Blood Brain Barrier Course – MSc BioPharmaceutical Sciences
Universiteit Leiden – Tamar Woudenberg
Content:
1. Basic Aspects of the BBB and Pharmacokinetic Principles
a. BBB Morphology, Anatomy, Physiology and Function
b. BBB Transport Modes
c. Factors in CNS Drug Effects
d. General Pharmacokinetic (PK) principles
e. BBB Transport: Rate and Extent. Impact on Brain vs Plasma PK
2. Methods for Investigating BBB Transport
a. In Vitro Methods (Cell Cultures, Monolayers)
b. In Situ Methods (Brain Sampling Techniques)
c. In Vivo Monitoring Methods: CSF & Microdialysis
d. In Vivo Monitoring Methods: PET
e. In Vitro Methods (Brain Slice and – Homogenate Uptake)
3. Integration and Translation of CNS Drug Delivery Principles
a. Sources of Variability in CNS Drug Distribution
b. Factors that Influence Unbound Brain Pharmacokinetics
c. Translational PK/PD Relationships of CNS Active Drugs
Learning objectives:
The aim of Caput BBB is to learn about:
- All factors that play a role in distribution of drugs into and within the brain
- The importance of distinguishing between total and free drug concentrations
- The importance of distinguishing between rate and extent of equilibration in brain
distribution
Be able to have three examples for:
• Conditions that change CSF
• Transporters
• Substrates for transporters
No need to know equations on receptor theory; however do understand that concentration-effect
relationship is dependent on the concentration-target occupancy as well as the target-occupancy effect
relationships. No need to learn all equations for the brain uptake index method (except for BUI%), brain
perfusion method; however do understand what is measured by what experimental approach (with
pros and cons).
The exam questions will be related to the slides and what has been added to the slides during the
lectures, and exercises.
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,Basic Aspects of the BBB and Pharmacokinetic Principles
BBB Morphology, Physiology and Function
Short history:
- 1885: water-soluble aniline dyes injected in human body stained all organs except the brain.
- 1913: dye injected into spinal fluid of the brain directly, brain was stained but the rest of the
body was not. This demonstrated the compartmentalization between brain and body.
- Lina Stern proposed the concept of blood brain barrier (then called hematoencephalic
barrier).
- 1941: two barrier systems observed, the Blood Brain Barrier (BBB) and the Blood Cerebrospinal
Fluid Barrier (BCSFB). Found that the barrier function is at the level of the capillary endothelial
cells and not the astrocytic end feet.
- 1942: highly lipid soluble dyes were able to transverse the BBB
The brain has a great capillary network; almost
every cell in the brain is in contact with a
capillary. The network consist of very small,
refined capillaries. The network could be
visualized by injecting horseradish peroxidase
a certain dye to colour the capillaries. The
capillaries are made of capillary endothelial
cells (ECs), which are covered by pericytes.
These pericytes are covered by astrocytes and
neurons. The diameter of capillaries are very
small, two red blood cells wouldn’t be able to
pass along side by side. As stated earlier: the
BBB acts on the level of the endothelial cells.
The function of the BBB is to control the brain environment: it protects the brain from variations in
“external” biochemical environment, it takes up nutrients and other necessary components for brain
function, and it discards waste products from brain activity (3x). Most important nutrient for the brain:
glucose.
Brain physiology:
- Weight of the human brain: 1400 g. Weight of rat brain: 1,8 g.
- There are 100 billion capillaries in human brain with total length of 644 km.
- The endothelial wall of the capillaries form the BBB. The thickness of this wall is 200-500 nm.
- The surface area of the human BBB is about 20m2.
- The total volume of all ECs in the human brain is about 5 mL
- Every neuron is virtually perfused by its own blood vessel.
- Distance between capillaries is about 25-40 μm.
- Average cerebral blood flow is 1 mL/min/g
- Mean transit time through a brain capillary is 1.4-2.8 sec.
- Volume of capillary blood in brain tissue is 3%
- Gray matter regions (containing neuronal cell bodies) has a higher capillary density than the
white matter (myelinated neuronal projections)
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,Three different barriers
The BBB is the barrier between blood plasma and brain tissue, at the level of capillary endothelial cells
by tight junction formation. It is by far the largest surface area for exchange (13-20 m2). No brain cell
is further that 25 μm away from a capillary, so once the BBB is crossed, the diffusion distances to
neurons and glial cell bodies are short. Targeting drugs across the BBB is therefore the favoured route
for drug delivery to the brain.
However, there are other barriers as well. The Blood Cerebrospinal Fluid Barrier (BCSFB) lies at the
choroid plexus in the lateral, third and fourth ventricles of the brain where tight junctions are formed
between epithelial cells at the CSF-facing surface (apical surface) of the epithelium. This is also a
barrier between blood and the brain but drugs diffuses into the cerebrospinal fluid. This barrier acts
at the level of the choroid plexus epithelial cells.
The last barrier is the arachnoid barrier. The brain is enveloped by the arachnoid membrane. The
arachnoid is avascular but lies close to the superior sagittal sinus and is separated from it by the dura.
The arachnoid is a multi-layered epithelium with tight junctions between cells of the inner layer that
form an effective seal. Arachnoid villi project into the sagittal sinus through the dura and a significant
amount of CSF drains into the sinus through these valve like villi, which only allow CSF movement out
of the brain to blood. Transport across the arachnoid membrane is not an important route for the
entry of solutes into the brain.
The neurovascular unit
This is a functional unit composed of groups of
neurons and their associated astrocytes, interacting
with smooth muscle cells and endothelial cells on
the micro vessels (arterioles) responsible for their
blood supply, capable of regulating the local blood
flow.
The gliovascular unit
Is a proposed functional unit composed of single
astrocytic glial cells and the neurons they surround,
interacting with local segments of blood vessels, and
capable of regulating blood flow at the arteriolar
level and BBB functions at the capillary level.
The exact distinction between both units is somewhat vague.
The endothelial cells are tightly bound to each other
via tight junctions. These tight junctions are
essential in BBB function. There are different
structures that function as tight junctions and the
different structures leave little space between the
endothelial cells. Drugs are able to pass though this
(intercellular) space between cells but to be able to
do so, the drug has to be really small. Examples of
tight junctions are: Claudin, Occludin and JAM
structures.
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, Astrocytes
Are a form of macroglial cells (non-neuronal “glue” cells) that envelop more than 90% of the BBB
endothelium. They interact with the ECs to help BBB function, morphology (tightness) and protein
expression. They provide support and nutrition, and maintain homeostasis. They remove excess ions
(potassium) and neurotransmitters and therefore regulate the external chemical environment of
neurons. They may regulate vasoconstriction and vasodilation of the capillaries by producing
substances such as arachidonic acid (vasoactive metabolites). They signal between each other via
calcium and IP3. In the human brain there is about 1 glia for every neuron.
Pericytes
Pericytes are cells that regulate endothelial cell proliferation, survival, migration, differentiation and
vascular branching. They cover approximately 20-30% of the microvascular circumference (so overlap
with astrocytes). There is evidence that pericytes control the blood flow in the microvasculature
(constriction of endothelial cells).
Basement membrane (Basal Lamina)
The basement membrane provides mechanical support for cell attachment and serves as a substratum
for cell migration. It separates adjacent tissue and is 20-60 nm thick. It is likely to function as a barrier
for the passage of macromolecules. The basement membrane is like a 3D mesh consisting of proteins
like integrins, laminin, perlecan, fibronectin, tenascin, collagens and proteoglycans. Cell adhesion
involves integrins (transmembrane receptors that bridge the cytoskeletal elements of a cell to the
extracellular matrix).
Glycocalyx
There is a negatively charged surface coat of proteoglycans,
glycosaminoglycans and absorbed plasma proteins lining the
luminal surface (inside) of the endothelial wall. This is the
glycocalyx. An intact glycocalyx is necessary for maintenance of
normal vascular function.
Important note: from now on “Brain Interstititial Fluid” (ISF) and
“Brain Extracellular Fluid” (ECF) will be used interchangeably.
The brain ISF/ECF is about 16-19% of the brain weight. 20% of the
brain weight is of proteins.
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