The objectives of this thesis are to produce radioactive antibody-conjugated gold nanoparticles to improve the efficacy of targeted radionuclide therapy for the treatment of cancer, and to demonstrate that this product can be produced at Texas A&M University. We have proposed a method for determining the distribution of radioactive nuclei per nanoparticle, which is critical for determining radiotherapeutic efficacy. Using the distribution of radioactive nuclei per nanoparticle, we have produced methods for calculating the radiative dose to tissue using nano-improved targeted radionuclide therapy, but more importantly we propose procedures to experimentally determine the efficacy of targeted radionuclide therapy improved by application of radioactive nanomaterials in combination with immunotherapy, nanomaterial cytotoxicity, and other cancer therapies such as chemotherapy. These methods can also be used to determine the efficacy of combinatory treatments as a function of time. Characterization of the antibody-nanoparticle attachment is critical; we have demonstrated successful antibody-nanoparticle conjugation using atomic force microscopy, dynamic light scattering, and agarose gel electrophoresis, providing more conclusive evidence of successful conjugation compared to flow cytometry. We provide a mathematical derivation from basic electron-transport principles which demonstrates the theoretical dosimetric advantages of applying radioactive nanomaterials to targeted radionuclide therapy. The general formulae can be applied to any tumor size, any radionuclide, and any pharmacokinetic nanoparticle distribution throughout the body, ultimately allowing a quick method of approximating the necessary activation time and treatment dosage parameters for a specific patient without burdensome Monte Carlo computational simulations. We further demonstrated that nano-TRT dosage to tumors should be considered as a function of radial position rather than average, as the dose across the tumor may be noticeably non-uniform causing some portions of the tumor to receive (potentially) significantly less dose than average.

京都大学 / 0048 / 新制・論文博士 / 博士(工学) / 乙第12923号 / 論工博第4116号 / 新制||工||1625(附属図書館) / 32133 / (主査)教授 木村 俊作, 教授 跡見 晴幸, 教授 岩田 博夫 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

No description available.

Atualmente os gliomas representam cerca de 81% dos tumores cerebrais malignos, com aumento na incidência tanto em crianças, como em adultos acima dos 45 anos. Um número elevado de receptores neuroquinina tipo 1 (NK-1) estão expressos em células de glioma, estando a ligação da Substância P (SP) a esses receptores, envolvida no desenvolvimento e progressão desse tipo de tumor. A SP quelada ao DOTA (SP-DOTA), radiomarcada, vem sendo testada para utilização na terapia de gliomas, sendo o lutécio-177 (177Lu), devido a seu menor alcance tecidual, o radioisótopo mais indicado para tumores localizados em áreas críticas do cérebro. No entanto, estudos indicam a necessidade da adição de um excesso de metionina para prevenção da oxidação peptídica da SP-DOTA-177Lu, visando aumentar a estabilidade e a capacidade de ligação às células tumorais. Para superar esse desafio, surge a perspectiva da utilização de um novo análogo da SP, com estrutura modificada, para prevenir a oxidação peptídica. Neste contexto, o objetivo desse trabalho foi estudar a marcação de um novo análogo da SP com 177Lu e caracterizar suas propriedades in vitro e in vivo, visando a obtenção de um radiofármaco inédito e com potencial aplicação na terapia de tumores cerebrais e realizar estudos preliminares de marcação deste novo análogo com Ítrio-90 (90Y). O novo análogo foi obtido pela troca do aminoácido metionina (Met) pelo aminoácido norleucina (Nle) na posição 11 da cadeia peptídica da SP, sendo esses peptídeos denominados respectivamente SP(Met11)-DOTA e SP(Nle11)-DOTA. Após análise da oxidação peptídica dos dois peptídeos, os parâmetros da radiomarcação da SP(Nle11)-DOTA, com 177LuCl3, foram estudados para determinar a melhor condição de marcação. As estabilidades in vitro da SP(Nle11)-DOTA-177Lu sob refrigeração (2-8°C), no freezer (-20°C) e em soro humano (37°C) foram determinadas após radiomarcação com alta atividade, quanto ao uso de agentes estabilizantes e após diluição. A SP(Nle11)-DOTA também foi radiomarcada com 90Y, utilizando-se a condição padrão determinada, sendo a estabilidade in vitro da SP(Nle11)-DOTA-90Y sob refrigeração (2-8°C) e em freezer (-20°C), avaliada após radiomarcação com alta atividade e quanto a utilização de agente estabilizante. A capacidade de ligação in vitro às células tumorais (U-87 MG e M059J) e a biodistribuição in vivo em camundongos BALB/c sadios foram determinadas para a SP(Nle11)-DOTA-177Lu e comparadas à SP(Met11)-DOTA-177Lu. A ligação às proteínas plasmáticas e a biodistribuição em camundongos Nude com modelo tumoral também foram avaliadas. Os resultados obtidos na análise da oxidação peptídica comprovaram a importância da adição de excesso de metionina para prevenção da oxidação peptídica e indicaram uma alta estabilidade da SP(Nle11)-DOTA, durante e após o processo de radiomarcação. A adição de 148 MBq (4 mCi) da solução de 177LuCl3 em HCl 0,05N à 10 μg de SP(Nle11)-DOTA diluída em tampão acetato de sódio 0,4 M pH 4,5 seguida pela incubação a uma temperatura de 90ºC por 30 minutos, sob agitação de 350 rpm foi definida com condição padrão de marcação. O congelamento (-20°C), o uso de agentes estabilizantes e a diluição apresentaram-se como métodos efetivos para garantir uma alta estabilidade in vitro da SP(Nle11)-DOTA-177Lu, após a marcação com alta atividade. Bons resultados também foram observados para a marcação da SP(Nle11)-DOTA com 90YCl3 e para a estabilidade in vitro da SP(Nle11)-DOTA-90Y, após congelamento (-20°C) e quando utilizado ácido gentísico como estabilizante. A SP(Nle11)-DOTA-177Lu apresentou uma boa especificidade pelas células tumorais, principalmente pelas células de glioma humano M059J, sugerindo que a substituição do aminoácido metionina por norleucina na posição 11 não compromete a capacidade de ligação da SP(Nle11) às células tumorais. Uma baixa porcentagem de ligação às proteínas plasmáticas e um rápido clareamento sanguíneo foram observados para a SP(Nle11)-DOTA-177Lu, sendo esse radiofármaco eliminado preferencialmente por via renal. A SP(Nle11)-DOTA-177Lu apresentou uma boa estabilidade in vivo e se mostrou incapaz de atravessar a barreira hematoencefálica, sendo seu uso indicado por injeção intratumoral ou intracavitária. O estudo de biodistribuição em animais com modelo tumoral, mostrou que esse radiofármaco se liga às células tumorais por ligações receptor específicas. Com base nesse dados conclui-se que a SP(Nle11)-DOTA-177Lu, apresenta-se como um radiofármaco inédito que devido às suas propriedades in vitro e in vivo favoráveis, apresenta potencial aplicação na terapia de tumores cerebrais, representando uma nova possibilidade dentro do limitado arsenal terapêutico para esse tipo de tumor. / Currently gliomas represent about 81% of malignant brain tumors with increased incidence in children and in adults over 45 years. A large number of type 1 neurokinin receptor (NK-1) are expressed in glioma cells, being the binding of substance P (SP) to these receptors, involved in the development and progression of this tumor type. The SP conjugated at DOTA chelator (SP-DOTA), radiolabeled, have been tested for use in the treatment of gliomas, and the lutetium-177 (177 Lu), due to its lower tissue range, has been the most suitable radioisotope for tumors located in critical areas brain. However, studies indicate the necessity of adding an excess of methionine to prevent the peptide SP-DOTA-177Lu oxidation in order to increase the stability and capacity to bind to tumor cells. To overcome this challenge, there is the prospect of using a new analog of SP with a modified structure, to prevent peptide oxidation. In this context, the aim of this work was study the labeling of a new analog of SP with 177Lu and characterize their properties in vitro and in vivo, in order to obtain a novel radiopharmaceutical with potential application in brain tumor therapy, and perform preliminary studies labeling of this new analog with yttrium-90 (90Y). The new analog was obtained by replacement of the amino acid methionine (Met) by the amino acid norleucine (Nle) at position 11 of the peptide chain of SP, and these peptides were called SP(Met11)-DOTA and SP(Nle11)-DOTA respectively. After analysis of the oxidation for the two peptides, the radiolabeling parameters of the SP(Nle11)-DOTA with 177LuCl3 were studied to determine the best labeling condition. The SP(Nle11)-DOTA was also radiolabeled with 90Y, using standard condition, and the stability in vitro of the SP(Nle11)-DOTA-90Y assessed under refrigeration (2-8 °C) and under freezing (-20° C), after radiolabeling with high activity and use of stabilizing agent. The stabilities in vitro of the SP (Nle11)-DOTA-177Lu under refrigeration (2-8 °C), under freezing (-20 °C) and in human serum (37 °C) were determined after radiolabeling with high activity, with use of stabilizing agents and after dilution. The ability of in vitro binding to tumor cells (U-87 MG and M059J) and the biodistribution in vivo in healthy BALB/c mice were determined for the 177Lu-DOTA-SP(Nle11) and compared to 177Lu-DOTA-SP(Met11). The plasma protein binding and biodistribution in Nude mice with tumor model were also evaluated. The results obtained from analysis of oxidation for the two peptides confirmed the importance of adding excess methionine to prevent peptide oxidation and indicated a high stability of the DOTA- SP(Nle11), during and after the radiolabeling process. The addition of 148 MBq (4 mCi) of 177LuCl3 solution in 0.05N HCl at 10 μg DOTA-SP(Nle11) diluted in 0.4 M sodium acetate buffer pH 4.5 followed by incubation at a temperature of 90 °C for 30 minutes under constant agitation to 350 rpm was defined as standard labeling condition. The freezing (-20 °C), the use of stabilizing agents and the dilution were presented as effective methods to ensure high stability in vitro 177Lu-DOTA-SP(Nle11), after labeling with high activity. Good results were also observed for labeling DOTA-SP(Nle11) with 90YCl3 and for stability in vitro of the 90Y-DOTA-SP(Nle11) after freezing (-20 °C) and when gentisic acid was used as a stabilizer. The 177Lu-DOTA-SP(Nle11) showed good specificity to tumor cells, particularly human glioma cells (M059J), suggesting that substitution of the amino acid norleucine for methionine at position 11 does not compromise the capacity of SP(Nle11) binding to tumor cells. A low percentage of plasma protein binding and rapid blood clearance were observed for the 177Lu-DOTA-SP(Nle11), being this radiopharmaceutical preferably eliminated by the kidney. The 177Lu-DOTA-SP(Nle11) showed good stability in vivo and inability to cross the blood brain barrier, being its use indicated through intratumoral or intracavitary injection. The biodistribution studies in animals with tumor model showed that the radiopharmaceutical binds to the tumor cells by specific receptor binding. Based on this data was concluded that the 177Lu-DOTA-SP(Nle11), can be presented as a novel radiopharmaceutical that due to its favorable properties in vitro and in vivo, presents a potential application in the therapy of brain tumors, representing a new possibility within the limited therapeutic options for this type of tumor.

O câncer de próstata é considerado o segundo mais comum na população masculina em todo o mundo e ocupa a 15ª posição em mortes por câncer, em homens, representando cerca de 6% do total de mortes por câncer no mundo. O antígeno de membrana prostático específico (PSMA) é uma glicoproteína tipo II transmembrânica superexpressa no câncer de próstata, suas metástases e em neovascularizações relacionadas a tumores sólidos, que tem estimulado o desenvolvimento de pequenas moléculas inibidoras do receptor de PSMA, que carreguem agentes terapêuticos. Este trabalho pretendeu estudar de forma inédita a marcação e estabilidade radioquímica do Glu-NH-CO-NH-Lys(Ahx)-DOTA com 177Lu (PSMA-DOTA-177Lu) e avaliar seu potencial para a terapia do câncer de próstata. O radiofármaco foi obtido com pureza radioquímica elevada (PR > 95%) em todas as condições estudadas e permaneceu inalterada em ≤ - 20 °C até 48 horas, mesmo em atividade específica alta (74 MBq/μg). O ensaio de ligação específica do PSMADOTA- 177Lu mostrou que a fração do peptídeo que se ligou às células LNCaP de tumor de próstata foi de 1,79 ± 0,21 %, 2,47 ± 0,03 %, 3,07 ± 0,01 % e 4,13 ± 0,27%, para as concentrações de 0,15 x 106, 0,3 x 106, 0,5 x 106 e 1 x 106 células, respectivamente. O ensaio de internalização do PSMA-DOTA-177Lu sugere que o maior percentual da ligação específica do radiofármaco às células LNCaP corresponde à fração da ligação de superfície (99,03 ± 0,84 %). Os parâmetros farmacocinéticos determinados no estudo in vivo em camundongos Balb/c são compatíveis com o rápido clareamento sanguíneo e excreção renal, além de apresentar apreciável captação tumoral in vivo (2,76 ± 1,21 % Al/g) após 4 horas de administração do radiofármaco. Os estudos em estabilidade em soro humano demonstram estabilidade alta do radiofármaco PSMA-DOTA-177Lu por um período de até 24 horas, que foi confirmada pela baixa captação óssea demonstrada nos estudos in vivo de biodistribuição. O estudo de variação da atividade possibilitou estabelecer a atividade específica ideal (MBq/μg), que será extrapolada para um piloto de produção do radiofármaco. Os resultados favoráveis deste estudo encorajam a perspectiva de realização de ensaio clínico controlado deste novo radiofármaco, avaliando seu potencial para aplicação no tratamento do câncer de próstata. / Prostate cancer is the second most frequently worldwide diagnosed cancer among males and ranks the 15th common cause of death from cancer in men, comprising 6% of the world\'s total cancer deaths. The prostate specific membrane antigen (PSMA) is a type II transmembrane glycoprotein, which is overexpressed in prostate cancer as well as in the neovasculature of solid tumors and metastasis. These features render PSMA an ideal target for developing small molecules inhibitors of PSMA and therapeutic approaches to targeted drugs delivery. This work intended to study in an unprecedented way the radiochemical labeling and stability of Glu-NHCO- NH-Lys (Ahx)-DOTA with 177Lu (177Lu-PSMA-DOTA) and its potential anti-tumor effects in prostate cancer. The radiopharmaceutical PSMA-177Lu was obtained with high radiochemical purity (RP > 95%) under all studied conditions and remained unchanged up to 48 hours (high at ≤ -20 °C), even at high specific activity (74 MBq / μg). The cellular uptake of PSMA-DOTA-177Lu was determined using PSMAexpressing LNCaP cells and showed a binding of 1.79 ± 0.21%, 2.47 ± 0.03%, 3.07 ± 0.01% and 4.13 ± 0.27%, to 0.15 × 106, 0.3 × 106, 0.5 × 106 and 1 × 106 LNCaP cells, respectively. The PSMA-DOTA-177Lu internalization assay revealed that the membrane-bound activity (non-internalized peptide) to LNCap cells, was 99.03 ± 0.84%. Moreover, the pharmacokinetic in vivo studies performed in Scid mice resulted in a rapid blood clearance and renal excretion, and showed significant tumor uptake (2.76 ± 1.21% lA/g) 4 hours after PSMA-DOTA-177Lu administration. Stability studies in human serum demonstrated high PSMA-DOTA-177Lu stability over 24 hours, which is in agreement with the low bone uptake obtained in the in vivo biodistribution studies. Furthermore, the comparative study aiming to establish the ideal PSMA-DOTA-177Lu specific activity (MBq / μg) will be considered for further radiopharmaceutical production. The pre-clinical data obtained from this study suggested a great potential for PSMA-DOTA-177Lu to be included in clinical trials and to make a major contribution to the treatment of prostate cancer.

Le mélanome cutané est un cancer très invasif, dont l'évolution est rapidement fatale au stade métastatique du fait d'une absence de thérapies réellement efficaces. Face à ce constat, une stratégie de multimodalité a été évaluée au moyen d'un vecteur spécifique des cellules de mélanome et présentant la double potentialité diagnostique (imagerie TEP) et thérapeutique (radiothérapie interne vectorisée (RIV)) en fonction du radioélément introduit. Dans ce but, des analogues iodés et fluorés d'ICF01012, un vecteur ciblant la mélanine surexprimée dans un grand nombre de mélanomes, ont été synthétisés puis radiomarqués (iode-125 d'une part et fluor-18 d'autre part). Les profils pharmacocinétiques des radiotraceurs ont par la suite été étudiés in vivo sur modèle murin de mélanome, par imagerie scintigraphique γ (125I) et par imagerie TEP (18F). A l'issue de ces études, le traceur 4 a été sélectionné comme molécule leader de cette série, en raison d'un tropisme tumoral spécifique, élevé et durable, associé à élimination rapide des tissus non-cibles. Ce composé a ensuite été radiomarqué à l'iode-131 pour une évaluation en RIV sur le même modèle murin. Le traitement avec [131I]4 a induit une inhibition significative de la croissance tumorale et une augmentation significative de la médiane de survie. Au vu des résultats prometteurs obtenus avec le traceur 4, des études de métabolisme sur le même modèle murin ont été effectuées. En conclusion, en termes de chimie, radiochimie, stabilités in vitro et in vivo, TEP et RIV, le traceur 4 a validé notre concept de multimodalité. A terme, ce composé pourrait être transféré pour des études cliniques afin : de sélectionner les patients présentant des lésions de mélanome pigmentées (18F/TEP) et donc susceptibles de répondre au protocole de RIV ; de traiter ces patients (131I/RIV) ; de suivre la réponse au traitement (18F/TEP). En parallèle a été développé le premier groupement prosthétique iodé et fluoré multimodal, le 4-fluoro-3-iodobenzoate de tétrafluorophényle 108, pour le marquage de vecteurs d'intérêt (peptides, protéines, nanoparticules…). Le composé 108 et les précurseurs de radiomarquages correspondants ont été synthétisés en peu d'étapes, avec de bons rendements. Les radiomarquages ont ensuite été mis au point, et ont permis d'obtenir [125I]108 et [18F]108 en des temps relativement courts, avec d'excellents rendements et puretés radiochimiques. Le groupement prosthétique a ensuite été couplé avec succès à un ligand organique et deux peptides. En conclusion, nous avons démontré que le composé multimodal 108 peut être utilisé pour la radiofluoration et la radioiodation de vecteurs d'intérêt, autorisant des applications à la fois diagnostiques (TEP/18F) et thérapeutiques (RIV/131I) pour la prise en charge de nombreux cancers. / Melanoma is the most serious form of skin cancer with a poor prognosis for patients with metastatic disease. Our project deals with a multimodal approach, using a single fluorinated and iodinated melanintargeting compound, and offering both imaging (PET/18F) and therapeutic (targeted radionuclide therapy (TRT)/131I) applications, depending on the radioisotope introduced. Furthermore, using PET imaging, our strategy allows a selection of TRT-responded patients (i.e. bearing pigmented metastases) as well as a monitoring of treatment response. In previous works, the iodinated quinoxaline-carboxamide compound ICF01012 was evaluated in a TRT protocol, using melanoma-bearing mice models. It demonstrated efficacy, with significant inhibition of tumoural growth and improvement of the median survival. Based on these results, iodinated and fluorinated analogs of ICF01012 were synthesized, for multimodality purposes. All new compounds were then radiolabelled with iodine-125 and fluorine-18 (fully automated radiosyntheses), with good radiochemical yields and excellent radiochemical purities. For pharmacokinetic profile studies on melanoma-bearing mice, [125I]radiotracers were evaluated by γ-scintigraphy and [18F]radiotracers by PET. Compound 4 emerged as the lead tracer, with a specific and long-lasting tumoural uptake and a fast clearance from non-specific tissues, leading to highly contrasted images. The tracer 4 was then radiolabelled with iodine-131, with excellent radiochemical yield and purity, to perform a TRT assay on the same melanoma model. Treatment with [131I]4 significantly inhibited tumoural growth and lung metastasis occurrence. Moreover, it significantly improved the median survival. As tracer 4 demonstrated promising results in PET imaging and TRT of melanoma, its metabolism was investigated with [125I]4 and [18F]4: radiotracers were found unchanged in melanin-containing tissues (tumour and eyes), while a fast breakdown was observed in excretion organs and fluids (four metabolites were identified). In conclusion, in terms of chemistry, radiochemistry, in vitro and in vivo stability, PET imaging and TRT, compound 4 validated our multimodality concept. We also developed the first bimodal fluorinated and iodinated prosthetic group, the 2,3,5,6-tetrafluorophenyl 4-fluoro-3-iodobenzoate (108), as a suitable acylating agent for the labelling of a large variety of compounds. In this approach, this new compound allows applications in diagnosis (PET imaging/18F) and therapy (TRT/131I). Compound 108 and its corresponding precursors for radiolabelling were synthesized in very few steps, with good yields. [125I]108 was then prepared in one-step starting from a perfluorostannane precursor, and purified by F-SPE cartridge to avoid time-consuming HPLC. As for [18F]108, it was produced by a fully automated three steps, two-pots radiosynthesis process. [125I]108 and [18F]108 were both obtained in a short time, with excellent radiochemical yields and purities. These prosthetic groups were then successfully used to radiolabel small organic ligand N,N-diethylethylenediamine and peptides NDP-MSH and PEG3[c(RGDyK)]2, in mild conditions, with good yields. In conclusion, we demonstrated that compound 108 could be a promising acylating bimodal prosthetic group for radiofluorination and radioiodination of small organic molecules, peptides, proteins, antibodies as well as nanoparticles.

Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues.  This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry. Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody molecule site specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized. Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of ZHER2:2395 molecules in the kidneys. Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties. Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using 188Re-ZHER2:V2 may be feasible. It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.

O câncer de próstata é considerado o segundo mais comum na população masculina em todo o mundo e ocupa a 15ª posição em mortes por câncer, em homens, representando cerca de 6% do total de mortes por câncer no mundo. O antígeno de membrana prostático específico (PSMA) é uma glicoproteína tipo II transmembrânica superexpressa no câncer de próstata, suas metástases e em neovascularizações relacionadas a tumores sólidos, que tem estimulado o desenvolvimento de pequenas moléculas inibidoras do receptor de PSMA, que carreguem agentes terapêuticos. Este trabalho pretendeu estudar de forma inédita a marcação e estabilidade radioquímica do Glu-NH-CO-NH-Lys(Ahx)-DOTA com 177Lu (PSMA-DOTA-177Lu) e avaliar seu potencial para a terapia do câncer de próstata. O radiofármaco foi obtido com pureza radioquímica elevada (PR > 95%) em todas as condições estudadas e permaneceu inalterada em ≤ - 20 °C até 48 horas, mesmo em atividade específica alta (74 MBq/μg). O ensaio de ligação específica do PSMADOTA- 177Lu mostrou que a fração do peptídeo que se ligou às células LNCaP de tumor de próstata foi de 1,79 ± 0,21 %, 2,47 ± 0,03 %, 3,07 ± 0,01 % e 4,13 ± 0,27%, para as concentrações de 0,15 x 106, 0,3 x 106, 0,5 x 106 e 1 x 106 células, respectivamente. O ensaio de internalização do PSMA-DOTA-177Lu sugere que o maior percentual da ligação específica do radiofármaco às células LNCaP corresponde à fração da ligação de superfície (99,03 ± 0,84 %). Os parâmetros farmacocinéticos determinados no estudo in vivo em camundongos Balb/c são compatíveis com o rápido clareamento sanguíneo e excreção renal, além de apresentar apreciável captação tumoral in vivo (2,76 ± 1,21 % Al/g) após 4 horas de administração do radiofármaco. Os estudos em estabilidade em soro humano demonstram estabilidade alta do radiofármaco PSMA-DOTA-177Lu por um período de até 24 horas, que foi confirmada pela baixa captação óssea demonstrada nos estudos in vivo de biodistribuição. O estudo de variação da atividade possibilitou estabelecer a atividade específica ideal (MBq/μg), que será extrapolada para um piloto de produção do radiofármaco. Os resultados favoráveis deste estudo encorajam a perspectiva de realização de ensaio clínico controlado deste novo radiofármaco, avaliando seu potencial para aplicação no tratamento do câncer de próstata. / Prostate cancer is the second most frequently worldwide diagnosed cancer among males and ranks the 15th common cause of death from cancer in men, comprising 6% of the world\'s total cancer deaths. The prostate specific membrane antigen (PSMA) is a type II transmembrane glycoprotein, which is overexpressed in prostate cancer as well as in the neovasculature of solid tumors and metastasis. These features render PSMA an ideal target for developing small molecules inhibitors of PSMA and therapeutic approaches to targeted drugs delivery. This work intended to study in an unprecedented way the radiochemical labeling and stability of Glu-NHCO- NH-Lys (Ahx)-DOTA with 177Lu (177Lu-PSMA-DOTA) and its potential anti-tumor effects in prostate cancer. The radiopharmaceutical PSMA-177Lu was obtained with high radiochemical purity (RP > 95%) under all studied conditions and remained unchanged up to 48 hours (high at ≤ -20 °C), even at high specific activity (74 MBq / μg). The cellular uptake of PSMA-DOTA-177Lu was determined using PSMAexpressing LNCaP cells and showed a binding of 1.79 ± 0.21%, 2.47 ± 0.03%, 3.07 ± 0.01% and 4.13 ± 0.27%, to 0.15 × 106, 0.3 × 106, 0.5 × 106 and 1 × 106 LNCaP cells, respectively. The PSMA-DOTA-177Lu internalization assay revealed that the membrane-bound activity (non-internalized peptide) to LNCap cells, was 99.03 ± 0.84%. Moreover, the pharmacokinetic in vivo studies performed in Scid mice resulted in a rapid blood clearance and renal excretion, and showed significant tumor uptake (2.76 ± 1.21% lA/g) 4 hours after PSMA-DOTA-177Lu administration. Stability studies in human serum demonstrated high PSMA-DOTA-177Lu stability over 24 hours, which is in agreement with the low bone uptake obtained in the in vivo biodistribution studies. Furthermore, the comparative study aiming to establish the ideal PSMA-DOTA-177Lu specific activity (MBq / μg) will be considered for further radiopharmaceutical production. The pre-clinical data obtained from this study suggested a great potential for PSMA-DOTA-177Lu to be included in clinical trials and to make a major contribution to the treatment of prostate cancer.

Atualmente os gliomas representam cerca de 81% dos tumores cerebrais malignos, com aumento na incidência tanto em crianças, como em adultos acima dos 45 anos. Um número elevado de receptores neuroquinina tipo 1 (NK-1) estão expressos em células de glioma, estando a ligação da Substância P (SP) a esses receptores, envolvida no desenvolvimento e progressão desse tipo de tumor. A SP quelada ao DOTA (SP-DOTA), radiomarcada, vem sendo testada para utilização na terapia de gliomas, sendo o lutécio-177 (177Lu), devido a seu menor alcance tecidual, o radioisótopo mais indicado para tumores localizados em áreas críticas do cérebro. No entanto, estudos indicam a necessidade da adição de um excesso de metionina para prevenção da oxidação peptídica da SP-DOTA-177Lu, visando aumentar a estabilidade e a capacidade de ligação às células tumorais. Para superar esse desafio, surge a perspectiva da utilização de um novo análogo da SP, com estrutura modificada, para prevenir a oxidação peptídica. Neste contexto, o objetivo desse trabalho foi estudar a marcação de um novo análogo da SP com 177Lu e caracterizar suas propriedades in vitro e in vivo, visando a obtenção de um radiofármaco inédito e com potencial aplicação na terapia de tumores cerebrais e realizar estudos preliminares de marcação deste novo análogo com Ítrio-90 (90Y). O novo análogo foi obtido pela troca do aminoácido metionina (Met) pelo aminoácido norleucina (Nle) na posição 11 da cadeia peptídica da SP, sendo esses peptídeos denominados respectivamente SP(Met11)-DOTA e SP(Nle11)-DOTA. Após análise da oxidação peptídica dos dois peptídeos, os parâmetros da radiomarcação da SP(Nle11)-DOTA, com 177LuCl3, foram estudados para determinar a melhor condição de marcação. As estabilidades in vitro da SP(Nle11)-DOTA-177Lu sob refrigeração (2-8°C), no freezer (-20°C) e em soro humano (37°C) foram determinadas após radiomarcação com alta atividade, quanto ao uso de agentes estabilizantes e após diluição. A SP(Nle11)-DOTA também foi radiomarcada com 90Y, utilizando-se a condição padrão determinada, sendo a estabilidade in vitro da SP(Nle11)-DOTA-90Y sob refrigeração (2-8°C) e em freezer (-20°C), avaliada após radiomarcação com alta atividade e quanto a utilização de agente estabilizante. A capacidade de ligação in vitro às células tumorais (U-87 MG e M059J) e a biodistribuição in vivo em camundongos BALB/c sadios foram determinadas para a SP(Nle11)-DOTA-177Lu e comparadas à SP(Met11)-DOTA-177Lu. A ligação às proteínas plasmáticas e a biodistribuição em camundongos Nude com modelo tumoral também foram avaliadas. Os resultados obtidos na análise da oxidação peptídica comprovaram a importância da adição de excesso de metionina para prevenção da oxidação peptídica e indicaram uma alta estabilidade da SP(Nle11)-DOTA, durante e após o processo de radiomarcação. A adição de 148 MBq (4 mCi) da solução de 177LuCl3 em HCl 0,05N à 10 μg de SP(Nle11)-DOTA diluída em tampão acetato de sódio 0,4 M pH 4,5 seguida pela incubação a uma temperatura de 90ºC por 30 minutos, sob agitação de 350 rpm foi definida com condição padrão de marcação. O congelamento (-20°C), o uso de agentes estabilizantes e a diluição apresentaram-se como métodos efetivos para garantir uma alta estabilidade in vitro da SP(Nle11)-DOTA-177Lu, após a marcação com alta atividade. Bons resultados também foram observados para a marcação da SP(Nle11)-DOTA com 90YCl3 e para a estabilidade in vitro da SP(Nle11)-DOTA-90Y, após congelamento (-20°C) e quando utilizado ácido gentísico como estabilizante. A SP(Nle11)-DOTA-177Lu apresentou uma boa especificidade pelas células tumorais, principalmente pelas células de glioma humano M059J, sugerindo que a substituição do aminoácido metionina por norleucina na posição 11 não compromete a capacidade de ligação da SP(Nle11) às células tumorais. Uma baixa porcentagem de ligação às proteínas plasmáticas e um rápido clareamento sanguíneo foram observados para a SP(Nle11)-DOTA-177Lu, sendo esse radiofármaco eliminado preferencialmente por via renal. A SP(Nle11)-DOTA-177Lu apresentou uma boa estabilidade in vivo e se mostrou incapaz de atravessar a barreira hematoencefálica, sendo seu uso indicado por injeção intratumoral ou intracavitária. O estudo de biodistribuição em animais com modelo tumoral, mostrou que esse radiofármaco se liga às células tumorais por ligações receptor específicas. Com base nesse dados conclui-se que a SP(Nle11)-DOTA-177Lu, apresenta-se como um radiofármaco inédito que devido às suas propriedades in vitro e in vivo favoráveis, apresenta potencial aplicação na terapia de tumores cerebrais, representando uma nova possibilidade dentro do limitado arsenal terapêutico para esse tipo de tumor. / Currently gliomas represent about 81% of malignant brain tumors with increased incidence in children and in adults over 45 years. A large number of type 1 neurokinin receptor (NK-1) are expressed in glioma cells, being the binding of substance P (SP) to these receptors, involved in the development and progression of this tumor type. The SP conjugated at DOTA chelator (SP-DOTA), radiolabeled, have been tested for use in the treatment of gliomas, and the lutetium-177 (177 Lu), due to its lower tissue range, has been the most suitable radioisotope for tumors located in critical areas brain. However, studies indicate the necessity of adding an excess of methionine to prevent the peptide SP-DOTA-177Lu oxidation in order to increase the stability and capacity to bind to tumor cells. To overcome this challenge, there is the prospect of using a new analog of SP with a modified structure, to prevent peptide oxidation. In this context, the aim of this work was study the labeling of a new analog of SP with 177Lu and characterize their properties in vitro and in vivo, in order to obtain a novel radiopharmaceutical with potential application in brain tumor therapy, and perform preliminary studies labeling of this new analog with yttrium-90 (90Y). The new analog was obtained by replacement of the amino acid methionine (Met) by the amino acid norleucine (Nle) at position 11 of the peptide chain of SP, and these peptides were called SP(Met11)-DOTA and SP(Nle11)-DOTA respectively. After analysis of the oxidation for the two peptides, the radiolabeling parameters of the SP(Nle11)-DOTA with 177LuCl3 were studied to determine the best labeling condition. The SP(Nle11)-DOTA was also radiolabeled with 90Y, using standard condition, and the stability in vitro of the SP(Nle11)-DOTA-90Y assessed under refrigeration (2-8 °C) and under freezing (-20° C), after radiolabeling with high activity and use of stabilizing agent. The stabilities in vitro of the SP (Nle11)-DOTA-177Lu under refrigeration (2-8 °C), under freezing (-20 °C) and in human serum (37 °C) were determined after radiolabeling with high activity, with use of stabilizing agents and after dilution. The ability of in vitro binding to tumor cells (U-87 MG and M059J) and the biodistribution in vivo in healthy BALB/c mice were determined for the 177Lu-DOTA-SP(Nle11) and compared to 177Lu-DOTA-SP(Met11). The plasma protein binding and biodistribution in Nude mice with tumor model were also evaluated. The results obtained from analysis of oxidation for the two peptides confirmed the importance of adding excess methionine to prevent peptide oxidation and indicated a high stability of the DOTA- SP(Nle11), during and after the radiolabeling process. The addition of 148 MBq (4 mCi) of 177LuCl3 solution in 0.05N HCl at 10 μg DOTA-SP(Nle11) diluted in 0.4 M sodium acetate buffer pH 4.5 followed by incubation at a temperature of 90 °C for 30 minutes under constant agitation to 350 rpm was defined as standard labeling condition. The freezing (-20 °C), the use of stabilizing agents and the dilution were presented as effective methods to ensure high stability in vitro 177Lu-DOTA-SP(Nle11), after labeling with high activity. Good results were also observed for labeling DOTA-SP(Nle11) with 90YCl3 and for stability in vitro of the 90Y-DOTA-SP(Nle11) after freezing (-20 °C) and when gentisic acid was used as a stabilizer. The 177Lu-DOTA-SP(Nle11) showed good specificity to tumor cells, particularly human glioma cells (M059J), suggesting that substitution of the amino acid norleucine for methionine at position 11 does not compromise the capacity of SP(Nle11) binding to tumor cells. A low percentage of plasma protein binding and rapid blood clearance were observed for the 177Lu-DOTA-SP(Nle11), being this radiopharmaceutical preferably eliminated by the kidney. The 177Lu-DOTA-SP(Nle11) showed good stability in vivo and inability to cross the blood brain barrier, being its use indicated through intratumoral or intracavitary injection. The biodistribution studies in animals with tumor model showed that the radiopharmaceutical binds to the tumor cells by specific receptor binding. Based on this data was concluded that the 177Lu-DOTA-SP(Nle11), can be presented as a novel radiopharmaceutical that due to its favorable properties in vitro and in vivo, presents a potential application in the therapy of brain tumors, representing a new possibility within the limited therapeutic options for this type of tumor.

To address the unmet clinical need to eradicate treatment-resistant CD133+ cancer stems within tumors, a CSC-targeted radionuclide therapy (TRT) and companion diagnostic imaging probes were developed utilizing CD133-targeting antibodies and antibody fragments. In Chapter 1, background research providing context for the work in this Thesis is presented. In Chapter 2, a CD133-targeting antibody, RW03IgG, underwent radiolabeling with lutetium-177 to synthesize [177Lu]Lu-DOTA-RW03IgG for CD133-TRT. The CD133-TRT was evaluated for pharmacokinetics and treatment of a CD133 expressing human colorectal tumor bearing mouse model. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG demonstrated notable uptake in the colorectal tumors and off-target organ uptake consistent with previously reported antibody-based TRTs. Confirmation that tumor uptake was mediated by antibody-antigen binding was verified through co-injection with an excess dose of unlabeled RW03IgG. A dose-escalation therapy trial using [177Lu]Lu-DOTA-RW03IgG for treatment of the colorectal cancer mouse model revealed a dose-dependent reduction in tumor growth rate at well-tolerated doses. The decrease in tumor growth rate observed due to [177Lu]Lu-DOTA-RW03IgG treatment, along with an improvement in overall mouse survival, demonstrate the therapeutic efficacy of CD133-TRT. Additionally, histopathological and immunohistochemical (IHC) analyses indicated low off-target organ toxicity and significant anti-tumor effects. These findings suggested the potential for enhanced overall survival benefits through multiple doses. However, results on multiple-dosed CD133-TRT on the tumor growth rate and overall mouse survival were inconclusive. In Chapter 3, an orthotopic patient-derived glioblastoma (GBM) mouse model was developed that replicates anatomical pharmacokinetic challenges and CSC populations observed in patient tumors. Stereotactic engraftment of the patient GBM cells was optimized to reproducibly deliver tumor cells to the thalamus and growth was monitored using bioluminescence imaging. Ex vivo analysis confirmed various key characteristics of patient GBM, including CD133 expression, hypercellularity, and invasiveness. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG using the PDX GBM mouse model indicate antibody-antigen driven tumor uptake, determined through co-injection an excess dose of unlabeled RW03IgG. Ex vivo autoradiography supported the biodistribution results and showed elevated uptake of [177Lu]Lu-DOTA-RW03IgG in tumor relative to non-tumor bearing brain tissue. Chapters 4 and 5 centered on the development and evaluation of companion diagnostic CD133-targeted immunoPET probes. Chapter 4 specifically explored probes derived from the full antibody, RW03IgG. The probes were synthesized by conjugating RW03IgG with DFO-NCS to produce DFO-RW03IgG at different chelator-to-antibody ratios. The various DFO-RW03IgG conjugates were then radiolabeled with zirconium-89 to obtain [89Zr]-DFO-RW03IgG. Biodistribution studies and PET imaging revealed promising tumor uptake of [89Zr]-DFO-RW03IgG, and it was observed that higher chelator-to-antibody ratios led to increased accumulation in off-target organs. Chapter 5 investigated a probe derived from an scFv-Fc fragment of RW03, [89Zr]-DFO-RW03scFv-Fc. Biodistribution studies and PET images of colorectal tumor-bearing mice administered [89Zr]-DFO-RW03scFv-Fc showed favorable tumor uptake and low off-target organ accumulation. In Chapter 6, a probe for CD133-Photoacoustic Imaging (PAI) was synthesized through conjugation of RW03IgG with IR-783, an organic dye recognized for its favorable photoacoustic properties. Challenges were encountered in isolating the product, (IR-783)-RW03IgG, at high degrees of labeling (DOL) due to product aggregation. In vitro binding assays indicated that (IR-783)-RW03IgG (DOL = 1) maintained a comparable binding affinity to native RW03IgG. In vivo, colorectal tumors in mice administered (IR-783)-RW03IgG (DOL = 1) did not exhibit significant contrast from the background tissue, and the tumor PA signal did not differ significantly compared to tumors in mice administered an IR-783 labeled isotype IgG. The results suggest that a higher concentration of dye is needed within colorectal tumors for effective tumor visualization than what was provided by IR-783-RW03IgG. Chapter 7 investigated the use of Imaging Mass Cytometry (IMC) to simultaneously visualize [177Lu]Lu-DOTA-RW03IgG and multiple tumor biomarkers in tissue specimens collected from colorectal tumor xenograft mice treated with CD133-TRT. IMC showed undetectable concentrations of hafnium-177 (the decay product of lutetium-177) in tumors treated with CD133-TRT. However, lutetium-176 and lutetium-175, sourced from the carrier-added [177Lu]LuCl3 used in the synthesis of [177Lu]Lu-DOTA-RW03IgG, were present at levels sufficient for IMC visualization. The distribution of lutetium-176, representing [177Lu]Lu-DOTA-RW03IgG, within tumors, was imaged concomitantly with CD133, DNA damage markers, and several additional biomarkers that describe elements of the tumor microenvironment. These collective results endorse IMC as a useful tool to assess the distribution of TRT within tumors and uncover changes to the microenvironment in response to treatment. / Thesis / Doctor of Philosophy (PhD) / Targeted radionuclide therapy (TRT) and molecular imaging strategies were developed to aid in the elimination of the rare and particularly resilient Cancer Stem Cell (CSC) population in tumors. A fully human monoclonal antibody and antibody fragments targeting CD133, a molecular biomarker for CSCs, provided the means to deliver radioactive isotopes for therapy and imaging to CD133+ cells in tumors. The therapeutic efficacy of CD133-TRT for treatment of a colorectal cell line-derived xenograft mouse model was promising, and the treatment showed uptake in orthotopic patient derived glioblastoma tumors engrafted in mice. ImmunoPET probes targeting CD133 were optimized and successfully delineated CD133 expressing tumors from background tissue, warranting further evaluation using patient-representative cancer models. A non-invasive CD133-targeting Photoacoustic Imaging (PAI) probe was synthesized through conjugation of the CD133-targeting antibody to an organic dye, IR-783, although further probe optimization is required to provide tumor contrast. Tumor specimens from mice treated with CD133-TRT were assessed by Imaging Mass Cytometry (IMC), which revealed detectable concentrations of carrier isotopes from the therapy in the tumors, implicating the discovery of a powerful new tool for multiplexed single-cell level resolution imaging for cellular-scale analysis of targeted radionuclide therapy. The CSC-therapy and select molecular imaging probes generated in this Thesis warrant further evaluation using patient-representative mouse models of cancer.