Development of novel radiotracers as tools for imaging the human brain

Introduction: Brain imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to study the processes underlying neurological and psychiatric disorders. In addition, in vivo brain imaging using SPECT or PET may provide new approaches for drug target identification, pre-clinical testing and occupancy studies, and therefore improve drug discovery. The utility of in vivo brain imaging using SPECT or PET relies on the ability of different radiotracers (typically organic compounds labelled with radionuclides) to bind to a wide variety of targets, including receptors, transporters and enzymes. Therefore the development of novel radiotracers for in vivo brain imaging using SPECT of PET is of vital importance. This thesis is focused on the process of developing novel radiotracers as tools for imaging the human brain, where the radiotracer discovery and development pipeline is discussed and each step prior to clinical trials investigated. Radiotracer discovery: Previously, discovery of novel brain radiotracers has largely relied on simplistic screening tools. Improved selection methods at the early stages of radiotracer discovery and an increased understanding of the relationships between in vitro physicochemical and in vivo radiotracer properties are needed. This thesis investigated if high performance liquid chromatography (HPLC) methodologies could provide criteria for lead candidate selection by comparing HPLC measurements with radiotracer properties in humans. In this study, ten molecules, previously used as radiotracers in humans, were analysed to obtain the following measures: partition coefficient (Log P); permeability (Pm); percentage of plasma protein binding (%PPB); and membrane partition coefficient (Km). Relationships between brain entry measurements (Log P, Pm and %PPB) and in vivo brain percentage injected dose (%ID); and Km and specific binding in vivo (BPND) were investigated. Results showed that HPLC measurements of Pm, %PPB and Km were potentially useful in predicting in vivo performance and hence allow evaluation and ranking of compound libraries for the selection of lead radiotracer candidates at early stages of radiotracer discovery. The HPLC tool developed provides information on in vivo non-specific binding and binding potential that is not possible using conventional screening methods. Another important finding reported in this thesis is that Log P should not be relied on as a predictor of brain entry. The HPLC tool developed, together with competition binding assays, was used to characterise a newly synthesised library of compounds for imaging of the translocator protein (TSPO) in brain using SPECT. Results showed that compound LS 1 was the most likely to succeed within the library investigated, but the high %PPB observed for LS 1 suggested novel compounds with improved %PPB were needed. Thus, a novel library of compounds for imaging of TSPO in brain using SPECT is currently been developed for future testing using the HPLC tool developed here and competition binding assays. Pre-clinical research: radiotracers for imaging the noradrenaline transporter (NAT) in brain using SPECT. In this thesis, NKJ64, a novel iodinated analogue of reboxetine, was successfully radiolabelled via electrophilic iododestannylation and evaluated as a potential SPECT radiotracer for imaging the NAT in brain using rodents and non-human primates. Biological evaluation of the novel radiotracer, 123/125I-NKJ64, in rodents included: in vitro ligand binding assays; in vitro and ex vivo autoradiography; in vivo biodistribution studies and ex vivo pharmacological blocking studies. In rats, 123/125I-NKJ64 displayed saturable binding with nanomolar affinity for the NAT in cortical homogenates, regional distribution consistent with the known density of NAT in the rodent brain and high maximum brain uptake of around 2.93 % of the injected dose. The specific: non-specific ratio (locus coeruleus:caudate putamen) of 123I-NKJ64 uptake was 2.8 at 30 minutes post intravenous injection and prior administration of reboxetine significantly reduced the accumulation of 123I-NKJ64 in the locus coeruleus (> 50% reduction). Data obtained using rodents indicated that further evaluation of 123I-NKJ64 in non-human primates was needed to determine its utility as a SPECT radiotracer for imaging of NAT in brain. Consequently, in vivo kinetic modelling studies using SPECT imaging with 123I-NKJ64 and two baboons were carried out to determine 123I-NKJ64 brain binding kinetics, brain distribution and plasma metabolism in non-human primates. Even though a high brain uptake of around 3.0% of the injected dose was determined, the high non-specific binding observed throughout the brain, a low binding potential (BPND<2) in NAT rich regions and a brain distribution that was inconsistent with the known NAT distribution in non-human primate brain precludes the translation of 123I-NKJ64 into humans. Another NAT radiotracer, 123I-INER, developed by Tamagnan and colleagues at Yale University and Institute for Degenerative Disorders, New Haven, USA, was also investigated as part of this thesis. Kinetic modelling analysis of 123I-INER in baboon brain was investigated for different models, namely invasive and reference tissue models. Bolus plus constant infusion experiments with displacement at equilibrium using six different doses of atomoxetine and four different doses of reboxetine were carried out in several baboons to obtain occupancy measurements as a function of injected dose (mg/kg) for the two NAT selective drugs. Results showed that reference tissue models were able to determine BPND values of 123I-INER in different brain regions. In addition the volume of distribution could be determined by dividing concentration in tissue by the concentration in venous blood at 3 hours post-injection. After administration of atomoxetine or reboxetine, dose-dependent occupancy was observed in brain regions known to contain high densities of NATs. Results supported the translation of 123I-INER into humans studies, despite the slow kinetics determined over the imaging period. Pharmacokinetic properties of 123I-INER described in this thesis may be used to simplify future data acquisition and image processing. Conclusion In conclusion, this thesis reported: (1) the development of novel radiotracers for brain imaging, namely NAT and TSPO; and (2) the development of a new methodology for aiding lead molecule identification at early stages of radiotracer discovery (i.e. prior to radiolabelling). In addition, an overview of radiotracer discovery and development process is provided in a single document, with a focus on brain radiotracers

Implement an in-store technology at the Dolce Vita pharmacy of grupo Reis Barata

The massive development of technologies combined with confinement caused consumer behavior to change. To ensure customer loyalty, pharmacies need to rethink new ways to improve the in-store customer experience in order to capture and retain customers. The main objective of this business plan was to identify which In-store Technology is suitable for Grupo Reis Barata's reality. After market consultation with companies specialized in this type of products and services, the pick-up robot was selected to be implemented at the Dolce Vita Pharmacy. From the group's app, the customer can make their purchases in the physical store without going to the cashier, they just have to place their purchase order in the app, make the online payment, and after entering the code in the pick-up&go area, just have to collect the purchase and leave the store, without having to make contact with any store employee. To ensure the feasibility of the project, three focus groups were held, each consisting of eight people between the ages of 18 and 60, living in the Lisbon area, literate and tech-friendly. The reality of In-store Technologies is already present in other sectors, and with the experience of other players in the market, we can replicate it for the pharmaceutical sector.O desenvolvimento massivo de tecnologias aliado ao confinamento fez com que o comportamento do consumidor mudasse. Para garantir a fidelização do cliente, as farmácias precisam repensar novas formas de melhorar a experiência do cliente na loja, a fim de captar e fidelizar o cliente. O principal objetivo deste plano de negócios foi identificar qual a In-store Tecnology adequada à realidade do Grupo Reis Barata. Após consulta de mercado com empresas especializadas neste tipo de produtos e serviços foi selecionado o pick-up robot para ser implementado na Farmácia Dolce Vita. A partir da app do grupo, o cliente poderá fazer as suas compras na loja física sem se dirigir à caixa, apenas tem que fazer o seu pedido de compra na app, efetuar o pagamento online, e após inserção de código na zona de pick-up&go apenas tem que recolher a compra e sair da loja, sem ter contato com nenhum colaborador da loja. Para garantir viabilidade do projeto foi realizado três focus group cada um composto por oito pessoas entre os 18 e os 60 anos, residentes na zona de Lisboa, literatos e tech-friendly. A realidade das In-store Tecnology já está presente em outros setores, e com a experiência de outros players no mercado podemos replicá-la para o setor farmacêutico

COMPUTATIONAL MODELING OF CELLULAR EFFECTS POST-IRRADIATION WITH LOW- AND HIGH-LET PARTICLES AND DIFFERENT ABSORBED DOSES

The use of computational methods to improve the understanding of biological responses to various types of radiation is an approach where multiple parameters can be modelled and a variety of data is generated. This study compares cellular effects modelled for low absorbed doses against high absorbed doses. The authors hypothesized that low and high absorbed doses would contribute to cell killing via different mechanisms, potentially impacting on targeted tumour radiotherapy outcomes. Cellular kinetics following irradiation with selective low- and high-linear energy transfer (LET) particles were investigated using the Virtual Cell (VC) radiobiology algorithm. Two different cell types were assessed using the VC radiobiology algorithm: human fibroblasts and human crypt cells. The results showed that at lower doses (0.01 to 0.2 Gy), all radiation sources used were equally able to induce cell death (p\u3e0.05, ANOVA). On the other hand, at higher doses (1.0 to 8.0 Gy), the radiation response was LET and dose dependent (p\u3c0.05, ANOVA). The data obtained suggests that the computational methods used might provide some insight into the cellular effects following irradiation. The results also suggest that it may be necessary to re-evaluate cellular radiation-induced effects, particularly at low doses that could affect therapeutic effectiveness

Avaliação, diagnóstico e tratamento da surdez: conhecimento e conduta de médicos com atuação em neonatologia e/ou pediatria de um hospital-escola terciário

CONTEXT AND OBJECTIVE: Infant hearing deficiency is a human disorder with devastating effects and serious implications for the development of speech and language. Early diagnosis of hearing loss should be the objective of a multidisciplinary team, and early-intervention programs should immediately follow this. The aim of this study was to investigate the knowledge and conduct of pediatricians and pediatric residents in a tertiary teaching hospital regarding deafness. DESIGN AND SETTING: Cross-sectional study in a tertiary hospital in the state of São Paulo, Brazil. METHODS: Eighty-eight questionnaires were randomly distributed to pediatricians and pediatric residents. RESULTS: Thirty-six questionnaires were analyzed. Most respondents (61.1%) were residents in pediatrics and/or neonatology. Eighty-three percent of them performed special procedures on babies presenting a high risk of deafness, and 55% reported that they had no knowledge of techniques for screening hearing. Most of them were unaware of the classifications of level and type of hearing loss. According to 47.2% of them, infants could begin to use a hearing aid at six months of age. Most of them reported that infants could undergo hearing rehabilitation during the first six months of life, and all respondents stated 's responsibilities. CONCLUSIONS: Even though most of the participants followed special procedures with babies presenting a high risk of deafness, they did not routinely investigate hearing. All respondents believed that it is a doctor's responsibility to be concerned about child communication.CONTEXTO E OBJETIVO: A deficiência auditiva na criança é uma das desordens humanas cujo impacto tem efeito devastador produzindo sérias conseqüências no desenvolvimento da fala e linguagem. O diagnóstico precoce da perda auditiva deve ser objetivo de uma equipe interdisciplinar e ser seguido imediatamente por programas de intervenção precoce. O objetivo foi investigar o conhecimento e condutas de pediatras e residentes em pediatria de um hospital-escola terciário em relação à surdez. TIPO DE ESTUDO E LOCAL: Estudo transversal, hospital terciário do Estado de São Paulo. MÉTODOS: Oitenta e oito questionários distribuídos para pediatras e residentes em pediatria. RESULTADOS: Analisados 36 questionários. A maioria dos entrevistados (61,1%) foi de residentes em pediatria e/ou neonatologia. Oitenta e três por cento realizavam alguma conduta especial com bebês de alto risco para surdez, e 55% referiram não ter conhecimento sobre técnicas de triagem auditiva. A maioria desconhecia as classificações de grau e tipo de perda auditiva. Para 47,2%, a criança poderia utilizar o aparelho auditivo a partir de seis meses. A maioria referiu que a criança poderia realizar reabilitação auditiva nos primeiros seis meses de vida e todos os entrevistados responderam que é função do médico ter preocupação com a comunicação da criança. CONCLUSÕES: Mesmo a maioria adotando condutas especiais com bebês de alto risco para surdez, não se investigava rotineiramente a audição. Todos os entrevistados julgaram ser função do médico se preocupar com a comunicação da criança

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Synthetic sentence identification (SSI) and contralateral acoustic stapedius reflex

BACKGROUND: the study of the relationship of the contralateral acoustic reflex with the auditory skill of closure. AIM: to analyze the identification of a speech signal in the presence of competitive sounds in subjects with absence of contralateral acoustic reflex. METHOD: application of the synthetic sentence identification (SSI) test under the conditions of competitive contralateral message (SSI-CCM), with the signal-to-noise ratio of 0 and -40dB, and ipsilateral competitive message (SSI-ICM), with the signal-to-noise ratio of 0, -10, -15 and -20dB, in 43 young adults (group A = 21 subjects with contralateral acoustic reflex present in all of the investigated frequencies, and group B = 22 subjects with contralateral reflex absent at the frequency of 500Hz, or in all of the investigated frequencies, or still in some of the investigated frequencies necessarily including 500Hz), of both gender, with no hearing, otologic or learning disabilities. RESULTS: the acoustic reflex threshold was above 100dB NA in 59% of the individuals in group B and in 14% of the individuals in group A. All subjects performed according to the normal pattern suggested in the specialized literature for the SSI test. The performance of group B in the SSI-ICM test was inferior to that of group A for all the signal-to-noise ratios used, although the difference was not statistically significant. Group B, which presented an acoustic reflex threshold higher than 100dB NA or the absence of the acoustic reflex, was also the group that presented the worse performance in the SSI test. CONCLUSION: the absence of the contralateral acoustic reflex seems to interfere in the identification of the speech signal in the presence of competitive noises.TEMA: estudo da relação do reflexo acústico contralateral na habilidade auditiva de fechamento auditivo. OBJETIVO: analisar a identificação do sinal de fala em presença de sons competitivos em sujeitos com ausência do reflexo acústico contralateral. MÉTODO: aplicação do teste de identificação de sentenças sintéticas (SSI) nas condições mensagem competitiva contralateral (SSI-MCC), na relação sinal-ruído de 0 e -40dB e mensagem competitiva ipsilateral (SSI-ICM), na relação sinal-ruído de 0, -10, -15 e -20dB, em 43 adultos-jovens (grupo A = 21 sujeitos com presença do reflexo acústico contralateral em todas as freqüências pesquisadas e grupo B = 22 sujeitos com ausência do reflexo na freqüência de 500Hz, em todas as freqüências pesquisadas ou ainda em algumas das freqüências pesquisadas, mas que incluísse 500Hz), de ambos os sexos sem queixas auditivas, otológicas ou de aprendizagem. RESULTADOS: o limiar do reflexo acústico esteve acima de 100dB NA em 59% dos indivíduos do grupo B e em 14% dos indivíduos do grupo A, todos os indivíduos apresentaram desempenho de acordo com o padrão de normalidade sugerido pela literatura especializada para o teste SSI, o desempenho do grupo B no teste SSI-ICM foi inferior ao grupo A em todas as relações sinal-ruído utilizadas, embora a diferença não tenha sido estatisticamente significante, o grupo B que apresentou limiar do reflexo acústico superior a 100dB NA ou ausência do reflexo acústico também foi o que apresentou pior desempenho no teste SSI. CONCLUSÃO: a ausência do reflexo acústico contralateral parece interferir na identificação do sinal de fala na presença de ruídos competitivos.Pontifícia Universidade Católica de São PauloUniversidade de São Paulo Faculdade de Medicina de Ribeirão Preto Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeça e PescoçoUniversidade Federal de São Paulo (UNIFESP)PUC-SP Departamento de Clínica FonoaudiológicaUNIFESPSciEL