Research Institute for Medicines

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Publications

A Critical Perspective on 3D Liver Models for Drug Metabolism and Toxicology Studies

Publication . Serras, Ana S.; Rodrigues, Joana S.; Cipriano, Madalena; Rodrigues, Armanda V.; Oliveira, Nuno G; Miranda, Joana P

The poor predictability of human liver toxicity is still causing high attrition rates of drug candidates in the pharmaceutical industry at the non-clinical, clinical, and post-marketing authorization stages. This is in part caused by animal models that fail to predict various human adverse drug reactions (ADRs), resulting in undetected hepatotoxicity at the non-clinical phase of drug development. In an effort to increase the prediction of human hepatotoxicity, different approaches to enhance the physiological relevance of hepatic in vitro systems are being pursued. Three-dimensional (3D) or microfluidic technologies allow to better recapitulate hepatocyte organization and cell-matrix contacts, to include additional cell types, to incorporate fluid flow and to create gradients of oxygen and nutrients, which have led to improved differentiated cell phenotype and functionality. This comprehensive review addresses the drug-induced hepatotoxicity mechanisms and the currently available 3D liver in vitro models, their characteristics, as well as their advantages and limitations for human hepatotoxicity assessment. In addition, since toxic responses are greatly dependent on the culture model, a comparative analysis of the toxicity studies performed using two-dimensional (2D) and 3D in vitro strategies with recognized hepatotoxic compounds, such as paracetamol, diclofenac, and troglitazone is performed, further highlighting the need for harmonization of the respective characterization methods. Finally, taking a step forward, we propose a roadmap for the assessment of drugs hepatotoxicity based on fully characterized fit-for-purpose in vitro models, taking advantage of the best of each model, which will ultimately contribute to more informed decision-making in the drug development and risk assessment fields.

2021-02-22Journal article

Open access

Development of Chitosan Particles Loaded with siRNA for Cystatin C to Control Intracellular Drug-Resistant Mycobacterium tuberculosis

Publication . Pires, David; Mandal, Manoj; Matos, Ana I.; Peres, Carina; Catalão, Maria João; Azevedo-Pereira, José M.; Satchi-Fainaro, Ronit; Florindo, Helena F; Anes, Elsa

The golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.

2023-04-08Journal article

Open access

New synthetic methodologies under flow conditions as tool for API synthesis

Publication . Cavaca, Lídia A. S.; Afonso, Carlos Alberto Mateus; Loureiro, Rui Manuel da Silva; Oliveira, Susana Dias Lucas de

The preparation of medicinally relevant small molecules from biomass is a field of interest due to: 1) structural diversity obtained from biomass modification; 2) being potentially more sustainable and environmentally friendly approach, and; 3) more readily available starting materials most likely lead to lower cost of final product. Biomass furanic platforms, including furfural, have attracted special attention from the scientific community. The transformation of furfural to trans-4,5-diamino-cyclopent-2-enones (DCP) has been extensively studied by several research groups, and different methodologies are now available to obtain these compounds. Being a class of molecules with medicinal interest, taking advantage of cyclopentenone rich scaffold is now an eminent strategy to explore other reactivities and obtain novel bioactive molecules. The transformation of furfural to DCP continues to be a current topic of research, and in this thesis, we studied the scalability of the process by developing a continuous flow methodology for easier scale up/out taking into consideration sustainability metrics. Moreover, the challenging preparation of some DCP derivatives bearing electron-poor anilines led us to develop a high-pressure methodology, which ended up to improve DCP yields. Our interest in producing bioactive scaffolds under sustainable conditions prompted us to explore the reactivity of DCP based on the valorisation of furfural. In this sense, we developed a continuous flow hydrogenation of DCP using H-Cube® Mini Plus apparatus with high selectivity towards unprecedent 2,3-diaminocyclopentanones and α-enaminones, including an ATP sensitive potassium channel agonist. The sustainability of the method was also accessed by green metrics. The α-enaminone core reactivity was further studied through photochemical transformations and Diels-Alder reaction using α-enaminone as diene. A series of photochemical reactions were investigated. The carboxylation of in-house prepared aminals and thioaminals was attempted. Thioaminals were prepared in this thesis. The photodecarbonylation of ketones, including 2,3-diaminocyclopentanones, was studied using environmentally friendly ionic liquids, PEG and deep eutectic systems.

2022-05Doctoral thesis

Open access

Topical ocular delivery of nanoparticles with epoetin beta in Wistar Hannover rats

Publication . Silva, Beatriz; Gonçalves, Lídia; Braz, Berta São; Delgado, Esmeralda

Topical instillation of drugs targeting the posterior ocular segment is an expanding area of research. Chitosan and hyaluronic acid have remarkable mucoadhesive properties and potentially enhance pre-corneal retention time after topical instillation. Bearing this in mind, we explored the possibility of delivering epoetin beta (EPOβ) to the posterior segment of the eye in a chitosan-hyaluronic acid (CS/HA-EPOβ) nanoparticulate system using the topical route of administration. Complete ophthalmological examinations, electroretinography and microhematocrit evaluations were performed in Wistar Hannover (WH) rats, before and after topical administration of nanoparticles. The right eye received CS/HA-EPOβ and the left eye received only empty nanocarriers (control). Animals were split into 6 groups and at designated timepoints, all animals from each group (n = 3) were euthanized and both eyes enucleated. Retinal morphology and EPOβ ocular distribution were assessed, respectively, through hematoxylin and eosin (HE) and immunofluorescence staining. After topical administration, no adverse ocular signs were noted and no significant changes either in microhematocrits nor in electroretinographies were detected. During the study, intraocular pressure (IOP) was always kept within physiological range bilaterally. No histological changes were detected in any of the ocular globes. Immunofluorescence enabled the identification of EPOβ in the retina 12 h after the administration, its presence still being detectable at day 21. In conclusion, CS/HA nanoparticles could efficiently deliver EPOβ to the retina of WH rats after topical instillation, being considered biologically safe. Topical administration of this nanoformulation could be a valuable tool for retinal neuroprotection, decreasing risks associated with more invasive routes of administration, being cost effective and also increasing long-term patients’ compliance.

2023-01-27Journal article

Open access

Phylogeography of hepatitis B virus: the role of Portugal in the early dissemination of HBV worldwide

Publication . Marcelino, Rute; Ezeonwumelu, Ifeanyi Jude; Janeiro, André; Mimoso, Paula; Matos, Sónia; Briz, Veronica; Pimentel, Victor; Pingarilho, Marta; Marinho, Rui; Maria Marcelino, José; Taveira, Nuno; Abecasis, Ana

In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.

2022Journal article

Open access