BBB L3

Question 1

What is a ‘trojan’?

Answer 1

disguise molecule that fools the BBB into uptake - typically using endogenous molecules such as Transferrin, Lipoproteins to target their respective receptors

Question 2

What are the two factors that clearance of drugs is dependent upon from the brain?

Answer 2

1) Size of the drug - if larger = longer time to clear

2) if taken up/bound by cells/neurones

Question 3

1) What is the cause for 80% of malignant brain tumours?

2) neurones can divide - true or false?

Answer 3

• gliomas e.g. glioblastoma

N.B name of tumour derived from type of cell

FALSE - neurons only cell (in humans) that cannot divide

Question 4

What is the median survival of patients with glioblastoma multiform?

Answer 4

12-18 months - very fast progressing

Question 5

Define and describe metastases

Answer 5

The origination of a cancer from a primary peripheral site e.g. skin, breast, lung, prostate, colon and travelled to a secondary site via the blood to the form a secondary tumour

Question 6

Give an example of a metastasis from a peripheral site that exists in the brain - and describe how it does this.

Answer 6

Breast cancer cells can release enzymes that are capable of degrading the BBB - hence gain access and metastasise in the brain tissue

Question 7

a) Name molecular targets to inhibit glioma growth and proliferation, whilst describing the purpose behind the reason to target these sites
b) Have these methods had success in treatment of brain tumours? Why?

Answer 7

a) 1) Target growth factor receptor - as only small amount needed to allow tumour growth. Hence, reduce growth factor slightly = significant affect on growth rate of tumour
2) Intracellular signalling pathway e.g. P53 - for apoptosis -hence if can up-regulate this = cause apoptosis - i.e. “cell suicide”
3) Block cell division/proliferation by inhibiting DNA replication e.g. with use of chemotherapeutics
b) NO - due to the difficulty of crossing the BBB

Question 8

Describe the function of cDNA, siRNA, and Chemotherapeutics in treatment of brain tumours

Answer 8

cDNA - used to cause up-regulation and expression of P53 - inducing apoptosis of brain tumour cells

siRNA - to knockdown expression of growth factor receptors = slows tumour growth

Chemotherapeutics e.g. Vincristine, Vinblastine, Paclitaxel, Daunorubicin, Doxorubicin, Irinotecan –> all act to prevent cell division and/or disrupt DNA synthesis

Question 9

Why don’t the cDNA, siRNA, and chemotherapy methods work as of yet for brain tumours?

Answer 9

Physical barrier:
Large hydrophilic drugs (RNA, DNA), cannot go through tight junctions, and hydrophilic drugs are not recognized by the barrier i.e. barrier is of lipid nature.

Metabolic barrier:
RNA and DNA = degraded by nucleases in endothelial cells and in plasma i.e. degraded by the metabolic enzymes that exist in the BBB but N.B: degraded nucleic acids are recycled and re-used by the endothelia.

Transport barrier:
large lipophilic drugs = substrates for BBB efflux transporters, which stop the drugs from entering the brain i.e. arrive at efflux pump – kicked out – all of chemotherapeutic agents are subject to this.

Question 10

What two problems d chemotherapeutics need to overcome?

Answer 10

1) Efflux transporters at the BBB
2) Efflux transportes at the tumour site
i. e. even if the molecule can surpass the ‘first line of defence’ at the BBB, the tumour cell also has it’s own efflux transporters that are able to kick-out the drug

Question 11

Name the three common efflux transporter families

Answer 11

1) BCRP (Breast Cancer Resistant Protein)
2) MRP 1,3,5
3) P-Glycoprotein

Question 12

Give two examples of chemotherapeutic agents from each of the following efflux transporter families:

BCRP (Breast Cancer Resistant Protein)

MRP 1,3,5

P-Glycoprotein

Answer 12

BRCP - Methotrexate, Irinotecan

MRP 1,3,5 - Daunorubicin, Doxorubicin

P-Glycoprotein - Paclitaxel, Vincristine

Question 13

What are the advantages of using nanoparticles for delivery of drugs?

Answer 13

Can encapsulate drug in order to:
a) Avoid metabolic barrier and efflux transporter barrier

b) Carry relatively large cargo to neurones
c) Carry variety of different drug types (almost all) = dependent on type of nanoparticle
d) High molecular weight drugs delivered i.e. large and complex drugs

Question 14

What are the disadvantages of using nanoparticles to deliver drugs?

Answer 14

a) Toxicity of the nanoparticle itself
b) Accumulation of nanoparticle by liver and spleen
c) Not only target brain i.e. will enter other organs = off-target effects
d) Clinical trials needed for both the therapeutic drug and nanoparticle itself = makes trials costly and time consuming – i.e. testing nanoparticle = almost like having to test entirely separate drug.

Question 15

what is the size of major types of nanoparticles?

Answer 15

<200 nm

Question 16

Name the main types of nanoparticle formulations and their respective advantages and disadvantages

Answer 16

Liposome: phospholipid sphere that self-assembles, hence easily made. The lipid soluble drug can be incorporated in the outer hydrophobic layer, whereas hydrophilic drugs can be incorporated in the centre of the particle.

Inorganic material: e.g. gold, silicon dioxide: often used for imaging dyes - not for drug delivery

Carbon nanomaterial: e.g. nanotubes –> becoming more useful as they are like ‘pins; that can penetrate the cell membrane = don’t need to be recognised by receptor on cell BUT major problem = loading of drug cargo is minimal onto the nanotube.

Polymer nanoparticle: composed of endogenous molecules e.g. glycolic acid, polysaccharides = not toxic, but ONLY for hydrophilic compounds

Question 17

How can the following factors affect nanoparticle design and hence drug delivery:

a) composition
b) size
c) shape
d) charge

Answer 17

a) lipid and hydrophilic moieties = compartmentalise drugs in different areas of the nanoparticle
b) too small i.e. <20 nm = filtered by the kidneys BUT too big = cannot get into the cell - hence optimal size = 50-100nm
c) cube = rare, normally round or rolled - where rolled = better penetration but round are easier to produce

d) charge - if positively charged = allow better cell penetration BUT positive charge would be degraded in plasma, hence need to maintain a neutral charge in the blood = do this by adding PEG
(optimal = neutral in blood and then positive when reach site of action)

Question 18

What was the purpose of formulating paclitaxel (PTX) as a nanoparticle formulation? Describe the effect on:

a) circulation time
b) the benefits of the size and use of PEG
c) the fact it accumulates in the spleen and lung tissues - is this preferable?

Answer 18

a) increased circulation time from <5 hr to 24 hr
b) size was 50-80nm (hence good for cell penetration) and PEG allowed neutral charged within the blood = aids circulation time
c) the spleen and lungs are not preferable - whereas it would be more preferable if the accumulation was in the liver - given the higher metabolic activity compared to other organs

Question 19

Give examples of nanoparticles in the clinic for delivery of chemotherapeutic agents

Answer 19

Onivvde MM-398 = liposomal NP+PEG, 100nm, Irinotecan cargo - for pancreatic cancer

Caelyx/Myocet = liposomal NP+PEG 180nm, Daunorubicin cargo - for ovarian cancer

Both show good properties i.e. good size, formulated with PEG - but worth noting that both are lipid soluble.

Question 20

What is the main method for optimisation of brain delivery?

Answer 20

Trojan drugs

Trojan = molecules attached to the NP that are recognised by the BBB receptors - hence they ‘fool’ the BBB into thinking they are an endogenous molecule

Question 21

What transport mechanism do Trojan drugs utilise?

Answer 21

Receptor Mediated Transport (RMT)

Question 22

Describe the process of transcytosis related to RMT

Answer 22

Includes the cellular movement process of both endo- and exocytosis.
RMT = specific - where receptors recognise a specific substrate which triggers an intra-cellular pathway to induce endocytosis. This is the mechanism by which trojan drugs can work i.e. by tagging the NP with a ‘trojan’ i.e. an endogenous molecule that is able to be recognised by a cell-receptor that enables endocytosis

Question 23

Give two examples of endogenous substrates used as trojans

Answer 23

• Transferrin (Targeting the transferrin receptor TfR)

- Lipoproteins (Targeting the LRP receptors)

Question 24

Describe Clathrin-mediated endocytosis

Answer 24

Clathrin-mediated endocytosis defined by stages of nucleation, cargo selection, coat assembly and scission – where clathrin proteins are within the cell = attracted to the surface and form a lattice. Nucleation = arrival of AP2 and clathrin. These further accumulate near the growing vesicle and promote clathrin lattice formation and subsequent invagination. This lattice supports the membrane invagination  promotes vesicle formation. Scission = needed for movement of vesicle and cargo into the cell. As well as the classic pit formation, clathrin can form extended flat lattices, which can be seen in the micrograph. The factors that regulate this organization and these nets protect the vesicle from metabolic enzymes = drug cargo remains intact.
The vesicle finally ‘pinches off’.
The initial lattice formation = triggered by binding of substrate to receptors on the surface.

Question 25

The receptors targeted with trojan molecules for RMT at the BBB are specific to the BBB receptors, true or false?

what is the impact of this outcome

Answer 25

False

This means the receptors do exist elsewhere in the body - hence there is the potential for off-target effects BUT it is important to note the receptors targeted e.g. the TfR and LRP - are expressed more at the BBB - hence is good to target these to minimise off-target effects

Question 26

The NP-PTX LRP targeting trojan molecule allows more drug to enter the brain -but what is the trade-off for this?

Why is the trade-off not a great impact considering the risk-benefit

Answer 26

trade-off = accumulates in the liver - hence may get some liver side effects and toxicity

the trade-off is not of great impact given this medication is usually for patients at end-of-like, hence accept the trade-off of liver s/e for prolonged life a little more

Question 27

What was the purpose of binding p53 to the TfR-NP molecule?

Answer 27

to induce cell apoptosis

Question 28

What is the benefit of targeting tumour cells with trojan molecules?

hint - relate to their functions for cell-mediated transport

Answer 28

the tumour cell only has an endocytosis function NOT exocytosis - hence once the therapeutic payload is in the cell - it can not come out - hence is able to remain in the cell = kill the cell

Question 29

Give an example of a drug in the pipeline that utilises the RMT and AMT mechanism

Answer 29

Dual modification: NP can be taken up by two BBB uptake systems i.e. improves efficiency of trying to get drug cargo into the cell/
AMT = more general method – not receptor mediated i.e. can occur anywhere on cell = increases chance of entering cell. But only occurs if +ve charge, whereas PEG coating = neutral charge, and inside shell = +ve. Therefore, make ’double-shell’ like system – where can remove PEG coating once at site of entry and then expose the +ve inner surface = done via using metabolic enzymes within the BBB to break the links between the two different charged layers.
These enzymes are known as MMP (matrix-metallo proteases) = breakdowns the linker between PEG and inner core  reveals +ve charged NP = then taken up by AMT – hence = using endogenous enzymes of BBB metabolic barrier to increase to increase uptake.

Question 30

Describe an example nanoparticle in the pipeline that utilises chemotaxis as a mechanism to improve the formulation

Answer 30

Uses glucose metabolism to generate an oxygen ‘jet’ i.e. part of the NP membrane = thinner, and inside the NP exists two enzymes – Glucose oxidase and/or Catalase.
Glucose = in NP  glucose oxidase metabolizes to H2O2 = can then be further converted to H2O and O2 (reactive oxygen species) = creates ‘oxygen jet’. This increases pressure in NP and is forced out of thin wall = causes random and rapid movement of the NP – theory = more likely to hit wall of capillary more times if moving randomly compared to movement with blood flow. This random movement allows increased interaction with endothelium  more likely to bind LRP receptor  undergo entry into cell.