 2018-06-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The purpose of this study was to assess the diagnostic performance of 68Ga?Prostate-specific Membrane Antigen (PSMA) Positron Emission Tomography (PET) for detection of prostate cancer and recurrent prostate cancer. Methods: A systematic search was performed in PubMed, Cochrane and Embase to identify relevant published studies reporting on the performance of 68Ga?Prostate-specific Membrane Antigen (PSMA) PET in patients with suspected, confirmed, untreated or recurrent Prostate Cancer. A composite standard included changing in PSA values, clinical follow?up and histopathological findings as reference standard. Results: Thirty-five studies including in total 3532 patients who underwent a 68Ga-PSMA PET met our inclusion criteria. We obtained a pooled positive likelihood ratio (LR) of 1.81 (0.75 to 4.36) with 0% heterogeneity. For patients with PSA <0.5ng/mL, the positive LR pooled estimate was 1.17 (0.37-3.73) and the sensitivity was 56% (0.42-0.68). Conclusion: This meta-analysis of available studies demonstrates that 68Ga-PSMA PET appears to provide good sensitivity and specificity. / O objetivo deste estudo foi avaliar o desempenho diagn?stico da tomografia por emiss?o de p?sitrons (PET) com ant?geno de membrana espec?fico da Pr?stata (PSMA) para detec??o de c?ncer de pr?stata e c?ncer de pr?stata recorrente. M?todos: uma pesquisa sistem?tica foi realizada em PubMed, Cochrane e Embase para identificar estudos relevantes publicados que relatam o desempenho do PSMA PET marcado com 68Ga em pacientes com c?ncer de pr?stata suspeito, confirmado, n?o tratado ou recorrente. Um composto padronizado incluiu altera??o nos valores de PSA, acompanhamento cl?nico e achados histopatol?gicos como padr?o de refer?ncia. Resultados: trinta e cinco estudos, que inclu?ram no total 3532 pacientes submetidos a um PET de 68Ga-PSMA, atendiam aos crit?rios de inclus?o. Obtivemos uma raz?o de verossimilhan?a positiva combinada (LR) de 1,81 (0,75 a 4,36) com 0% de heterogeneidade. Para pacientes com PSA <0,5ng/mL, a estimativa de LR combinada positiva foi de 1,17 (0,37-3,73) e a sensibilidade foi de 56% (0,42-0,68). Conclus?o: Esta meta-an?lise de estudos dispon?veis demonstra que o PET de 68Ga-PSMA parece fornecer boa sensibilidade e especificidade.