Fluorine-Modifications Contribute to Potent Antiviral Activity against Highly Drug-Resistant HIV-1 and Favorable Blood-Brain Barrier (BBB) Penetration Property of Novel Central Nervous System (CNS)-targeting HIV-1 Protease Inhibitors In Vitro .

To date, there are no specific treatment regimens for the HIV-1-related central nervous system (CNS) complications, such as HIV-1-associated neurocognitive disorders (HAND). In the present study, we report that two newly generated CNS-targeting HIV-1 protease inhibitors (PIs), GRL-08513 and GRL-08613, which have P1-3,5- bis -fluorophenyl- or P1- para -monofluorophenyl-ring, and P2-tetrahydropyrano-tetrahydrofuran ( Tp -THF) with a sulfonamide isostere, are potent against wild-type HIV-1s and multiple clinically isolated HIV-1s (EC 50 : 0.0001∼0.0032 μM). As assessed with HIV-1 variants that had been selected in vitro to propagate at 5 μM concentration of each HIV-1 PI (atazanavir, lopinavir, or amprenavir), GRL-08513 and GRL-08613 efficiently inhibited the replication of these highly-PI-resistant variants (EC 50 : 0.003∼0.006 μM). GRL-08513 and GRL-08613 also maintained their antiviral activity against HIV-2 ROD as well as severe multi-drug-resistant clinical HIV-1 variants. Additionally, when we assessed with the in vitro blood-brain barrier (BBB) reconstruction system, GRL-08513 and GRL-08613 showed the most promising properties of CNS-penetration among the evaluated compounds including the majority of FDA-approved cART drugs. In the crystallographic analysis of compound-protease (PR) complexes, it was demonstrated that the Tp -THF rings at the P2 moiety of GRL-08513 and GRL-08613 form robust hydrogen-bond interactions with the active-site of HIV-1 PR. Furthermore, both the P1-3,5- bis -fluorophenyl- and P1- para -monofluorophenyl-rings sustain greater contact surfaces and form stronger van der Waals interactions with PR compared to the case of darunavir-PR complex. Taken together, these results strongly suggest that GRL-08513 and GRL-08613 have favorable features for the patients infected with wild-type/multi-drug-resistant HIV-1s, and might serve as candidates of preventive and/or therapeutic for HAND and other CNS complications.

Special delEVery: Extracellular Vesicles as Promising Delivery Platform to the Brain

The treatment of central nervous system (CNS) pathologies is severely hampered by the presence of tightly regulated CNS barriers that restrict drug delivery to the brain. An increasing amount of data suggests that extracellular vesicles (EVs), i.e., membrane derived vesicles that inherently protect and transfer biological cargoes between cells, naturally cross the CNS barriers. Moreover, EVs can be engineered with targeting ligands to obtain enriched tissue targeting and delivery capacities. In this review, we provide a detailed overview of the literature describing a natural and engineered CNS targeting and therapeutic efficiency of different cell type derived EVs. Hereby, we specifically focus on peripheral administration routes in a broad range of CNS diseases. Furthermore, we underline the potential of research aimed at elucidating the vesicular transport mechanisms across the different CNS barriers. Finally, we elaborate on the practical considerations towards the application of EVs as a brain drug delivery system.

Chimeric CNS-Targeting-Peptide Engineered Exosomes for Experimental Allergic Encephalomyelitis Therapy

Background: Multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), leads to demyelination, neuronal injury, and loss of white matter, yet still can't be cured. Exosomes are double-layered membrane vesicles of 30–200 nm in size, which can easily penetrate the blood–brain barrier (BBB). Exosomes derived from umbilical cord mesenchymal stem cells exosomes (UMSC-exos) has been shown to treat experimental autoimmune encephalomyelitis (EAE) through the action of anti-inflammatory and immunomodulatory, but its clinical translation has been hampered by their inefficacious accumulation in CNS. Therefore, we developed a TAxI-peptide-chimeric UMSC-exos termed TAxI-exos for CNS-specific accumulation and curative effect in EAE.Methods: We used an EAE model in vivo, and actived T cells and BV-2 cells models in vitro. After two immunizations to establish the EAE model, UMSC-exos, TAxI-exos or DiR labeled exosomes were administered to EAE mice EAE mice for one dose (150μg) before the peak at day 15. On day 30, the mice were sacrificed to collect spinal cords, spleens, and blood for analysis of demyelination, inflammation, microglia, the proportions of T-cell subsets, and the expression of inflammatory cytokines. In vitro, for immune mechanism analyses, PBMCs and splenocytes isolated from healthy C57BL/6 mice, were activated and incubated with 0.15mg/mL UMSC-exos or TAxI-exos. Activated BV-2 cells were used to explore the targeting-ability and polarization-regulating ability of UMSC-exos and TAxI-exos.Results: As expected, TAxI-exos had significant curative effects in EAE mice compared with UMSC-exos via an enhanced targeting-ability. The treatment alleviated inflammation, facilitated microglial cell polarization from M1 to M2, reduced the proportions of T-cell subsets, increased the expression levels of IL-4, IL-10, TGF-β, and IDO-1, and decreased the levels of IL-2, IL-6, IL-17A, IFN-γ, and TNF-α. Conclusions: TAxI-exos have a great CNS-targeting ability and suppress pathological processes in EAE mice, which have a great potential therapeutic utility for MS and other CNS diseases.

Enhanced CNS transduction from AAV.PHP.eB infusion into the cisterna magna of older adult rats compared to AAV9

The development of high efficiency, central nervous system (CNS) targeting AAV-based gene therapies is necessary to address challenges in both pre-clinical and clinical investigations. The engineered capsids, AAV.PHP.B and AAV.PHP.eB, show vastly improved blood-brain barrier penetration compared to their parent serotype, AAV9, but with variable effect depending on animal system, strain, and delivery route. As most characterizations of AAV.PHP variants have been performed in mice, it is currently unknown whether AAV.PHP variants improve CNS targeting when delivered intrathecally in rats. We evaluated the comparative transduction efficiencies of equititer doses (6 × 1011vg) of AAV.PHP.eB-CAG-GFP and AAV9-CAG-GFP when delivered into the cisterna magna of 6–9-month old rats. Using both quantitative and qualitative assessments, we observed consistently superior biodistribution of GFP+ cells and fibers in animals treated with AAV.PHP.eB compared to those treated with AAV9. Enhanced GFP signal was uniformly observed throughout rostrocaudal brain regions in AAV.PHP.eB-treated animals with matching GFP protein expression detected in the forebrain, midbrain, and cerebellum. Collectively, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.