Dodecyl creatine ester therapy: from promise to reality.

Pathogenic variants in SLC6A8, the gene which encodes creatine transporter SLC6A8, prevent creatine uptake in the brain and result in a variable degree of intellectual disability, behavioral disorders (e.g., autism spectrum disorder), epilepsy, and severe speech and language delay. There are no treatments to improve neurodevelopmental outcomes for creatine transporter deficiency (CTD). In this spotlight, we summarize recent advances in innovative molecules to treat CTD, with a focus on dodecyl creatine ester, the most promising drug candidate.

Brain organoids for hypoxic-ischemic studies: from bench to bedside.

Our current knowledge regarding the development of the human brain mostly derives from experimental studies on non-human primates, sheep, and rodents. However, these studies may not completely simulate all the features of human brain development as a result of species differences and variations in pre- and postnatal brain maturation. Therefore, it is important to supplement the in vivo animal models to increase the possibility that preclinical studies have appropriate relevance for potential future human trials. Three-dimensional brain organoid culture technology could complement in vivo animal studies to enhance the translatability of the preclinical animal studies and the understanding of brain-related disorders. In this review, we focus on the development of a model of hypoxic-ischemic (HI) brain injury using human brain organoids to complement the translation from animal experiments to human pathophysiology. We also discuss how the development of these tools provides potential opportunities to study fundamental aspects of the pathophysiology of HI-related brain injury including differences in the responses between males and females.

Targeting the Multiple Complex Processes of Hypoxia-Ischemia to Achieve Neuroprotection.

Hypoxic-ischemic encephalopathy (HIE) is a major cause of newborn brain damage stemming from a lack of oxygenated blood flow in the neonatal period. Twenty-five to fifty percent of asphyxiated infants who develop HIE die in the neonatal period, and about sixty percent of survivors develop long-term neurological disabilities. From the first minutes to months after the injury, a cascade of events occurs, leading to blood-brain barrier (BBB) opening, neuronal death and inflammation. To date, the only approach proposed in some cases is therapeutic hypothermia (TH). Unfortunately, TH is only partially protective and is not applicable to all neonates. This review synthesizes current knowledge on the basic molecular mechanisms of brain damage in hypoxia-ischemia (HI) and on the different therapeutic strategies in HI that have been used and explores a major limitation of unsuccessful therapeutic approaches.

Molecular Heterogeneity of the Brain Endothelium.

The blood-brain barrier (BBB) is part of a neurovascular structure located in the brain's micro vessels, that is essential to maintain brain homeostasis, but prevents the brain uptake of most drugs. Because of its importance in neuro-pharmacotherapy, the BBB has been the subject of extensive research since its discovery over 100 years ago. Major advances in understanding the structure and function of the barrier have been made. Drugs are re-designed to cross the BBB. However, despite these efforts, overcoming the BBB efficiently to treat brain diseases safely remains challenging. The majority of BBB research studies focus on the BBB as a homogenous structure throughout the different brain regions. However, this simplification may lead to an inadequate understanding of the BBB function with significant therapeutic consequences. From this perspective, we analyzed the gene and protein expression profiles of the BBB in the micro vessels from the brains of mice that were isolated from two different brain regions, namely the cortex and the hippocampus. The expression profile of the inter-endothelial junctional protein (claudin-5), three ABC transporters (P-glycoprotein, Bcrp and Mrp-1), and three BBB receptors (lrp-1, TRF and GLUT-1) were analyzed. Our gene and protein analysis showed that the brain endothelium in the hippocampus exhibits different expression profiles compared to the brain cortex. Specifically, brain endothelial cells (BECs) of the hippocampus express higher gene levels of abcb1, abcg2, lrp1, and slc2a1 compared to the BECs of the cortex regions with a trend of increase for claudin-5, while BECs of the cortex express higher gene levels of abcc1 and trf compared to the hippocampus. At the protein levels, the P-gp expression was found to be significantly higher in the hippocampus compared to the cortex, while TRF was found to be up-regulated in the cortex. These data suggest that the structure and function of the BBB are not homogeneous, and imply that drugs are not delivered similarly among the different brain regions. Appreciation of the BBB heterogeneity by future research programs is thus critical for efficient drug delivery and the treatment of brain diseases.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

PURPOSE: Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. METHODS: Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. RESULTS: We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. CONCLUSION: These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

Tissue biodistribution of intravenously administrated titanium dioxide nanoparticles revealed blood-brain barrier clearance and brain inflammation in rat.

BACKGROUND: Notwithstanding increasing knowledge of titanium dioxide nanoparticles (TiO2 NPs) passing through biological barriers, their biodistribution to the central nervous system (CNS) and potential effects on blood-brain barrier (BBB) physiology remain poorly characterized. METHODS: Here, we report time-related responses from single-dose intravenous (IV) administration of 1 mg/kg TiO2 NPs to rats, with particular emphasis on titanium (Ti) quantification in the brain. Ti content in tissues was analyzed using inductively coupled plasma mass spectrometry. Integrity and functionality of the BBB as well as brain inflammation were characterized using a panel of methods including RT-PCR, immuno-histo chemistry and transporter activity evaluation. RESULTS: Biokinetic analysis revealed Ti biopersistence in liver, lungs and spleen up to one year after TiO2 NPs administration. A significant increase of Ti in the brain was observed at early end points followed by a subsequent decrease. In-depth analysis of Ti in the total brain demonstrated quantitative Ti uptake and clearance by brain microvasculature endothelial cells (BECs) with minimal translocation in the brain parenchyma. The presence of Ti in the BECs did not affect BBB integrity, despite rapid reversible modulation of breast cancer resistance protein activity. Ti biopersistence in organs such as liver was associated with significant increases of tight junction proteins (claudin-5 and occludin), interleukin 1ß (IL-1ß), chemokine ligand 1 (CXCL1) and γ inducible protein-10 (IP-10/CXCL10) in BECs and also increased levels of IL-1ß in brain parenchyma despite lack of evidence of Ti in the brain. These findings mentioned suggest potential effect of Ti present at a distance from the brain possibly via mediators transported by blood. Exposure of an in vitro BBB model to sera from TiO2 NPs-treated animals confirmed the tightness of the BBB and inflammatory responses. CONCLUSION: Overall, these findings suggest the clearance of TiO2 NPs at the BBB with persistent brain inflammation and underscore the role of Ti biopersistence in organs that can exert indirect effects on the CNS dependent on circulating factors.

Titanium dioxide nanoparticle impact and translocation through ex vivo, in vivo and in vitro gut epithelia.

BACKGROUND: TiO2 particles are commonly used as dietary supplements and may contain up to 36% of nano-sized particles (TiO2-NPs). Still impact and translocation of NPs through the gut epithelium is poorly documented. RESULTS: We show that, in vivo and ex vivo, agglomerates of TiO2-NPs cross both the regular ileum epithelium and the follicle-associated epithelium (FAE) and alter the paracellular permeability of the ileum and colon epithelia. In vitro, they accumulate in M-cells and mucus-secreting cells, much less in enterocytes. They do not cause overt cytotoxicity or apoptosis. They translocate through a model of FAE only, but induce tight junctions remodeling in the regular ileum epithelium, which is a sign of integrity alteration and suggests paracellular passage of NPs. Finally we prove that TiO2-NPs do not dissolve when sequestered up to 24 h in gut cells. CONCLUSIONS: Taken together these data prove that TiO2-NPs would possibly translocate through both the regular epithelium lining the ileum and through Peyer's patches, would induce epithelium impairment, and would persist in gut cells where they would possibly induce chronic damage.

Epigenetic alterations in creatine transporter deficiency: a new marker for dodecyl creatine ester therapeutic efficacy monitoring.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study, we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients' cells. We also investigated the effect of dodecyl creatine ester (DCE), a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation, unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy.

A Fab of trastuzumab to treat HER2 overexpressing breast cancer brain metastases.

Despite major therapeutic advances for two decades, including the most recently approved anti-HER2 drugs, brain metastatic localizations remain the major cause of death for women with metastatic HER2 breast cancer. The main reason is the limited drug passage of the blood-brain barrier after intravenous injection and the significant efflux of drugs, including monoclocal antibodies, after administration into the cerebrospinal fluid. We hypothesized that this efflux was linked to the presence of a FcRn receptor in the blood-brain barrier. To overcome this efflux, we engineered two Fab fragments of trastuzumab, an anti-HER2 monoclonal antibody, and did a thorough preclinical development for therapeutic translational purpose. We demonstrated the safety and equal efficacy of the Fabs with trastuzumab in vitro, and in vivo using a patient-derived xenograft model of HER2 overexpressing breast cancer. For the pharmacokinetic studies of intra-cerebrospinal fluid administration, we implemented original rat models with catheter implanted into the cisterna magna. After intraventricular administration in rats, we demonstrated that the brain-to-blood efflux of Fab was up to 10 times lower than for trastuzumab, associated with a two-fold higher brain penetration compared to trastuzumab. This Fab, capable of significantly reducing brain-to-blood efflux and enhancing brain penetration after intra-cerebrospinal fluid injection, could thus be a new and original effective drug in the treatment of HER2 breast cancer brain metastases, which will be demonstrated by a phase I clinical trial dedicated to women in resort situations.

P-glycoprotein, breast cancer resistance protein, Organic Anion Transporter 3, and Transporting Peptide 1a4 during blood-brain barrier maturation: involvement of Wnt/ß-catenin and endothelin-1 signaling.

Our current knowledge about drug transporters in the maturational brain is very limited. In this study, we provide a comprehensive overview of the expression and activity profile of P-glycoprotein (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters during blood-brain barrier (BBB) maturation. Gene and protein expressions of the analyzed transporters increase as the brain matures, with no variation in their activity for P-gp and bcrp, while the transport activity of oat3 and oatp1a4 increases during brain maturation from preterm up to adulthood. For the first time, we illustrate a downregulation of nuclear ß-catenin expression in brain capillaries when bcrp, P-gp, oat3, and oatp1a4 transporters are at their highest expression levels. In vivo activation of ß-catenin in rat brains, by intracerebroventricular (ICV) injection of a GSK-3 inhibitor, enhances the activity of P-gp, bcrp, oat3, and oatp1a4. Interestingly, in an in vitro BBB model consisting of a coculture of primary endothelial brain cells with astrocytes or in vivo, activation of ß-catenin enhances the mRNA expression of ET-1. Interestingly, blocking the ETA receptor for endothelin-1 in vivo by ICV injection of a ETA antagonist decreases transporter activity mediated by the activation of ß-catenin. These findings shed light on the role of an interaction between ß-catenin and endothelin-1 signaling in the regulation of these transporters at the BBB.

Exposure to vehicle emissions results in altered blood brain barrier permeability and expression of matrix metalloproteinases and tight junction proteins in mice.

BACKGROUND: Traffic-generated air pollution-exposure is associated with adverse effects in the central nervous system (CNS) in both human exposures and animal models, including neuroinflammation and neurodegeneration. While alterations in the blood brain barrier (BBB) have been implicated as a potential mechanism of air pollution-induced CNS pathologies, pathways involved have not been elucidated. OBJECTIVES: To determine whether inhalation exposure to mixed vehicle exhaust (MVE) mediates alterations in BBB permeability, activation of matrix metalloproteinases (MMP) -2 and -9, and altered tight junction (TJ) protein expression. METHODS: Apolipoprotein (Apo) E(-/-) and C57Bl6 mice were exposed to either MVE (100 µg/m(3) PM) or filtered air (FA) for 6 hr/day for 30 days and resulting BBB permeability, expression of ROS, TJ proteins, markers of neuroinflammation, and MMP activity were assessed. Serum from study mice was applied to an in vitro BBB co-culture model and resulting alterations in transport and permeability were quantified. RESULTS: MVE-exposed Apo E(-/-) mice showed increased BBB permeability, elevated ROS and increased MMP-2 and -9 activity, compared to FA controls. Additionally, cerebral vessels from MVE-exposed mice expressed decreased levels of TJ proteins, occludin and claudin-5, and increased levels of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1ß in the parenchyma. Serum from MVE-exposed animals also resulted in increased in vitro BBB permeability and altered P-glycoprotein transport activity. CONCLUSIONS: These data indicate that inhalation exposure to traffic-generated air pollutants promotes increased MMP activity and degradation of TJ proteins in the cerebral vasculature, resulting in altered BBB permeability and expression of neuroinflammatory markers.

Proteolytically Stable Diaza-Peptide Foldamers Mimic Helical Hot Spots of Protein-Protein Interactions and Act as Natural Chaperones.

A novel class of peptidomimetic foldamers based on diaza-peptide units are reported. Circular dichroism, attenuated total reflection -Fourier transform infrared, NMR, and molecular dynamics studies demonstrate that unlike the natural parent nonapeptide, the specific incorporation of one diaza-peptide unit at the N-terminus allows helical folding in water, which is further reinforced by the introduction of a second unit at the C-terminus. The ability of these foldamers to resist proteolysis, to mimic the small helical hot spot of transthyretin-amyloid ß (Aß) cross-interaction, and to decrease pathological Aß aggregation demonstrates that the introduction of diaza-peptide units is a valid approach for designing mimics or inhibitors of protein-protein interaction and other therapeutic peptidomimetics. This study also reveals that small peptide foldamers can play the same role as physiological chaperone proteins and opens a new way to design inhibitors of amyloid protein aggregation, a hallmark of more than 20 serious human diseases such as Alzheimer's disease.

Dodecyl creatine ester improves cognitive function and identifies key protein drivers including KIF1A and PLCB1 in a mouse model of creatine transporter deficiency.

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8-/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.

Deciphering neuronal deficit and protein profile changes in human brain organoids from patients with creatine transporter deficiency.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3ß, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3ß, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.

Susceptibility of juvenile and adult blood-brain barrier to endothelin-1: regulation of P-glycoprotein and breast cancer resistance protein expression and transport activity.

BACKGROUND: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play a critical role in keeping neurotoxic substances from entering the brain. We and others have previously reported an impact of inflammation on the regulation of adult blood-brain barrier (BBB) efflux transporters. However, studies in children have not been done. From the pediatric clinical perspective, it is important to understand how the central nervous system (CNS) and BBB drug efflux transporters differ in childhood from those of adults under normal and inflammatory conditions. Therefore, we examined and compared the regulation of P-gp and BCRP expression and transport activity in young and adult BBB and investigated the molecular mechanisms underlying inflammatory responses. METHODS: Rats at postnatal day (P) P21 and P84, corresponding to the juvenile and adult stages of human brain maturation, respectively, were treated with endothelin-1 (ET-1) given by the intracerebroventricular (icv) route. Twenty-four hours later, we measured P-gp and BCRP protein expression in isolated brain capillary by immunoblotting as well as by transport activity in vivo by measuring the unbound drug partitioning coefficient of the brain (K(p,uu,brain)) of known efflux transporter substrates administered intravenously. Glial activation was measured by immunohistochemistry. The release of cytokines/chemokines (interleukins-1α, 1-ß (IL-1ß), -6 (IL-6), -10 (IL-10), monocyte chemoattractant protein (MCP-1/CCL2), fractalkine and tissue inhibitor of metalloproteinases-1 (TIMP-1)) were simultaneously measured in brain and serum samples using the Agilent Technology cytokine microarray. RESULTS: We found that juvenile and adult BBBs exhibited similar P-gp and BCRP transport activities in the normal physiological conditions. However, long-term exposure of the juvenile brain to low-dose of ET-1 did not change BBB P-gp transport activity but tended to decrease BCRP transport activity in the juvenile brain, while a significant increase of the activity of both transporters was evidenced at the BBB in the adult brain. Moreover, juvenile and adult brain showed differences in their expression profiles of cytokines and chemokines mediated by ET-1. CONCLUSIONS: BBB transporter activity during neuroinflammation differs between the juvenile and adult brains. These findings emphasize the importance of considering differential P-gp and BCRP transport regulation mechanisms between adult and juvenile BBB in the context of neuroinflammation.

In vivo effects of zearalenone on the expression of proteins involved in the detoxification of rat xenobiotics.

Zearalenone (ZEN) is a lactone derivative of the resorcylic acid produced by various Fusarium species that are widely found in foods and animal feeds. ZEN exerts species-specific estrogenic effects, possibly because of the metabolism differences arising from reduction, hydroxylation, or glucuro-conjugation. The main objective of this study was to determine the levels of expression of rat proteins that are involved in the ZEN detoxification pathway upon acute ZEN treatment. This was achieved by monitoring the mRNA associated with 25 genes using RT-PCR upon ZEN uptake. These genes code for a variety of proteins that are involved in cellular detoxifying pathways, transporters, cytochromes P450 (CYPs), hydroxysteroid dehydrogenases, and transferases, and receptors that are involved in CYP expression or steroid metabolism. Liver samples from rats treated with ZEN were compared to untreated rats or animals treated with classical CYP inducers (phenobarbital, dexamethasone, ß-naphtoflavone, and clofibrate). Significant changes of mRNA expression were observed for the efflux transporter, P-glycoprotein, monooxygenases (CYP2C7, CYP2E1, CYP3A1, CYP3A2, and aromatase), steroid dehydrogenases, and Uridine diphospho-glucuronyl transferases (UGTs). Following a single ZEN treatment, the initial modifications in mRNA levels indicate a close association with microsomal enzyme activity of the CYP2B, CYP2C, and CYP3A protein families.

Regulation of P-glycoprotein by miR-27a-3p at the Brain Endothelial Barrier.

Multi-drug resistance P-glycoprotein (P-gp/MDR1) is one of the most clinically relevant ABC transporters, highly enriched at the blood-brain barrier (BBB) with a broad substrate spectrum including therapeutic drugs and metabolic waste products. Altered P-gp transport function has been implicated in multi-drug resistance and in the pathogenesis and progression of neurological diseases. Recent studies have shown that P-gp expression is modulated by micro-RNAs in peripheral organs. Particularly, miR-27a-3p has been shown to play a critical role in the regulation of P-gp in multi-drug resistant cancer cells. In brain disorders, altered levels of miR-27a-3p were reported in several diseases associated with alterations in P-gp expression at the BBB. However, effect of altered miR-27a-3p expression on P-gp expression at the BBB remains to be determined. In this study, we investigated the role of miR-27a-3p in the regulation of P-gp expression and activity at the brain endothelium. Levels of miR-27a-3p were modulated by mimic and inhibitor transfection in an in-vitro model of human brain endothelial hCMEC/D3 cells. Effect of miR-27a-3p modulation on P-gp expression and activity was examined and the underlying regulatory mechanisms explored. Our results showed that transfection of hCMEC/D3 cells with miR-27a-3p mimic induces expression and activity of P-gp while miR-27a-3p inhibition exerted opposite effects. Mechanistic studies revealed that miR-27a-3p regulates P-gp by mediating Glycogen Synthase Kinase 3 Beta (GSK3ß) inhibition and activating Wnt/ß-catenin signaling. These findings shed light on miR-27a-3p/GSK3ß/ß-catenin as a novel axis that could be exploited to modulate P-gp efflux activity at the brain endothelium and help improving CNS diseases treatment or brain protection.

miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability.

BACKGROUND: The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer's disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. METHODS: Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. RESULTS: Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. CONCLUSION: For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier's function.

Barriers to the Intestinal Absorption of Four Insulin-Loaded Arginine-Rich Nanoparticles in Human and Rat.

Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 ± 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa.

In vitro primary human and animal cell-based blood-brain barrier models as a screening tool in drug discovery.

Brain penetration is characterized by its extent and rate and is influenced by drug physicochemical properties, plasma exposure, plasma and brain protein binding and BBB permeability. This raises questions related to physiology, interspecies differences and in vitro/in vivo extrapolation. We herein discuss the use of in vitro human and animal BBB model as a tool to improve CNS compound selection. These cell-based BBB models are characterized by low paracellular permeation, well-developed tight junctions and functional efflux transporters. A study of twenty drugs shows similar compound ranking between rat and human models although with a 2-fold higher permeability in rat. cLogP < 5, PSA < 120 Å, MW < 450 were confirmed as essential for CNS drugs. An in vitro/in vivo correlation in rat (R² = 0.67; P = 2 × 10⁻4) was highlighted when in vitro permeability and efflux were considered together with plasma exposure and free fraction. The cell-based BBB model is suitable to optimize CNS-drug selection, to study interspecies differences and then to support human brain exposure prediction.