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.

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.

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.

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.

Development of radioimmunoconjugate for diagnosis and management of head-and-neck subclinical cancer and colorectal carcinoma

ABSTRACT Scientific innovations in diagnostic methods are important drivers of cancer control and prevention. Noninvasive imaging of the epidermal growth factor receptor (EGFR) in head-and-neck squamous, cell carcinoma and colorectal cancer could be valuable to select patients for EGFR-targeted therapy, as well as to monitor the efficacy and occurrence of resistance to immunotherapy. In order to develop the first Brazilian radioimmunoconjugate for diagnosis, Cetuximab has been conjugated to p-SCN-Bn-DTPA chelator and radiolabeled with Indium-111. The conjugation methodology was optimized using different mAb:DTPA molar ratios, time was then reduced for immunoconjugate preparation, besides the protein recovery' percentage increased after purification (m = 83.8 ± 0.91 %). The stability of Cetuximab-DTPA at - 20 oC was evaluated for six months, and its integrity was greater than 90% (m =93.9 ± 1.5%, N = 24). The radioimmunoconjugate with specific activity of 185 MBq/mg showed radiochemical purity above 95% (m=96.8 ± 1.31 %, N = 15). We conclude that the radioimmunoconjugate 111In-DTPA-cetuximab is stable and may be applied to the diagnosis of EGFR-positive tumors.

Diretriz para Cintilografia de Perfusão Miocárdica de Repouso e Estresse / Guideline for Rest and Stress Myocardial Perfusion Scintigraphy

Esse artigo trata das Diretrizes da Cintilografia Miocárdica de Repouso e Esforço. Foi desenvolvido e escrito pela Sociedade Brasileira de Medicina Nuclear para que sirva como guia das melhores práticas usadas em Medicina Nuclear. Sua função é ser uma ferramenta educacional para ajudar os serviços de Medicina Nuclear do Brasil a garantirem um atendimento de qualidade aos pacientes

Application of 17% EDTA Enhances Diffusion of (45)Ca-labeled OH(-) and Ca(2+) in Primary Tooth Root Canal.

Proper cleaning of the root canal is key to the success of endodontic treatment as it allows more effective diffusion of medication throughout the dentinal tubules. The aim of this in vitro study was to investigate the efficacy of 17% ethylenediaminetetraacetic acid (EDTA) in enhancing diffusion of hydroxyl (OH(-)) and calcium ions (Ca(2+)) throughout the root canal in primary teeth. The canals of 25 primary tooth roots were cleaned with endodontic files and 1% sodium hypochlorite. Three groups (G) were then established: GI, in which final irrigation was performed with 1% sodium hypochlorite; GII, in which 17% EDTA was used; and GIII, in which no irrigation was performed. The roots canals in GI and GII were filled with a calcium hydroxide-based paste labeled with the radioisotope calcium-45. Diffusion of OH(-) was detected with pH strips and Ca(2+) analyzed by measuring radioactivity in counts per min. Group II differed statistically from the other groups in diffusion of OH(-) at 24 hr (p<0.05), but no significant difference among groups was found at the day 7 evaluation; GII also differed statistically from the other groups in diffusion of Ca(2+) at 24 hr (p<0.05). These results suggest that application of 17% EDTA in primary tooth enhances diffusion of OH(-) and Ca(2+).

Garantia da qualidade aplicada à produção de radiofármacos: [revisão] / Quality assurance in radiopharmaceutical production: [review]

Radiofármacos são produzidos e distribuídos no Brasil há mais de 40 anos pelos Institutos da Comissão Nacional de Energia Nuclear (CNEN), particularmente o Instituto de Pesquisas Energéticas e Nucleares (IPEN), para uso em procedimentos diagnósticos e terapêuticos em Medicina Nuclear. Alguns aspectos da produção, distribuição e utilização dos radiofármacos são bastante particulares, diferenciando-se dos fármacos convencionais, tornando necessário estabelecer regulamentação específica para tais produtos. Neste sentido, existem orientações da Organização Mundial de Saúde (OMS) bem como regulamentações de órgãos sanitários de diversos países que já fazem distinção aos radiofármacos nas legislações específicas.

Radiopharmaceuticals have been produced and distributed in Brazil for almost 40 years, by the Institutes of the Comissão Nacional de Energia Nuclear (CNEN) particularly the Instituto de Pesquisas Energéticas e Nucleares (IPEN), and applied in Nuclear Medicine in diagnostic and therapeutic procedures. Some aspects related to the production, distribution and use of radiopharmaceuticals are very uncommon and different from the conventional drugs, making necessary the introduction of an specific regulation for these radioactive drugs. In this way, the World Health Organization (WHO) and health regulatory agencies from different countries have specific legislations to radiopharmaceuticals production and use.

Comparative biodistribution profile of [131I]VIP and [131I]VIP10-28

Various tumor cells express significantly higher amounts of VIP receptors (VIPR) that provided the basis for the clinical use of radiolabeled VIP for the in vivo localization of tumors. This work studied the labeling of VIP and VIP10-28 with iodine-131 to compare the biological distribution of the labeled compounds in Nuce mice and the affinity for tumor cells. Both VIP and VIP10-28 peptides contain two tyrosine residues, in positions 10 and 22, that are theoretically equally susceptible to radioiodination employing oxidative electrophilic substitution using oxidizing agents like Chloramine T. Radiochemical purity of the reaction mixture was determined by electrophoresis and HPLC. The VIP peptide and the fragment were labeled with radioiodine with good radiochemical yield (above 96 percent). Suitable, but important differences can be observed in biological distribution studies. Comparatively, blood clearance was faster for labeled VIP and perhaps because of this, the uptake in tumor was lower, especially during the first hour. These differences observed in the biological distribution of the compounds can be related to the lipophilicity of the labeled compounds.

Várias células tumorais expressam significantemente uma alta quantidade de receptores VIP (VIPR) que determinam a base para o uso clínico de VIP radiomarcado para localização de tumores in vivo. Foi estudado neste trabalho a marcação do VIP e do fragmento VIP10-28 com iodo-131 comparando a distribuição biológica dos compostos marcados em camundongos Nude e sua afinidade pelas células tumorais. Ambos os peptídeos, VIP e VIP10-28. contém dois resíduos de tirosina nas posições 10 e 22, que teoricamente são igualmente susceptíveis pela substituição eletrofílica oxidativa do radioiodo utilizando Cloramina T como agente oxidante. A pureza radioquímica da mistura de reação foi determinada por eletroforese e cromatografia líquida de alta eficiência (CLAE). O VIP e fragmento foram marcados com radioiodo com bom rendimento radioquímico (superior a 96 por cento). Importantes diferenças foram observadas nos estudos de distribuição biológica. Comparativamente, o clareamento sanguíneo foi mais rápido para VIP marcado e por esta razão, a captação no tumor foi inferior, especialmente na primeira hora. Estas diferenças observadas na distribuição biológica dos compostos podem estar relacionadas com a lipofilicidade dos compostos marcados.

Labeling of DOTA-Tyr3-octreotate with 177Lu - stability and biodistribution study

The labeling with 177Lu and quality control procedures to produced DOTA (1,4,7,10-N,N´,N´´,N´´´, tetraazaciclododecane tetraacetic acid) coupled octreotate labeled peptide was evaluated. The labeling was performed using 30µg of DOTA-Tyr3-octreotate and 500 and 1110 MBq of 177Lu, buffered with sodium acetate/acetic acid 0.4M pH 4.5 for 20 minutes at 100ºC. The radiochemical purity was determined by ITLC na HPLC. Biological distribution studies were performed on Nude mices with tumours (AR42J rat pancreatic tumour cells) by invasive method. The stability of the 177Lu-DOTA-Tyr3-octreotate was followed by 7 days, and in both labeling procedures, the radiochemical purity were superior than 98 percent. Biodistribution studies showed fast blood clearance and the kidneys were the critical organs. The uptake in tumour were significant after 24 hours and the labeled peptide showed high in vivo stability.

Spectrophotometric determination of Sn+2 in lyophilized kit for labeling with 99mTc

The preparation of 99mTc labeled radiopharmaceuticals depends on the reduction of the technetium pertechnetate, commonly by stannous chloride (SnCl2). The determination of the Sn+2 contents in the lyophilized preparations represents an important quality control procedure that may be applied to the process and to the final product. The objective os this work is the optimization of an spectrophotometric assay to the determination os Sn+2 contents in a citrate-stannous lyophilized kit for 99mTc labeling. The spectrophotometric methodology employed is based in the colour development when Sn+2 reacts with sodium molibdate in the presence of potasium thiocianate in chloridric medium. The colourfull reaction studied showed high stability after 60 minutes of the mixtures preparation. The sequence of reagents introduction in the reaction mixture was determinant to the assay. The molibdenium-stannous-tiocianate sequence produces calibration curves with good correlations (R2 > 0.99). The concentrations of the molibdenium solution was also studied, in order to determine a ideal concentration for the Sn+2 range. The spectrophotometric method studied was usefull to the determination of Sn+2 content in different batches of citrate-stannous preparations. The method was fast and easy and can be applied to different stages of the production process, in order to guarantee the content of Sn+2 in the preparations.

Direct labeling of chemotactic peptide ForNleLFNleYK with radioiodine in vivo stability evaluation

Some peptides are naturally occuring inflammatory mediators which specifically bind to receptors abundantly present in the area of inflammation, and owing to their small size, they rapidly clear from all non-target tissues. ForNleLFNleYK is a synthetic chemotactic peptide with high affinity to receptors on the white blood cell membranes. This hexapeptide contains a tyrosine residue susceptible to iodination by oxidative eletrophilic substitution - direct labeling. The aim of this study was the radioiodination of ForNleLFNleYK using the direct method (chloramine T) and its in vivo stability evaluation. The labeled compound was obtained in a short reaction time with high radiochemical purity (96.8 ± 0.84 por ciento) and remained stable over 48 hours when stored at low temperature. Biological distribution studies showed an uptake in inflammated tight significantly greater than the normal tight (p < 0.05, Student t test), and some in vivo dehalogenation of the compound.

Radioligantes para neurorreceptores benzodiazepínicos / Radioligands for benzodiazepine neuroreceptors

Os inúmeros avanços técnico-científicos das últimas décadas possibilitaram identificar e caracterizar a estrutura de uma variedade de neurotransmissores e de seus receptores no cérebro, bem como estudar suas interações. Nesse artigo serão relacionados os radiofármacos utilizados em medicina nuclear diagnóstica para acesso a neurorreceptores. Radioligantes cerebrais são moléculas marcadas com isótopos emissores de pósitron ou emissores de fótons (gama emissores) que se ligam seletivamente a sítios receptores específicos no SNC (Sistema Nervoso Central). Para utilização in vivo esses radioligantes não devem apresentar toxicidade e, também, não devem apresentar atividade farmacológica na dose administrada...

Optimization of labeling conditions to produce N-isopropyl-p-[123I]-iodoamphetamine: a radiopharmaceutical for brain perfusion imaging

Brain SPECT tracers, such as [123I]-labeled amines and technetium-labeled compounds, offer high resolution SPECT images after intravenous injection using conventional rotating gamma cameras. The aim of this study was to optimize labeling conditions to produce N-isopropyl-p-[123I]-iodoamphetamine) and to propose an easy, rapid and inexpensive chromatography quality control procedure to determine radiochemical purity. [123I]-IMP was produced by isotopic exchange in presence of Cu(II) as catalyst and an excess of ascorbic acid as reducing agent. In order to obtain good radiochemical yield in a minimal reaction time, labeling reaction parameters were evaluated, with the study of different reaction temperatures and time, mass of ascorbic acid, reaction pH and [131I]NaI/IMP ratio. [123I]-IMP was obtained with radiochemical yield greater than 97 per cent using 3 mg of IMP.HCl, 3.15 mol of Cu++ and an excess of ascorbic acid in acid medium (pH 2.0) at 175oC and 30 minutes. Radiochemical purity was determined by paper chromatography with high resolution. Biological distribution studies in animals showed high and persistent cerebral uptake and in vivo stability of the compound

Influência do anestésico (éter etílico e uretana) sobre alguns radiofármacos renais / Anesthetics influence (ether-ethilic and urethane) on some renal radiopharmaceuticals

Fêz-se um estudo comparativo entre os anestésicos éter etílico e uretana, em ratos (Wistar). Observou-se uma variaçäo significativa nos resultados apresentados, quando investigados radiofármacos para vias renais. Com uretana, a captaçäo renal aumenta progressivamente em virtude da inibiçäo da filtraçäo renal e esta começa a ser restabelecida com a eliminaçäo do efeito da anestesia. Utilizando-se éter etílico observa-se que o radiofármaco é eliminado rapidamente dos rins (filtraçäo tubular ou glomerular), evidenciando a preservaçäo da funçäo renal