Blood brain barrier pharmacology and drug delivery

Question 1

What are the four protective mechanisms of the brain and spinal cord?

Answer 1

• The skill and vertebral common: the skull (cranium) protects the braun and the vertebral column (vertebrae) protect the spinal cord
• The meninges: protective membranes that surround the brain and SC
• Cerebrospinal fluid (CSF): Found in the ventricles and central column of the spinal cord- it cushions the brain and acts as a shock absorber.
• Blood-brain barrier (BBB): the barrier between blood vessels and the neuronal tissue of the brain. This is protective as it is highly selective over what can enter the brain e.g. glucose and amino acids, and what can’t.

Question 2

Describe the structure of the meninges.

Answer 2

These are membranes that surround the brain for protection- they are NOT part of the BBB.
- They are made up of 3 membranes:
Dura mater: Outer, strong membrane, which holds it all together
Arachnoid mater: Highly vascularised
Pia mater: Fine, thin, innermost layer that adheres to the brain tissue

Between the Pia mater and the arachnoid mater is the subarachnoid spaces

In the area below the cranium, the dura mater is made of 2 membranes; 1 below the skill and one outside the brain tissue. In-between these two membranes is a fluid-filled area (blood) called the Dural sinus

Question 3

Discuss the fluid compartments of the brain.

Answer 3

These compartments are highly controlled- this is critical for function as neurones need a controlled environment as fluctuations in ion concentrations for example, can cause depolarisation and alter neurone firing.

2 main EC compartments:
- CSF- in the ventricles and central; canal of spinal cord
- Interstitial compartment: space containing interstitial fluid surrounding the cells of the CNS.

These compartments are continuous (not highly separate) so allows some exchange between the 2- hence both need to be tightly regulated

Question 4

What are the 2 barriers in the brain and what are they made-up of?

Answer 4

• Blood:CSF barrier: made of choroid epithelium that produces CSF
• Blood:brain barrier: Endothelial cells- production of interstitial fluid

Question 5

Where are the 4 ventricles filled with CSF fluid located?

Answer 5

• 2 x lateral- left and right, beneath the cerebral hemisphere
• 3rd: in the central line of the Brain
• 4th: Within the brainstem

Question 6

What are the ventricles lined with?

Answer 6

Ependymal cells. These are ciliated and so beat to allow the flow of CSF

Question 7

How much CSF is in our brains?

Answer 7

125-150mL - it is constantly being turnover but needs to remain balanced for pressure maintenance

Question 8

What is the density of the CSF like?

Answer 8

It is the same as the brain, to allow the brain to float in the CSF.

Question 9

What secretes CSF?

Answer 9

Epithelial cells in the chloroid plexus in the ventricles.
it then is drained into the subarachnoid space and out via the arachnoid villi into the dural sinus. The drainage is in the 4th ventricle.

Question 10

What is the BBB?

Answer 10

The barrier between the blood and the interstitial fluid of the brain. Is formed from cerebral microvascular endothelial cells and associated cells/structures.
- It is highly vascualr- need lots of blood and oxygen to the brain

Question 11

What does it mean that the BBB is highly regulated?

Answer 11

Is tight regulation of what can enter the interstitial fluid and what can’t.
e.g. keeps out;
- Circulating neurotransmitters e.g. glycine, glutamate, hormones from inside the body
- xenobiotics- drugs from outside the body

Question 12

Compare general capillaries with capillaries in the brain.

Answer 12

General (in periphery):
- Allows substances to move in and out of blood easily.
- Has aqueous pores between endothelial cells (~4nm) = paracellular pathway
- May have fenestrations- holes on the endothelial cells (20-100nm)
- High capacity for vesicular transport via pinocytotic vesicles- allow larger molecules to move in and out of blood

Brain capillary:
- Cerebral microvascular endothelial cells have very tight junctions between them- cells are so tightly held together- no pores for the paracellular pathway and things to move through
- Have pericytes to help control movement
- Have astrocytes with foot processes that surround the capillary to maintain the tight junctions
- No intercellular pores or fenestrations
- No pinocytosis/endocytosis

LOOK AT DIAGRAMS OF THE 2

Question 13

What are 2 examples of parts of the brain that are ‘outside’ the BBB and why?

Answer 13

These are known as the circumventricular organs.
- Posterior pituitary
- The area postrema (CTZ)

These are areas where substances have to get in to out of the brain that would be unable to cross the BBB.
- Posterioir pituita: secretes protein hormones that need to enter the periphery so vessels need to be leaky- no BBB
- Area postrem - has the CTZ which induces vomitting to remove toxic substances from the GIT

they are isolated from the rest of the brain via tanocytes that make up a tanycytic membrane that surrounds the circumventricular organs to separate them from the rest of the brain.

Question 14

How does the BBB prevent the uptake of most pharmaceuticals>

Answer 14

Because of its very tight junctions between capillary endothelial cells- too hard to cross

Question 15

What drugs is lipid-mediated diffusion possible for?

Answer 15

VERY few drugs can utilise this.
- Some small drugs, MW less than 400 MW
- Need to be hydrophobic - less than 8 hydrogen bonds
- Excludes all large and hydrophilic drugs

Question 16

What properties must drugs have to be able to under go passive lipid-mediated diffusion?

Answer 16

• Small drugs, MW less than 400 MW
• Need to be hydrophobic - less than 7 hydrogen bonds
• highly lipid- solublele, high logP

Only 6-12% of drugs meet this criteria

Question 17

Why is transporting drugs across the CSF to the brain not efficient?

Answer 17

Direct diffusion across CSF to the brain is inefficient (the CSF:Brain interface) because it is diffusion-mediated.

The drug that penetrates the barrier is proportional to the distance in the brain squared.
So drug concentration decreases (rapidly) logarithmically with the distance into the brain by a 10-20 fold decrease per mm increase in penetration distance into the brain.
- This is okay of the brain is small e.g. in mice, but not in humans.
In humans, the diffusion of drug is limited from brain:csf interface as only a small amount of the brain would be penetrated (5mm) but parts of our brain is greater than 50mm from the interface.
Therefore the concentration of drug needed to force enough drug into the brain, would ne at least 5x orders of magnitudes times the therapeutic dose. This would cause an increase in side effects and toxicity risk.

Therfore, this isn’t a widely used route of drug delivery to the CNS!!

NOTE- this doesn’t apply to IV drug delivery.

PLEASE LOOK AT THE BRAIN:BLOOD:CSF INTERFACES DIAGRAM

Question 18

Of the 5 routes used to deliver drugs to the GIT, which 2 can be used to deliver drugs to the CNS?

Answer 18

• The transcellular route: passive diffusion
• Transcellular route: Active transporter utilisation (carrier-mediated and receptor mediated transport)

Cant use:
- Paracellular route via tight junctions- too tight in brain
- Lipid absorption via micelles/bile salts- no bile or micelles
- Particulate absorption via GALT- no GALT

Question 19

What percentage of drugs can’t cross the BBB?

Answer 19

98% of small drugs and all large drugs can’t cross the bbb.

Question 20

What are the 3 methods of intravascular trans-BBB delivery?

Answer 20

• Transcellular lipophilic pathway
• Carrier-mediated transport (usually smaller molecules)
• Receptor-mediated transcytosis
  (usually larger molecules)

Question 21

What happens to the permeability of drugs across the BBB via lipid-mediated diffusion as the MW increases?

Answer 21

Ideal is less than 400MW
But there is a 100-fold decrease in permeation across the BBB from MW of 300 to 450

Question 22

What happens to the permeability of drugs across the BBB via lipid-mediated diffusion as the polar surface area changes? and what is polar SA?

Answer 22

As the polar surface area increases, permeation decreases.
Polar SA is the surface sum of all polar atoms- mainly oxygen and nitrogen and their attached hydrogens.
If low SA- more likely to penetrate
If high SA: less likely to penetrate and will stay in blood
A graphs of polar surface area against Log Brain/blood- shows a downward diagonal line

Question 23

What happens to the permeability of drugs across the BBB via lipid-mediated diffusion as the LogP of the drug changes?

Answer 23

Increasing the LogP increases the permeability causing more drug to cross the BBB.
A graph of LogP vs permeability would show a straight line through the origin- low logP e.g. -5 is hydrophilic and has low permeability. High LogP e.g. +5 is hydrophobic and shows good permeability.

Question 24

What is the value for polar surface area of drugs that are able to penetrate the BBB?

Answer 24

CNS drugs that penetrate the brain by passive transport have a polar surface area below 70 Å2
- Most oral non-CNS drugs have a larger value of up to 120 and so won’t cross the BBB

Question 25

What are the drug examples used to illustrate the passive lipid-diffusion transport route?

Answer 25

Morphine and Heroin
Morphine has a LogP of 0.99
Heroin has a LogP of 2.3 = is more hydrophobic and so is transports across the BBB better-taken up about 100x more than Morphine

• Morphine has two -OH groups
• In heroin, these OH groups have been acetylated ( O-C=0-CH3).
  This acetylated groups means heroin has no more H-bonding ability and so increases permeability across the BBB.

(LOOK AT DIGRAMS OF EACH DRUG PLEASE)

With this theory in mind, it may appear that removing hydrogen bonds may be a useful route to drug modification for drugs being able to cross the BBB. BUT, removing H-bonds did not provide a useful route for drug modification for 2 reasons:
- The groups used to block these hydrogen bonds are often still easily hydrolysed and removed, meaning hydrogen bonding can be restored = back to the starting position (won’t permeate as too polar)
- Also changing these groups, affect the drugs activity and so even if it does permeate, to doesn’t actually work to provide a therapeutic effect
= therefore this path isn’t effective to expoloit.

Question 26

Why is removing hydrogen bonds not an effective drug modification to enhance BBB permeability via lipid-mediated diffusion?

Answer 26

BUT, removing H-bonds did not provide a useful route for drug modification for 2 reasons:
- The groups used to block these hydrogen bonds are often still easily hydrolysed and removed, meaning hydrogen bonding can be restored = back to the starting position (won’t permeate as too polar)
- Also changing these groups, affect the drugs activity and so even if it does permeate, to doesn’t actually work to provide a therapeutic effect
= therefore this path isn’t effective to expoloit.

Question 27

What is the idea of carrier-mediated diffusion for drugs crossing the BBB?

Answer 27

This is the adaptation of the drug to increase its affinity for an endogenous transporter. Part of the drug or a modification acts as a carrier of the drug and is taken up by a transporter.

Question 28

Discuss glucose transport via carrier-mediated transport.

Answer 28

Glucose ( and other hexoses e.g. mannose, galactose) is transported by GLUT-1 (Glucose transporter type 1)- a uniport transmembrane protein transporter- 1 way only
- Glucose is the brains energy source and is needed 100x more than amino acids.
- Glucose transport by GLUT-1 is 50,000 x faster than by transmembrane diffusion

Question 29

What kinetics does the GLUT-1 transporter follow and what does this mean?

Answer 29

GLUT-1 follows Michaelis-Menten kinetics:

V = Vmax [S]/[S]+Km
describes the rate of transport across the membrane
Vmax is high for GLUT1 (1420) compared to just 91 in MCT1 as the brain needs a lot of glucose
Vmax:Cap: is how many molecules per second each transporter can transport. For Glut1: 600 and in MCAT1 and LAT1 this is 2300 and 3000 respectively. but as GLUT-1 has so many transporters there is still such a high conc of glucose entering the brain so is fine.

Question 30

Give some examples of other transporters than gLUT-1.

Answer 30

LAT1 (large neutral aa): E.g. phenylalanine, large neutral AAs
CAT1 (Cationic aa): Arginine, Lysine, Orthinine
MCT1 (Monocarboxylic acid): Lactate, pyruvate, mono carboxylic acids
CNT1 (Nucleotides): Adenosine, guanosine, inosine, uridine

Question 31

What is a route to exploit for drug delivery via carrier-mediated transport, with examples?

Answer 31

Can design a drug to have a high affinity for exploiting an endogenous transporter to allow it to cross the BBB into the brain.

Example (look at structures please):
L-Dopa used in Parkinson’s disease, has a strong affinity for the LAT1 transporter (large, neutral AA transporter) as it is a large AA with a similar structure to phenylalanine.

BUT, predicting affinity from structure can be hard.
For examples in Gabapentin, which can also be used in PD.
lOOK AT STRUCTURES HERE PLS
- Gabapentin is also transported by LAT-1 but has a very different structure to L-dopa and phenylalanine.
LAT-1 is specific for 𝛼-amino acids like phenyl alanine. These are amino acids where the amine group is on the first carbon after the carboxylic acid group.
BUT, in gabapentin, the amine is on the 𝜸-carbons (alpha= C1, beta= C2, gamma= C3). BUT, as gabapentin is a cyclic molecule, the amine and COOH positions can rotate and flip to mimic that of an alpha- amino acid and then have affinity for LAT-1.

Question 32

What methods have been tried for if a drug doesn’t have a natural affinity for a carrier-mediated transporter?

Answer 32

• Engineering of a drug that does have affinity e.g. by coupling the drug molecule to a glucose molecule to exhibit affinity for the GLUT-1 transporter
  BUT, this was a FAIL, as the glucose-drug conjugate was not recognised but the GLUT-1 transporter- didn’t recognise the glucose binding site. Therefore this conjugation destroys glucose affinity for GLUT-1.
• Also, have tried coupling a drug to L-Cysteine- a small, neutral amino acid with low affinity for LAT-1. This was a SUCCESS- As adding the drug to form a conjugate makes it larger so now has a high affinity for the LAT-1 (Large amino transporter 1)

NOTE: Overall, specifies for CMTs are so specific that any conjugation disrupts them, so this drug design method is difficult!

Question 33

What are examples of active efflux transporters?

Answer 33

P-glycoprotein (P-GP)
CYP 450 esp 3A4
These remove molecules back out of the cell.

Question 34

What are the roles of P-GP and CYP-P450 transporters?

Answer 34

These are efflux transporters that remove drugs back out of the cell.
They are both found in brain capillary endothelium.
- P-GP acts to remove the drug ad any metabolites back out into the blood
- CYP 3A4: Acts to decrease the conc of drug that is absorbed across the BBB
= these together, decrease the systemic exposure to the drug showing less therapeutic effects.

Question 35

What is a potential drug target for efflux transporters an why is this cautioned?

Answer 35

Can inhibit the P-glycoprotien (can inhibit cyp to probably) to increase the concentration of drug inside the cell (prevents P-GP from being able to remove it).
BUT, these efflux transporters are needed for many other physiological functions and so inhibiting then can lead to neurotoxicity and side effects so need to be careful with the degree to which you inhibit it.

Also, would be potential for interactions if co-administered with any drugs that are substrates/inducers/inhibitors of P-GP or CYP-3A4

Question 36

What is the idea of receptor-mediated transocytosis for drugs crossing the BBB?

Answer 36

Binding of a drug to a receptor on the apical (blood-facing) side of the cell and is then vesicularised (put into a vesicle), they can transocytose through the cell to the basolateral face (brain-facing) and release the drug into the brain.

Putting drugs into vesicles allows much larger molecules (compared to lipid-mediated diffusion or CMT) to be able to cross the BBB, such as insulin, Nanoparticles, antibodies

Question 37

Give an example of receptor-mediated transocytosis and how this works.

Answer 37

Transferrin transports iron into the brain via a transferrin receptor.
- Transferin binds Fe3+ to form a HOLO-Transferrin complex
- This complex is able to bind to the transferrin reception a pit in the membrane surface. This pit is surrounded by Clathrin molecules which induce the formation of a vesicle that incorporates the complex and the transferrin receptor
- This vesicle undergoes endosomal acidification- a change in pH from neutral to 5.5. This causes the iron to be releases out of the vesicle as Fe2+ as the Fe3+ is reduced via membrane oxioreductase.
- Now the transferrin, no longer has iron bound and so is known as Apo-transferrin. It remains bound to the receptor and is recycled to the cell surface to bind another iron bound transferring molecule.

Free Fe2+ in endoscope is transported into the cytoplasm via DMT-1
It is then transported across the basolateral membrane into the brain via ferroprotein-1

• Drug development: can engineer a drug coupled to a ligand e.g. monoclonal antibody with an affinity for a receptor. This is known as the molecular Trojan horse approach
  e.g. generate Anti-TFR antibodies that have high affinity for this transferrin receptor. The anti-bodies then go through the vesicularisation process and can cross the cell into the brain.
  Examples e.g. Transferrin, insulin, Leptin (satiety)

BUT: We don’t want these drugs to have too high affinity for the transferrin receptor as once it has bound, it will then not be released again into the brain side (isn’t freed from the receptor inside the vesicle)
If, have a lower affinity, it may be taken up less strongly, but once it is taken up and transported it will be easily released into the brain.

Question 38

Why don’t we want drug utilising the receptor-mediated transport (e.g. binding to ferritin) to have a too high affinity for the receptor?

Answer 38

We don’t want these drugs to have too high affinity for the transferrin receptor as once it has bound, it will then not be released again into the brain side (isn’t freed from the receptor inside the vesicle)
If, have a lower affinity, it may be taken up less strongly, but once it is taken up and transported it will be easily released into the brain.

Question 39

What is a positive of the RMT design in terms of the drug itself?

Answer 39

This properties of the drug itself is less important than CMT, as the drug doesn’t have to be recognised at the right place AND have a therapeutic effect. It just needs to have a therapeutic effect as it is taken to the right place by an antibody vehicle.