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Browsing by Subject "blood-brain barrier (BBB)"

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  • Degradation of amyloid-β peptide with a blood-brain barrier penetrating protein drug 
    Suominen, Laura (2020)
    Background: Alzheimer’s disease (AD) is a worldwide challenge for health care professionals and researchers. Every year, AD causes dementia for millions of patients. No preventive or curative medication is available despite continuous research. Amyloid-beta (Aβ) deposits in brain are one of the main pathological findings in AD. Accumulating Aβ peptides are thought to be the reason behind further disease progression. If the Aβ accumulation could be restricted or Aβ degradation increased their toxic effects would be prevented. Soluble oligomers and protofibrils are the most toxic species of Aβ. Most of the Aβ targeting drugs developed so far have not specifically targeted these toxic species. Neprilysin (NEP) is a major Aβ degrading enzyme that targets mostly the smallest species (monomers and dimers) of Aβ. Another common challenge for protein drugs has been passing the blood-brain barrier (BBB). Different strategies, such as utilising transferrin receptor (TfR) mediated transcytosis, have been studied for drug transport. For example, a rat anti-mouse TfR antibody, 8D3, or its fragments can be used for drug transportation. Objectives: To produce a recombinant protein, sNEP-scFv8D3, combining soluble NEP and single chain variable fragment of 8D3. Testing its ability to degrade different species and isoforms of Aβ in vitro and study in vivo brain uptake. Evaluate whether it is a promising model for future AD treatments. Methods: The recombinant protein was expressed in Expi293 cells and purified with affinity chromatography. The TfR binding was studied with TfR ELISA and enzymatic activity with MCA assay. Aβ ELISA was used for determining the Aβ degradation. Recombinant protein was compared to sNEP. In in vivo studies the brain uptake and blood half-life of radiolabeled sNEP-scFv8D3 of were studied on NLGF mice. Immunohistochemical analyses of brain cryo sections were done to evaluate the co-localisation of Aβ aggregates and sNEP-scFv8D3. Results and discussion: sNEP-scFv8D3 bound to TfR and showed similar enzymatic activity as sNEP. Both sNEP-scFv8D3 and sNEP were able to degrade monomeric Aβ-40 and Aβ-42 but no significant effect was seen on larger aggregates. In mice brain, sNEP-scFv8D3 was detected in same areas as Aβ aggregates. Compared to sNEP, our recombinant protein had better brain uptake. The blood half-life of sNEP-scFv8D3 was approximately 9.5 h and it was cleared fast from the brain. Already 6 h post injection, levels in the brain had dropped more than by half. Further studies are needed to determine whether sNEP-scFv8D3 is effectively transported across the BBB and if it can reduce brain Aβ levels in vivo. Conclusions: In the future, sNEP-scFv8D3 or its improved version could be used at the earliest stages of AD to prevent disease progression. Since sNEP-scFv8D3 degrades only small Aβ aggregates it could be combined with another drug targeting larger oligomers. Together they would decrease the total Aβ deposition in brain.
  • Investigating Drug Transport at the Blood-Brain Barrier and the Effects of P-glycoprotein on the Brain Distribution of Drugs (Literature Review) : Modeling P-Glycoprotein Mediated Efflux at the Blood-Brain Barrier : Possibilities from In Vivo to In Vitro (Experimental Work) 
    Sjöstedt, Noora (2011)
    The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.

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