Blood brain barrier

Question 1

Which types of drugs are able to undergo lipid-mediated diffusion across the CNS?

Answer 1

Small hydrophobic drugs, with a molecular weight under 400 and less than 8 hydrogen bonds.

Question 2

Which transport systems need to be exploited to enable delivery of drugs across the BBB?

Answer 2

Receptor mediated transport
Carrier mediated transport

Question 3

Outline structural differences between endothelial cell barrier in a general capillary and a brain capillary.

Answer 3

In general capillaries:
-Pores present
-Fenestrations
-Vesicular transport

In brain capillaries:
-Above aren’t present
- Tight junctions exist

Question 4

Compare the two interfaces present in the brain.

Answer 4

The BBB separates the lumen of the brain capillaries from the brain parenchyma. The main contribution to the BBB property of reduced permeability comes from the tight junctions (TJs) among endothelial cells lining of the capillaries. The BCSFB (Blood CSF barrier) is at each ventricle’s choroid plexus epithelial cells, joined together by TJs. Unlike the endothelium in the brain parenchyma, capillaries of the choroid plexus have no TJs and are fenestrated; therefore, they are relatively leaky and permeable to small molecules.

Question 5

Describe the route of drug delivery across the interfaces.

Answer 5

Drugs tend to cross the BCSFB from the CSF into the blood and then across the BBB into the brain parenchyma. Although there is an opportunity for cells to cross directly from the CSF to the brain passing both interface - this does not occur due to it being a diffusion mediated process which is equivalent to (1/d squared) meaning there is a 10-20 fold decrease per mm and to have any therapeutic effect this would mean administering the drug at a dosage 5x the therapeutic level. Due to the nature of the drugs (anti-psychotics) this would cause toxicity so instead the drugs cross the two interfaces from the CSF to the brain parenchyma.

Question 6

Recap the five methods of drug delivery across the intestinal epithelia.

Answer 6

A. Transcellular route: passive diffusion
B. Paracellular route (tight junctions): passive diffusion
C. Transcellular route: active transporter utilization
D. Lipid absorption via micelles / bile salts
E. Particulate absorption via GALT (gut-associated lymphatic tissue)

Question 7

Out of the 5 methods of drug delivery across the intestinal epithelia which are used across the BBB.

Answer 7

A. Transcellular route: passive diffusion
C. Transcellular route: active transporter utilization

The paracellular route is unable in the BBB, due to the presence of tight junctions and both the micelle and GALT method is specific only to the intestinal epithelia.

Question 8

How many drugs are able to cross the BBB using the passive transcellular route?

Answer 8

98% of small drugs and 100% of large ones are unable to cross the BBB under passive transcellular diffusion.

Question 9

What are the three mechanisms of intravascular trans-BBB delivery?

Answer 9

Transcellular lipophilic pathway
Carrier mediated transport
Receptor mediated transcytosis

Question 10

What is the criteria for Lipid mediated transport?

Answer 10

-Must be small molecules under 400MW with a high Log P and low H-Bonding (ideally CNS drugs should have fewer than 7 hydrogen bonds which reflect the degree of polarity).
BBB permeation decreases 100-fold going from MW = 300 to MW = 450
This reflects a polar surface area change of 50 A to 100 A.

Question 11

Describe the relationship between permeability and Log P across the BBB.

Answer 11

The more hydrophobic the drug the higher degree of permeability across the BBB, this generally when plotted forms a straight line - proportional relationship between hydrophobicity and permeability. The only exception is glucose, which is relatively hydrophilic but has high permeation, this is due to however glucose being carrier mediated across the CNS.

Question 12

Explain the concept of polar surface area in relation to permeability across the BBB.

Answer 12

Polar surface area of a molecule can be calculated by the sum of all the polar atoms of a molecules and the area they occupy of the surface which can then be used to predict the likelihood of the molecule being able to cross the BBB. Computational studies can be used to determine this rather than having to experimentally produce them.
Again this can be plotted; the dynamic surface area of the molecule against Log (Brain/Blood) - so the ratio of drug occupying in the brain compared to the blood to determine whether it is capable of crossing the BBB.

Question 13

What can be deduced from graphs plotting the dynamic surface area of the molecule against Log (Brain/Blood)?

Answer 13

Smaller molecules with a reduced polar surface area have greater permeation across the BBB compared to larger molecules with a greater polar surface area and are more likely to stay in the blood.

Question 14

What is the cut off points for polar surface area is required for CNS and non-CNS administration?

Answer 14

CNS drugs that penetrate brain by passive transport have polar surface area below 70 Å2
* Most orally administered non-CNS can have larger values up to 120 Å2

Question 15

Describe how differences in the structures of Morphine and Heroin relates to its permeation across the BBB.

Answer 15

The chemical structure of Morphine contains two hydroxyl groups; in codeine one is acetylated and in Heroin both are acetylated meaning they are unable to participate in hydrogen bonding, which increases the permeation across the BBB by 100x.
The LogP of Morphine is 0.99
The LogP of Heroin is 2.3

Question 16

However why is removing hydrogen bonds not proved a useful method of drug delivery across the CNS?

Answer 16

Removing H-bonds has not generally proved a useful route to drug modification as H-bond blocking groups are either easily hydrolysable or affect drug activity - it then doesn’t work in the CNS and is unable to bond to its target.

Question 17

In consideration of the inefficiency of removing hydrogen bonds to exploit transcellular passive diffusion, what is a more effective method of drug delivery across the CNS?

Answer 17

Modifying drugs to increase their affinity for endogenous transporters so that then the drug molecule or part of the drug molecule can act as a carrier for itself and is uptaken by one of many transporters.

Question 18

Which transporter is responsible for glucose uptake across the BBB?

Answer 18

The GLUT-1 transporter which transports glucose alongside other hexoses including mannose and galactose. The rate of transport is 50,000 times faster than transmembrane diffusion - this is crucial as glucose is the brain’s only source of energy.

Question 19

What type of transporter is GLUT-1?

Answer 19

It is a uniporter transmembrane protein meaning that it only transports substances one way.

Question 20

What kinetics does the rate of carrier mediated diffusion does GLUT-1 obey?

Answer 20

Michaelis–Menten kinetics:
v = Vmax / Km + S

This describes the rate of transport across the membrane whereas diffusion limited transport is based upon J= -D / dC/dX

Question 21

State the other carrier mediated transporters.

Answer 21

Large neutral amino acid transporter 1 (LAT1)
Cationic amino acid transporter 1 (CAT1)
MonoCarboxylic acid transporter 1 (MCT1)
Concentrative Nucleoside transporter 2 (CNT2)

Question 22

What are some of the substrates for LAT1?

Answer 22

Phenylalanine and some other large and small amino acids

Question 23

What are some of the substrates for CAT1?

Answer 23

Lysine, arginine, ornithine

Question 24

What are some of the substrates for MCT?

Answer 24

Lactate, pyruvate, monocarboxylic

Question 25

What are some of the substrates for CNT2?

Answer 25

Adenosine, guanosine, inosine, uridine

Question 26

Explain an example of a drug which has been designed to exploit one of the transporters.

Answer 26

Levodopa a precursor of dopamine which is used in the treatment of Parkinson’s disease. It binds to and exploits LAT (large neutral amino acid transporter) in which it is transferred across the BBB due to its high resemblance of the amino acid Phenylalanine (just contains two extra hydroxyl groups on the aromatic ring).

Question 27

Describe how Gabapentin also exploits LAT1.

Answer 27

Gabapentin (which can also be used in the treatment of PD?) has demonstrated affinity for LAT1 despite not structurally mimicking an amino acid that usually exploits the binding site like Levodopa. LAT1 binding site has a preference for alpha-amino acids (these are ones which the amine is attached to the first carbon adjacent to the carboxylic acid). In Gabapentin despite the amine being attached to the gamma nitrogen the ability to undergo bond rotation allows the structure to adopt a low energy confirmation in which are similar to alpha amino acids and hence are able to bind to binding sites on the transporters.

Question 28

Is using a glucose-drug conjugate effective for facilitating carrier mediated transports?

Answer 28

Although it appeared a promising technique for drugs that were unable to be engineered to possess affinity for the GLUT-1 transporter, any affinity for glucose is destroyed by conjugation.

Question 29

What is an effective conjugate for carrier mediated transports?

Answer 29

Using cysteine which usually has a low affinity for the large neutral amino acid transporter. By combining with a drug now as a large molecule not only does it retain its affinity but it is now actually greater as the whole drug complex is larger.

However using conjugates in carrier mediated diffusion is usually quite extensive process as there is no screening tool and specificities for CMTs are so precise that conjugation significantly disrupts them.

Question 30

Are active efflux transporters also found in the brain?

Answer 30

Yes both p-gp and CYP450 is also present in the brain capillary endothelia and acts again similarly to their presence in the GI tract as a barrier to drug delivery across the BBB.

Question 31

How does p-gp work to inhibit drug delivery?

Answer 31

Has a multi-drug resistance mechanism where it transports the drug back into the blood.

Question 32

Would it be an effective transport mechanism by inhibiting this process?

Answer 32

Although it is an alternative therapeutic route by inhibiting this protein has it will lead to higher accumulation of drug inside the cell, they will be more likely to partition across the cell membrane into the cerebral tissue. However it is not effective to inhibit these proteins as they are responsible for carrying out a variety of roles and lead to neurotoxicity.

Question 33

Briefly outline the role of p-gp and CYP3A4 in reducing the absorption across the BBB.

Answer 33

Both p-gp and CYP 3A4 are found in brain capillary endothelia. CYP 3A4 reduces the amount of drug absorbed through the BBB and p-gp as an efflux transporter protein pumps drugs and their metabolites back into the bloodstream, overall both act to reduce the overall systemic exposure.

Question 34

Where is OATP found and what are its substrates?

Answer 34

OATP stands for organic anion transporter polypeptide family. It is found on both the apical and basolateral surfaces of the brain endothelial cells lining the blood brain barrier and transports anions such as anions such as bile acids, cholate, oestrogen
conjugates, digoxin, opioid peptides.

Question 35

What does OAT stand for?

Answer 35

Organic anion transporter family and it has a broad substrate range.

Question 36

What does ENT stand for?

Answer 36

Equilibrative nucleoside transporter which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections.

Question 37

How does receptor mediated transcytosis differ from carrier mediated transcytosis?

Answer 37

This involves the substrate binding to a receptor on the apical surface of the brain endothelial cell, in which it then becomes enclosed within a vesicle and transported across the cell and released on the basolateral side.

Question 38

Which type of molecules are able to be facilitated by this mechanism (receptor mediated transport)?

Answer 38

This vesiculation allows much larger molecules, such as insulin, nanoparticles and antibodies, to cross the BBB.
Example: Transferrin transport of iron into brain by Transferrin receptor (TfR)

Question 39

Describe the process of transferrin transport across the BBB.

Answer 39

Transferrin binds to Iron 3+ which then forms Holo-Tf. At neutral pH transferrin receptor is able to bind to Holo-Tf. This then sits in a pit surrounded by clathrin molecules inducing vesicularisation lined with clathrin molecules.
Endosomal acidification of pH 5.5 causes the iron to be released and out of the vesicle. The transferrin molecule now called Apo-transferrin molecule still bound to its receptor is recycled to the cell surface and allows the receptor to be exposed again once exposed at physiological pH.

Question 40

Is it more beneficial to have a high or low affinity anti-transferrin receptor antibody?

Answer 40

This is the concept that exploits the transferrin receptor to facilitate endocytosis. Although a drug that has a high receptor affinity at trace doses demonstrates greater uptake its inability to dissociate with the receptor on the basolateral surface and hence to be recycled means that at a therapeutic concentration antibodies with a lower affinity to the receptor actually demonstrate greater uptake due to their ability to dissociate and then be recycled back onto the surface.

Question 41

What are some examples of endogenously transported molecules?

Answer 41

Transferrin, leptin, insulin
Potentially - are lot more modifications/freedoms are permitted with RMT than CMT. This is because the properties of the drug are much less important for its delivery - now transported by vehicles so only needs to bind to its therapeutic target (the antibody which is able to distinguish the receptor).

Question 42

What is meant by the molecular trojan horse concept?

Answer 42

Molecular Trojan horses are genetically engineered proteins that cross the blood-brain barrier (BBB) via endogenous receptor-mediated transport processes. The horse is the antibody and the BBB is the trojan that will take the drug into the cell - become vesicularised and then released on to the other side of the cell.

Question 43

Explain why modifying drugs to exploit the RMT is easier than the CMT.

Answer 43

In receptor mediated transport across the blood brain barrier is much easier to exploit as the drug itself does not have to be modified in order to bind directly to the receptor to facilitate its transport across the BBB and therefore only has to ensure affinity for its biological target. However in carrier mediated transport they have to ensure affinity to both the carrier which is highly specific and to also ensure affinity for their biological target. Therefore engineering drugs to exploit receptor mediated transport is easier.