Synthesis and biological evaluation of potential acetylcholinesterase inhibitors based on a benzoxazine core.

With the purpose of expanding the structural variety of chemical compounds available as pharmacological tools for the treatment of Alzheimer's disease, we synthesized and evaluated a novel series of indole-benzoxazinones (Family I) and benzoxazine-arylpiperazine derivatives (Family II) for potential human acetylcholinesterase (hAChE) inhibitory properties. The most active compounds 7a and 7d demonstrated effective inhibitory profiles with Ki values of 20.3 ± 0.9 µM and 20.2 ± 0.9 µM, respectively. Kinetic inhibition assays showed non-competitive inhibition of AChE by the tested compounds. According to our docking studies, the most active compounds from both series (Families I and II) showed a binding mode similar to donepezil and interact with the same residues.

Ronapreve (REGN-CoV; casirivimab and imdevimab) reduces the viral burden and alters the pulmonary response to the SARS-CoV-2 Delta variant (B.1.617.2) in K18-hACE2 mice using an experimental design reflective of a treatment use case.

With some exceptions, global policymakers have recommended against the use of existing monoclonal antibodies in COVID-19 due to loss of neutralization of newer variants. The purpose of this study was to investigate the impact of Ronapreve on compartmental viral replication using paradigms for susceptible and insusceptible variants. Virological efficacy and impact on pathogenicity was assessed in K18-hACE2 mice inoculated with either the Delta or BA.1 Omicron variants. Ronapreve reduced sub-genomic viral RNA levels in lung and nasal turbinate, 4 and 6 days post-infection, for the Delta variant but not the Omicron variant. It also blocked brain infection, which is seen with high frequency in K18-hACE2 mice after Delta variant infection. At day 6, the inflammatory response to lung infection with the Delta variant was altered to a multifocal granulomatous inflammation in which the virus appeared to be confined. The current study provides evidence of an altered tissue response to SARS-CoV-2 after treatment with a monoclonal antibody combination that retains neutralization activity. These data demonstrate that experimental designs that reflect treatment use cases are achievable in animal models for monoclonal antibodies. Extreme caution should be taken when interpreting prophylactic experimental designs that may not be representative of treatment.IMPORTANCEFollowing the emergence of the SARS-CoV-2 Omicron variant, the WHO recommended against the use of Ronapreve in its COVID-19 treatment guidelines due to a lack of efficacy based on current pharmacokinetic-pharmacodynamic understanding. However, the continued use of Ronapreve, specifically in vulnerable patients, was advocated by some based on in vitro neutralization data. Here, the virological efficacy of Ronapreve was demonstrated in both the lung and brain compartments using Delta as a paradigm for a susceptible variant. Conversely, a lack of virological efficacy was demonstrated for the Omicron variant. Comparable concentrations of both monoclonal antibodies were observed in the plasma of Delta- and Omicron-infected mice. This study made use of a reliable murine model for SARS-CoV-2 infection, an experimental design reflective of treatment, and demonstrated the utility of this approach when assessing the effectiveness of monoclonal antibodies.

Understanding the Degradation of Core-Shell Nanogels Using Asymmetrical Flow Field Flow Fractionation.

Nanogels are candidates for biomedical applications, and core-shell nanogels offer the potential to tune thermoresponsive behaviour with the capacity for extensive degradation. These properties were achieved by the combination of a core of poly(N-isopropylmethacrylamide) and a shell of poly(N-isopropylacrylamide), both crosslinked with the degradable crosslinker N,N'-bis(acryloyl)cystamine. In this work, the degradation behaviour of these nanogels was characterised using asymmetric flow field flow fractionation coupled with multi-angle and dynamic light scattering. By monitoring the degradation products of the nanogels in real-time, it was possible to identify three distinct stages of degradation: nanogel swelling, nanogel fragmentation, and nanogel fragment degradation. The results indicate that the core-shell nanogels degrade slower than their non-core-shell counterparts, possibly due to a higher degree of self-crosslinking reactions occurring in the shell. The majority of the degradation products had molecule weights below 10 kDa, which suggests that they may be cleared through the kidneys. This study provides important insights into the design and characterisation of degradable nanogels for biomedical applications, highlighting the need for accurate characterisation techniques to measure the potential biological impact of nanogel degradation products.

Chemoprophylactic Assessment of Combined Intranasal SARS-CoV-2 Polymerase and Exonuclease Inhibition in Syrian Golden Hamsters.

Pibrentasvir (PIB) has been demonstrated to block exonuclease activity of the SARS-CoV-2 polymerase, protecting favipiravir (FVP) and remdesivir (RDV) from post-incorporation excision and eliciting antiviral synergy in vitro. The present study investigated the chemoprophylactic efficacy of PIB, FVP, RDV, FVP with PIB, or RDV with PIB dosed intranasally twice a day, using a Syrian golden hamster contact transmission model. Compared to the saline control, viral RNA levels were significantly lower in throat swabs in FVP (day 7), RDV (day 3, 5, 7), and RDV+PIB (day 3, 5) treatment groups. Similarly, findings were evident for nasal turbinate after PIB and RDV treatment, and lungs after PIB, FVP, and FVP+PIB treatment at day 7. Lung viral RNA levels after RDV and RDV+PIB treatment were only detectable in two animals per group, but the overall difference was not statistically significant. In situ examination of the lungs confirmed SARS-CoV-2 infection in all animals, except for one in each of the RDV and RDV+PIB treatment groups, which tested negative in all virus detection approaches. Overall, prevention of transmission was observed in most animals treated with RDV, while other agents reduced the viral load following contact transmission. No benefit of combining FVP or RDV with PIB was observed.

Evaluation of Nafamostat as Chemoprophylaxis for SARS-CoV-2 Infection in Hamsters.

The successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention that is implementable alongside vaccination programmes. Nafamostat is a serine protease inhibitor that inhibits SARS-CoV-2 entry in vitro, but it has not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and has an extremely short plasma half-life. This study sought to determine whether intranasal dosing of nafamostat at 5 mg/kg twice daily was able to prevent the airborne transmission of SARS-CoV-2 from infected to uninfected Syrian Golden hamsters. SARS-CoV-2 RNA was detectable in the throat swabs of the water-treated control group 4 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, throat swabs from the intranasal nafamostat-treated hamsters remained SARS-CoV-2 RNA negative for the full 4 days of cohabitation. Significantly lower SARS-CoV-2 RNA concentrations were seen in the nasal turbinates of the nafamostat-treated group compared to the control (p = 0.001). A plaque assay quantified a significantly lower concentration of infectious SARS-CoV-2 in the lungs of the nafamostat-treated group compared to the control (p = 0.035). When taken collectively with the pathological changes observed in the lungs and nasal mucosa, these data are strongly supportive of the utility of intranasally delivered nafamostat for the prevention of SARS-CoV-2 infection.

ß-Cyclodextrin-Based Nanosponges Inclusion Compounds Associated with Gold Nanorods for Potential NIR-II Drug Delivery.

This article describes the synthesis and characterization of two nanocarriers consisting of ß-cyclodextrin-based nanosponges (NSs) inclusion compounds (ICs) and gold nanorods (AuNRs) for potential near-infrared II (NIR-II) drug-delivery systems. These nanosystems sought to improve the stability of two drugs, namely melphalan (MPH) and curcumin (CUR), and to trigger their photothermal release after a laser irradiation stimulus (1064 nm). The inclusion of MPH and CUR inside each NS was confirmed by field emission scanning electron microscopy (FE-SEM), Raman spectroscopy, Fourier transform infrared spectroscopy, (FT-IR) differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and proton nuclear magnetic resonance (1H-NMR). Furthermore, the association of AuNRs with both ICs was confirmed by FE-SEM, energy-dispersive spectroscopy (EDS), TEM, dynamic light scattering (DLS), ζ-potential, and UV-Vis. Moreover, the irradiation assays demonstrated the feasibility of the controlled-photothermal drug release of both MPH and CUR in the second biological window (1000-1300 nm). Finally, MTS assays depicted that the inclusion of MPH and CUR inside the cavities of NSs reduces the effects on mitochondrial activity, as compared to that observed in the free drugs. Overall, these results suggest the use of NSs associated with AuNRs as a potential technology of controlled drug delivery in tumor therapy, since they are efficient and non-toxic materials.

Transfersomes as alternative topical nanodosage forms for the treatment of skin disorders.

Topical drug delivery is a promising approach to treat different skin disorders. However, it remains a challenge mainly due to the nature and rigidity of the nanosystems, which limit deep skin penetration, and the unsuccessful demonstration of clinical benefits; greater penetration by itself, does not ensure pharmacological success. In this context, transfersomes have appeared as promising nanosystems; deformability, their unique characteristic, allows them to pass through the epidermal microenvironment, improving the skin drug delivery. This review focuses on the comparison of transfersomes with other nanosystems (e.g., liposomes), discusses recent therapeutic applications for the topical treatment of different skin disorders and highlights the need for further studies to demonstrate significant clinical benefits of transfersomes compared with conventional therapies.

The Influence of Size and Chemical Composition of Silver and Gold Nanoparticles on in vivo Toxicity with Potential Applications to Central Nervous System Diseases.

The physicochemical and optical properties of silver nanoparticles (SNPs) and gold nanoparticles (GNPs) have allowed them to be employed for various biomedical applications, including delivery, therapy, imaging, and as theranostic agents. However, since they are foreign body systems, they are usually redistributed and accumulated in some vital organs, which can produce toxic effects; therefore, this a crucial issue that should be considered for potential clinical trials. This review aimed to summarize the reports from the past ten years that have used SNPs and GNPs for in vivo studies on the diagnosis and treatment of brain diseases and those related to the central nervous system, emphasizing their toxicity as a crucial topic address. The article focuses on the effect of the nanoparticle´s size and chemical composition as relevant parameters for in vivo toxicity. At the beginning of this review, the general toxicity and distribution studies are discussed separately for SNPs and GNPs. Subsequently, this manuscript analyzes the principal applications of both kinds of nanoparticles for glioma, neurodegenerative, and other brain diseases, and discusses the advances in clinical trials. Finally, we analyze research prospects towards clinical applications for both types of metallic nanoparticles.

Functionalization with PEG/Angiopep-2 peptide to improve the delivery of gold nanoprisms to central nervous system: in vitro and in vivo studies.

One of main drawbacks for the treatment of neurodegenerative pathologies is ensuring the delivery of therapeutic agents into the central nervous system (CNS). Nowadays, gold nanoprisms (GNPr) have become an emerging nanomaterial with a localized surface plasmon resonance in the biological window, showing applications in both detection and treatment of diseases. In this work, GNPr were functionalized with polyethylene glycol (PEG) and Angiopep-2 (Ang2) peptide to obtain a new highly stable nanomaterial and evaluate its toxicity and ability to cross the blood-brain barrier (BBB) in a zebrafish larvae model. The success in the functionalization was confirmed by a full characterization that showed the physicochemical changes at each step. In turn, the colloidal stability of GNPr-PEG-Ang2 in biologically relevant media also was demonstrated. The toxicity assays of GNPr-PEG-Ang2 performed on SH-SY5Y neuroblastoma cell line and on zebrafish larvae showed no effects both in vitro and in vivo. GNPr delivery to the CNS was studied in zebrafish larvae by immersion. We confirmed that functionalization with PEG-Ang2 improved the crossing through the BBB in this model compared with GNPr functionalized only with PEG. Notably, our nanomaterial was not detected in the CNS of zebrafish larvae 24 h after exposure that correlates with an adequate clearance of GNPr-PEG-Ang2 from the brain. This report is the first study of GNPr in the in vivo model of zebrafish larvae demonstrating that its functionalization with Ang2 allows the crossing of the BBB. Moreover, considering the stability achieved of the GNPr-PEG-Ang2 and the results of in vitro and in vivo studies, this work becomes a high contribution to the design of new nanomaterials with potential biomedical applications for CNS-related diseases.

The curvature of gold nanoparticles influences the exposure of amyloid-ß and modulates its aggregation process.

Gold nanoparticles (GNP) are tunable nanomaterials that can be used to develop rational therapeutic inhibitors against the formation of pathological aggregates of proteins. In the case of the pathological aggregation of the amyloid-ß protein (Aß), the shape of the GNP can slow down or accelerate its aggregation kinetics. However, there is a lack of elementary knowledge about how the curvature of GNP alters the interaction with the Aß peptide and how this interaction modifies key molecular steps of fibril formation. In this study, we analysed the effect of flat gold nanoprisms (GNPr) and curved gold nanospheres (GNS) on in vitro Aß42 fibril formation kinetics by using the thioflavin-based kinetic assay and global fitting analysis, with several models of aggregation. Whereas GNPr accelerate the aggregation process and maintain the molecular mechanism of aggregation, GNS slow down this process and modify the molecular mechanism to one of fragmentation/secondary nucleation, with respect to controls. These results can be explained by a differential interaction between the Aß peptide and GNP observed by Raman spectroscopy. While flat GNPr expose key hydrophobic residues involved in the Aß peptide aggregation, curved GNS hide these residues from the solvent. Thus, this study provides mechanistic insights to improve the rational design of GNP nanomaterials for biomedical applications in the field of amyloid-related aggregation.

Intranasal administration of gold nanoparticles designed to target the central nervous system: Fabrication and comparison between nanospheres and nanoprisms.

The presence of the blood-brain barrier (BBB) limit gold nanoparticles (GNP) accumulation in central nervous system (CNS) after intravenous (IV) administration. The intranasal (IN) route has been suggested as a good strategy for circumventing the BBB. In this report, we used gold nanoprisms (78 nm) and nanospheres (47 nm), of comparable surface areas (8000 vs 7235 nm2) functionalized with a polyethylene glycol (PEG) and D1 peptide (GNPr-D1 and GNS-D1, respectively) to evaluate their delivery to the CNS after IN administration. Cell viability assay showed that GNPr-D1 and GNS-D1 were not cytotoxic at concentrations ranged between 0.05 and 0.5 nM. IN administration of GNPr-D1 and GNS-D1 demonstrated a significant difference between the two types of GNP, in which the latter reached the CNS in higher levels. Pharmacokinetic study showed that the peak brain level of gold was 0.75 h after IN administration of GNS-D1. After IN and IV administrations of GNS-D1, gold concentrations found in brain were 55 times higher via the IN route compared to IV administration. Data revealed that the IN route is more effective for targeting gold to the brain than IV administration. Finally, no significant difference was observed between the IN and IV routes in the distribution of GNS-D1 in the various brain areas.

Improving gold nanorod delivery to the central nervous system by conjugation to the shuttle Angiopep-2.

AIM: To improve the in vivo delivery of gold nanorods (GNRs) to the central nervous system of rats, these gold nanoparticles were conjugated to Angiopep-2, a shuttle peptide that can cross the blood-brain barrier. MATERIALS & METHODS: GNRs were synthesized and modified using polyethylene glycol and Angiopep-2 (GNR-PEG-Angiopep-2). The physicochemical properties, in vitro cytotoxicity and ex vivo biodistribution of the conjugate were examined. RESULTS: GNR-PEG-Angiopep-2 was stable over the following days, and the different concentrations that were tested did not affect the viability of microvascular endothelial cells. The conjugation of Angiopep-2 to GNRs enhanced the endocytosis of these particles (in vitro) and the accumulation in brains (in vivo), when compared with GNRs modified only with PEG. CONCLUSION: This study provides evidence that Angiopep-2 improves the delivery of GNRs to the brain parenchyma. This property is highly relevant for future applications of GNRs as platforms for photothermal and theranostic purposes.

Peptides and proteins used to enhance gold nanoparticle delivery to the brain: preclinical approaches.

An exciting and emerging field in nanomedicine involves the use of gold nanoparticles (AuNPs) in the preclinical development of new strategies for the treatment and diagnosis of brain-related diseases such as neurodegeneration and cerebral tumors. The treatment of many brain-related disorders with AuNPs, which possess useful physical properties, is limited by the blood-brain barrier (BBB). The BBB highly regulates the substances that can permeate into the brain. Peptides and proteins may represent promising tools to improve the delivery of AuNPs to the central nervous system (CNS). In this review, we summarize the potential applications of AuNPs to CNS disorders, discuss different strategies based on the use of peptides or proteins to improve the delivery of AuNPs to the brain, and examine the intranasal administration route, which bypasses the BBB. We also analyze the potential neurotoxicity of AuNPs and the perspectives and new challenges concerning the use of peptides and proteins to enhance the delivery of AuNPs to the brain. The majority of the work described in this review is in a preclinical stage of experimentation, or in select cases, in clinical trials in humans. We note that the use of AuNPs still requires substantial study before being translated into human applications. However, for further clinical research, the issues related to the potential use of AuNPs must be analyzed.