This thesis is focused on the development, preclinical evaluation, and optimization of radiotracers for the detection of gastrin-releasing peptide receptor (GRPR)-expressing tumors. The work is divided into three distinct parts: (1) the development of bombesin (BN) antagonist (RM26)-based imaging radiotracers for the detection of GRPR-expressing tumors using different positron emission tomography (PET) and single photon emission computed tomography (SPECT) radionuclides (68Ga, 18F and 111In), (2) the establishment of a method to monitor the ligand-G protein-coupled receptor (GPCR) interaction in real time without requiring purification and stabilization of the receptors, and (3) the evaluation of radiopeptide structure-related factors (length of mini-PEG linker and composition of chelator for metal labeling) affecting the in vitro and in vivo characteristics of RM26-based tracers. We demonstrated the possibility of high-contrast in vivo imaging of GRPR-expressing xenografts despite the physiological expression of GRPR in abdominal organs. Fast radioactivity clearance from the blood and healthy organs, including receptor-positive organs, and long retention in the tumors resulted in high tumor-to-background ratios. A novel real-time assay for measuring the kinetics of the radiotracers targeting GPCR was evaluated. Living cells were used instead of purified receptors in this technology, bringing the developmental work one step closer to the true target environment (imaging in living systems). The comparative study of 68Ga-labeled NOTA-PEGn-RM26 with di-, tri-, tetra- and hexaethylene glycol chains demonstrated that the addition of only a few units of ethylene glycol to the spacer is insufficient to appreciably affect the biodistribution of the radiopeptide. Finally, a comparative study of 68Ga-labeled PEG2-RM26 analogs N-terminally conjugated to NOTA, NODAGA, DOTA or DOTAGA highlighted the influence of the chelator on the targeting properties of the radiopeptide. The main conclusion that can be drawn from this thesis is that 68Ga-NOTA-PEG2-RM26 has favorable biodistribution properties, such as rapid clearance from blood and tissues with physiological GRPR expression levels and long retention in GRPR-expressing tumors, and that this radiopeptide is potentially suitable for initial clinical investigation.

臨床上止癢藥物的發展有限,故尋找具有廣泛止癢效果的藥物及探討癢覺傳遞的 神經受體機制是相當重要的。從過去研究中發現,bombesin 能在嚙齒類動物引發強 烈的搔抓行為。另外,bombesin 對 bombesin receptor family 中的 gastrin- releasing peptide receptor(GRPr)和 neuromedin B receptor(NMBr)也有高度親 和力。本研究主要目的為釐清 GRPr 和 NMBr 是否具有獨立調控癢覺搔抓行為的能 力。藉由側腦室於大白鼠中樞給藥並量化大白鼠的後肢搔抓行為,建立 GRP 與 NMB 這兩種胜肽在大白鼠上引發搔抓行為的基本藥理特性,並透過 GRPr 與 NMBr 這兩種受體的專一性拮抗劑探討受體間對於調控癢覺搔抓行為的獨立性。透過每天連 續施打 bombesin、GRP 與 NMB 這三種胜肽探討大白鼠搔抓行為的耐受性及三種胜 肽引發之搔抓行為在長時間觀測上的比較。最後,利用 GRPr 與 NMBr 這兩種受體 的專一性拮抗劑來探討 bombesin 引發搔抓行為的機制。利用側腦室給予 GRP (0.03 - 0.3 nmol)與 NMB(0.1-1 nmol)皆引發劑量相關性的搔抓行為反應。GRPr 的拮抗劑 RC-3095 (0.1-1 nmol)可以劑量相關性地阻斷 GRP 所引發的搔抓行 為,但不能阻斷 NMB 所引發的搔抓行為。相對而言,NMBr 的拮抗劑 PD168368 (0.3-3 nmol)可以劑量相關性地阻斷 NMB 引發的搔抓行為,但不能阻斷 GRP 所引 發的搔抓行為。藉由這些拮抗劑實驗,証實了 GRPr 與 NMBr 兩受體皆調控癢覺的訊 息傳遞並獨立不互相影響。Bombesin、GRP 與 NMB 都能引發搔抓行為,但程度與 持續時間上都有差異。但是每天施打這三種胜肽皆引發搔抓行為的耐受性。然而, RC-3095 與 PD168368 的有效劑量或兩個拮抗劑的組合並不能阻斷 bombesin 所引 發的搔抓行為,意指 bombesin 所引發的搔抓行為可能由其他受體調控。由本研究得 知,GRPr 與 NMBr 分別獨立調控中樞神經系統的癢覺訊息傳遞,並有程度與持續時 間上的差異。更重要的是,綜合先前文獻研究讓我們瞭解 bombesin-recognized neurons 中還有其他重要的受體機制調控於中樞神經系統的癢覺傳遞。 / Bombesin is a pruritogenic agent that causes intense itch-scratching activity in rodents. Bombesin has high affinity for the gastrin-releasing peptide receptor (GRPr) and the neuromedin B receptor (NMBr). The aim of this study was to investigate pharmacologically the ability of GRPr and NMBr to themselves elicit scratching behavior in rats. The intracerebroventricular (i.c.v.) route was selected for drug delivery because the study focused on supraspinal sites of action. The magnitude and duration of scratching produced by the naturally occurring peptides GRP and NMB were characterized. Antagonists selective for GRPr (RC-3095) and NMBr (PD168368) were used to define the role of GRPr and NMBr in the scratching response. After i.c.v. administration, GRP (0.03-0.3 nmol) and NMB (0.1-1 nmol) dose-dependently elicited marked scratching. There was a tolerance to scratching elicited by daily repeated administration of bombesin, GRP, or NMB. Presession administration of RC-3095 (0.1-1 nmol) and PD168368 (0.3-3 nmol) dose- dependently antagonized scratching elicited by GRP and NMB, respectively. More important, 1 nmol of RC-3095 failed to block NMB- elicited scratching and 3 nmol of PD168368 failed to block GRP-elicited scratching. In addition, pretreatment with effective doses of RC-3095 or PD168368 alone or in combination did not block bombesin-elicited scratching. Through the use of the selective antagonists RC-3095 and PD168368, this study demonstrates that central GRPr and NMBr may act independently to elicit scratching behavior. Although the receptor mechanisms underlying bombesin-elicited scratching elude us, this study provides a pharmacological basis for GRPr and NMBr antagonists as potential antipruritics.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-123).

Introdução: A incidência de melanoma maligno (MM) está aumentando mundialmente e o manejo de pacientes com doença avançada representa um difícil problema. Durante décadas, a quimioterapia foi o tratamento padrão no tratamento de pacientes com MM metastático. Entretanto, essa modalidade tem produzido resultados desapontadores. Recentemente, com os avanços no conhecimento sobre os eventos moleculares relacionados ao desenvolvimento do MM, novas drogas dirigidas a alvos moleculares de relevância na doença têm sido identificadas. O peptídeo liberador de gastrina (GRP) é um peptídeo neuroendócrino, o qual possui efeito estimulador no crescimento em vários tipos de neoplasias murinas e humanas. Poucos dados são conhecidos em relação à expressão de receptores de GRP (GRPR) em materiais de biópsia de pacientes portadores de MM. A identificação de uma expressão elevada destes receptores no MM poderá permitir uma maior compreensão sobre a biologia desta neoplasia, bem como embasar estudos com o uso de moduladores desta via de sinalização com potencial ação terapêutica. Objetivos: O objetivo desse estudo foi determinar a expressão de receptores de GRP em biópsias de pacientes com MM, bem como buscar eventuais correlações entre os níveis de expressão de GRPR e fatores prognósticos reconhecidos nesta doença. Métodos: Foi realizado um estudo imuno-histoquímico (IHQ) em blocos fixados em formalina e embebidos em parafina, contendo material de biópsia de 51 pacientes portadores de MM cutâneo. Utilizou-se um anticorpo policlonal de coelho anti-GRPR (OPA1-15619, Affinity Bioreagents, USA). Após a quantificação da expressão de GRPR nas amostras, foram analisadas as diferenças de expressão entre subgrupos prognósticos, com a aplicação do teste exato de Fisher. Resultados: A expressão de GRPR foi demonstrada no citoplasma de 42 das 51 (82,4%) amostras de MM cutâneo. A expressão foi considerada forte em 30 amostras (58,9%). Não foi observada diferença significativa na expressão de GRPR quando foram analisadas amostras de MM em sítio primário versus metastático. Foram correlacionados os escores da expressão de GRPR com os achados patológicos associados ao prognóstico do MM cutâneo primário, não tendo sido detectadas diferenças significativas com relação aos níveis de Clark (p=0,35) e Índice espessura de Breslow (p= 0,175). Conclusão: Nosso trabalho mostrou uma expressão de GRPR em amostras de MM cutâneo na vasta maioria dos casos (82,4%). Em 30 amostras (58.3%), a expressão de GRPR foi considerada de forte intensidade. Não houve associação entre a intensidade de expressão de GRPR quando comparadas amostras de sítio primário versus metastático, níveis de Clark ou índices de Breslow. Este estudo é um dos primeiros na literatura a descrever uma expressão de GRPR elevada em amostras obtidas de pacientes portadores de MM cutâneo. Estudos subsequentes, preferencialmente com um maior número amostral, são necessários para confirmar estes achados e permitir melhor análise da expressão deste receptor em distintos subgrupos prognósticos. Se confirmados, os nossos dados podem justificar a realização de estudos que explorem novas estratégias terapêuticas utilizando agentes moduladores da expressão de GRPR em pacientes com MM refratário ao tratamento convencional. / Background: The incidence of malignant melanoma (MM) is increasing worldwide and the management of patients with disseminated disease is a difficult problem. Chemotherapy was the treatment of choice in metastatic melanomas for many decades. However, this option produces disappointing results. Recently, the better understanding about molecular events related do the development of MM allowed the development of new drugs directed against specific molecular targets. The gastrin-releasing peptide (GRP) is a neuroendocrine peptide shown to have growth-stimulatory effects on many types of murine and human cancers. Few data are available about GRP receptor (GRPR) expression in MM. The understanding about the molecular biology of MM may allow the identification of novel intracellular pathways of relevance in this disease, and potential GRPR modulators of therapeutic application in patients with refractory MM. Objectives: The aim of this study was to determine the GRPR expression in biopsy samples of patients with cutaneous MM, as well as to correlate its expression with known prognostic factors of relevance in this disease. Methods: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsy samples from 51 patients with cutaneous MM. A rabbit polyclonal anti-GRPR antibody (OPA1-15619, Affinity Bioreagents, USA) was used. Following the quantification of GRPR expression in the samples, the differences in GRPR expression among distinct prognostic MM subgroups were analyzed, using the Fisher´s test. Results: GRPR immunoexpression was demonstrated in cytoplasm of 42/51 (82.4%) cutaneous MM cases. It was strongly expressed in 30 (58.9%) of the samples. No significant differences between GRPR expression neither in relation to the primary or metastatic site, nor among known prognostic subgroups Clark´s level (p=0.35) and Breslow index ( p= 0.175) was observed. Conclusion: Our study has demonstrated the occurrence of a high GRPR expression in tumor specimens obtained from patients with cutaneous MM (82,4%). In 30 samples, a strong intensity of expression was documented (58.3%). No correlation was observed between the level of GRPR expression in primary or metastatic sites, nor for distinct Clark´s levels or Breslow index. This is one of the first studies demonstrating a high GRPR expression in tumor samples from patients with MM. Further studies are warranted, preferably including a larger patient population, to allow a better analysis of the expression of these receptors in different prognostic subgroups. If these observations are confirmed, the therapeutic use of GRPR inhibitors should be considered in patients with advanced MM who failed conventional treatments.

Introdução: A incidência de melanoma maligno (MM) está aumentando mundialmente e o manejo de pacientes com doença avançada representa um difícil problema. Durante décadas, a quimioterapia foi o tratamento padrão no tratamento de pacientes com MM metastático. Entretanto, essa modalidade tem produzido resultados desapontadores. Recentemente, com os avanços no conhecimento sobre os eventos moleculares relacionados ao desenvolvimento do MM, novas drogas dirigidas a alvos moleculares de relevância na doença têm sido identificadas. O peptídeo liberador de gastrina (GRP) é um peptídeo neuroendócrino, o qual possui efeito estimulador no crescimento em vários tipos de neoplasias murinas e humanas. Poucos dados são conhecidos em relação à expressão de receptores de GRP (GRPR) em materiais de biópsia de pacientes portadores de MM. A identificação de uma expressão elevada destes receptores no MM poderá permitir uma maior compreensão sobre a biologia desta neoplasia, bem como embasar estudos com o uso de moduladores desta via de sinalização com potencial ação terapêutica. Objetivos: O objetivo desse estudo foi determinar a expressão de receptores de GRP em biópsias de pacientes com MM, bem como buscar eventuais correlações entre os níveis de expressão de GRPR e fatores prognósticos reconhecidos nesta doença. Métodos: Foi realizado um estudo imuno-histoquímico (IHQ) em blocos fixados em formalina e embebidos em parafina, contendo material de biópsia de 51 pacientes portadores de MM cutâneo. Utilizou-se um anticorpo policlonal de coelho anti-GRPR (OPA1-15619, Affinity Bioreagents, USA). Após a quantificação da expressão de GRPR nas amostras, foram analisadas as diferenças de expressão entre subgrupos prognósticos, com a aplicação do teste exato de Fisher. Resultados: A expressão de GRPR foi demonstrada no citoplasma de 42 das 51 (82,4%) amostras de MM cutâneo. A expressão foi considerada forte em 30 amostras (58,9%). Não foi observada diferença significativa na expressão de GRPR quando foram analisadas amostras de MM em sítio primário versus metastático. Foram correlacionados os escores da expressão de GRPR com os achados patológicos associados ao prognóstico do MM cutâneo primário, não tendo sido detectadas diferenças significativas com relação aos níveis de Clark (p=0,35) e Índice espessura de Breslow (p= 0,175). Conclusão: Nosso trabalho mostrou uma expressão de GRPR em amostras de MM cutâneo na vasta maioria dos casos (82,4%). Em 30 amostras (58.3%), a expressão de GRPR foi considerada de forte intensidade. Não houve associação entre a intensidade de expressão de GRPR quando comparadas amostras de sítio primário versus metastático, níveis de Clark ou índices de Breslow. Este estudo é um dos primeiros na literatura a descrever uma expressão de GRPR elevada em amostras obtidas de pacientes portadores de MM cutâneo. Estudos subsequentes, preferencialmente com um maior número amostral, são necessários para confirmar estes achados e permitir melhor análise da expressão deste receptor em distintos subgrupos prognósticos. Se confirmados, os nossos dados podem justificar a realização de estudos que explorem novas estratégias terapêuticas utilizando agentes moduladores da expressão de GRPR em pacientes com MM refratário ao tratamento convencional. / Background: The incidence of malignant melanoma (MM) is increasing worldwide and the management of patients with disseminated disease is a difficult problem. Chemotherapy was the treatment of choice in metastatic melanomas for many decades. However, this option produces disappointing results. Recently, the better understanding about molecular events related do the development of MM allowed the development of new drugs directed against specific molecular targets. The gastrin-releasing peptide (GRP) is a neuroendocrine peptide shown to have growth-stimulatory effects on many types of murine and human cancers. Few data are available about GRP receptor (GRPR) expression in MM. The understanding about the molecular biology of MM may allow the identification of novel intracellular pathways of relevance in this disease, and potential GRPR modulators of therapeutic application in patients with refractory MM. Objectives: The aim of this study was to determine the GRPR expression in biopsy samples of patients with cutaneous MM, as well as to correlate its expression with known prognostic factors of relevance in this disease. Methods: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsy samples from 51 patients with cutaneous MM. A rabbit polyclonal anti-GRPR antibody (OPA1-15619, Affinity Bioreagents, USA) was used. Following the quantification of GRPR expression in the samples, the differences in GRPR expression among distinct prognostic MM subgroups were analyzed, using the Fisher´s test. Results: GRPR immunoexpression was demonstrated in cytoplasm of 42/51 (82.4%) cutaneous MM cases. It was strongly expressed in 30 (58.9%) of the samples. No significant differences between GRPR expression neither in relation to the primary or metastatic site, nor among known prognostic subgroups Clark´s level (p=0.35) and Breslow index ( p= 0.175) was observed. Conclusion: Our study has demonstrated the occurrence of a high GRPR expression in tumor specimens obtained from patients with cutaneous MM (82,4%). In 30 samples, a strong intensity of expression was documented (58.3%). No correlation was observed between the level of GRPR expression in primary or metastatic sites, nor for distinct Clark´s levels or Breslow index. This is one of the first studies demonstrating a high GRPR expression in tumor samples from patients with MM. Further studies are warranted, preferably including a larger patient population, to allow a better analysis of the expression of these receptors in different prognostic subgroups. If these observations are confirmed, the therapeutic use of GRPR inhibitors should be considered in patients with advanced MM who failed conventional treatments.

Introdução: A incidência de melanoma maligno (MM) está aumentando mundialmente e o manejo de pacientes com doença avançada representa um difícil problema. Durante décadas, a quimioterapia foi o tratamento padrão no tratamento de pacientes com MM metastático. Entretanto, essa modalidade tem produzido resultados desapontadores. Recentemente, com os avanços no conhecimento sobre os eventos moleculares relacionados ao desenvolvimento do MM, novas drogas dirigidas a alvos moleculares de relevância na doença têm sido identificadas. O peptídeo liberador de gastrina (GRP) é um peptídeo neuroendócrino, o qual possui efeito estimulador no crescimento em vários tipos de neoplasias murinas e humanas. Poucos dados são conhecidos em relação à expressão de receptores de GRP (GRPR) em materiais de biópsia de pacientes portadores de MM. A identificação de uma expressão elevada destes receptores no MM poderá permitir uma maior compreensão sobre a biologia desta neoplasia, bem como embasar estudos com o uso de moduladores desta via de sinalização com potencial ação terapêutica. Objetivos: O objetivo desse estudo foi determinar a expressão de receptores de GRP em biópsias de pacientes com MM, bem como buscar eventuais correlações entre os níveis de expressão de GRPR e fatores prognósticos reconhecidos nesta doença. Métodos: Foi realizado um estudo imuno-histoquímico (IHQ) em blocos fixados em formalina e embebidos em parafina, contendo material de biópsia de 51 pacientes portadores de MM cutâneo. Utilizou-se um anticorpo policlonal de coelho anti-GRPR (OPA1-15619, Affinity Bioreagents, USA). Após a quantificação da expressão de GRPR nas amostras, foram analisadas as diferenças de expressão entre subgrupos prognósticos, com a aplicação do teste exato de Fisher. Resultados: A expressão de GRPR foi demonstrada no citoplasma de 42 das 51 (82,4%) amostras de MM cutâneo. A expressão foi considerada forte em 30 amostras (58,9%). Não foi observada diferença significativa na expressão de GRPR quando foram analisadas amostras de MM em sítio primário versus metastático. Foram correlacionados os escores da expressão de GRPR com os achados patológicos associados ao prognóstico do MM cutâneo primário, não tendo sido detectadas diferenças significativas com relação aos níveis de Clark (p=0,35) e Índice espessura de Breslow (p= 0,175). Conclusão: Nosso trabalho mostrou uma expressão de GRPR em amostras de MM cutâneo na vasta maioria dos casos (82,4%). Em 30 amostras (58.3%), a expressão de GRPR foi considerada de forte intensidade. Não houve associação entre a intensidade de expressão de GRPR quando comparadas amostras de sítio primário versus metastático, níveis de Clark ou índices de Breslow. Este estudo é um dos primeiros na literatura a descrever uma expressão de GRPR elevada em amostras obtidas de pacientes portadores de MM cutâneo. Estudos subsequentes, preferencialmente com um maior número amostral, são necessários para confirmar estes achados e permitir melhor análise da expressão deste receptor em distintos subgrupos prognósticos. Se confirmados, os nossos dados podem justificar a realização de estudos que explorem novas estratégias terapêuticas utilizando agentes moduladores da expressão de GRPR em pacientes com MM refratário ao tratamento convencional. / Background: The incidence of malignant melanoma (MM) is increasing worldwide and the management of patients with disseminated disease is a difficult problem. Chemotherapy was the treatment of choice in metastatic melanomas for many decades. However, this option produces disappointing results. Recently, the better understanding about molecular events related do the development of MM allowed the development of new drugs directed against specific molecular targets. The gastrin-releasing peptide (GRP) is a neuroendocrine peptide shown to have growth-stimulatory effects on many types of murine and human cancers. Few data are available about GRP receptor (GRPR) expression in MM. The understanding about the molecular biology of MM may allow the identification of novel intracellular pathways of relevance in this disease, and potential GRPR modulators of therapeutic application in patients with refractory MM. Objectives: The aim of this study was to determine the GRPR expression in biopsy samples of patients with cutaneous MM, as well as to correlate its expression with known prognostic factors of relevance in this disease. Methods: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsy samples from 51 patients with cutaneous MM. A rabbit polyclonal anti-GRPR antibody (OPA1-15619, Affinity Bioreagents, USA) was used. Following the quantification of GRPR expression in the samples, the differences in GRPR expression among distinct prognostic MM subgroups were analyzed, using the Fisher´s test. Results: GRPR immunoexpression was demonstrated in cytoplasm of 42/51 (82.4%) cutaneous MM cases. It was strongly expressed in 30 (58.9%) of the samples. No significant differences between GRPR expression neither in relation to the primary or metastatic site, nor among known prognostic subgroups Clark´s level (p=0.35) and Breslow index ( p= 0.175) was observed. Conclusion: Our study has demonstrated the occurrence of a high GRPR expression in tumor specimens obtained from patients with cutaneous MM (82,4%). In 30 samples, a strong intensity of expression was documented (58.3%). No correlation was observed between the level of GRPR expression in primary or metastatic sites, nor for distinct Clark´s levels or Breslow index. This is one of the first studies demonstrating a high GRPR expression in tumor samples from patients with MM. Further studies are warranted, preferably including a larger patient population, to allow a better analysis of the expression of these receptors in different prognostic subgroups. If these observations are confirmed, the therapeutic use of GRPR inhibitors should be considered in patients with advanced MM who failed conventional treatments.

Prostate cancer is one of the most common types of cancer worldwide and claims hundreds of thousands of lives annually. Currently the most common treatment for prostate cancer is external beam radiotherapy, however, this treatment comes with serious side effects since it lacks selectivity for the cancer cells. Therefore, less harmful treatments are needed and sought for, such as targeted treatments that are intended to only affect cancer cells and thereby reduce the side effects. Targeted treatments require a target that differentiates the cancer cells from healthy cells. A promising target candidate that has gained attention in recent years is gastrin releasing peptide receptor (GRPR), a protein commonly overexpressed in prostate cancer cells. Furthermore, a targeting molecule intended to bind to the target is also required. For this purpose, the bombesin analogue RM26, a high affinity GRPR binder, shows promise. Previous studies have led to the development of RM26-conjugates for the purpose of targeted prostate cancer radiotherapy. In these conjugates RM26 has been linked to a DOTA-chelator for radiolabeling, and an albumin binding domain (ABD) to prolong the conjugate’s half-life in vivo by binding to human serum albumin (HSA). The idea is that the RM26-conjugate will bind to both HSA in the blood and to GRPR on the prostate cancer cells and eliminate the cancer cells with the radiation from the radionuclide attached to the DOTA-chelator. Although these earlier studied conjugates have been very promising some improvements of certain aspects need to be achieved, mainly to improve the biodistribution with retained GRPR binding affinity. Therefor the purpose of this project was to produce three new versions of previous RM26- conjugates and evaluate if they are suitable for further prostate cancer therapy studies. The three RM26-conjugates were developed with primarily recombinant expression in E. coli cells and solid phase peptide synthesis (SPPS). The characterization phase in this project was carried out with mainly five different methods: matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization- mass spectrometry (ESI-MS), circular dichroism (CD), surface plasmon resonance (SPR) and flow cytometry. The results showed that all three new RM26-conjugates were possible to produce and yielded final products corresponding to the expected molecular weights. Furthermore, the results indicate that all three RM26-conjuagtes are stable and maintain their structural properties under in vivo- temperatures and that they have high binding affinity for HSA. Further studies need to be conducted before drawing any certain conclusions regarding GRPR binding affinity. / Prostatacancer är en av de mest vanligt förekommande cancertyperna världen över och skördar hundratusentals liv årligen. I nuläget är extern strålbehandling det vanligaste terapialternativet mot prostatacancer, men denna behandling kommer med allvarliga biverkningar på grund av att den saknar selektivitet för cancerceller. Därför finns ett stort behov av mindre skadliga behandlingsformer, såsom riktade behandlingar som endast är avsedda att påverka cancerceller och därigenom minska biverkningarna. Riktade behandlingar kräver ett mål som skiljer cancercellerna från friska celler. En lovande målkandidat som har uppmärksammats de senaste åren är gastrinfrisättande peptidreceptor (GRPR), ett protein som vanligtvis överuttrycks i prostatacancerceller. I tillägg så krävs också en målsökande molekyl avsedd att binda till målet. För detta ändamål visar bombesinanalogen RM26, en GRPR-bindare med hög affinitet, sig vara lovande. Tidigare studier har utvecklat RM26-konjugat för målinriktad strålbehandling av prostatacancer. Dessa konjugat består av en RM26-peptid bunden till en DOTA-kelator för radioinmärkning och en albuminbindande domän (ABD) för att förlänga konjugatens halveringstid in vivo genom att binda till humant serumalbumin (HSA). Syftet med RM26- konjugaten är att de ska binda till både HSA i blodet och GRPR på prostatacancercellerna, och därmed eliminera cancercellerna med strålning från den radioinmärkta DOTA-kelatorn. Även om de tidigare RM26-konjugaten har varit mycket lovande krävs det att vissa förbättringar av några aspekter uppnås, främst affiniteten för GRPR. Syftet med detta projekt var därför att producera tre nya versioner av tidigare RM26-konjugat och utvärdera ifall de uppvisar tillfredsställande egenskaper. De tre RM26-konjugaten utvecklades primärt rekombinant i E. coli-celler och fastfas- peptidsyntes (SPPS). Karaktäriseringsfasen i detta projekt genomfördes med huvudsakligen fem olika metoder: MALDI-TOF-MS, elektrosprejjonisering-masspektrometri (ESI-MS), cirkulär dikroism (CD), ytplasmonresonans (SPR) och flödescytometri. Resultaten visade att alla tre nya RM26-konjugat var möjliga att producera och gav slutprodukter motsvarande de förväntade molekylvikterna. Vidare indikerar resultaten att alla tre RM26-konjugat är stabila och bibehåller sina strukturella egenskaper under in vivo-temperaturer och att de har hög affinitet för HSA. Ytterligare studier bör utföras innan säkrare slutsatser kan dras angående GRPR-bindningsaffinitet.

Submitted in fulfillment of the requirements of the Degree of Doctor of Technology: Chemistry, Durban University of Technology, 2011. / Understanding the dynamics and mechanism of protein folding continues to be one of the central problems in molecular biology. Peptide folding experiments characterize the dynamics and molecular mechanisms of the early events of protein folding. However, generally the highly flexible nature of peptides makes their bioactive conformation assessment reasonably difficult as peptides fold at very fast rates experimentally, requiring probing on the nanosecond time resolution. On the other hand, determining the bioactive conformation of biological peptides is a requirement for the design of peptidomimetics in computer-aided drug design. Peptides offer a unique opportunity to bridge the gap between theoretical and experimental understanding of protein folding. Therefore, the present work focuses on the exploration of the conformational space of biologically active neuropeptides with the aim of characterizing their conformational profile. Specifically, bombesin, neuromedin B (NMB) and neuromedin C (NMC), have been chosen for the current investigations. These peptides are widely distributed in the gastrointestinal tract, spinal cord and brain, and are known to elicit various physiological effects, including inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. These peptides act as a growth factor in a wide range of tumours including carcinomas of the pancreas, stomach, breast, prostate, and colon. This work is intended to get some insight into the performance of different procedures used to explore the configurational space to provide an adequate atomic description of these systems. Different methodological studies involving utilization of molecular dynamics (MD), multicanonical replica exchange molecular dynamics (REMD) and simulate annealing (SA) are undertaken to explore the folding characteristics and thermodynamics of these neuropeptides. MD and REMD calculations on bombesin peptide have revealed its dual conformational behaviour never discovered before and is described in chapter 3. These results explain the known structure-activity studies and open the door to the understanding of the affinity of this peptide to two different receptors: BB1 and BB2. In the case of NMC, REMD calculations are carried out in explicit and implicit solvents, using the Generalized Born (GB) surface area, and are then complemented with two additional MD simulations performed using Langevin and Berendsen thermostats. The results obtained clearly reveal that REMD, performed under explicit solvent conditions, is more efficient and samples preferentially folded conformations with a higher content of  and γ turns. Moreover, these results show good agreement with the experimental results supporting the role of two -turns for its biological action, as reported in the literature. Finally, the results obtained from MD, REMD and SA calculations on NMB reveal that the peptide has a tendency to adopt both turns and helices suggesting its two different receptor recognizing and binding conformations during its biological action. Hence, the present work provides comprehensive information about the conformational preferences of neuropeptides which could lead to a better understanding of their native conformations for future investigations and point the way towards developing their new antagonists.

Têm sido estudadas novas modalidades para o diagnóstico e tratamento de câncer de próstata avançado baseado em peptídeos. Receptores para o peptídeo liberador de gastrina (GRPr) são superexpressos em vários tipos de células cancerígenas, incluindo câncer de mama e próstata. A bombesina é um análogo do peptídeo GRP de mamíferos que se liga com alta especificidade e afinidade a GRPr. Várias pesquisas têm sido realizadas para desenvolver e radiomarcar um análogo da bombesina com lutécio-177 com interesse para terapia de tumores pequenos ou metástases, devido às características físicas e viabilidade comercial do radionuclídeo. O objetivo deste trabalho foi estudar o processo de marcação e controle de qualidade de derivados da bombesina, usando como agente quelante DTPA e DOTA e espaçador aminoacídico Gly5, e realizar estudos de estabilidade e de biodistribuição de modo a avaliar o potencial de aplicação em estudos clínicos. Os estudos demonstraram que os análogos da bombesina estudados podem ser marcados com 177Lu com alto rendimento de marcação e alta atividade específica. Os estudos de estabilidade in vitro sugerem que os derivados DOTA-conjugados apresentam maior estabilidade que o DTPA-conjugados quando armazenados sob refrigeração e em soro humano. O coeficiente de partição do PG5N-DOTA-177Lu indicou maior lipofilicidade quando comparado com o derivado PG5M-DOTA-177Lu , porém sem influência sobre o clareamento sanguíneo ou captação hepática do composto. Os compostos DOTA-conjugados apresentam menor ligação às proteínas plasmáticas, corroborado pelo rápido clareamento sanguíneo observado nos estudos in vivo. Os estudos de biodistribuição com os compostos PG5M-DOTA-177Lu e PG5N-DOTA-177Lu mostraram o rápido clareamento sanguíneo dos compostos e excreção renal, e baixa captação óssea, indicando estabilidade in vivo dos derivados 177Lu-DOTA. O composto PG5M-DOTA-177Lu apresentou maior captação no pâncreas e intestinos, órgãos com maior porcentagem de receptores para bombesina. Entretanto, a ligação dos compostos às células tumorais (PC-3) in vitro foi similar para ambos os compostos. / New therapy modalities for the treatment of advanced prostate cancer based on peptide analogues are reviewed. It has been shown that gastrin releasing peptide receptors (GRPr) are overexpressed in various types of cancer cells including prostate and breast cancer. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPr. Significant research efforts have been devoted to the design bombesin analogues labeled with 177Lu considering the excellent radiophysical properties and commercial availability of the radionuclide. The aim of this work was to study the labeling and the quality control of a bombesin analogues with Lu-177 using DOTA and DTPA as chelate group and an aminoacidic spacers Gly5 and study the stability and biodistribution properties in order to evaluate the potencial for clinical application. The studies showed that the bombesin analogs were labeled with 177Lu with high radiochemical yield and high specific activity. In vitro stability studies showed that DOTA-conjugated peptides were more stable than DTPA-conjugated, when stored under refrigeration or in human serum. The partition coefficient values showed that 177LuDOTA-PG5N was more lipophilic when compared with 177LuDOTA-PG5M but without influence on blood clearance and liver uptake. The DOTA-conjugated peptides presented low binding to plasma proteins, that contributes for fast blood clearance in vivo. Biodistribution studies of 177LuDOTA-PG5M and 177LuDOTA-PG5N, showed significant renal uptake, as a result of the urinary excretion and low bone uptake, that indicated the in vivo stability of the 177Lu-DOTA-complexes. The compound 177LuDOTA-PG5M showed higher uptake on pancreas and intestinses and these organs presents high density of BBN receptors. But the in vitro binding assays (PC-3 tumors cells) showed similar results for both compounds.