Assessment of bioactive peptides derived from laminin-111 as prospective breast cancer-targeting agents.

Breast cancer remains a pressing public health issue primarily affecting women. Recent research has spotlighted bioactive peptides derived from laminin-111, implicated in breast tumor development. Remarkably, the sequences IKVAV, YIGSR, and KAFDITYVRLKF from the α1, ß1, and γ1 chains, respectively, have garnered significant attention. This study aims to assess the potential of these radiolabeled peptides as targeting agents for breast cancer. The three peptides were synthesized using the Fmoc strategy, purified via reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized through mass spectrometry. Iodine-131 (131I) radiolabeling was performed using the chloramine T method, exhibiting high radiochemical yield and stability for [131I]I-YIKVAV and [131I]I-YIGSR. Conversely, [131I]I-KAFDITYVRLKF demonstrated low radiochemical yield and stability and was excluded from the biological studies. The lipophilicity of the compounds ranged from - 2.12 to - 1.10. Serum protein binding assay for [131I]I-YIKVAV and [131I]I-YIGSR reached ≅ 48% and ≅ 25%, respectively. Affinity for breast cancer cells was evaluated using MDA-MB-231 and MCF-7 tumor cell lines, indicating the affinity of the radiopeptides with these tumor cells. Ex vivo biodistribution profiles of the radiopeptides were assessed in the MDA-MB-231 breast tumor animal model, revealing tumor tissue accumulation, supported by a high tumor-to-contralateral muscle ratio and autoradiography. These results signify the effective penetration of YIKVAV and YIGSR into tumor tissue. Therefore, the synthesized α1 and ß1 peptide fragments exhibit favorable characteristics as potential breast cancer-targeting agents, promising future exploration as radiopharmaceuticals for breast cancer.

Anti-HER2 monoclonal antibody based-radioimmunoconjugates: Assessment of the chelating agent influence.

In the present work, the radioimmunoconjugates 111In-DTPA-trastuzumab and 177Lu-DOTA-trastuzumab were evaluated regarding the influence of the chelating agents on the physical-chemical parameters and human epidermal growth factor receptor 2 (HER2) tumor cell binding. Data showed that both chelating agents, at predetermined molar ratios (antibody:chelator - 1:10 and 1:20), did not influence the immunoconjugates integrity, the radiolabeling process and the radiolabeled antibodies stability. However, differences were observed in the lipophilic feature between DOTA and DTPA radioimmunoconjugates and in the specific binding to SK-BR-3 tumor cells (HER2 positive). Therefore, this study showed the importance of assessing the influence of chelating agents and their molar ratios in the development process of radioimmunoconjugates.

Radiosynthesis Standardization and Preclinical Assessment of the [68Ga]Ga-DOTA-Ubiquicidin29-41: A Translational Study Targeting Differential Diagnosis of Infectious Processes.

Human bacterial infections significantly contribute to the increase in healthcare-related burdens. This scenario drives the study of novel techniques for the early and precise diagnosis of infectious processes. Some alternatives include Nuclear Medicine- and Molecular Imaging-based strategies. However, radiopharmaceuticals that are available for routine assessments are not specific to differentiating infectious from aseptic inflammatory processes. In this context, [68Ga]Ga-DOTA-Ubiquicidin29-41 was synthesized using an automated module and radiochemical; in vivo and in vitro studies were performed. The radiopharmaceutical remained stable in saline (up to 180 min) and in rodent serum (up to 120 min) with radiochemical purities > 99 and 95%, respectively. Partition coefficient and serum protein binding at 60 min were determined (-3.63 ± 0.17 and 44.06 ± 1.88%, respectively). Ex vivo biodistribution, as well as in vivo microPET/CT images in mice, showed rapid blood clearance with renal excretion and reduced uptake in other organs in Staphylococcus aureus-infected animals. Higher uptake was observed in the target as compared to the non-target tissue (p < 0.0001) at 60 min post administration. The presented in-human clinical case demonstrates uptake of the radiopharmaceutical by Staphyloccocus aureus bacteria. These results indicate the potential of [68Ga]Ga-DOTA-Ubiquicidin29-41 as a radiopharmaceutical that can be obtained in a hospital radiopharmacy for the diagnosis of infectious processes using PET/CT.

Radiochemical and biological assessments of a PSMA-I&S cold kit for fast and inexpensive 99mTc-labeling for SPECT imaging and radioguided surgery in prostate cancer.

The expression of prostate-specific membrane antigen (PSMA) is upregulated in prostate cancer (PCa) cells and PSMA-ligands have been radiolabeled and used as radiopharmaceuticals for targeted radionuclide therapy (TRT), single photon emission computed tomography (SPECT) or positron emission tomography (PET) molecular imaging, and radioguided surgery in PCa patients. Herein, we aimed at radiolabeling the PSMA-I&S cold kit with 99mTc, resulting in a radiopharmaceutical with high radiochemical yield (RCY) and stability for SPECT imaging and radioguided surgery in PCa malignancies. Various pre-clinical assays were conducted to evaluate the [99mTc]Tc-PSMA-I&S obtained by the cold kit. These assays included assessments of RCY, radiochemical stability in saline, lipophilicity, serum protein binding (SPB), affinity for LNCaP-PCa cells (binding and internalization studies), and ex vivo biodistribution profile in naive and LNCaP-PCa-bearing mice. The radiopharmaceutical was obtained with good RCY (92.05% ± 2.20%) and remained stable for 6 h. The lipophilicity was determined to be -2.41 ± 0.06, while the SPB was ∼97%. The binding percentages to LNCaP cells were 9.41% ± 0.57% (1 h) and 10.45% ± 0.45% (4 h), with 63.12 ± 0.93 (1 h) and 65.72% ± 1.28% (4 h) of the bound material being internalized. Blocking assays, employing an excess of unlabeled PSMA-I&S, resulted in a reduction in the binding percentage by 2.6 times. The ex vivo biodistribution profile confirmed high accumulation of [99mTc]Tc-PSMA-I&S in the tumor and the tumor-to-contralateral muscle ratio was ∼6.5. In conclusion, [99mTc]Tc-PSMA-I&S was successfully obtained by radiolabeling the cold kit using freshly eluted [99mTc]NaTcO4, exhibiting good RCY and radiochemical stability. The preclinical assays demonstrated that the radiopharmaceutical shows favorable characteristics for SPECT imaging and radioguided surgery in PCa patients.

Radioimmunotheranostic Pair Based on the Anti-HER2 Monoclonal Antibody: Influence of Chelating Agents and Radionuclides on Biological Properties.

The oncogene HER2 is an important molecular target in oncology because it is associated with aggressive disease and the worst prognosis. The development of non-invasive imaging techniques and target therapies using monoclonal antibodies is a rapidly developing field. Thus, this work proposes the study of the radioimmunotheranostic pair, [111In]In-DTPA-trastuzumab and [177Lu]Lu-DOTA-trastuzumab, evaluating the influence of the chelating agents and radionuclides on the biological properties of the radioimmunoconjugates (RICs). The trastuzumab was immunoconjugated with the chelators DTPA and DOTA and radiolabeled with [111In]InCl3 and [177Lu]LuCl3, respectively. The stability of the RICs was evaluated in serum, and the immunoreactive and internalization fractions were determined in SK-BR-3 breast cancer cells. The in vivo pharmacokinetics and dosimetry quantification and the ex vivo biodistribution were performed in normal and SK-BR-3 tumor-bearing mice. The data showed that there was no influence of the chelating agents and radionuclides on the immunoreactive and internalization fractions of RICs. In contrast, they influenced the stability of RICs in serum, as well as the pharmacokinetics, dosimetry and biodistribution profiles. Therefore, the results showed that the nature of the chelating agent and radionuclide could influence the biological properties of the radioimmunotheranostic pair.

Comparative overview of innovation and patent filings in Radiopharmacy.

OBJECTIVE: To expose the current situation of the Brazilian Nuclear Medicine in relation to innovation, taking into account the Intellectual Property protection and the particularities of this field. METHODS: The number and the origin of patents filings from Brazil, United States and European Patent Convention countries were retrospectively compared in a 20-year period. RESULTS: The number of accumulated patents filings of conventional pharmaceuticals was ten times higher compared to the radiopharmaceuticals in the three regions studied. CONCLUSION: The largest number of Brazilian patents filings corresponded to the international patent applications, which is related to the country development conditions, as well as to the difficulties in the process of patent filing.

Kinetic modeling of 68Ga-PSMA-11 and validation of simplified methods for quantification in primary prostate cancer patients.

BACKGROUND: The positron emission tomography (PET) ligand 68Ga-Glu-urea-Lys(Ahx)-HBED-CC (68Ga-PSMA-11) targets the prostate-specific membrane antigen (PSMA), upregulated in prostate cancer cells. Although 68Ga-PSMA-11 PET is widely used in research and clinical practice, full kinetic modeling has not yet been reported nor have simplified methods for quantification been validated. The aims of our study were to quantify 68Ga-PSMA-11 uptake in primary prostate cancer patients using compartmental modeling with arterial blood sampling and to validate the use of standardized uptake values (SUV) and image-derived blood for quantification. RESULTS: Fifteen patients with histologically proven primary prostate cancer underwent a 60-min dynamic 68Ga-PSMA-11 PET scan of the pelvis with axial T1 Dixon, T2, and diffusion-weighted magnetic resonance (MR) images acquired simultaneously. Time-activity curves were derived from volumes of interest in lesions, normal prostate, and muscle, and mean SUV calculated. In total, 18 positive lesions were identified on both PET and MR. Arterial blood activity was measured by automatic arterial blood sampling and manual blood samples were collected for plasma-to-blood ratio correction and for metabolite analysis. The analysis showed that 68Ga-PSMA-11 was stable in vivo. Based on the Akaike information criterion, 68Ga-PSMA-11 kinetics were best described by an irreversible two-tissue compartment model. The rate constants K1 and k3 and the net influx rate constants Ki were all significantly higher in lesions compared to normal tissue (p < 0.05). Ki derived using image-derived blood from an MR-guided method showed excellent agreement with Ki derived using arterial blood sampling (intraclass correlation coefficient = 0.99). SUV correlated significantly with Ki with the strongest correlation of scan time-window 30-45 min (rho 0.95, p < 0.001). Both Ki and SUV correlated significantly with serum prostate specific antigen (PSA) level and PSA density. CONCLUSIONS: 68Ga-PSMA-11 kinetics can be described by an irreversible two-tissue compartment model. An MR-guided method for image-derived blood provides a non-invasive alternative to blood sampling for kinetic modeling studies. SUV showed strong correlation with Ki and can be used in routine clinical settings to quantify 68Ga-PSMA-11 uptake.

A new model of experimental hemispherotomy in young adult Rattus norvegicus: a neural tract tracing and SPECT in vivo study.

OBJECTIVE: The objective of this study was to describe a new experimental model of hemispherotomy performed on laboratory animals. METHODS: Twenty-six male young adult Wistar rats were distributed into two groups (surgery and control). The nonfluorescent anterograde neurotracer biotinylated dextran amine (BDA; 10,000 MW) was microinjected into the motor cortex area (M1) according to The Rat Brain in Stereotaxic Coordinates atlas to identify pathways and fibers disconnected after the experimental hemispherectomy. SPECT tomographic images of 99mTc hexamethylpropyleneamine oxime were obtained to verify perfusion in functioning areas of the disconnected and intact brain. A reproducible and validated surgical procedure is described in detail, including exact measurements and anatomical relationships. An additional 30 rodents (n = 10 rats per group) were divided into naïve, sham, and hemispherotomy groups and underwent the rotarod test. RESULTS: Cortico-cortical neural pathways were identified crossing the midline and contacting neuronal perikarya in the contralateral brain hemisphere in controls, but not in animals undergoing hemispherotomy. There was an absence of perfusion in the left side of the brain of the animals undergoing hemispherotomy. Motor performance was significantly affected by brain injuries, increasing the number of attempts to maintain balance on the moving cylinder in the rotarod test at 10 and 30 days after the hemispherotomy, with a tendency to minimize the motor performance deficit over time. CONCLUSIONS: The present findings show that the technique reproduced neural disconnection with minimal resection of brain parenchyma in young adult rats, thereby duplicating the hemispherotomy procedures in human patients.

Preclinical molecular imaging: development of instrumentation for translational research with small laboratory animals.

OBJECTIVE:: To present the result of upgrading a clinical gamma-camera to be used to obtain in vivo tomographic images of small animal organs, and its application to register cardiac, renal and neurological images. METHODS:: An updated version of the miniSPECT upgrading device was built, which is composed of mechanical, electronic and software subsystems. The device was attached to a Discovery VH (General Electric Healthcare) gamma-camera, which was retired from the clinical service and installed at the Centro de Imagem Pré-Clínica of the Hospital Israelita Albert Einstein. The combined system was characterized, determining operational parameters, such as spatial resolution, magnification, maximum acceptable target size, number of projections, and acquisition and reconstruction times. RESULTS:: Images were obtained with 0.5mm spatial resolution, with acquisition and reconstruction times between 30 and 45 minutes, using iterative reconstruction with 10 to 20 iterations and 4 projection subsets. The system was validated acquiring in vivo tomographic images of the heart, kidneys and brain of normal animals (mice and adult rats), using the radiopharmaceuticals technetium-labeled hexakis-2-methoxy-isobutyl isonitrile (99mTc-Sestamibi), technetium-labeled dimercaptosuccinic acid (99mTc-DMSA) and technetium-labeled hexamethyl propyleneamine oxime (99mTc-HMPAO). CONCLUSION:: This kind of application, which consists in the adaptation for an alternative objective of already existing instrumentation, resulted in a low-cost infrastructure option, allowing to carry out large scale in vivo studies with enhanced quality in several areas, such as neurology, nephrology, cardiology, among others. OBJETIVO:: Apresentar o resultado da adaptação de uma gama câmara clínica para uso dedicado na obtenção de imagens tomográficas in vivo de órgãos de pequenos animais de experimentação, e de sua aplicação na obtenção de imagens cardíacas, renais e neurológicas. MÉTODOS:: Foi construída uma versão atualizada do dispositivo de adaptação miniSPECT, composto por três subsistemas: mecânico, eletrônico e de software. O dispositivo foi montado em uma câmara Discovery VH da General Electric Healthcare, retirada do serviço clínico e instalada no Centro de Imagem Pré-Clínica do Hospital Israelita Albert Einstein. O sistema combinado foi caracterizado, determinando parâmetros de funcionamento como resolução espacial, magnificação, limites de tamanho dos alvos de estudo, número de projeções, tempo de registro e tempo de reconstrução das imagens tomográficas. RESULTADOS:: Foram obtidas imagens com resolução espacial de até 0,5mm, com tempos de registro e reconstrução de 30 a 45 minutos, utilizando reconstrução iterativa com 10 a 20 iterações e 4 subconjuntos de projeções. O sistema foi validado obtendo imagens tomográficas in vivo do coração, dos rins e do cérebro de animais normais (camundongos e ratos adultos), utilizando os radiofármacos hexaquis-2-metoxi-isobutil-isonitrila marcado com 99mTc (Sestamibi-99mTc), ácido dimercaptosuccínico marcado com 99mTc (DMSA-99mTc) e hexametil-propileno-amina-oxima marcada com 99mTc (HMPAO-99mTc). CONCLUSÃO:: Este tipo de aplicação, que consiste na adaptação para um objetivo alternativo de instrumentação já existente, constituiu-se em uma opção de infraestrutura de baixo custo, que permite realizar estudos in vivo em larga escala, com qualidade aprimorada, em áreas diversas, como neurologia, nefrologia, cardiologia, entre outras.

Comparative overview of innovation and patent filings in Radiopharmacy / Panorama comparativo da inovação e do depósito de patentes em Radiofarmácia

ABSTRACT Objective To expose the current situation of the Brazilian Nuclear Medicine in relation to innovation, taking into account the Intellectual Property protection and the particularities of this field. Methods The number and the origin of patents filings from Brazil, United States and European Patent Convention countries were retrospectively compared in a 20-year period. Results The number of accumulated patents filings of conventional pharmaceuticals was ten times higher compared to the radiopharmaceuticals in the three regions studied. Conclusion The largest number of Brazilian patents filings corresponded to the international patent applications, which is related to the country development conditions, as well as to the difficulties in the process of patent filing.

RESUMO Objetivo Evidenciar a situação atual da Medicina Nuclear brasileira em relação à inovação, considerando a proteção à Propriedade Intelectual e suas particularidades na área. Métodos Foi realizado um levantamento comparativo do número e da origem de depósitos de patentes relacionadas a essa área no Brasil, nos Estados Unidos e em países participantes da European Patent Convention , em um período de 20 anos retroativos. Resultados O número de depósitos acumulados de patentes de medicamentos convencionais foi dez vezes maior quando comparado às famílias relacionadas aos processos que envolvem radiofármacos, nas três regiões estudadas. Conclusão O maior número de depósitos de patentes brasileiras correspondeu aos pedidos de patentes internacionais, refletindo as condições de desenvolvimento do país, bem como as dificuldades encontradas no processo de depósito de uma patente.

Current Approach in Radiochemical Quality Control of the 99mTc-Radiopharmaceuticals: a mini-review

Abstract To present optimized chromatographic systems for radiochemical purity (RCP) evaluation of 99mTc-eluate and 99mTc-radiopharmaceuticals, as well as to assess doses calibrator reliability for routine purposes in hospital radiopharmacies. RCP was determined by different systems and radioactivity was quantified by TLC-scanner, doses calibrator and gamma-counter. Suitable and optimized systems were presented for RCP analyses. No significant differences were observed between radioactivity counting devices and, thus, doses calibrator showed reliability for RCP determination in hospital radiopharmacies.

Preclinical molecular imaging: development of instrumentation for translational research with small laboratory animals / Imagem molecular pré-clínica: desenvolvimento de instrumentação para pesquisa translacional com pequenos animais

ABSTRACT Objective: To present the result of upgrading a clinical gamma-camera to be used to obtain in vivo tomographic images of small animal organs, and its application to register cardiac, renal and neurological images. Methods: An updated version of the miniSPECT upgrading device was built, which is composed of mechanical, electronic and software subsystems. The device was attached to a Discovery VH (General Electric Healthcare) gamma-camera, which was retired from the clinical service and installed at the Centro de Imagem Pré-Clínica of the Hospital Israelita Albert Einstein. The combined system was characterized, determining operational parameters, such as spatial resolution, magnification, maximum acceptable target size, number of projections, and acquisition and reconstruction times. Results: Images were obtained with 0.5mm spatial resolution, with acquisition and reconstruction times between 30 and 45 minutes, using iterative reconstruction with 10 to 20 iterations and 4 projection subsets. The system was validated acquiring in vivo tomographic images of the heart, kidneys and brain of normal animals (mice and adult rats), using the radiopharmaceuticals technetium-labeled hexakis-2-methoxy-isobutyl isonitrile (99mTc-Sestamibi), technetium-labeled dimercaptosuccinic acid (99mTc-DMSA) and technetium-labeled hexamethyl propyleneamine oxime (99mTc-HMPAO). Conclusion: This kind of application, which consists in the adaptation for an alternative objective of already existing instrumentation, resulted in a low-cost infrastructure option, allowing to carry out large scale in vivo studies with enhanced quality in several areas, such as neurology, nephrology, cardiology, among others.

RESUMO Objetivo: Apresentar o resultado da adaptação de uma gama câmara clínica para uso dedicado na obtenção de imagens tomográficas in vivo de órgãos de pequenos animais de experimentação, e de sua aplicação na obtenção de imagens cardíacas, renais e neurológicas. Métodos: Foi construída uma versão atualizada do dispositivo de adaptação miniSPECT, composto por três subsistemas: mecânico, eletrônico e de software. O dispositivo foi montado em uma câmara Discovery VH da General Electric Healthcare, retirada do serviço clínico e instalada no Centro de Imagem Pré-Clínica do Hospital Israelita Albert Einstein. O sistema combinado foi caracterizado, determinando parâmetros de funcionamento como resolução espacial, magnificação, limites de tamanho dos alvos de estudo, número de projeções, tempo de registro e tempo de reconstrução das imagens tomográficas. Resultados: Foram obtidas imagens com resolução espacial de até 0,5mm, com tempos de registro e reconstrução de 30 a 45 minutos, utilizando reconstrução iterativa com 10 a 20 iterações e 4 subconjuntos de projeções. O sistema foi validado obtendo imagens tomográficas in vivo do coração, dos rins e do cérebro de animais normais (camundongos e ratos adultos), utilizando os radiofármacos hexaquis-2-metoxi-isobutil-isonitrila marcado com 99mTc (Sestamibi-99mTc), ácido dimercaptosuccínico marcado com 99mTc (DMSA-99mTc) e hexametil-propileno-amina-oxima marcada com 99mTc (HMPAO-99mTc). Conclusão: Este tipo de aplicação, que consiste na adaptação para um objetivo alternativo de instrumentação já existente, constituiu-se em uma opção de infraestrutura de baixo custo, que permite realizar estudos in vivo em larga escala, com qualidade aprimorada, em áreas diversas, como neurologia, nefrologia, cardiologia, entre outras.