Brain Bioavailability Research Articles

Abstract LB327: Discovery and characterization of ABSK112, a next-generation and potential best-in-class EGFR Exon20 mutant inhibitor with superior selectivity and brain penetration ability

Abstract Introduction: EGFR Exon20 mutations are clinically validated oncogenic alterations including a wide spectrum of mutations occurring in lung cancer and various other cancer types. Although several EGFR Exon20 inhibitors have reached clinical stage or received approval, there still leave large room for improvement in safety and efficacy, likely due to their limited selectivity against wild-type EGFR or other kinases, suboptimal mutation coverage, and lack brain penetrating ability. Herein, we have discovered a novel and next-generation EGFR Exon20 mutation inhibitor, ABSK112. It showed high selectivity over wild-type EGFR and other kinases, as well as a more comprehensive coverage over majority of EGFR Exon20 mutations in comparison with other EGFR Exon20 inhibitors. Method: Anti-proliferation experiments in cell lines harboring various EGFR Exon20 mutations were used to evaluate potency and spectrum of coverage for ABSK112 and other inhibitors. Efficacy studies and PK/PD study in multiple tumor models confirmed its in vivo activities. Cell proliferation and in vivo efficacies studies in models harboring mutated or wild type EGFR were used to demonstrate its selectivity. Kinome selectivity, safety profiles, PK and ADME profiles were also characterized. Results: ABSK112 showed potent inhibition of proliferation in multiple EGFR Exon20 mutation cell lines and superior wild-type EGFR selectivity compare to other inhibitors including mobocertinib (TAK788). In xenograft mouse models with various EGFR Exon20 mutations, oral dose of ABSK112 showed strong and dose-dependent anti-tumor efficacy. Moreover, ABSK112 exhibited much reduced in vivo inhibition against xenograft tumors with wild-type EGFR, compared to mobocertinib, confirming its superior selectivity. PK/PD analysis showed good correlation between ABSK112 exposure and inhibition of EGFR signaling. ABSK112 also demonstrated excellent preclinical brain penetration, bioavailability, and safety profile. Conclusion: ABSK112 is a novel and next-generation EGFR Exon20 mutation inhibitor with improved selectivity over wild-type EGFR and strong brain penetrating ability. It has demonstrated superior in vivo efficacy in several xenograft models with various EGFR Exon20 mutations, and excellent drug-like properties supporting its further development into clinical studies. Citation Format: Mei Ning, Shuqun Yang, Juan Peng, Shuangyun Hu, Weijia Lu, Cheng Dai, Baowei Zhao, Mingming Zhang, Hongping Yu, Zhui Chen, Yao-chang Xu. Discovery and characterization of ABSK112, a next-generation and potential best-in-class EGFR Exon20 mutant inhibitor with superior selectivity and brain penetration ability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB327.

Selective PPAR-Delta/PPAR-Gamma Activation Improves Cognition in a Model of Alzheimer's Disease.

Background: The continuously increasing association of Alzheimer's disease (AD) with increased mortality rates indicates an unmet medical need and the critical need for establishing novel molecular targets for therapeutic potential. Agonists for peroxisomal proliferator activating receptors (PPAR) are known to regulate energy in the body and have shown positive effects against Alzheimer's disease. There are three members of this class (delta, gamma, and alpha), with PPAR-gamma being the most studied, as these pharmaceutical agonists offer promise for AD because they reduce amyloid beta and tau pathologies, display anti-inflammatory properties, and improve cognition. However, they display poor brain bioavailability and are associated with several adverse side effects on human health, thus limiting their clinical application. Methods: We have developed a novel series of PPAR-delta and PPAR-gamma agonists in silico with AU9 as our lead compound that displays selective amino acid interactions focused upon avoiding the Tyr-473 epitope in the PPAR-gamma AF2 ligand binding domain. Results: This design helps to avoid the unwanted side effects of current PPAR-gamma agonists and improve behavioral deficits and synaptic plasticity while reducing amyloid-beta levels and inflammation in 3xTgAD animals. Conclusions: Our innovative in silico design of PPAR-delta/gamma agonists may offer new perspectives for this class of agonists for AD.

Abstract 6263: Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma

Abstract Background: Glioblastoma (GBM) patients face a dismal prognosis, with median survival of ~15 months and 5-year survival rate of 6.8%. Standard of care includes surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Unfortunately, all GBM patients succumb to their disease. Glioma stem cells (GSCs), a GBM subpopulation, possess enhanced DNA repair that contributes to chemoradiotherapy resistance. The epigenetic modifier lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in GBM and emerging studies show KDM1A is implicated in DNA damage response. This study aims to determine the significance of KDM1A to promote DNA repair in GSCs. We hypothesize KDM1A inhibition augments therapeutic efficacy of TMZ via attenuation of DNA repair pathways. Methods: Patient-derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination KDM1A knockdown or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR), and non-homologous end-joining (NHEJ) reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ was determined using orthotopic murine GBM models. Results: GBM patient data sets showed KDM1A is elevated in recurrent GBM versus primary tumors. KDM1A knockdown, knockout, or pharmacological inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies showed KDM1A is enriched at the promoters of DNA repair genes. RNA-seq studies confirmed that KDM1A inhibition reduced expression of DNA repair-related genes. RT-qPCR and Western blotting validated downregulation of DNA double-strand break repair genes. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay demonstrated increased TMZ-mediated DNA damage in NCD38-treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of mice bearing orthotopic GBM tumors. Conclusions: Our results provide compelling evidence KDM1A is essential for DNA repair in GSCs and KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination therapy of KDM1A inhibitor NCD38 with TMZ is a potential novel therapeutic strategy to improve GBM outcomes. Citation Format: Salvador Alejo, Bridgitte Palacios, Prabhakar Pitta Venkata, He Yi, Wenjing Li, Jessica Johnson, Sridharan Jayamohan, Weixing Zhao, Siyuan Zheng, Takayoshi Suzuki, Rajeshwar R. Tekmal, Andrew Brenner, Ratna K. Vadlamudi, Gangadhara R. Sareddy. Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6263.

301 Inhibition of lysine-specific histone demethylase 1A (KDM1A/LSD1) attenuates DNA double strand break repair and enhances efficacy of temozolomide in glioblastoma

OBJECTIVES/GOALS: Glioblastoma (GBM) patients face a poor prognosis. Glioma stem cells (GSCs), a chemo resistant GBM subpopulation, possess enhanced DNA repair and elevated levels of epigenetic modifier KDM1A. This study aims to establish the significance of KDM1A in DNA repair and determine the potential of novel KDM1A inhibitor NCD38 to enhance TMZ efficacy in GSCs. METHODS/STUDY POPULATION: Patient derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination of KDM1A knockdown, knockout, or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR) or non-homologous end-joining (NHEJ) DNA repair reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ treatment was determined using orthotopic murine GBM models. RESULTS/ANTICIPATED RESULTS: KDM1A knockdown, knockout, or inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies revealed KDM1A is enriched at DNA repair gene promoters. RNA-seq studies suggest KDM1A inhibition reduces DNA double strand break repair gene expression, with these findings validated using RT-qPCR and Western blotting. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay support findings of increased DNA damage in NCD38/TMZ combination treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of orthotopic GBM tumor-bearing mice. DISCUSSION/SIGNIFICANCE: Our results show compelling evidence that KDM1A is essential for DNA repair in GSCs and that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination of KDM1A inhibitor NCD38 with TMZ could serve as a promising novel therapeutic strategy that can be translated to improve GBM patient outcomes.

Exploring temozolomide encapsulated PEGylated liposomes and lyotropic liquid crystals for effective treatment of glioblastoma: in-vitro, cell line, and pharmacokinetic studies

Temozolomide (TMZ) is one of the best choices for treating glioblastoma. However, due to the short plasma half-life, only 20–30 % brain bioavailability can be achieved using traditional formulations. In the present study, PEGylated liposomes and lyotropic liquid crystals (LLCs) were developed and investigated to prolong the plasma circulation time of TMZ. Industrially feasible membrane extrusion and modified hot melt emulsification techniques were utilized during the formulation. Liposomes and LLCs in the particle size range of 80–120 nm were obtained with up to 50 % entrapment efficiency. The nanocarriers were found to show a prolonged release of up to 72 h. The cytotoxicity studies in glioblastoma cell lines revealed a ∼1.6-fold increased cytotoxicity compared to free TMZ. PEGylated liposomes and PEGylated LLCs were found to show a 3.47 and 3.18-fold less cell uptake in macrophage cell lines than uncoated liposomes and LLCs, respectively. A 1.25 and 2-fold increase in the plasma t1/2 was observed with PEGylated liposomes and PEGylated LLCs, respectively, compared to the TMZ when administered intravenously. Extending plasma circulation time of TMZ led to significant increase in brain bioavailability. Overall, the observed improved pharmacokinetics and biodistribution of TMZ revealed the potential of these PEGylated nanocarriers in the efficient treatment of glioblastoma.

Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.

Nanosystems for Brain Targeting of Antipsychotic Drugs: An Update on the Most Promising Nanocarriers for Increased Bioavailability and Therapeutic Efficacy

Orally administered antipsychotic drugs are the first-line treatment for psychotic disorders, such as schizophrenia and bipolar disorder. Nevertheless, adverse drug reactions jeopardize clinical outcomes, resulting in patient non-compliance. The design formulation strategies for enhancing brain drug delivery has been a major challenge, mainly due to the restrictive properties of the blood-brain barrier. However, recent pharmacokinetic and pharmacodynamic in vivo assays confirmed the advantage of the intranasal route when compared to oral and intravenous administration, as it allows direct nose-to-brain drug transport via neuronal pathways, reducing systemic side effects and maximizing therapeutic outcomes. In addition, the incorporation of antipsychotic drugs into nanosystems such as polymeric nanoparticles, polymeric mixed micelles, solid lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, nanoemulgels, nanosuspensions, niosomes and spanlastics, has proven to be quite promising. The developed nanosystems, having a small and homogeneous particle size (ideal for nose-to-brain delivery), high encapsulation efficiency and good stability, resulted in improved brain bioavailability and therapeutic-like effects in animal models. Hence, although it is essential to continue research in this field, the intranasal delivery of nanosystems for the treatment of schizophrenia, bipolar disorder and other related disorders has proven to be quite promising, opening a path for future therapies with higher efficacy.

Hyperoside alleviates toxicity of β-amyloid via endoplasmic reticulum-mitochondrial calcium signal transduction cascade in APP/PS1 double transgenic Alzheimer's disease mice

Alzheimer's disease is a neurodegenerative disorder characterized by a decline in cognitive function. The β-amyloid (Aβ) hypothesis suggests that Aβ peptides can spontaneously aggregate into β-fragment-containing oligomers and protofibrils, and this activation of the amyloid pathway alters Ca2+ signaling in neurons, leading to neurotoxicity and thus apoptosis of neuronal cells. In our study, a blood-brain barrier crossing flavonol glycoside hyperoside was identified with anti-Aβ aggregation, BACE inhibitory, and neuroprotective effect in cellular or APP/PSEN1 double transgenic Alzheimer's disease mice model. While our pharmacokinetic data confirmed that intranasal administration of hyperoside resulted in a higher bio-availability in mice brain, further in vivo studies revealed that it improved motor deficit, spatial memory and learning ability of APP/PSEN1 mice with reducing level of Aβ plaques and GFAP in the cortex and hippocampus. Bioinformatics, computational docking and in vitro assay results suggested that hyperoside bind to Aβ and interacted with ryanodine receptors, then regulated cellular apoptosis via endoplasmic reticulum-mitochondrial calcium (Ca2+) signaling pathway. Consistently, it was confirmed that hyperoside increased Bcl2, decreased Bax and cyto-c protein levels, and ameliorated neuronal cell death in both in vitro and in vivo model. By regulating Aβ-induced cell death via regulation on Ca2+ signaling cascade and mitochondrial membrane potential, our study suggested that hyperoside may work as a potential therapeutic agent or preventive remedy for Alzheimer's disease.

Investigating the Targeting Power to Brain Tissues of Intranasal Rasagiline Mesylate-Loaded Transferosomal In Situ Gel for Efficient Treatment of Parkinson's Disease.

Rasagiline mesylate (RSM) is a hydrophilic drug with poor oral bioavailability (36%) because of hepatic first-pass metabolism. The present study focuses on delivering RSM directly to the brain through its inclusion within transferosomal in situ gel administered through the intranasal (IN) route. Transferosomes were formed by the thin-film hydration method with the aid of Design-Expert® software by varying the edge activator (EA) type in the absence or presence of cholesterol. By desirability calculations, the optimum formulation was composed of phosphatidylcholine and sodium deoxycholate as an EA (5:1% w/w) with no cholesterol. The optimum formulation was 198.63 ± 34.98 nm in size and displayed an entrapment efficiency of 95.73 ± 0.09%. Transmission electron microscopy revealed discrete and spherical vesicles. Optimized transferosomes were further incorporated into an in situ gel composed of 0.5% pectin, 15% Pluronic® F-127, and 5% Pluronic® F-68 and tested for the in vivo performance. The systemic as well as brain kinetics were assessed in rats by comparing the IN-administered in situ gel to the IV aqueous solution. The optimum in situ gel showed safety and biocompatibility on rats' nasal mucosa with enhanced brain bioavailability (131.17%). Drug targeting efficiency and direct transport percentage indices (304.53% and 67.16%, respectively) supported successful brain targeting offering direct nose-to-brain drug delivery.

Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments the efficacy of temozolomide in glioblastoma.

Efficient DNA repair in response to standard chemo and radiation therapies often contributes to glioblastoma (GBM) therapy resistance. Understanding the mechanisms of therapy resistance and identifying the drugs that enhance the therapeutic efficacy of standard therapies may extend the survival of GBM patients. In this study, we investigated the role of KDM1A/LSD1 in DNA double-strand break (DSB) repair and a combination of KDM1A inhibitor and temozolomide (TMZ) in vitro and in vivo using patient-derived glioma stem cells (GSCs). Brain bioavailability of the KDM1A inhibitor (NCD38) was established using LS-MS/MS. The effect of a combination of KDM1A knockdown or inhibition with TMZ was studied using cell viability and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, RT-qPCR, western blot, homologous recombination (HR) and non-homologous end joining (NHEJ) reporter, immunofluorescence, and comet assays. Orthotopic murine models were used to study efficacy in vivo. TCGA analysis showed KDM1A is highly expressed in TMZ-treated GBM patients. Knockdown or knockout or inhibition of KDM1A enhanced TMZ efficacy in reducing the viability and self-renewal of GSCs. Pharmacokinetic studies established that NCD38 readily crosses the blood-brain barrier. CUT&Tag-seq studies showed that KDM1A is enriched at the promoters of DNA repair genes and RNA-seq studies confirmed that KDM1A inhibition reduced their expression. Knockdown or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity and enhanced TMZ-mediated DNA damage. Acombination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced the survival of tumor-bearing mice. Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.

Translational Significance of GMF-β Inhibition by Indazole-4-yl-methanol in Enteric Glial Cells for Treating Multiple Sclerosis

In the emerging context of gut-brain control of multiple sclerosis (MS), developing therapeutics targeting proinflammatory proteins controlling the gut-brain immunomodulation is welcoming. One such immunomodulator is glia maturation factor-β (GMF-β). GMF-β activation following GMF-β-ser-83 phosphorylation upregulates proinflammatory responses and exacerbates experimental autoimmune encephalomyelitis (EAE). Notably, GMF-β-/- mice exhibited no EAE symptoms. Thus, we identified 1H-indazole-4-yl-methanol (GMFBI.1) inhibitor which blocked GMF-β-ser-83 phosphorylation critical in EAE suppression. To establish gut GMF-β's role in EAE in the context of gut-brain involvement in neurodegenerative diseases, we altered gut GMFBI.1 bioavailability as an index of EAE suppression. At first, we identified Miglyol 812N as a suitable biocompatible GMFBI.1 carrier compared to other FDA-approved carriers using in silico molecular docking analysis. GMFBI.1 administration in Miglyol 812N enhanced its retention/brain permeability. Subsequently, we administered GMFBI.1-Miglyol 812N by subcutaneous/oral routes at different doses with differential GMFBI.1 bioavailability in gut and brain to assess the role of local GMFBI.1 bioavailability in EAE reversal by a pharmacokinetic approach. Deprival of gut GMFBI.1 bioavailability led to partial EAE suppression despite having sufficient GMFBI.1 in circulation to inhibit brain GMF-β activity. Restoration of gut GMFBI.1 bioavailability led to complete EAE reversal. Molecular pathology behind partial/full EAE reversal was associated with differential GMF-β-Ser-83 phosphorylation/GM-CSF expression levels in enteric glial cells owing to GMFBI.1 bioavailability. In addition, we observed leaky gut reversal, tight junction protein ZO-1 restoration, beneficial gut microbiome repopulation, recovery from gut dysbiosis, and upregulation of Treg cells. GMFBI.1's dual gut/brain targeting of GMF-β has therapeutical/translational potential in controlling autoimmunity in MS.

Nano and Microemulsions for the Treatment of Depressive and Anxiety Disorders: An Efficient Approach to Improve Solubility, Brain Bioavailability and Therapeutic Efficacy.

Most drugs used for the treatment of depression, anxiety and related disorders have low absorption, high metabolism, low brain targeting and/or low water solubility, which can make it hard to formulate them at high strength and can also lead to decreased bioavailability. Incorporating these drugs into nanometric emulsions can solve these issues. Hence, the aim of the present review was to assess the potential of nano and micro emulsions for the delivery of antidepressant and anxiolytic drugs. The results from several studies showed that nanometric emulsions were able to increase drug strength up to 20,270-fold (compared to aqueous solubility). Moreover, in general, the formulations showed droplet size, polydispersity index, zeta potential, viscosity, osmolality, pH, in vitro drug release and ex vivo drug permeation as adequate for the intended effect and administration route. In vivo animal pharmacokinetic experiments showed that nanometric emulsions improved systemic drug bioavailability and/or brain targeting, and in vivo pharmacodynamic studies showed that they had antidepressant and/or anxiolytic effects, also being apparently safe. Hence, the current review provides proof of the potential of nano and microemulsions for improving solubilization and increasing the overall bioavailability of antidepressant and/or anxiolytic drugs, providing evidence of a possible useful strategy for future therapies.

Polyphenol bioavailability and modulatory potential on brain antioxidative markers in C57BL/6 mouse

Background and purpose: Prunus spinose L. is a traditionally consumed, recently scientifically reexamined plant. Brain bioavailability and functionality of polyphenols (PPH) of blackthorn flower extract (PSE) was investigated.Materials and methods: C57BL/6 mice received oral daily repeated doses of 25 mg/kg body weight of total PSE polyphenols for 28 days. Brain concentrations of individual polyphenols from PSE were determined by UPLC/MS on 1st,7th, 14th, 21st, and 28th day. Brain antioxidative defense markers were examined as indicators of functionality after bioaccumulation.Results: A total of 68.7% PPH present in PSE were detected in the brain. Higher (p≤0.05) Cmax/AUClast in the PSE treatment vs. control group was recorded for 59.1% of brain detected compounds, indicating relatively good bioaccumulation in the brain. The highest present compounds in PSE were not necessarily the ones mostly bioapsorbed in the brain. Kaempherols were not significantly distributed, opposite to phenolic acids, quercetins or epicgaloatechin-3-gallate. The compounds with the highest concentrations on 28th day were 4-p-coumaroylquinonic acid, (-)-epicatechin, quercetin-3-O-rutinoside, quercetin–rhamnoside, kaempherol-3-rutinoside and quercetin-3-gucoside. Brain lipid peroxidation (MDA) decreased (p<0.05) on the 21st and 28th day in the PSE group. Increase (p<0.05) in GSH concentration was observed on the 21st and 28th and SOD activity on the 28th day. Catalase activity was unchanged. It could be hypothesized that highest PPH concentration-ratios, caused reduction of lipid peroxidation by radical scavenging and simultaneous induction of glutathione and SOD pathways.Conclusions: Screened compounds could be candidates for examining or creation of brain targeted “neuro-nutriceuticals” polyphenol mixtures.

Nose-to-brain delivery of rotigotine redispersible nanosuspension: In vitro and in vivo characterization

Rotigotine (RTG) is an anti-Parkinson's drug approved by the USFDA to treat motor and non-motor symptoms of both early and advanced stages of the disease. However, issues like poor aqueous solubility, low oral bioavailability (<1%), first-pass metabolism, low systemic bioavailability (∼37%) and low brain bioavailability demean the therapy. This work developed poloxamer-stabilized RTG-Nanosuspension for intranasal (i.n.) administration. RTG-Nanosuspension was optimized using Box-Behnken design with critical variables affecting the responses, viz. particle size and PDI. The optimized RTG-Nanosuspension showed mean particle size of 73 nm and PDI of 0.286. Lyophilized RTG-Nanosuspension was also characterized for drug crystallinity, interactions, and morphology. The lyophilized RTG-Nanosuspension showed good stability and was a porous structure. The first 15 min of in vitro dissolution showed 95% cumulative drug dissolved from RTG-Nanosuspension formulation. RTG-Nanosuspension showed 20-fold increase in nasal permeation. The nasal ciliotoxicity study showed both RTG-Nanosuspension and drug dispersion were safe for i.n. delivery. An in vivo study showed that optimized RTG-Nanosuspension helped target RTG to the brain following i.n. administration. Overall, the RTG-Nanosuspension formulation showed potential in nose-to-brain delivery for targeted application in Parkinson's disease.

Identification of a Cardiac Glycoside Exhibiting Favorable Brain Bioavailability and Potency for Reducing Levels of the Cellular Prion Protein.

Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets Na,K-ATPases (NKAs) with cardiac glycosides (CGs), causing cells to respond with the degradation of these pumps and nearby molecules, including PrPC. Because the therapeutic window of widely used CGs is narrow and their brain bioavailability is low, we set out to identify a CG with improved pharmacological properties for this indication. Starting with the CG known as oleandrin, we combined in silico modeling of CG binding poses within human NKA folds, CG structure-activity relationship (SAR) data, and predicted blood-brain barrier (BBB) penetrance scores to identify CG derivatives with improved characteristics. Focusing on C4'-dehydro-oleandrin as a chemically accessible shortlisted CG derivative, we show that it reaches four times higher levels in the brain than in the heart one day after subcutaneous administration, exhibits promising pharmacological properties, and suppresses steady-state PrPC levels by 84% in immortalized human cells that have been differentiated to acquire neural or astrocytic characteristics. Finally, we validate that the mechanism of action of this approach for reducing cell surface PrPC levels requires C4'-dehydro-oleandrin to engage with its cognate binding pocket within the NKA α subunit. The improved brain bioavailability of C4'-dehydro-oleandrin, combined with its relatively low toxicity, make this compound an attractive lead for brain CG indications and recommends its further exploration for the treatment of prion diseases.

Valproic Acid-Induced Upregulation of Multidrug Efflux Transporter ABCG2/BCRP via PPARα-Dependent Mechanism in Human Brain Endothelial Cells

Despite the progress made in the development of new antiepileptic drugs (AEDs), poor response to them is a rising concern in epilepsy treatment. Of several hypotheses explaining AED treatment failure, the most promising theory is the overexpression of multidrug transporters belonging to ATP-binding cassette (ABC) transporter family at blood-brain barrier. Previous data show that AEDs themselves can induce these transporters, in turn affecting their own brain bioavailability. Presently, this induction and the underlying regulatory mechanism involved at human blood-brain barrier is not well elucidated. Herein, we sought to explore the effect of most prescribed first- and second-line AEDs on multidrug transporters in human cerebral microvascular endothelial cells, hCMEC/D3. Our work demonstrated that exposure of these cells to valproic acid (VPA) induced mRNA, protein, and functional activity of breast cancer resistance protein (BCRP/ABCG2). On examining the substrate interaction status of AEDs with BCRP, VPA, phenytoin, and lamotrigine were found to be potential BCRP substrates. Furthermore, we observed that siRNA-mediated knockdown of peroxisome proliferator-activated receptor alpha (PPARα) or use of PPARα antagonist, resulted in attenuation of VPA-induced BCRP expression and transporter activity. VPA was found to increase PPARα expression and trigger its translocation from cytoplasm to nucleus. Findings from chromatin immunoprecipitation and luciferase assays showed that VPA enhances the binding of PPARα to its response element in the ABCG2 promoter, resulting in elevated ABCG2 transcriptional activity. Taken together, these in vitro findings highlight PPARα as the potential molecular target to prevent VPA-mediated BCRP induction, which may have important implications in VPA pharmacoresistance. SIGNIFICANCE STATEMENT: Induction of multidrug transporters at blood-brain barrier can largely affect the bioavailability of the substrate antiepileptic drugs in the brains of patients with epilepsy, thus affecting their therapeutic efficacy. The present study reports a mechanistic pathway of breast cancer resistance protein (BCRP/ABCG2) upregulation by valproic acid in human brain endothelial cells via peroxisome proliferator-activated receptor alpha involvement, thereby providing a potential strategy to prevent valproic acid pharmacoresistance in epilepsy.

The inhibition of NCS-1 binding to Ric8a rescues fragile X syndrome mice model phenotypes

Fragile X syndrome (FXS) is caused by the loss of function of Fragile X mental retardation protein (FMRP). FXS is one of the leading monogenic causes of intellectual disability (ID) and autism. Although it is caused by the failure of a single gene, FMRP that functions as an RNA binding protein affects a large number of genes secondarily. All these genes represent hundreds of potential targets and different mechanisms that account for multiple pathological features, thereby hampering the search for effective treatments. In this scenario, it seems desirable to reorient therapies toward more general approaches. Neuronal calcium sensor 1 (NCS-1), through its interaction with the guanine-exchange factor Ric8a, regulates the number of synapses and the probability of the release of a neurotransmitter, the two neuronal features that are altered in FXS and other neurodevelopmental disorders. Inhibitors of the NCS-1/Ric8a complex have been shown to be effective in restoring abnormally high synapse numbers as well as improving associative learning in FMRP mutant flies. Here, we demonstrate that phenothiazine FD44, an NCS-1/Ric8a inhibitor, has strong inhibition ability in situ and sufficient bioavailability in the mouse brain. More importantly, administration of FD44 to two different FXS mouse models restores well-known FXS phenotypes, such as hyperactivity, associative learning, aggressive behavior, stereotype, or impaired social approach. It has been suggested that dopamine (DA) may play a relevant role in the behavior and in neurodevelopmental disorders in general. We have measured DA and its metabolites in different brain regions, finding a higher metabolic rate in the limbic area, which is also restored with FD44 treatment. Therefore, in addition to confirming that the NCS-1/Ric8a complex is an excellent therapeutic target, we demonstrate the rescue effect of its inhibitor on the behavior of cognitive and autistic FXS mice and show DA metabolism as a FXS biochemical disease marker.

Alzheimer's Disease: Treatment Strategies and Their Limitations.

Alzheimer's disease (AD) is the most frequent case of neurodegenerative disease and is becoming a major public health problem all over the world. Many therapeutic strategies have been explored for several decades; however, there is still no curative treatment, and the priority remains prevention. In this review, we present an update on the clinical and physiological phase of the AD spectrum, modifiable and non-modifiable risk factors for AD treatment with a focus on prevention strategies, then research models used in AD, followed by a discussion of treatment limitations. The prevention methods can significantly slow AD evolution and are currently the best strategy possible before the advanced stages of the disease. Indeed, current drug treatments have only symptomatic effects, and disease-modifying treatments are not yet available. Drug delivery to the central nervous system remains a complex process and represents a challenge for developing therapeutic and preventive strategies. Studies are underway to test new techniques to facilitate the bioavailability of molecules to the brain. After a deep study of the literature, we find the use of soft nanoparticles, in particular nanoliposomes and exosomes, as an innovative approach for preventive and therapeutic strategies in reducing the risk of AD and solving problems of brain bioavailability. Studies show the promising role of nanoliposomes and exosomes as smart drug delivery systems able to penetrate the blood-brain barrier and target brain tissues. Finally, the different drug administration techniques for neurological disorders are discussed. One of the promising therapeutic methods is the intranasal administration strategy which should be used for preclinical and clinical studies of neurodegenerative diseases.

Meta-Analysis: A Convenient Tool for the Choice of Nose-to-Brain Nanocarriers.

The intranasal route represents a high promising route of administration aiming for brain delivery. Yet, it represents one of the most difficult and complicated routes. Accordingly, scientists are in a continuous search for novel drug delivery vehicles such as the lipid and polymeric nanoparticles that are apt to enhance the bioavailability of the administered drugs to reach the brain. In this study, a certain number of publications were selected from different databases and literature. Meta-analysis studies using two different algorithms (DerSimonian-Laird and inverse variance) followed aiming to explore the published studies and confirm by evidence the superiority of nanocarriers in enhancing the brain bioavailability of various drugs. Furthermore, the quantitative comparison of lipid versus polymeric nanosystems was performed. The area under the curve (AUC) as an important pharmacokinetic parameter extracted from in vivo animal studies was designated as the "effect" in the performed meta-analysis after normalization. Forest plots were generated. The meta-analysis confirmed the augmentation of the AUC after the comparison with traditional preparations such as solutions and suspensions. Most importantly, lipid nanoparticles were proven to be significantly superior to the polymeric counterparts.

Nose to brain delivery of selegiline loaded PLGA/lipid nanoparticles: Synthesis, characterisation and brain pharmacokinetics evaluation

Parkinson's disease (PD) is the second most common progressive neurodegenerative disease that promotes neuronal cell death. The primary treatment strategy for Parkinson's disease involves the therapy of an MAO-B inhibitor molecule, selegiline. The present research aims to fabricate selegiline loaded polymeric (PLGA) nanoparticles (SPNPs) and selegiline loaded lipid-PLGA hybrid nanoparticles (SLPNPs) while using the single emulsion solvent evaporation method. The prepared SPNPs and SLPNPs formulations were subjected to preliminary optimisation using particle size, zeta-potential, and entrapment efficiency (EE%), and the selected formulations were subjected to further characterisation. Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM) confirmed the particles' 2-D spherical morphology and size. ATR-FTIR was used to evaluate the physical interactions among the formulation ingredients. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) studies confirm that SPNPs and SLPNPs were in an amorphous state. Ex-vivo studies showed 77.56% and 80% of drug permeation in 24 h for optimised formulations of SPNPs-2 and SLPNPs-1, respectively, while pure selegiline showed 65% Permeation. In-vivo study revealed a steady and controlled release profile with a half-life of 13.5 h, and AUC0-24 was observed at 130327.56 ± 231.6 ng/ml*h for SPNPs-2. For SLPNPs-1 an immediate release was observed with a very short half-life and AUC0-24 47548.57 ± 434.8 ng/ml*h compared with pure selegiline HCl solution with 2.3 h and AUC0-24 11116.52 ± 345.7 ng/ml*h. SLPNPs-1 has a shorter half-life due to its immediate release properties with the presence of lipid. Meanwhile, SPNPs-2, which only has a polymeric layer, has a longer half-life due to its sustained release properties. Overall, SPNPs-2 and SLPNPs-1 are promising carriers for selegiline nasal uptake and increase its brain bioavailability compared to oral absorption by pure selegiline.