Entry of therapeutics into the brain: Influence of exposed polarity calculated in silico and measured in vitro by supercritical fluid chromatography

Glioblastoma is the most malignant brain tumor among adults. Despite multimodality treatment, it remains incurable, mainly because of its extensive heterogeneity and infiltration in the brain parenchyma. Recent evidence indicates dysregulation of the expression of the Promyelocytic Leukemia Protein (PML) in primary Glioblastoma samples. PML is implicated in various ways in cancer biology. In the brain, PML participates in the physiological migration of the neural progenitor cells, which have been hypothesized to serve as the cell of origin of Glioblastoma. The role of PML in Glioblastoma progression has recently gained attention due to its controversial effects in overall Glioblastoma evolution. In this work, we studied the role of PML in Glioblastoma pathophysiology using the U87MG cell line. We genetically modified the cells to conditionally overexpress the PML isoform IV and we focused on its dual role in tumor growth and invasive capacity. Furthermore, we targeted a PML action mediator, the Enhancer of Zeste Homolog 2 (EZH2), via the inhibitory drug DZNeP. We present a combined in vitro–in silico approach, that utilizes both 2D and 3D cultures and cancer-predictive computational algorithms, in order to differentiate and interpret the observed biological results. Our overall findings indicate that PML regulates growth and invasion through distinct cellular mechanisms. In particular, PML overexpression suppresses cell proliferation, while it maintains the invasive capacity of the U87MG Glioblastoma cells and, upon inhibition of the PML-EZH2 pathway, the invasion is drastically eliminated. Our in silico simulations suggest that the underlying mechanism of PML-driven Glioblastoma physiology regulates invasion by differential modulation of the cell-to-cell adhesive and diffusive capacity of the cells. Elucidating further the role of PML in Glioblastoma biology could set PML as a potential molecular biomarker of the tumor progression and its mediated pathway as a therapeutic target, aiming at inhibiting cell growth and potentially clonal evolution regarding their proliferative and/or invasive phenotype within the heterogeneous tumor mass.

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Brain physiome: A concept bridging in vitro 3D brain models and in silico models for predicting drug toxicity in the brain

Bioactive Materials ◽

10.1016/j.bioactmat.2021.11.009 ◽

2021 ◽

Author(s):

Yoojin Seo ◽

Seokyoung Bang ◽

Jeongtae Son ◽

Dongsup Kim ◽

Yong Jeong ◽

...

Keyword(s):

In Silico ◽

Drug Toxicity ◽

Brain Models ◽

In Silico Models ◽

The Brain

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Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00774.2015 ◽

2016 ◽

Vol 310 (11) ◽

pp. H1702-H1714 ◽

Cited By ~ 19

Author(s):

Ádám Nyúl-Tóth ◽

Maria Suciu ◽

Judit Molnár ◽

Csilla Fazakas ◽

János Haskó ◽

...

Keyword(s):

Endothelial Cells ◽

Cerebral Cortex ◽

White Matter ◽

Blood Brain Barrier ◽

In Silico ◽

Grey Matter ◽

Ex Vivo ◽

Cortical Grey Matter ◽

The Brain

The blood-brain barrier (BBB) is the main interface controlling molecular and cellular traffic between the central nervous system (CNS) and the periphery. It consists of cerebral endothelial cells (CECs) interconnected by continuous tight junctions, and closely associated pericytes and astrocytes. Different parts of the CNS have diverse functions and structures and may be subject of different pathologies, in which the BBB is actively involved. It is largely unknown, however, what are the cellular and molecular differences of the BBB in different regions of the brain. Using in silico, in vitro, and ex vivo techniques we compared the expression of BBB-associated genes and proteins (i.e., markers of CECs, brain pericytes, and astrocytes) in the cortical grey matter and white matter. In silico human database analysis (obtained from recalculated data of the Allen Brain Atlas), qPCR, Western blot, and immunofluorescence studies on porcine and mouse brain tissue indicated an increased expression of glial fibrillary acidic protein in astrocytes in the white matter compared with the grey matter. We have also found increased expression of genes of the junctional complex of CECs (occludin, claudin-5, and α-catenin) in the white matter compared with the cerebral cortex. Accordingly, occludin, claudin-5, and α-catenin proteins showed increased expression in CECs of the white matter compared with endothelial cells of the cortical grey matter. In parallel, barrier properties of white matter CECs were superior as well. These differences might be important in the pathogenesis of diseases differently affecting distinct regions of the brain.

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In vitro, in vivo and in silico models of drug distribution into the brain

Journal of Pharmacokinetics and Pharmacodynamics ◽

10.1007/s10928-013-9303-7 ◽

2013 ◽

Vol 40 (3) ◽

pp. 301-314 ◽

Cited By ~ 19

Author(s):

Scott G. Summerfield ◽

Kelly C. Dong

Keyword(s):

In Silico ◽

Drug Distribution ◽

In Silico Models ◽

The Brain

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Modelling the impact of clot fragmentation on the microcirculation after thrombectomy

10.1101/2020.11.30.403808 ◽

2020 ◽

Author(s):

Wahbi K. El-Bouri ◽

Andrew MacGowan ◽

Tamás I. Józsa ◽

Matthew J. Gounis ◽

Stephen J. Payne

Keyword(s):

Blood Flow ◽

Ischaemic Stroke ◽

Computational Model ◽

In Silico ◽

Large Scale ◽

In Vitro Experiments ◽

Mechanical Removal ◽

The Impact ◽

The Brain

1AbstractMany ischaemic stroke patients who have a mechanical removal of their clot (thrombectomy) do not get reperfusion of tissue despite the thrombus being removed. One hypothesis for this ‘no-reperfusion’ phenomenon is micro-emboli fragmenting off the large clot during thrombectomy and occluding smaller blood vessels downstream of the clot location. This is impossible to observe in-vivo and so we here develop an in-silico model based on in-vitro experiments to model the effect of micro-emboli on brain tissue. Through in-vitro experiments we obtain, under a variety of clot consistencies and thrombectomy techniques, micro-emboli distributions post-thrombectomy. Blood flow through the microcirculation is modelled for statistically accurate voxels of brain microvasculature including penetrating arterioles and capillary beds. A novel micro-emboli algorithm, informed by the experimental data, is used to simulate the impact of micro-emboli successively entering the penetrating arterioles and the capillary bed. Scaled-up blood flow parameters – permeability and coupling coefficients – are calculated under various conditions. We find that capillary beds are more susceptible to occlusions than the penetrating arterioles with a 4x greater drop in permeability per volume of vessel occluded. Individual microvascular geometries determine robustness to micro-emboli. Hard clot fragmentation leads to larger micro-emboli and larger drops in blood flow for a given number of micro-emboli. Thrombectomy technique has a large impact on clot fragmentation and hence occlusions in the microvasculature. As such, in-silico modelling of mechanical thrombectomy predicts that clot specific factors, interventional technique, and microvascular geometry strongly influence reperfusion of the brain. Micro-emboli are likely contributory to the phenomenon of no-reperfusion following successful removal of a major clot.2Author summaryAfter an ischaemic stroke - one where a clot blocks a major artery in the brain - patients can undergo a procedure where the clot is removed mechanically with a stent - a thrombectomy. This reopens the blocked vessel, yet some patients don’t achieve blood flow returning to their tissue downstream. One hypothesis for this phenomenon is that the clot fragments into smaller clots (called micro-emboli) which block smaller vessels downstream. However, this can’t be measured in patients due to the inability of clinical imaging resolving the micro-scale. We therefore develop a computational model here, based on experimental thrombectomy data, to quantify the impact of micro-emboli on blood flow in the brain after the removal of a clot. With this model, we found that micro-emboli are a likely contributor to the no-reflow phenomenon after a thrombectomy. Individual blood vessel geometries, clot composition, and thrombectomy technique all impacted the effect of micro-emboli on blood flow and should be taken into consideration to minimise the impact of micro-emboli in the brain. Furthermore, the computational model developed here allows us to now build large-scale models of blood flow in the brain, and hence simulate stroke and the impact of micro-emboli on the entire brain.

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P.7.c.002 An in vitro, in vivo, in silico approach to investigate effects of OAT3 inhibition on bumetanide levels in the brain

European Neuropsychopharmacology ◽

10.1016/s0924-977x(14)71157-5 ◽

2014 ◽

Vol 24 ◽

pp. S718

Author(s):

M.D. Donovan ◽

F.E. O'Brien ◽

G.B. Boylan ◽

B.T. Griffin ◽

J.F. Cryan

Keyword(s):

In Silico ◽

The Brain

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Comparison of liquid chromatography and supercritical fluid chromatography coupled to compact single quadrupole mass spectrometer for targeted in vitro metabolism assay

Journal of Chromatography A ◽

10.1016/j.chroma.2014.10.055 ◽

2014 ◽

Vol 1371 ◽

pp. 244-256 ◽

Cited By ~ 35

Author(s):

Dany Spaggiari ◽

Florence Mehl ◽

Vincent Desfontaine ◽

Alexandre Grand-Guillaume Perrenoud ◽

Szabolcs Fekete ◽

...

Keyword(s):

Liquid Chromatography ◽

Supercritical Fluid ◽

Mass Spectrometer ◽

Supercritical Fluid Chromatography ◽

In Vitro Metabolism ◽

Quadrupole Mass Spectrometer ◽

Quadrupole Mass ◽

Single Quadrupole Mass Spectrometer

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5-(Hydroxyphenyl)-γ-Valerolactone-Sulfate, a Key Microbial Metabolite of Flavan-3-ols, Is Able to Reach the Brain: Evidence from Different in Silico, In Vitro and In Vivo Experimental Models

Nutrients ◽

10.3390/nu11112678 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2678 ◽

Cited By ~ 13

Author(s):

Donato Angelino ◽

Diogo Carregosa ◽

Cristina Domenech-Coca ◽

Monia Savi ◽

Inês Figueira ◽

...

Keyword(s):

In Silico ◽

Experimental Models ◽

In Vivo Studies ◽

Neuroprotective Effects ◽

Physical Chemical ◽

In Silico Study ◽

Polar Metabolites ◽

The Brain

Phenolic compounds have been recognized as promising compounds for the prevention of chronic diseases, including neurodegenerative ones. However, phenolics like flavan-3-ols (F3O) are poorly absorbed along the gastrointestinal tract and structurally rearranged by gut microbiota, yielding smaller and more polar metabolites like phenyl-γ-valerolactones, phenylvaleric acids and their conjugates. The present work investigated the ability of F3O-derived metabolites to cross the blood-brain barrier (BBB), by linking five experimental models with increasing realism. First, an in silico study examined the physical-chemical characteristics of F3O metabolites to predict those most likely to cross the BBB. Some of these metabolites were then tested at physiological concentrations to cross the luminal and abluminal membranes of brain microvascular endothelial cells, cultured in vitro. Finally, three different in vivo studies in rats injected with pure 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, and rats and pigs fed grapes or a F3O-rich cocoa extract, respectively, confirmed the presence of 5-(hydroxyphenyl)-γ-valerolactone-sulfate (3′,4′ isomer) in the brain. This work highlighted, with different experimental models, the BBB permeability of one of the main F3O-derived metabolites. It may support the neuroprotective effects of phenolic-rich foods in the frame of the “gut-brain axis”.

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Effect of pH and inhibitors on beta-amyloid fibril assembly

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166221 ◽

1996 ◽

Vol 54 ◽

pp. 752-753

Author(s):

Beverly E. Maleeff ◽

Timothy K. Hart ◽

Stephen J. Wood ◽

Ronald Wetzel

Keyword(s):

Amyloid Fibril ◽

Peptide Fragment ◽

Hydrophobic Peptides ◽

Amyloid Fibril Formation ◽

Effects Of Ph ◽

Severity Of The Disease ◽

Kinetics Of ◽

The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

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