Liposomes as nanosystems for the transport and delivery of bioactive agents

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2011The concept of pharmaceutical nanobiotechnology was originated in the 1970s when liposomes started to be used as nano-drug delivery systems (NanoDDS) to incorporate lipophilic and hydrophilic drugs. Since then liposomes have been the most widely investigated nano-carrier system aiming to achieve controlled drug delivery. The inability of several conventional therapies to deliver the therapeutic dose of the active agents to the diseased tissues at the desired time and concomitantly avoid causing severe toxic effects to healthy tissues or organs has brought considerable attention to the development and clinical use of NanoDDS. In the work developed in this thesis we intended to use liposomes as NanoDDS for cytosolic delivery of bioactive agents aiming to target either diseases of the Mononuclear Phagocytic System (MPS) or cancer. Simultaneously we explored the flexibility and the multifunctional nature of liposomes in different aspects. Special attention was given on the potential of liposomes to carry new bioactive agents with distinctive physicochemical features: small molecules (anti-parasitic drugs) or macromolecules (oligonucleotides) and their ability to target different types of cells, such as macrophages (phagocytic cells) and tumour cells (non-phagocytic). In order to achieve our aims we chose two disease models: Leishmaniasis and Small Cell Lung Cancer (SCLC). The development of liposomal formulations of dinitroanilines for the treatment of leishmaniasis was addressed in Chapter II. Dinitroanilines have proved in vitro anti-leishmanial activity but they are not used in clinical practice as chemotherapeutics for the treatment of leishmaniasis. Nevertheless, they hold great potential in the treatment of this disease due to a selective mechanism of action against parasite tubulins and to the absence of toxicity to mammals. To reach this aim we chose two complementary strategies. The first (Part A) consisted in the association of one dinitroaniline, trifluralin (TFL) with conventional liposomes. The second (Part B) consisted in the incorporation of chemical derivatives of TFL (TFL-D) prepared by organic chemistry hemi-synthesis methods, in order to further improve the chemical stability and biological activity. Conventional liposomes were used as solvents for these hydrophobic and difficult to handle dinitroanilines (either the TFL or the TFL-D) and also because they are naturally cleared from the circulation by the MPS favouring their choice to target intracellular infections of this system, such as leishmaniasis. xiv In Part A, after achieving an efficient incorporation and stabilization of TFL in liposomal formulations (stability on storage up to 2 years in lyophilized form) their therapeutic activity in appropriated animal models of visceral and cutaneous leishmaniasis was evaluated. All TFL liposomal formulations were active against different strains of Leishmania, showing significant reduction in the levels of visceral and cutaneous infections in mice. A superior activity (at least 2-fold) was observed for liposomal TFL as compared to the free drug. A selected TFL liposomal formulation also improved the clinical condition of experimentally infected dogs and reduced the parasite load. In Part B, two new dinitroaniline derivatives were used. This approach was pursued mainly to circumvent several disadvantages of TFL such as unfavourable physicochemical properties and difficulties on handling. Selected conventional liposomes were optimised for the incorporation of these TFL-Ds. The anti-leishmanial activity of TFL-D liposomal formulations was evaluated both in vitro and in vivo. The in vitro biological evaluation of the TFL-D liposomal formulations has demonstrated their activity against Leishmania parasites in culture without revealing signs of toxicity. In addition, extensive parasite load inhibition (> 90%) was observed after treatment with one of the TFL-D liposomal formulations in a murine model of zoonotic visceral leishmaniasis. The use of liposomes as NanoDDS in cancer therapy was addressed in Chapter III. The association of conventional anti-cancer drugs with liposomes has been particularly investigated not only because it increases their concentration in the tumour tissue and reduces their negative side effects, but also because of the extensive application of gene therapy protocols in the treatment of cancer. In fact, antisense oligonucleotides (asODN) or other nucleic acid molecules are considered a new class of anti-cancer drugs since they are able to selectively inhibit the expression of a gene. They act by binding to a complementary region of the mRNA causing its degradation with the consequent down-regulation of the corresponding protein. However, nucleic acids molecules need adequate NanoDDS to be efficiently delivered into the cytosol of the tumour cells due to their poor stability in physiological fluids, high susceptibility to nuclease degradation and limited ability to penetrate through cellular membranes. Based on this rationale, Chapter III is focused on the development of a targeted-liposome delivery system containing an asODN for the treatment of SCLC. For this purpose, long circulating (PEG-grafted) cationic liposomes were used for the encapsulation of the asODN. The attachment of a targeting ligand for selective xv cellular delivery, on the outer surface of this long circulating formulation, makes it a specific delivery system for SCLC cells. Two different cationic liposomal formulations, Coated Cationic Liposomes (CCL) and Stabilized Antisense Lipid Particles (SALP), were selected for the encapsulation of an asODN that inhibits the expression of c-myc oncogene, associated with SCLC cell proliferation. The hexapeptide antagonist G was chosen as the targeting ligand to promote internalization of these formulations. The effect of the peptide coupling method, conventional and post-insertion, on the loading capacity and size of both formulations was assessed. The post-insertion coupling method applied both to CCL and SALP liposomes containing as(c-myc), developed in Chapter III resulted in antagonist G-targeted formulations with the necessary characteristics for evaluation of in vitro delivery of asODN to SCLC. The strategy of using antagonist G as the targeting ligand proved to be successful as it increased the uptake of both formulations as compared to the non-targeted counterparts, in particular in a variant SCLC cell line characterised by being resistant to conventional chemotherapy. The presence of the antagonist G at the surface of SALP did not affect the long circulation characteristics of the SALP liposomes as shown in pharmacokinetic studies. In addition, the preferential accumulation of this formulation in the lungs, substantiate the rationale behind the design of these targeted liposomes for in vivo intracellular delivery of nucleic acids. Overall, the main objectives of this work were reached. Throughout its experimental development new and important issues were identified and remain open. These issues may be an interesting starting point for future research.O conceito de nanobiotecnologia no campo farmacêutico teve a sua origem na década de 1970 quando os lipossomas foram, pela primeira vez, utilizados como sistemas de transporte e entrega de fármacos (NanoDDS, do inglês Nano Drug Delivery Systems) para a incorporação quer de moléculas hidrófobas quer hidrófilas. Desde essa altura e tendo como objectivo a entrega de fármacos de forma controlada, os lipossomas têm sido os sistemas de transporte in vivo mais estudados. O desenvolvimento de NanoDDS tem sido alvo de grande interesse como alternativa a algumas terapias convencionais nos casos em que se demonstra incapacidade de entrega da dose terapêutica de fármacos nos seus locais de acção e em tempo útil, sem que se verifiquem, em simultâneo efeitos adversos nos tecidos sãos. O trabalho aqui desenvolvido refere-se à concepção e desenvolvimento de nano-formulações, via incorporação em lipossomas, para transporte intracelular de agentes bioactivos não convencionais e de natureza diversa, quer nas suas características físico-químicas, quer nos alvos terapêuticos a que se destinam: moléculas de baixo peso molecular, comercializadas ou obtidos por hemi-síntese, dirigidas a macrófagos infectados por Leishmania e uma macromolécula (oligonucleótido) destinada a células do cancro das pequenas células do pulmão (SCLC, do inglês Small Cell Lung Cancer). O Capítulo II refere-se ao desenvolvimento de formulações lipossomais de dinitroanilinas para o tratamento da leishmaniose. As dinitroanilinas são uma nova classe de compostos que, embora evidenciem actividade anti-parasitária, não fazem parte da prática clínica do tratamento desta doença. No entanto, possuem um mecanismo de acção selectivo nas tubulinas dos parasitas que, juntamente com ausência de toxicidade para os mamíferos, lhes conferem grande potencial como fármacos leishmanicidas. Para atingir este objectivo foram utilizadas duas abordagens complementares, a primeira das quais (Parte A) consistiu na incorporação de uma dinitroanilina, a trifluralina (TFL), em lipossomas convencionais. A segunda abordagem (Parte B) consistiu na incorporação em lipossomas de moléculas hemi-sintéticas derivadas da TFL (TFL-D). Esta estratégia teve como objectivo melhorar a estabilidade química e a actividade biológica da TFL. Os lipossomas convencionais foram utilizados como solventes para as dinitroanilinas (TFL e TFL-D), que são compostos hidrofóbicos e de difícil manuseamento. Além disso e uma vez que este tipo de lipossomas é xviii naturalmente eliminado da circulação sanguínea através do sistema fagocitário mononuclear (MPS, do inglês Monoculear Phagocytic System) a sua utilização para o tratamento de infecções de células e órgãos desse sistema, tal como a leishmaniose, parece pertinente. Na Parte A deste estudo, foram obtidas formulações lipossomais de TFL com elevada estabilidade (até 2 anos na forma liofilizada) e que apresentam parâmetros de incorporação apropriados. Estas formulações foram avaliadas em termos da sua actividade terapêutica em modelos animais de infecção de leishmaniose visceral e cutânea (Leishmania donovani e L. major). Observou-se uma acentuada redução dos níveis de infecção nos animais tratados com as formulações lipossomais de TFL, as quais demonstraram ser, pelo menos, 2 vezes superiores à do fármaco na forma livre. Do mesmo modo, verificou-se uma acentuada melhoria do quadro clínico, devida à grande redução da carga parasitária em cães experimentalmente infectados com leishmaniose e tratados com uma formulação lipossomal de TFL. Na Parte B deste trabalho foram utilizados dois novos derivados da TFL. Esta abordagem constitui uma forma de ultrapassar as propriedades físico químicas da TFL, tais como a sua baixa solubilidade aquosa e a dificuldade de manuseamento deste fármaco. Após optimização das formulações de lipossomas convencionais contendo dois TFL-D foi avaliada a sua actividade leishmanicida quer em ensaios in vitro quer em modelos animais. A avaliação biológica in vitro das formulações lipossomais de TFL-D demonstrou serem activas contra parasitas de Leishmania em cultura, sem revelarem sinais de toxicidade em relação a culturas de células de mamífero. Por outro lado, em estudos num modelo animal de leishmaniose visceral zoonótica, foi observada uma elevada redução (> 90%) da carga parasitária após tratamento com estas mesmas formulações. A utilização de NanoDDS tem sido investigada com particular interesse no tratamento do cancro, com o objectivo de a associação de fármacos anti-tumorais com lipossomas possa aumentar a concentração daqueles no tecido tumoral, reduzindo simultaneamente, a sua acumulação em tecidos normais. Esta estratégia tem sido seguida não só com fármacos anti-tumorais convencionais como também e com maior incidência nas últimas décadas, em protocolos de terapia génica. De facto moléculas como os oligonucleótidos antisentido (asODN, do inglês antisense oligonucleotides) ou outros ácidos nucleicos são considerados uma nova classe de fármacos anti-tumorais uma vez que são capazes de inibir a expressão de um gene. Os asODN actuam através da ligação a uma região complementar de um determinado mRNA provocando a sua xix degradação e consequente inibição da expressão da proteína correspondente. No entanto, para os ácidos nucleicos atingirem os seus alvos intracelulares necessitam de NanoDDS uma vez que são pouco estáveis em fluidos biológicos, apresentam uma elevada susceptibilidade à degradação por nucleases e não atravessam facilmente as membranas biológicas. Assim o Capítulo III refere-se ao desenvolvimento de formulações lipossomais apropriadas para a encapsulação de asODN e direccionadas por ligandos acoplados à superfície. Neste sentido, foram utilizados lipossomas catiónicos de longo tempo de circulação aos quais foi acoplado um ligando capaz de promover a internalização celular selectiva deste sistema por parte das células do SCLC. Dois tipos de lipossomas catiónicos designados por CCL (do inglês Coated Cationic Liposomes) e SALP (do inglês Stabilized Antisense Lipid Particles) foram seleccionados para a encapsulação de um asODN, designado por as(c-myc) que inibe a expressão do oncogene c-myc, associado à elevada proliferação celular que caracteriza o SCLC. O ligando responsável pelo direccionamento e internalização utilizado foi o hexapéptido designado por antagonista G. Dois métodos de acoplamento do péptido (convencional e pós-inserção) foram comparados e foi avaliado o efeito de cada um em parâmetros como a eficácia de encapsulação e o tamanho médio dos lipossomas. Os lipossomas CCL e SALP direccionados com o antagonista G possuem as características necessárias para a avaliação da entrega de as(c-myc) a células de SCLC em cultura. A estratégia da utilização do antagonista G como ligando para a entrega dirigida de as(c-myc) mostrou um aumento da internalização de ambas as formulação, em particular no caso de uma linha celular de SCLC caracterizada por ser resistente a fármacos convencionais. Por outro lado, a presença do antagonista G à superfície dos SALP não afectou negativamente as características de longo tempo de circulação no sangue apresentadas por esta formulação. Para além disso, a acumulação preferencial desta formulação direccionada nos pulmões, sustenta esta estratégia de desenho de sistemas especificamente dirigidos para a entrega intracelular de ácidos nucleicos neste órgão. Dum modo geral, os principais objectivos deste trabalho foram atingidos. Ao longo do seu desenvolvimento experimental novas e importantes questões foram identificadas que permanecem em aberto e que constituem um ponto de partida interessante para trabalhos futuros

Persistent primary hyperparathyroidism: an uncommon location for an ectopic gland: Case report and review

Primary hyperparathyroidism (PHPT) is a common endocrine disorder that mainly affects middle-aged women. Patients are usually asymptomatic. The disease might be ascribable to hyperplasia, carcinoma, and single or multiple adenomas. PHPT may be sporadic or familial, the latter comprising multiple endocrine neoplasia type 1 or 2A, familial benign hypocalciuria hypercalcemia, and hyperparathyroidism-jaw tumor syndrome. The most common causes for persistent PHPT are multiglandular disease, and missed abnormal ectopic or orthotopic parathyroid glands. Imaging localization studies should precede a new surgical intervention. Ectopic parathyroid glands are rarely located at the aortopulmonary window. For diagnosis confirmation, 99mTc-sestamibi SPECT/CT seems to be an advantageous test. Another possibility is to perform 99mTc-sestamibi followed by thoracic CT or MRI. Parathyroidectomy may be performed by means of median sternotomy, thoracotomy, or video-assisted thoracoscopy. We describe a case of persistent primary hyperparathyroidism due to the presence of an ectopic parathyroid gland found at the aortopulmonary window. As the investigation necessary to clarify the etiology of recurrent nephrolithiasis proceeded, the diagnosis of PHPT was determined. The patient underwent subtotal parathyroidectomy; nevertheless, PHPT persisted. Genetic syndromes that could account for this condition were excluded. Imaging studies available at that time were not able to locate abnormal glands; moreover, the patient refused to undergo surgical exploration. Later, the patient underwent 99mTc-sestamibi SPECT/CT, which revealed a parathyroid gland at the aortopulmonary window

Novel Antiretroviral Therapeutic Strategies for HIV.

When the first cases of HIV infection appeared in the 1980s, AIDS was a deadly disease without any therapeutic alternatives. Currently, there is still no cure for most cases mainly due to the multiple tissues that act as a reservoir for this virus besides the high viral mutagenesis that leads to an antiretroviral drug resistance. Throughout the years, multiple drugs with specific mechanisms of action on distinct targets have been approved. In this review, the most recent phase III clinical studies and other research therapies as advanced antiretroviral nanodelivery systems will be here discussed. Although the combined antiretroviral therapy is effective in reducing viral loading to undetectable levels, it also presents some disadvantages, such as usual side effects, high frequency of administration, and the possibility of drug resistance. Therefore, several new drugs, delivery systems, and vaccines have been tested in pre-clinical and clinical trials. Regarding drug delivery, an attempt to change the route of administration of some conventional antiretrovirals has proven to be successful and surpassed some issues related to patient compliance. Nanotechnology has brought a new approach to overcoming certain obstacles of formulation design including drug solubility and biodistribution. Overall, the encapsulation of antiretroviral drugs into nanosystems has shown improved drug release and pharmacokinetic profile.This work was supported with Gilead GÉNESE Ref. PGG/006/2016.info:eu-repo/semantics/publishedVersio

Estudo fatorial exploratório das dimensões comportamentais de adesão em pessoas com diabetes mellitus tipo 1 e tipo 2

Background & Aim: The current study sought out to conduct an exploratory factor analysis in order to examine behavioral dimensions associated with adherence in diabetes mellitus (DM) patients. Additionally, our goal was to test a theoretical model consisting of internal and relational factors. Method: Three hundred and forty-seven patients attending consultations at the Serviço de Endocrinologia, Diabetes e Metabolismo of the Centro Hospitalar Universitário de Coimbra and at the Associação Portuguesa de Diabéticos de Portugal. The self-report instruments were Rosenberg Self-esteem Scale, WHOQOL-Bref, Escala de Satisfação com o Suporte Social, Medida de Adesão aos Tratamentos, Attitudes Towards Doctors and Medicine, Beck Depression Inventory, Diabetes Health Profile e Questionário de Autocuidados da Diabetes. Exploratory factor analysis was used to assess the instrument's dimensionality with Kaiser-Meyer-Olkin and Bartlett’s Sphericity Test. Results: For type 1 DM the factor structure found included six components with a total explained variance of 70.8%. For type 2 DM (insulin treatment) seven components were found explaining a total variance of 74.9%.  For type 2 diabetes (oral treatment) six factors accounting for 73.4% of the total explained variance were found. Conclusions: The highest component loadings were found in constructs that should theoretically belong to the corresponding factors. In the case of type 1 DM the therapeutic factor disappears, combining the self-care factor, and in the case of type 2 DM (oral), this component emerged as a dimension. A forced two-dimentional factor analysis indicated that social support load in internal factor. These findings led us to reflect on the suitability of the instrument for the assessment of social support, instead of the model itself. An explanation based on the multidimensionality of therapeutic adherence was achieved considering that internal structure and semantics conditioned our results.Contexto e Objetivos: Vários têm sido os modelos ou teorias explicativas na identificação das dimensões comportamentais que determinam a tendência dos indivíduos para aderirem ou não às recomendações terapêuticas na diabetes. Neste trabalho pretendemos, através de análise fatorial exploratória, analisar quais as dimensões comportamentais associadas à adesão em pessoas com diabetes mellitus (DM). Adicionalmente, foi objetivo testar o modelo teórico composto por três grandes fatores: internos, relacionais e externos ao paciente. Método: 347 doentes frequentando consultas de diabetes do Serviço de Endocrinologia, Diabetes e Metabolismo no Centro Hospitalar Universitário de Coimbra e na Associação Portuguesa de Diabéticos de Portugal que, voluntariamente e informadamente, aceitaram colaborar no preenchimento de uma bateria de testes constituída por instrumentos de autorresposta. Resultados: A estrutura fatorial encontrada no caso da DM tipo-1 é de seis componentes com uma variância total explicada de 70,84% na DM tipo-2 com tratamento insulínico é de sete componentes com variância total explicada de 74,94% e no caso da DM tipo 2 com tratamento oral o número de componentes é de seis com variância total explicada de 73,42%. Conclusões: Concluiu-se que as saturações mais elevadas correspondem aos construtos a que teoricamente deveriam pertencer. Na DM tipo-1 o fator "terapêutico" desaparece, associando-se ao “autocuidado” e na DM tipo-2 oral, esta componente emerge como uma dimensão. Da análise forçada a dois fatores concluímos que o "suporte social" não satura adequadamente no fator relacional, mas sim no fator interno. Levou-nos então a refletir não sobre o modelo propriamente dito, mas da pertinência da escolha do instrumento para a mensuração do suporte social. Apesar das excelentes qualidades psicométricas, o instrumento apresenta itens redigidos para a avaliação da satisfação social percebida e não tanto sobre as relações sociais. Conseguimos encontrar uma explicação baseada na multidimensionalidade do atributo onde a estrutura interna e semântica condicionaram os resultados

Insights on Host–Parasite Immunomodulation Mediated by Extracellular Vesicles of Cutaneous Leishmania shawi and Leishmania guyanensis

Funding Information: This study was supported by FCT-Foundation for Science and Technology, I.P., through research grant PTDC/CVT-CVT/28908/2017 and PTDC/CVT-CVT/0228/2020 and by national funds within the scope of Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA, UIDB/00276/2020) and Global Health and Tropical Medicine (GHTM, UID/04413/2020). Publisher Copyright: © 2023 by the authors.Leishmaniasis is a parasitic disease caused by different species of Leishmania and transmitted through the bite of sand flies vector. Macrophages (MΦ), the target cells of Leishmania parasites, are phagocytes that play a crucial role in the innate immune microbial defense and are antigen-presenting cells driving the activation of the acquired immune response. Exploring parasite–host communication may be key in restraining parasite dissemination in the host. Extracellular vesicles (EVs) constitute a group of heterogenous cell-derived membranous structures, naturally produced by all cells and with immunomodulatory potential over target cells. This study examined the immunogenic potential of EVs shed by L. shawi and L. guyanensis in MΦ activation by analyzing the dynamics of major histocompatibility complex (MHC), innate immune receptors, and cytokine generation. L. shawi and L. guyanensis EVs were incorporated by MΦ and modulated innate immune receptors, indicating that EVs cargo can be recognized by MΦ sensors. Moreover, EVs induced MΦ to generate a mix of pro- and anti-inflammatory cytokines and favored the expression of MHCI molecules, suggesting that EVs antigens can be present to T cells, activating the acquired immune response of the host. Since nano-sized vesicles can be used as vehicles of immune mediators or immunomodulatory drugs, parasitic EVs can be exploited by bioengineering approaches for the development of efficient prophylactic or therapeutic tools for leishmaniasis.publishersversionpublishe

Targeting cancer resistance via multifunctional gold nanoparticles

POCI-01-0145-FEDER-007728 SFRH/BD/120030/2016 PD/BD/105734/2014 Pest-OE/UID/DTP/04138/2013Resistance to chemotherapy is a major problem facing current cancer therapy, which is continuously aiming at the development of new compounds that are capable of tackling tumors that developed resistance toward common chemotherapeutic agents, such as doxorubicin (DOX). Alongside the development of new generations of compounds, nanotechnology-based delivery strategies can significantly improve the in vivo drug stability and target specificity for overcoming drug resistance. In this study, multifunctional gold nanoparticles (AuNP) have been used as a nanoplatform for the targeted delivery of an original anticancer agent, a Zn(II) coordination compound [Zn(DION)2]Cl2 (ZnD), toward better efficacy against DOX-resistant colorectal carcinoma cells (HCT116 DR). Selective delivery of the ZnD nanosystem to cancer cells was achieved by active targeting via cetuximab, NanoZnD, which significantly inhibited cell proliferation and triggered the death of resistant tumor cells, thus improving efficacy. In vivo studies in a colorectal DOX-resistant model corroborated the capability of NanoZnD for the selective targeting of cancer cells, leading to a reduction of tumor growth without systemic toxicity. This approach highlights the potential of gold nanoformulations for the targeting of drug-resistant cancer cells.publishersversionpublishe

Persistent primary hyperparathyroidism: an uncommon location for an ectopic gland -Case report and review Hiperparatiroidismo primário persistente: glândula ectópica em localização rara -Caso clínico e revisão

SUMMARY Primary hyperparathyroidism (PHPT) is a common endocrine disorder that mainly affects middleaged women. Patients are usually asymptomatic. The disease might be ascribable to hyperplasia, carcinoma, and single or multiple adenomas. PHPT may be sporadic or familial, the latter comprising multiple endocrine neoplasia type 1 or 2A, familial benign hypocalciuria hypercalcemia, and hyperparathyroidism-jaw tumor syndrome. The most common causes for persistent PHPT are multiglandular disease, and missed abnormal ectopic or orthotopic parathyroid glands. Ima ging localization studies should precede a new surgical intervention. Ectopic parathyroid glands are rarely located at the aortopulmonary window. For diagnosis confirmation, Tc-sestamibi SPECT/CT seems to be an advantageous test. Another possibility is to perform 99m Tc-sestamibi followed by thoracic CT or MRI. Parathyroidectomy may be performed by means of median sternotomy, thoracotomy, or video-assisted thoracoscopy. We describe a case of persistent primary hyperparathyroidism due to the presence of an ectopic parathyroid gland found at the aortopulmonary window. As the investigation necessary to clarify the etiology of recurrent nephrolithiasis proceeded, the diagnosis of PHPT was determined. The patient underwent subtotal parathyroi dectomy; nevertheless, PHPT persisted. Genetic syndromes that could account for this condition were excluded. Imaging studies available at that time were not able to locate abnormal glands; moreover, the patient refused to undergo surgical exploration. Later, the patient underwent 99m Tc-sestamibi SPECT/CT, which revealed a parathyroid gland at the aortopulmonary window. Arq Bras Endocrinol Metab. 2012;56(6):393-403 SUMÁRIO O hiperparatiroidismo primário (HPP) é uma endocrinopatia frequente que afeta maioritariamente mulheres de meia-idade e é geralmente assintomática. A doença pode ser atribuível a hiperplasia, carcinoma, adenomas únicos ou múltiplos. O HPP inclui formas esporádicas e familiares. As formas familiares englobam neoplasia endócrina múltipla tipo 1 ou 2A, hipercalcemia hipocalciúrica familiar e síndrome hiperparatiroidismo/tumor mandibular-maxilar. As causas mais frequentes de HPP persistente são a presença de doença multiglandular ou de paratiroide anômala ectópica ou ortotópica não identificada previamente. É recomendável que a localização imagiológica preceda a reintervenção cirúrgica. A janela aortopulmonar é uma localização ectópica rara, sendo o 99m Tcsestamibi SPECT/TC um exame de confirmação vantajoso ou, alternativamente o 99m Tc-sestamibi seguido de TC ou RM torácica. A paratiroidectomia pode ser efetuada por meio de esternotomia mediana, toracotomia ou toracoscopia videoassistida. Descrevemos um caso de HPP persistente atribuível à presença de uma glândula paratiroide ectópica localizada à janela aortopulmonar. O diagnóstico de HPP foi estabelecido na sequência da investigação requisitada para esclarecimento etiológico da nefrolitíase recidivante constatada nessa doente. Foi submetida à paratiroidectomia subtotal; não obstante, o HPP persistiu. Excluíram-se síndromes genéticas que pudessem justificar esse quadro clínico. Os exames imagiológicos disponíveis (à época) revelaram-se infrutíferos na detecção de paratiroides anômalas; adicionalmente, a doente recusou exploração cirúrgica. Posteriormente, a doente foi submetida a 99m Tc-sestamibi SPECT/TC, que revelou a presença de uma paratiroide na janela aortopulmonar. Arq Bras Endocrinol Metab. 2012;56(6):393-40

Quercetin Liposomal Nanoformulation for Ischemia and Reperfusion Injury Treatment

PD/BD/135264/2017 UID/DTP/04138/2020 UIDP/04138/2020 UIDP/04378/2020 UIDB/04378/2020 LA/P/0140/2020 UIDB/50006/2020 UIDB/00100/2020Ischemia and reperfusion injury (IRI) is a common complication caused by inflammation and oxidative stress resulting from liver surgery. Current therapeutic strategies do not present the desirable efficacy, and severe side effects can occur. To overcome these drawbacks, new therapeutic alternatives are necessary. Drug delivery nanosystems have been explored due to their capacity to improve the therapeutic index of conventional drugs. Within nanocarriers, liposomes are one of the most successful, with several formulations currently in the market. As improved therapeutic outcomes have been demonstrated by using liposomes as drug carriers, this nanosystem was used to deliver quercetin, a flavonoid with anti-inflammatory and antioxidant properties, in hepatic IRI treatment. In the present work, a stable quercetin liposomal formulation was developed and characterized. Additionally, an in vitro model of ischemia and reperfusion was developed with a hypoxia chamber, where the anti-inflammatory potential of liposomal quercetin was evaluated, revealing the downregulation of pro-inflammatory markers. The anti-inflammatory effect of quercetin liposomes was also assessed in vivo in a rat model of hepatic IRI, in which a decrease in inflammation markers and enhanced recovery were observed. These results demonstrate that quercetin liposomes may provide a significant tool for addressing the current bottlenecks in hepatic IRI treatment.publishersversionpublishe

TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas

Context: Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers. Objectives: We aimed to evaluate the frequency of TERT promoter mutations in thyroid lesions and to investigate the prognostic significance of such mutations in a large cohort of patients with differentiated thyroid carcinomas (DTCs). Design: This was a retrospective observational study. Setting and Patients: We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed in five university hospitals were included. Mean follow-up (±SD) was 7.8 ± 5.8 years. Main Outcome Measures: Predictive value of TERT promoter mutations for distant metastasization, disease persistence at the end of follow-up, and disease-specific mortality. Results: TERT promoter mutations were found in 7.5% of papillary carcinomas (PTCs), 17.1% of follicular carcinomas, 29.0% of poorly differentiated carcinomas, and 33.3% of anaplastic thyroid carcinomas. Patients with TERT-mutated tumors were older (P < .001) and had larger tumors (P = .002). In DTCs, TERT promoter mutations were significantly associated with distant metastases (P < .001) and higher stage (P < .001). Patients with DTC harboring TERT promoter mutations were submitted to more radioiodine treatments (P = .009) with higher cumulative dose (P = .004) and to more treatment modalities (P = .001). At the end of follow-up, patients with TERT-mutated DTCs were more prone to have persistent disease (P = .001). TERT promoter mutations were significantly associated with disease-specific mortality [in the whole FCDTC (P < .001)] in DTCs (P < .001), PTCs (P = .001), and follicular carcinomas (P < .001). After adjusting for age at diagnosis and gender, the hazard ratio was 10.35 (95% confidence interval 2.01–53.24; P = .005) in DTC and 23.81 (95% confidence interval 1.36–415.76; P = .03) in PTCs. Conclusions: TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent prognostic value in DTC and, notably, in PTC.We acknowledge GENZYME for funding our work through a research project. This study was supported by the Portuguese Foundation for Science and Technology through PhD Grant SFRH/BD/81940/2011 (to J.V.); PhD Grant SFRH/BD/87887/2012 (to C.T.); PhD Grant SFRH/BD/79135/2011 (to A.A.); and the Scientific Investigation Project PIC/IC/83037/2007. Further funding was obtained from the project “Microenvironment, Metabolism and Cancer,” partially supported by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referência Estratégico Nacional, and through the European Regional Development Fund. The work of J.M.C.-T. was supported by Grant PI12/00749-FEDER from the Instituto de Salud Carlos III, the Ministry of Economy and Competitiveness (Madrid, Spain). The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an associate laboratory of the Portuguese Ministry of Science, Technology, and Higher Education, which is partially supported by the Foundation for Science and Technology

TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas

Funding: This study was supported by the Portuguese Foundation for Science and Technology through PhD Grant SFRH/BD/81940/ 2011 (to J.V.); PhD Grant SFRH/BD/87887/2012 (to C.T.); PhD Grant SFRH/BD/79135/2011 (to A.A.); and the Scientific Investigation Project PIC/IC/83037/2007. Further funding was obtained from the project “Microenvironment, Metabolism and Cancer,” partially supported by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referência Estratégico Nacional, and through the European Regional Development Fund. The work of J.M.C.-T. was supported by Grant PI12/00749-FEDER from the Instituto de Salud Carlos III, the Ministry of Economy and Competitiveness (Madrid, Spain). The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an associate laboratory of the Portuguese Ministry of Science, Technology, and Higher Education, which is partially supported by the Foundation for Science and Technology.Context: Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers. Objectives:Weaimed to evaluate the frequency of TERT promoter mutations in thyroid lesions and to investigate the prognostic significance of such mutations in a large cohort of patients with differentiated thyroid carcinomas (DTCs). Design: This was a retrospective observational study. Setting and Patients: We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed in five university hospitals were included. Mean follow-up (±SD) was 7.8 ± 5.8 years. Main Outcome Measures: Predictive value of TERT promoter mutations for distant metastasization, disease persistence at the end of follow-up, and disease-specific mortality. Results: TERT promoter mutations were found in 7.5% of papillary carcinomas (PTCs), 17.1% of follicular carcinomas, 29.0% of poorly differentiated carcinomas, and 33.3% of anaplastic thyroid carcinomas. Patients with TERT-mutated tumors were older (P < .001) and had larger tumors (P = .002). In DTCs, TERT promoter mutations were significantly associated with distant metastases (P< .001) and higher stage (P < .001). Patients with DTC harboring TERT promoter mutations were submitted to more radioiodine treatments (P = .009) with higher cumulative dose (P = .004) and to more treatment modalities (P=.001). At the end of follow-up, patients with TERT-mutated DTCs were more prone to have persistent disease (P=.001). TERT promoter mutations were significantly associated with disease-specific mortality [in the whole FCDTC (P < .001)] in DTCs (P < .001), PTCs (P = .001), and follicular carcinomas (P < .001). After adjusting for age at diagnosis and gender, the hazard ratio was 10.35 (95% confidence interval 2.01-53.24; P = .005) in DTC and 23.81 (95% confidence interval 1.36-415.76; P = .03) in PTCs. Conclusions: TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent prognostic value in DTC and, notably, in PTC.publishersversionpublishe