A European regulatory perspective on cystic fibrosis: current treatments, trends in drug development and translational challenges for CFTR modulators

In this article we analyse the current authorised treatments and trends in early drug development for cystic fibrosis (CF) in the European Union for the time period 2000–2016. The analysis indicates a significant improvement in the innovation and development of new potential medicines for CF, shifting from products that act on the symptoms of the disease towards new therapies targeting the cause of CF. However, within these new innovative medicines, results for CF transmembrane conductance regulator (CFTR) modulators indicate that one major challenge for turning a CF concept product into an actual medicine for the benefit of patients resides in the fact that, although pre-clinical models have shown good predictability for certain mutations, a good correlation to clinical end-points or biomarkers (e.g. forced expiratory volume in 1 s and sweat chloride) for all mutations has not yet been achieved. In this respect, the use of alternative end-points and innovative nonclinical models could be helpful for the understanding of those translational discrepancies. Collaborative endeavours to promote further research and development in these areas as well as early dialogue with the regulatory bodies available at the European competent authorities are recommended

Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity

The expression of acid ceramidase (AC) - a cysteine amidase that hydrolyses the proapoptotic lipid ceramide - is abnormally high in several human tumors, which is suggestive of a role in chemoresistance. Available AC inhibitors lack, however, the potency and drug-likeness necessary to test this idea. Here we show that the antineoplastic drug carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Modifications in the chemical scaffold of carmofur yield new AC inhibitors that act synergistically with standard antitumoral drugs to prevent cancer cell proliferation. These findings identify AC as an unexpected target for carmofur, and suggest that this molecule can be used as starting point for the design of novel chemosensitizing agents

Graphene Oxide Upregulates the Homeostatic Functions of Primary Astrocytes and Modulates Astrocyte-to-Neuron Communication

Graphene-based materials are the focus of intense research efforts to devise novel theranostic strategies for targeting the central nervous system. In this work, we have investigated the consequences of long-term exposure of primary rat astrocytes to pristine graphene (GR) and graphene oxide (GO) flakes. We demonstrate that GR/GO interfere with a variety of intracellular processes as a result of their internalization through the endolysosomal pathway. Graphene-exposed astrocytes acquire a more differentiated morphological phenotype associated with extensive cytoskeletal rearrangements. Profound functional alterations are induced by GO internalization, including the upregulation of inward-rectifying K+ channels and of Na+-dependent glutamate uptake, which are linked to the astrocyte capacity to control the extracellular homeostasis. Interestingly, GO-pretreated astrocytes promote the functional maturation of co-cultured primary neurons by inducing an increase in intrinsic excitability and in the density of GABAergic synapses. The results indicate that graphene nanomaterials profoundly affect astrocyte physiology in vitro with consequences for neuronal network activity. This work supports the view that GO-based materials could be of great interest to address pathologies of the central nervous system associated with astrocyte dysfunctions

An Increase in Membrane Cholesterol by Graphene Oxide Disrupts Calcium Homeostasis in Primary Astrocytes

The use of graphene nanomaterials (GNMs) for biomedical applications targeted to the central nervous system is exponentially increasing, although precise information on their effects on brain cells is lacking. In this work, the molecular changes induced in cortical astrocytes by few-layer graphene (FLG) and graphene oxide (GO) flakes are addressed. The results show that exposure to FLG/GO does not affect cell viability or proliferation. However, proteomic and lipidomic analyses unveil alterations in several cellular processes, including intracellular Ca2+ ([Ca2+ ]i ) homeostasis and cholesterol metabolism, which are particularly intense in cells exposed to GO. Indeed, GO exposure impairs spontaneous and evoked astrocyte [Ca2+ ]i signals and induces a marked increase in membrane cholesterol levels. Importantly, cholesterol depletion fully rescues [Ca2+ ]i dynamics in GO-treated cells, indicating a causal relationship between these GO-mediated effects. The results indicate that exposure to GNMs alters intracellular signaling in astrocytes and may impact astrocyte-neuron interactions

Physiological adaptations affecting drug pharmacokinetics in space: what do we really know? A critical review of the literature

As human spaceflight progresses with extended mission durations, the demand for effective and safe drugs will necessarily increase. To date, the accepted medications used during missions (for space motion sickness, sleep disturbances, allergies, pain, and sinus congestion) are administered under the assumption that they act as safely and efficaciously as on Earth. However, physiological changes have been documented in human subjects in spaceflight involving fluid shifts, muscle and bone loss, immune system dysregulation, and adjustments in the gastrointestinal tract and metabolism. These alterations may change the pharmacokinetics (PK) and pharmacodynamics of commonly used medications. Frustratingly, the information gained from bed rest studies and from in‐flight observations is incomplete and also demonstrates a high variability in drug PK. Therefore, the objectives of this review are to report (i) the impact of the space environmental stressors on human physiology in relation to PK; (ii) the state‐of‐the‐art on experimental data in space and/or in ground‐based models; (iii) the validation of ground‐based models for PK studies; and (iv) the identification of research gaps

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT(+) hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT(+) hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn(F7/F7) mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT(+) hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355

Chemical perturbation of oncogenic protein folding: from the prediction of locally unstable structures to the design of disruptors of Hsp90-Client interactions

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein‐90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co‐chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c‐Abl, c‐Src, Cdk4, B‐Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90‐system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein–protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation

Advances in the discovery of N-acylethanolamine acid amidase inhibitors

N-acylethanolamine acid amidase (NAAA) is a cysteine amidase that hydrolyzes saturated or monounsaturated fatty acid ethanolamides, such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA has been shown to exert analgesic and anti-inflammatory effects by engaging peroxisome proliferator-activated receptor-α. Like other fatty acid ethanolamides, PEA is not stored in cells, but produced on demand from cell membrane precursors, and its actions are terminated by intracellular hydrolysis by either fatty acid amide hydrolase or NAAA. Endogenous levels of PEA and OEA have been shown to decrease during inflammation. Modulation of the tissue levels of PEA by inhibition of enzymes responsible for the breakdown of this lipid mediator may represent therefore a new therapeutic strategy for the treatment of pain and inflammation. While a large number of inhibitors of fatty acid amide hydrolase have been discovered, few compounds have been reported to inhibit NAAA activity. Here, we describe the most representative NAAA inhibitors and briefly highlight their pharmacological profile. A recent study has shown that a NAAA inhibitor attenuated heat hyperalgesia and mechanical allodynia caused by local inflammation or nerve damage in animal models of pain and inflammation. This finding encourages further exploration of the pharmacology of NAAA inhibitors