Tezacaftor is a direct inhibitor of sphingolipid delta-4 desaturase enzyme (DEGS).

BACKGROUND: We recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) METHODS: hBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis. RESULTS: We demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain. CONCLUSIONS: We believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance.

Establishment of an orthotopic glioblastoma mouse model for preclinical studies.

Glioblastoma accounts almost 50% of all brain cancers, being the most common and lethal brain tumor in adults. Despite the current standard gold treatment based on surgery, chemotherapy, and radiotherapy, other treatment strategies are needed. Different in vitro models are currently used, including commercial cell lines, patient-derived cell lines, organoids, as well as in vivo models, being orthotopic xenografts the most used ones. In this chapter, we describe a standard protocol for the intracranial inoculation of glioblastoma cells in immunodeficient mice, and how to follow up the tumor progression and analyze the data.

Drug-Loaded Lipid Magnetic Nanoparticles for Combined Local Hyperthermia and Chemotherapy against Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a devastating tumor of the central nervous system, currently missing an effective treatment. The therapeutic gold standard consists of surgical resection followed by chemotherapy (usually with temozolomide, TMZ) and/or radiotherapy. TMZ does not, however, provide significant survival benefit after completion of treatment because of development of chemoresistance and of heavy side effects of systemic administration. Improvement of conventional treatments and complementary therapies are urgently needed to increase patient survival and quality of life. Stimuli-responsive lipid-based drug delivery systems offer promising prospects to overcome the limitations of the current treatments. In this work, multifunctional lipid-based magnetic nanovectors functionalized with the peptide angiopep-2 and loaded with TMZ (Ang-TMZ-LMNVs) were tested to enhance specific GBM therapy on an in vivo model. Exposure to alternating magnetic fields (AMFs) enabled magnetic hyperthermia to be performed, that works in synergy with the chemotherapeutic agent. Studies on orthotopic human U-87 MG-Luc2 tumors in nude mice have shown that Ang-TMZ-LMNVs can accumulate and remain in the tumor after local administration without crossing over into healthy tissue, effectively suppressing tumor invasion and proliferation and significantly prolonging the median survival time when combined with the AMF stimulation. This powerful synergistic approach has proven to be a robust and versatile nanoplatform for an effective GBM treatment.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer.

CDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here, we use computer-aided drug design to discover a chemical entity (ARN22089) that has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in 3D vascularized microtumor models (VMT) in vitro. Additionally, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. ARN22089 selectively blocks CDC42 effector interactions without affecting the binding between closely related GTPases and their downstream effectors. Taken together, we identify a class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

Lipid-Based Nanocarriers for The Treatment of Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant neoplasia having origin in the brain. The current treatments involve surgery, radiotherapy, and chemotherapy, being complete surgical resection the best option for the patient survival chances. However, in those cases where a complete removal is not possible, radiation and chemotherapy are applied. Herein, the main challenges of chemotherapy, and how they can be overcome with the help of nanomedicine, are approached. Natural pathways to cross the blood-brain barrier (BBB) are detailed, and different in vivo studies where these pathways are mimicked functionalizing the nanomaterial surface are shown. Later, lipid-based nanocarriers, such as liposomes, solid lipid nanoparticles, and nanostructured lipid carriers, are presented. To finish, recent studies that have used lipid-based nanosystems carrying not only therapeutic agents, yet also magnetic nanoparticles, are described. Although the advantages of using these types of nanosystems are explained, including their biocompatibility, the possibility of modifying their surface to enhance the cell targeting, and their intrinsic ability of BBB crossing, it is important to mention that research in this field is still at its early stage, and extensive preclinical and clinical investigations are mandatory in the close future.

Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors.

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Novel, Potent, and Druglike Tetrahydroquinazoline Inhibitor That Is Highly Selective for Human Topoisomerase II α over ß.

We disclose a novel class of 6-amino-tetrahydroquinazoline derivatives that inhibit human topoisomerase II (topoII), a validated target of anticancer drugs. In contrast to topoII-targeted drugs currently in clinical use, these compounds do not act as topoII poisons that enhance enzyme-mediated DNA cleavage, a mechanism that is linked to the development of secondary leukemias. Instead, these tetrahydroquinazolines block the topoII function with no evidence of DNA intercalation. We identified a potent lead compound [compound 14 (ARN-21934) IC50 = 2 µM for inhibition of DNA relaxation, as compared to an IC50 = 120 µM for the anticancer drug etoposide] with excellent metabolic stability and solubility. This new compound also shows ~100-fold selectivity for topoIIα over topoß, a broad antiproliferative activity toward cultured human cancer cells, a favorable in vivo pharmacokinetic profile, and the ability to penetrate the blood-brain barrier. Thus, ARN-21934 is a highly promising lead for the development of novel and potentially safer topoII-targeted anticancer drugs.

Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indoles as a Novel Class of CFTR Potentiators.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

From fabric to tissue: Recovered wool keratin/polyvinylpyrrolidone biocomposite fibers as artificial scaffold platform.

Keratin extracted from wool fibers has recently gained attention as an abundant source of renewable, biocompatible material for tissue engineering and drug delivery applications. However, keratin extraction and processing generally require a copious use of chemicals, not only bearing consequences for the environment but also possibly compromising the envisioned biological outcome. In this study, we present, for the first time, keratin-PVP biocomposite fibers obtained via an all-water co-electrospinning process and explored their properties modulation as a result of different thermal crosslinking treatments. The protein-based fibers featured homogenous morphologies and average diameters in the range of 170-290 nm. The thermomechanical stability and response to a wet environment can be tuned by acting on the curing time; this can be achieved without affecting the 3D fibrous network nor the intrinsic hydrophilic behavior of the material. More interestingly, our protein-based membranes treated at 170 °C for 18 h successfully sustained the attachment and growth of primary human dermal fibroblasts, a cellular model which can recapitulate more faithfully the physiological human tissue conditions. Our proposed approach can be viewed as pivotal in designing tunable protein-based scaffolds for the next generation of skin tissue growth devices.

Design, Synthesis, Dynamic Docking, Biochemical Characterization, and in Vivo Pharmacokinetics Studies of Novel Topoisomerase II Poisons with Promising Antiproliferative Activity.

We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known topoII blockers. Here, we report on the expansion of this hybrid molecular scaffold and present 16 more hybrid derivatives that have been designed, synthesized, and characterized for their ability to block topoII and for their overall drug-like profile. Some of these compounds act as topoII poison and exhibit good solubility, metabolic (microsomal) stability, and promising cytotoxicity in three cancer cell lines (DU145, HeLa, A549). Compound 3f (ARN24139) is the most promising drug-like candidate, with a good pharmacokinetics profile in vivo. Our results indicate that this hybrid new chemical class of topoII poisons deserves further exploration and that 3f is a favorable lead candidate as a topoII poison, meriting future studies to test its efficacy in in vivo tumor models.

Engineering shape-defined PLGA microPlates for the sustained release of anti-inflammatory molecules.

Over the years, nanoparticles, microparticles, implants of poly(D,l-lactide-co-glycolide) (PLGA) have been demonstrated for diverse biomedical applications. Yet, initial burst release and optimal modulation of the release profiles limit their clinical use. Here, shape-defined PLGA microPlates (µPLs) were realized for the sustained release of two anti-inflammatory molecules, the natural polyphenol curcumin (CURC) and the corticosteroid dexamethasone (DEX). Under the electron microscope, µPLs appeared as square prisms with an edge length of 20 µm. The top-down fabrication process allowed the authors to vary, readily and systematically, the µPL height from 5 to 10 µm and the PLGA mass from 1 to 5, 10 and 20 mg. 'Taller' particles realized with higher PLGA concentrations encapsulated more drug reaching on average values of about 150 pg/µPL, for both CURC and DEX. The µPL height and PLGA concentration had major effects on drug release, too. Under sink conditions, DEX release from tall µPLs at 1 h reduced from 50% to 10% and 2% for the 5, 10 and 20 mg PLGA configurations, respectively. Also, DEX was released more slowly from taller as compared to short µPLs. The opposite trend was observed for CURC, possibly for its lower hydrophobicity and molecular weight as compared to DEX. This was also confirmed by quantifying the free energy of translocation for the two drugs via molecular dynamics simulations. Finally, the anti-inflammatory activity of µPLs was tested in vitro on LPS-stimulated rat monocytes and in vivo on a murine model of UVB-induced skin burns. Both in vitro and in vivo, the expression of pro-inflammatory cytokines (IL-6, IL-1ß, and TNF-α) was significantly reduced by the application of µPLs as compared to the free compounds. In vivo, one single topical deposition of CURC-µPLs outperformed multiple, free CURC applications. This work demonstrates that geometry and polymer density can be effectively used to modulate the pharmacological performance of microparticles and mitigate the initial burst release.

Biokinetics and clearance of inhaled gold ultrasmall-in-nano architectures.

Among an organism's entry portals, the respiratory tract is one of the most promising routes for non-invasive administration of therapeutics for local and systemic delivery. On the other hand, it is the subtlest to protect from environmental pollution and microbial occurrences. Here, the biokinetics, distribution, and clearance trends of gold ultrasmall-in-nano architectures administered through a single intranasal application have been quantitatively evaluated. Apart from reaching the lung parenchyma, the (bio)degradable nano-architectures are able to translocate as well to secondary organs and be almost completely excreted within 10 days. These findings further support the clinical relevance of plasmonic nanomaterials for oncology and infectious disease treatment and management. Notably, this investigation also provides crucial information regarding the associated risks as a consequence of the pulmonary delivery of nanoparticles.

Biosafety and Biokinetics of Noble Metals: The Impact of Their Chemical Nature.

Effective excretion of nanostructured noble metals is still one of the most challenging bottlenecks for their employment in clinical practice. Besides the persistence issue, the clinical translation of inorganic nanomaterials is also affected by a bewildering lack of investigations regarding their quantitative biokinetics. Here, we have quantitatively correlated the chemical nature of the three most interesting noble metals for biomedical applications to their biosafety and biokinetics in, respectively, zebrafish and murine models. Gold, silver, and platinum ultrasmall-in-nano architectures with comparable size elicit, after intravenous administration, different excretion pathways depending on their intrinsic metallic nature. Understanding the in vivo fate of noble metal nanoparticles is a significant breakthrough to unlock their clinical employment for the establishment of treatments for neoplasms, infectious diseases, and neurological disorders.

Combining dietary phenolic antioxidants with polyvinylpyrrolidone: transparent biopolymer films based on p-coumaric acid for controlled release.

Polyvinylpyrrolidone (PVP) has probably been one of the most utilized pharmaceutical polymers with applications ranging from a blood plasma substitute to nanoparticle drug delivery, since its synthesis in 1939. It is a highly biocompatible, non-toxic and transparent film forming polymer. Although high solubility of PVP in aqueous environment is advantageous, it still poses several problems for some applications in which sustained targeting and release are needed or hydrophobic drug inclusion and delivery systems are to be designed. In this study, we demonstrate that a common dietary phenolic antioxidant, p-coumaric acid (PCA), can be combined with PVP covering a wide range of molar ratios by solution blending in ethanol, forming new transparent biomaterial films with antiseptic and antioxidant properties. PCA not only acts as an effective natural plasticizer but also establishes H-bonds with PVP increasing its resistance to water dissolution. PCA could be released in a sustained manner up to a period of 3 days depending on the PVP/PCA molar ratio. Sustained drug delivery potential of the films was studied using methylene blue and carminic acid as model drugs, indicating that the release can be controlled. Antioxidant and remodeling properties of the films were evaluated in vitro by free radical cation scavenging assay and in vivo on a murine model, respectively. Furthermore, the material resorption of films was slower as PCA concentration increased, as observed from the in vivo full-thickness excision model. Finally, the antibacterial activity of the films against common pathogens such as Escherichia coli and Staphylococcus aureus and the effective reduction of inflammatory agents such as matrix metallopeptidases were demonstrated. All these properties suggest that these new transparent PVP/PCA films can find a plethora of applications in pharmaceutical sciences including skin and wound care.

Antimicrobial, antioxidant, and waterproof RTV silicone-ethyl cellulose composites containing clove essential oil.

Ethyl cellulose (EC)/polydimethylsiloxane (PDMS) composite films were prepared at various concentrations of PDMS in the films (0, 5, 10, 15, and 20 wt.%). Morphological and chemical analysis by EDX-SEM and ATR-FTIR showed that EC-rich matrices and PDMS-rich particles were formed, with the two polymers interacting through Hbonds. The number and diameter of particles in the composite depended on the PDMS content and allowed a fine tuning of several properties such as opacity, hydrophobicity, water uptake, and water permeability. Relative low amounts of clove essential oil were also added to the most waterproof composite material (80 wt.% ethyl cellulose and 20 wt.% PDMS). The essential oil increased the flexibility and the antioxidant capacity of the composite. Finally, the antimicrobial properties were tested against common pathogens such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The presence of clove essential oil reduced the biofilm formation on the composites.

Transparent ciprofloxacin-povidone antibiotic films and nanofiber mats as potential skin and wound care dressings.

Water insoluble monohydrochloride monohydrate free ciprofloxacin (Cipro) antibiotic was incorporated in polyvinylpyrrolidone (PVP) polymer matrix by using acetic acid co-solvent in water. The resultant solutions were cast into fully transparent antimicrobial films. Proper concentrations of acetic acid eliminated in situ crystallization of the antibiotic and the resultant phase separation upon solvent evaporation. The solutions could also be electrospun into nanofiber mats (non-transparent). Presence of residual PVP-bound acetic acid in dry PVP films induced unprecedented levels of plasticity (stretching capacity) and softness to the films. Additionally, PVP-bound acetic acid also acted as an antiseptic. Antibacterial properties of the films and fiber mats were confirmed on Escherichia coli and Bacillus subtilis (growth and viability). Films and nanofiber mats demonstrated promising wound resorption characteristics by using in vivo full-thickness excisional skin wound healing mice model. Nanofiber mats were resorbed much faster than transparent films. Wound exudate absorption in the films and resorption rate of the nanofiber mats were dependent on the starting acetic acid concentrations. The fact that PVP/Cipro solutions in aqueous acetic acid can be used either to produce transparent soft films or nanofiber mats renders this process highly suitable for the fabrication of new-generation potential dressings for wound management and care.

Effects of Fatty Acid Amide Hydrolase (FAAH) Inhibitors in Non-Human Primate Models of Nicotine Reward and Relapse.

Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but it has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1'-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: (1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and α-type peroxisome proliferator-activated (PPAR-α) receptors; (2) shifted nicotine self-administration dose-response functions in a manner consistent with reduced nicotine reward; (3) blocked reinstatement of nicotine seeking induced by reexposure to either nicotine priming or nicotine-associated cues; and (4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB1 receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither of the FAAH inhibitors induced dopamine release in the nucleus accumbens shell--consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation because of their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement.

A glycosylated, labionin-containing lanthipeptide with marked antinociceptive activity.

Among the growing family of ribosomally synthesized, post-translationally modified peptides, particularly intriguing are class III lanthipeptides containing the triamino acid labionin. In the course of a screening program aimed at finding bacterial cell wall inhibitors, we discovered a new lanthipeptide produced by an Actinoplanes sp. The molecule, designated NAI-112, consists of 22 amino acids and contains an N-terminal labionin and a C-terminal methyl-labionin. Unique among lanthipeptides, it carries a 6-deoxyhexose moiety N-linked to a tryptophan residue. Consistently, the corresponding gene cluster encodes, in addition to the LanKC enzyme characteristic of this lanthipeptide class, a glycosyl transferase. Despite possessing weak antibacterial activity, NAI-112 is effective in experimental models of nociceptive pain, reducing pain symptoms in mice in both the formalin and the chronic constriction injury tests. Thus, NAI-112 represents, after the labyrinthopeptins, the second example of a lanthipeptide effective against nociceptive pain.