Blood Brain Barrier (II) Flashcards
What is the % brain tumours are primary. Give example.
80% of primary malignant brain tumours are gliomas e.g.
Glioblastoma
Astrocytoma
What is the median survival rate of patients with glioblastoma
Median survival of patients with glioblastoma multiform is only 12-18 months.
What % of cancer patients developing brain metastases.
25%, that originating from skin, lung, breast, colon, prostate.
What are the tumour molecular targets used in clinical practice to inhibit glioma growth and proliferation.
Growth factor receptors: GFR highly populated in tumour cells=inhibiting this slows growth.
Intracellular signalling pathways to cause apoptosis: activation p53
Block cell division / prevent mitosis / inhibit DNA replication.
What are the treatments used in peripheral tumours and why don’t they work for CNS tumours?
What makes drug delivery difficult with tumour cells in general.
Tumours have a transport barrier: over expression of efflux transporters .
What are the advantages of delivering drugs as nanoparticles across the BBB. (Preston Cristina Catherine Heather)
Advantages:
Encapsulate drug to:
- Protect it from Metabolic barrier and Efflux Transport barrier.
- Carry a relatively large drug cargo to the brain
- Can carry almost any type of drug: gene modifiers (cDNA, siRNA) carried in the hydrophilic core, chemotherapeutic agents carried in lipid membrane.
- High molecular weight drugs delivered e.g. paclitaxel molecular weight 853.9.
- Inorganic NPs are easier to track by microscopy techniques (e.g.
magnetic resonance imaging (MRI), TEM) or analytical techniques).
What are the disadvantages of delivering drugs as nanoparticles across the BBB (Teddy, Amelia, Derek, Callie).
- Toxicity of the nanoparticles itself
- Accumulation of nanoparticles by liver and spleen
- Does not only target brain (will enter other organs too)
- Clinical trials needed for both the therapeutic drug and the nanoparticles itself making trials costly and time consuming.
- Liposomes in comparison to polymeric nanoparticles are reported to have inferior stability and cargo-retention efficiency.
Give examples of major types of nano-carrier mediated cancer therapy.
Liposomes, polymer micelles, polymer nanoparticles, inorganic nanomaterial, dendrimer.
What are the main features to enhance of nanoparticle:
Composition
Drugs (covalently bound, adsorbed, entrapped).
Size
Shape
Charge
Ligands
In terms of composition what is the preferred in manufacture of NPs and why.
Natural polymers are selected because it avoids immune system, liver and kidney clearance. Especially those made from phospholipids (like normal cell walls).
Liposomes: good, flexible drug loading; lipid soluble drug in the shell, hydrophilic drug in the core.
Why are synthetic polymers not used in manufacture of NPs.
Synthetic polymers tend to be cleared by the kidney and liver quickly. Polymers: easy to manufacture but have few interaction sites and drug-loading areas.
Why are inorganic polymers not used in manufacture of NPs
Inorganic NPs are stable and easy to manufacture but can bring about side effects such as disturbed BBB structure, induce inflammation and clearance is of a concern.
What is the optimal size range to produce NP and why.
a. 40-80 nm
b. 60-120 nm
c. 50-100 nm
d. 20-60 nm
(c) 50-100 nm is optimum nanoparticle range as smaller NP penetrate BBB the best and also avoid rapid clearance by the kidney.
At what size do you see NPs rapidly being excreted by the kidneys among other unfavourable properties….
(a) < 30 nm
(b) < 20 nm
(c) <10 nm
(d) <50 nm
(b) <20 nm is where you see rapid NP clearance; limited capacity to carry drug and drug is released quickly.
What is the optimum shape for NPs drug delivery into the brain and why isn’t it seen in literature as much.
Nanorods have high brain accumulation and lower clearance rate with long-rods of 500 nm. However, its manufacture is complex so spherical NPs are use.
What shape of NPs are seen in literature.
Spherical NPs are the easiest to prepare and most numerous.
What can be adsorb onto the NP when in circulation.
Protein adsorption can take place were they coat the NPs surface forming a protein coating known as protein corona.
What are the implications of NPs having protein corona.
Alters the surface chemistry of NPs along with its aggregation state. NPs will be cleared rapidly by the reticuloendothelial system, decrease the NP dose available for accumulation in the brain.
How can NP protein corona be mitigated.
To overcome this poly ethylene glycol (PEG) coating is used to minimise protein fouling, improve stability in the blood, decrease clearance and increase biocompatibility.
What charge is optimal for NP delivery into the brain and why.
For reasons of low toxicity and low non-specific uptake, neutral or negative charged NPs are preferred, or NPs that can change their charge from neutral in the plasma, to positive.
What charge causes untoward side effects.
Positively charged NPs: more easily penetrate cell membranes (which have negative charge), but are more toxic.
What are the benefits or using neutral or negatively charged NPs.
Neutral or negatively charged NPs: stable, retain their drug, long blood circulation half-life, low rate of nonspecific cellular uptake.
How can ligands be used to optimise NPs drug delivery to the brain.
Ligands, such as transferrin, lipoprotein-1 (LRP-1) and Angiopep-2 receptor, added to improve BBB targeting and transport with the use of trojan technology.
Give example of nanoparticle in clinical trials.
Onivyde MM-398
What does the trojan horse technology offer.
Attach molecule to the NP that are recognised by natural BBB uptake transport, the most successful on being receptor mediated transcytosis. Fool the blood brain barrier into taking up the whole NP.
Describe the formation of transcytosis vesicles from Clathrin lattice. Benefit of NPs using transcytosis.?
Clathrin induces membrane curvature, thereby leading to membrane invagination and vesicle formation. By Trojan NPs using transcytosis it evades the metabolic barrier and goes around the physical barrier limitation of the blood brain barrier.
For Low density lipoprotein receptor-related protein (LRP-1) what ligand must be bound to it to trigger transcytosis.
Angiopep-2
Give an example (with the author) of nanoparticle that delivers DNA (p53 gene plasmid) to brain to stop cell division.
Kim et al. and group synthesised a self-assembling nanoparticle that encapsulated p53 gene, called scL-p53.
What is scL-p53 being used for and why.
Temozolomide (TMZ)-resistance in glioblastoma multi-forme (GBM) has been linked to up regulation of O6-methylguanine-DNA methyltransferase (MGMT). Wild type (wt) p53 was previously shown to down-modulate MGMT.
Label the properties of scL-p53.
How does scL-p53 enter the glioma cells.
What are the in vivo findings concerning scL-p53.
Kim et al. reported that in mouse brain tumour cells the relative level of p53 in untreated tumour showed a significant increase in the expression of p53 as early as 6 h postinjection of scL-p53. The tumour volume slow down, for the first 10 days only.
Label the diagram
What is the benefit of adding Trojan ligand to NP, in terms of distribution,, for the ANG-NP synthesised by Xin et al.
In vivo fluorescence imaging of tumour-bearing mice revealed that trojan technology changes the distribution of NP to more brain and liver and less in the spleen and lungs.
What was the in vivo findings with Ang-NP: Xin et al.
Tumour volume reduced most with ANG-NP compared to paclitaxel delivered alone. Life expectancy of mice improved.
Describe how nanoparticles taken up by two BBB uptake systems by RMT and AMT works…
- NP taken up by RMT using LRP trojan (ANG)
- NP taken up by adsorptive mediated transcytosis: NP is masked with negatively charged PEGylated cleavable lipopetide (PCL), which contains a recognition sequence for metalloproteinases (MMPs).
- MMPs cleaves the PEG coating, providing a charge switch that triggers a shift of the NP to from weakly negative to positive (-7 mV to +2 mV), thus favouring adsorptive mediated transcytosis.
- In tumour cells where RMT+AMT used there is a 2-fold increase in comparison to just RMT method only.
What were the findings with nanoparticles that can move, chemotaxis (fill the slide).
Rat brain tissues had high levels of chemotaxis LRP-NP compared to LRP-NP and NP. Almost 4-fold difference between injected dose chemotaxis LRP-NP vs LRP-NP.
Give the table of general characteristic of small molecules vs Biologics
What characteristic of antibody makes its delivery harder.
Describe the construction for Bi-specific antibody.
○ Antigen-binding fragment (Fabs) from two different polyclonal sera were re-associated into bi-specific (Fab) molecules.
○ One side is a trojan antibody to target the Transferrin receptor (TfR) for RMT (one light and one heavy chain).
○ The other side is the therapeutic antibody to inhibit amyloid-beta production using anti-BACE1 enzyme (one light and one heavy chain).
What have bi-specific antibody shown in vivo mice studies.
Anti-TfR/BACE bi-specific antibody showed increased uptake in the brain after 12 hours, compared to plain therapy antibody (BACE1). The therapy part of antibody works: amyloid in brain decreased after 24 hours. Bi-specific antibody binds to BACE 1 enzyme and inhibits amyloid synthesis.
In vivo studies results with dFab and sFab anti-amyloid/TfR antibody.
dFab and sFab anti-amyloid/TfR antibody
Give an example of how ATV has been used and in vivo studies to back it.
ATV seen in literature is an anti-BACE-1 Fab region combined to Fc region with engineered anti-transferrin receptor site. There was reduced amyloid in the brain of cynomolgus monkeys after 4 weeks. Inhibition of amyloid synthesis was mediated by BACE-1 binding.
