Health Sciences Research Centre

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The promise of prodrugs and nanosystems in nose-to-brain delivery of poorly soluble drugs

Publication . Pires, Patrícia Sofia Cabral; Santos, Adriana Oliveira dos; Alves, Gilberto Lourenço; Rodrigues, Márcio José de Abreu Marques

In brain-targeted drug delivery, the intranasal route can be a good alternative to parenteral administration. In addition to being associated with a greater comfort for the patient, intranasal drug delivery can reduce systemic drug distribution, resulting in an increased safety, and can allow direct drug transport to the brain, resulting in an increased therapeutic efficacy. For example, benzodiazepines administration for the treatment of acute epileptic episodes has been proven to be at least as effective as their intravenous administration. Nevertheless, their solubilization requires substantial amounts of organic solvents, which can cause lacrimation and nose and throat irritation. Additionally, benzodiazepines can cause somnolence, deleterious cognitive effects and dependence/tolerance. Phenytoin is also an antiepileptic drug, being non-inferior in efficacy, while not having these adverse effects. Although its systemic administration can cause other adverse events (such as cardiovascular complications or liver toxicity), its intranasal administration could increase its safety and even efficacy compared to other non-invasive routes in the treatment of status epilepticus. Yet, phenytoin has low aqueous solubility, being difficult to formulate at a high strength. However, its hydrophilic prodrug, fosphenytoin, has high water solubility. Hence, aqueous liquid water-based formulations of fosphenytoin for intranasal administration were developed. Pharmacokinetic results in mice showed that a fosphenytoin formulation containing hydroxypropyl methylcellulose and albumin prolonged drug concentration in the brain, also producing a high absolute drug bioavailability. The study demonstrated that phosphate ester prodrugs (such as fosphenytoin) can be an efficient strategy to increase the intranasal bioavailability of low solubility drugs (such as phenytoin). In addition, we hypothesized that if there was phenytoin in the formulation, in the active and diffusible form, brain drug delivery could be increased and/or made faster. Thus, nano and microemulsions containing phenytoin (internalized in the oil droplets) and fosphenytoin (solubilized in the aqueous phase) in combination were developed. A microemulsion having good characteristics (reasonably homogeneous, with small droplet size and physically stable for at least 1 week) was selected for pharmacokinetic evaluation in mice. In addition to the intranasal administration of this selected microemulsion, containing both phenytoin and fosphenytoin, a second microemulsion was also administered intranasally, having an identical composition but without phenytoin (with fosphenytoin only), for comparison purposes. Despite the existence of a small amount of phenytoin in the formulation not inducing accelerated brain drug delivery, it led to prolonged and increased drug levels. Moreover, the intranasal administration of the microemulsion containing both drugs led to a maximum brain concentration that was similar to that obtained with the intravenous fosphenytoin solution, also leading to prolonged drug retention. The microemulsion containing both drugs also had a higher bioavailability than any of the intranasally administered formulations containing fosphenytoin only (microemulsion without phenytoin, and simple fosphenytoin formulations). Furthermore, both microemulsions (the one containing both drugs and the one containing fosphenytoin only) led to higher drug concentrations at initial time points than those obtained with the simple intranasal fosphenytoin solution, which suggests that the microemulsion had a drug permeation enhancement effect. Thus, in general this work allowed to prove that the use of phosphate ester prodrugs can be an effective strategy in increasing the intranasal bioavailability of low solubility drugs, albumin is a good strategy to prolong brain targeting, the existence of a small amount of active drug (in addition to the prodrug), in an emulsified form, can increase drug levels at longer time points, and the use of microemulsions can increase brain drug delivery at shorter time points.

2021-03Doctoral thesis

Open access

New Steroidal Arylidene Derivatives Potentially Useful in the Treatment of Prostatic Diseases

Publication . Brito, Vanessa Sofia Figueiredo de; Almeida, Paulo Jorge da Silva; Silvestre, Samuel Martins; Alves, Gilberto Lourenço

The prostate is a male-specific hormone-responsive gland with a crucial role in the male reproductive system and requires androgenic hormones and an androgen receptor (AR) for proper growth and development. Generally, this gland is mainly affected by three pathologies, namely prostatitis, benign prostatic hyperplasia (BPH), and malignant prostate cancer (PCa). These disorders constitute a source of significant morbidity and mortality for men worldwide. Thus, pharmacological therapy's relevance in managing these prostatic disorders is unquestionable. Currently, it is well established that the importance of steroidal hormones in the pathophysiology of BPH and PCa. In this context, it is known that the abnormally high 5α-reductase (5AR) activity in humans results in excessively high 5α-dihydrotestosterone (DHT) levels in peripheral tissues, which have been mainly implicated in the pathogenesis of BPH and PCa. Therefore, the role of DHT had contributed to the interest in finding 5AR inhibitors (5ARIs), comprising an important and commonly applied strategy principally in the treatment of BPH. Considering the unmet medical need, it is recognized that the 5ARIs clinically used, such as finasteride, exhibit low potency and several adverse side effects that significantly reduce the quality of life of patients. Furthermore, the effect of these drugs in the PCa treatment is controversial, although it seems a valid and logical approach to explore. In this context, arylidenosteroids, a class of modified steroids, have been reported with important antiproliferative activity and also with potential 5AR inhibitory activity. In the present work, the preparation of novel steroidal arylidene derivatives potentially useful in prostatic diseases, displaying inhibitory capacity against 5AR and/or antiproliferative properties against tumoral cells was intended. Essentially, this thesis aimed the discover of hit compounds for further development in the context of BPH and PCa. To achieve this goal, different synthetic strategies were exploited and new series of steroidal arylidene derivatives were obtained, and their antitumoral and 5AR inhibitory activities were evaluated. Moreover, molecular docking simulations against important targets of steroidal molecules, 5AR type 2, estrogen receptor α (ERα), AR, steroid 17α-hydroxylase/17,20 lyase (CYP17A1), and aromatase, were performed to understand the possible affinity and interactions. This thesis also contemplates the conclusion of the biological evaluation of arylidene-4-azasteroids (VB 4a-g and VB 7a-g) synthesized at the Master’s project and a comprehensive three-dimensional quantitative structure-activity relationship (3D-QSAR study considering the antiproliferative activity in prostate cells, since this work fits into the context of this thesis. In relation to this last part, robust 3D-QSAR models generated to elucidate the relation of the proliferation of LNCaP (androgen-dependent) and PC-3 (non-androgen dependent) cells and the structure of tested compounds showed the importance of the presence of bulky groups and electronegative atoms. Concerning the doctoral project, after several failed attempts to prepare new 3-,4- and 6-azasteroids modified at D-ring with fused/attached heterocycles, a new synthetic strategy was studied and applied and three distinct series of novel arylidene oxidized steroids were successfully obtained from dehydroepiandrosterone. The chemical structures and high purity of the new 16E-arylidene-5α,6α-epoxyepiandrosterone, 16E-arylidene-3β,5α,6β-trihydroxyandrosten-17-one, and 16E-arylidene-androst-4-ene-3,6,17-trione derivatives were corroborated by melting point determination, infrared spectroscopy, nuclear magnetic resonance spectroscopy, and high-resolution mass spectroscopy. Then, to determine their antitumoral potential, the activity of all synthesized analogs over the viability of tumoral cells was evaluated against several human tumoral and non-tumoral cell lines were assessed by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of compounds on cell proliferation was evaluated against prostate cancer androgen-dependent cells (LNCaP), prostate cancer non-androgen dependent cells (PC-3), breast cancer cell line (MCF-7), normal prostate epithelium cells (PNT1A), and normal human dermal fibroblasts (NHDF). Furthermore, preliminary studies of the mechanism of action of the most promising derivatives were performed in MCF-7, LNCaP, and PC-3 cell lines using techniques, such as immunocytochemistry, fluorescence microscopy, and caspase activity assessment. Lastly, the potential 5AR inhibitory activity of all compounds, including the arylidene-4-azasteroids synthesized during the Master’s project, were determined through testosterone quantification in mice liver microsomes by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). This method was previously adapted, partially developed and validated, also in the context of the present project. Moreover, from the results of this assay, the main interactions of the best compounds against 5AR type 2 was also evaluated in the attempt of understanding the structure-activity relationship. Several of these new steroidal derivatives exhibited an interesting growth-inhibition effect on human tumoral cells, and generally, these tested compounds revealed to be less cytotoxic to non-tumoral cells producing higher IC50 values. The most relevant reduction of cell proliferation was observed with compounds VB 9e in MCF-7 cells (IC50= 3.47μM), VB 10e in PC-3 cells (IC50= 6.96 μM), and VB 11c in LNCaP cells (IC50= 6.48 μM). Moreover, the determined IC50 values of these compounds are very similar to the positive control, 5-fluouracil (5-FU). Additional biological studies showed that steroidal derivatives VB 9e and VB 11c seemed to trigger apoptosis in MCF-7 and LNCaP cells, respectively. VB 9e caused significant alterations in Ki67 (↓) and propidium iodide (↑) staining and modifications in nuclear morphology and cell distribution analysis in MCF-7 cells. On the other hand, VB 11c caused the same morphological and cell distribution alterations, and also seemed to increase the activity of caspase-3/7 in the LNCaP cell line. Molecular docking studies showed that these new derivatives presented stronger affinity to 5AR type 2 and CYP17A1, and poor affinity to ERα and AR. Relative to aromatase, it was verified higher affinity for steroidal 4-ene-3,6,17-triones. To finish, the effect of the steroidal arylidene derivatives on 5AR activity and the main results revealed that compound VB 11c seemed to be the most potent inhibitor (IC50= 6.12 nM). In addition, the in silico interactome study was performed and it was observed that this steroid displayed a higher number of interactions in common with finasteride, the reference, compared with other tested compounds. Interestingly, in this case, there is a correlation between in silico and experimental data. Taken together, the results showed that steroid VB 11c seemed to be the most promising molecule to be considered as a hit compound, presenting selectivity to tumoral prostate cells, potentially triggering the apoptotic cell death mechanism in the LNCaP cell line, and being the most potent against 5AR. In conclusion, the research work constitutes an important contribution to the knowledge concerning steroidal arylidene derivatives as agents with antiproliferative and 5AR inhibitory properties. Therefore, new paths were opened, showing the possibility of successfully developing potential new inhibitors based on steroid molecules for further development in the context of BPH and PCa.

2023-07-04Doctoral thesis

Embargoed access

The sex bias of cancer

Publication . Costa, Ana Raquel; Cruz, Inês; Oliveira, Mariana Lança de; Gonçalves, Isabel; Cascalheira, José; Santos, Cecilia

In cancers of hormone-dependent organs like women breast and reproductive organs, endometrium and ovaries, and men’s prostate and testicular cancer, the roles of sex hormones and deregulation of hormone axes are well-documented. More strikingly, epidemiological data highlights significant differences between sexes in the incidence of various cancers in non-reproductive organs, where the role of sex hormones has been less studied. In an era when personalised medicine is gaining recognition, understanding molecular, cellular and biological differences between men and women is timely for developing more appropriate therapeutic interventions according to gender. In this review we show that sex hormones also shape much of the deregulated cellular and molecular pathways leading to cell proliferation and cancer in nonreproductive organs.

2020Journal article

Open access

Phytochemical Characterization, Bioactivities Evaluation and Synergistic Effect of Arbutus unedo and Crataegus monogyna Extracts with Amphotericin B

Publication . Coimbra, Alexandra; Luís, Ângelo; Batista, Maria Teresa; Ferreira, Susana; Duarte, A. P.

The increased resistance to drugs by pathogens is a serious problem, with plants showing to be promising sources for the development of new drugs or the improvement of the effect of existing antimicrobial agents. Considering this, we aimed to evaluate the bioactivities of Arbutus unedo and Crataegus monogyna. Thus, the leaves were first extracted with methanol and then fractionated with different solvents. Phenolic compound profiles were assessed by HPLC-PDA-MSn and the antioxidant activity was evaluated using DPPH method and β-carotene bleaching assay. The antimicrobial activity of extracts was tested against several microorganisms. A. unedo contained mainly galloyl esters, hydrolysable tannins, and flavonoids, while in C. monogyna, 5-caffeoylquinic acid and flavonoids were the most representative polyphenols. Crude extracts showed antioxidant activity and the extracts and fractions displayed a weak antibacterial activity; however inhibiting the growth of Candida tropicalis and C. lusitaniae to which A. unedo extract showed higher activity. Most of the extracts and fractions demonstrated synergistic or additive interactions with amphotericin B against Candida spp. Therefore, the present study revealed significant bioactive properties of the extracts and fractions of A. unedo and C. monogyna, such as antioxidant and antifungal activities.

2020Journal article

Restricted access

Role of astrocytes in an in vitro model of ischemic stroke

Publication . Roque, Cláudio André Martins; Baltazar, Graça Maria Fernandes

Ischemic stroke (IS) is the leading cause of complex and serious long-term disability in developed countries, and after decades of effort there are no effective clinical treatments for IS, especially in the subacute and chronic phases. Currently, in these stages of the IS there is no alternative to promote the recovery of brain tissues affected by the ischemic injury. Most of the treatments (e.g., physical therapy, speech therapy, occupational therapy) are applied with the aim of reducing the sequelae left, or to controlling modifiable risk factors (e.g., hypertension, diabetes, coagulopathies). This leads to a need to develop new approaches to recover those areas, reduce the neurological deficits and, if possible, enhance the functions regulated by the affected brain regions. In this context, this work intends to explore two approaches that hypothetically could induce the recovery of the areas affected by ischemia. The first is related to the potent physiological effects of estrogens on central nervous system (CNS) and its participation in several processes such as, neurogenesis, the expression of neuroprotective factors and antioxidant mechanisms, through the evaluation of the potential beneficial effects induced by the selective activation of G protein–coupled estrogen receptor 1 (GPER or GPR30). The second, by evaluating the potential protective effects induced by high frequency repetitive magnetic stimulation (HF-rMS), an approach that has been described as having the ability to correct maladaptive brain plasticity and to enhance neuronal communication during rehabilitation. In both cases the ability to induce neuroprotection in neurodegenerative disorders, such as, Alzheimer´s disease, Parkinson’s disease, and mood disorders, was already demonstrated. To standardize the ischemic damage and evaluate the potential beneficial effects induced by these two approaches several in vitro models of ischemia were developed and characterized. Neuron-enriched, neuron-glia, and astrocyte-enriched primary cortical cultures subjected to oxygen and glucose deprivation (OGD) followed by a reperfusion period, were used as models. The evaluation of the effects induced by GPER activation and by HF-rMS was performed through the assessment of several parameters related cell survival and proliferation, GPER expression, calcium imaging, as well as neurite morphometric and synaptic modifications. Concerning the role of GPER on the ischemic injury, we observe that ischemia did not change the levels of GPER in neurons and astrocytes. Moreover, GPER selective activation had no impact in neuronal survival, whereas it induced the apoptosis of astrocytes, being this effect meditated by the activation of phospholipase C pathway, and the subsequent intracellular calcium rise. These data indicate a direct impact of GPER on the viability of astrocytes, and the coupling of GPER to different signaling pathways in astrocytes and neurons. Our data also shows that HF-rMS reduces the neuronal loss, the initial neurite degeneration and the loss of synaptic markers triggered by ischemia. Interestingly the protective effect triggered by HF-rMS required the presence of astrocytes. Taken together the data obtained suggests that HF-rTMS has the potential to be used as a therapeutic approach to reduce neuronal death and neuronal damage, by limiting neurite degeneration and enhance functional connectivity and synaptic plasticity in the areas affected by the ischemia. Furthermore, our results also suggest that astrocytes play a crucial role on ischemic injury. Astrocytes were more resistant to ischemic periods than neurons in all experiments performed and when they were present the injury was smaller, which indicate an active role in the neuronal protection against ischemia-induced injury. Taking into account their preponderant role in neuronal physiology and the fact that their presence is crucial for the observed beneficial effects induced by HF-rMS it seems evident that astrocytes could have a substantial impact on the protection and recovery of ischemia-induced lesion. Thereby, we hypothesize that astrocytes could be a potential therapeutic target for the treatment of cerebral ischemia and any methodology/approach that potentiate their beneficial effects may be a promising therapeutic approach.

2020-02-07Doctoral thesis

Open access