Epithelial Cell Junctions

&
Efflux Transporter Systems
I n relevance to
Oral Drug Absorption and Bioavailability
Chandra Teja U
Department of Pharmaceutics
KVSR SCOPS
By
Y13MPPC140002
What They Are;

How They are Important;

Their Types; and

Relevance in Oral Drug Absorption
We Will be Discussing
Epithelial Cell Junctions
Cell Junctions are the structures where long term association
between neighbouring cells and cells with ECM are established.

They

Hold the cells together with enough stability to compose tissue.

Are composed of Membrane-Associated Structures, usually
Transmembrane Proteins.

Also help in communication between cells.
What Are Cell Junctions!?
How Many Types Are There!?
Cell Junctions are of the following types
1. Tight/Occluding junctions: Seal cells together into sheets.
(forming an impermeable barrier)
2. Adhering/Anchoring junctions: Attach cells to other cells or
ECM. (providing mechanical support)
3. Gap/Communicating junctions: Allow exchange of chemical or
electrical information between cells
Tight or Occluding Junctions
The junction forms a
band around the cell
fusing cells together
and closing
intercellular space.

The number of fusion
sites often correlate
to the leakiness of the
tissue.
In renal tubule of the kidney (filtrating area) few fusion sites are
present enabling leakability.
In urinary bladder (contents should not leak out) numerous tight
junctions are present to form a tight seal between cells.
These junction greatly restricts the passage of water, electrolytes and
other small molecules across the epithelium.
They block the movement
of integral membrane
proteins (red and green)
between the apical and baso-
lateral surfaces of the cell.

Thus the special functions of
each surface, like
Receptor-mediated
endocytosis at the apical
surface
Exocytosis at the
basolateral surface
can be preserved.
Anchoring Junctions
Cells must be anchored to one another and to components of the ECM.

Junctional complexes called anchoring proteins extend through the
plasma membrane to link cytoskeletal proteins in one cell to that in
other cells as well as to the ECM.

Three types of anchoring junctions are observed:
Adherens Junctions
Desmosomes
Hemidesmosomes

Anchoring junctions are most abundant in tissues that are subjected to
severe mechanical stress, such as heart, muscle, and epidermis.
a) Adherens Junctions or Zonula Adherens
Situated at close proximity to the
apex but somewhat below the tight
junctions.

They are relatively permeable as
there are no fusion sites that
completely seal off the paracellular
space.

They often form a continuous
adhesion belt/band.
Their transmembrane anchors are composed of Cadherins to anchor with
other cells and Integrins to anchor with ECM.

Adherens junctions share the characteristic of anchoring cells through
their cytoplasmic contractile bundle of actin filaments.
b) Desmosomes. a.k.a. Macula Adherens
Can be visualized as rivets through
the plasma membrane of adjacent cells.

Can be situated anywhere on the lateral
surface of the cell.

It is a disk-shaped structure matched with
an identical structure at the surface of the
adjacent cell.

Their transmembrane anchors are
Cadherins.
Its adhesion function is further empowered by groups of intermediate
Keratin filaments which provides firm adhesion.
The membranes in this region are usually further apart (30nm).
c) Hemidesmosomes
Like desmosomes, they tie to intermediate filaments in the cytoplasm, but
their transmembrane anchors are Integrins rather than Cadherins.
Hemidesmosomes are like Desmosomes, but form links between Cells
and the basal lamina that underlie epithelia.
Gap Junctions

Are simply gaps (~2nm) between
the neighbouring cell membranes.
Contains protein units called
Connexin with a hydrophilic pore
of 1.5nm diameter that acts as an
ion channel between cells.
Present along the lateral surface of the epithelial cell.
A Gap-Junction is made up of six Transmembrane Connexin Subunits.
Inorganic ions, and small water soluble molecules (<1000kDa) pass
between the cells. Large Molecules like Proteins cannot pass through.
Efflux Transporter Systems
(Multi Drug Resistance)
MDR Transporters belong to the family of the ATP Binding Cassette
(ABC) proteins & are present in all living organisms.

The key physiological task of the MDR-ABC transporter network is to
provide general xenobiotic resistance, probably evolved as Complex
Cellular Defence Systems.

Hundreds of structurally diverse therapeutic agents are substrates to
these and they impedes the absorption, permeability, and retention of the
drugs, extruding them out of the cells.
In humans, 3 major types of MDR proteins are present, that are the
members of the subfamilies
The ABCB (P-glycoprotein/ABCB1/MDR1),
The ABCC (ABCC1/MRP1, ABCC2/MRP2), and
The ABCG (ABCG2/MXR/BCRP).
The major practical causes for studying MDR-ABC transporter
expression and function are
Predicting the fate of pharmaceutical agents in our body.
The relevance of in vitro experiments to the in vivo role of
MDR-ABC transporters.
Related to cancer & infection drug resistance.
MDR1 (P-gp) preferentially extrudes large hydrophobic molecules,
while ABCC1 and ABCG2 can transport both hydrophobic drugs and
intracellularly formed metabolites, e.g., Drug Conjugates.
P-glycoprotein is primarily found in epithelial cells lining the Small
I ntestine, Colon, pancreatic & bile ducts, kidney proximal tubules, and
also in the BBB.

The highest MRP1 levels are reported at testis, cardiomyocytes,
placenta, prostate, lung, thymus and kidney. It is found in lower levels
along the GIT.

Significant expression of ABCG2 was observed in the Placental
Barrier, and at the blood-testis barrier, the BBB, and the stem cells.
Hence, P-gp is of greatest importance when talking about Oral BA of
Drugs.
The P-GlycoProtein
The most extensively studied and experimented MDR Protein
P-gp is encoded by the ABCB1 gene.
It contributes greatly in the extrusion of many drugs from the blood into
the intestinal lumen.
It is also responsible for enhancing the excretion of drugs out of
hepatocytes and renal tubules into bile and urine.
It can recognize and transport numerous structurally diverse drugs over
a molecular weight range of 250 g/mol (cimetidine) to 1200 g/mol
(cyclosporin).
Therefore, P-gp can potentially reduce the absorption and oral
bioavailability and decrease the retention time of a number of drugs.
The transporter is also overexpressed on the surface of many
neoplastic cells and makes the chemotherapy almost ineffective in many
cases.
Location of P-gp Function
GI Epithelial Cells
Efflux of Cellular waste and Drugs
into lumen
Hepatocytes
Secretion of drugs into bile and re-
entry into Intestine
(Entero-Hepatic Circulation)
Renal Epithelial Cells
Efflux of Drugs into Tubular
Lumen, easily Excreted
Placenta, Brain, Testis Barrier Action for Entry of Drugs
Cancer Cells Resistance to Agents
Interactions of P-gp with Drugs
The substrate specificity of P-gp and CYP450 enzyme system has
overlap extensively.
Clinically Significant reduction in Oral Absorption and BA is seen with

Antacids
Cimetidine
Ranitidine
Antibiotics
Erythromycin
Levofloxacine
Cardiac Drugs
Digoxin

Antitumour Agents
Placitaxel
Doxorubicin
Vinblastin
Ca
2+
Channel Blockers
Diltiazem
Immunosuppressants
Cyclosporine A

Anti-retrovirals
Ritonavir
Indinavir
Steroids
Dexamethasone
Opioids
Morphine
Fentanyl

P-gp also has Inducers and Inhibitors
An I nducer further reduces the absorption of other substrates.
An I nhibitor generally increases the absorption of other drugs by
reducing P-gp activity.
I nducers
Avasimibe
Carbamazepine
Phenytoin
Rifampin
Morphine
Dexamithazone
I nhibitors
Quinidine
Reserpine
Cyclosporine
Verapamil
Azithromycin
Atorvastatin
P-gp Follows Saturable Kinetics
In the case of fast absorbing drugs having larger doses, efflux by P-
gp poses less impact on drug absorption and therefore is not
important in terms of bioavailability or pharmacokinetic properties.

This is because the transport activity of P-gp becomes saturated by
high concentrations of drug in the intestinal lumen.

This is best explained by Michaelis Menten Plot & Equation.
Enhancement of BA by P-gp Inhibition
P-gp mediated drug efflux greatly interferes with the delivery of Potent
Drugs or the drugs with slow dissolution and diffusion rates.
This decrease in drug absorption of those small amounts of drugs can be
life threatening at times as sufficient plasma levels of drug cannot be
reached.
Moreover, it can make the sustained release dosage forms of the
substrates completely ineffective by limiting their absorptions.
This is even more important in case of MDR exhibiting Cancer.
Hence, P-gp inhibition has gained a lot of importance in Research field
and many approaches are being postulated.
In general, P-gp can be inhibited by three mechanisms:
(i) blocking the pump competitively or non-competitively;
(ii) interfering with ATP hydrolysis; and
(iii) altering integrity of cell membrane lipids.
P-gp inhibitors are classified into three generations based on their specificity,
affinity, and toxicity.
1
st
gen. inhibitors are pharmacologically active substances which are
clinically used but have the ability to inhibit P-gp.
2
nd
gen. inhibitors pharmacologically inactive. But, they inhibit CYP
enzymes and ABC transporters leading to complicated pharmacokinetic
alterations. (Eg Dexverapamil, valspodar, etc)
3
rd
gen. inhibitors are under clinical development, aiming for higher
specificity and low toxicity (Eg Laniquidar, Tariquidar, etc)
Other approaches like Liposomes (Active Targeting), Use of P-gp bypassing
Polymers, Drug-Macromoecule Conjugates, etc are being studied.
References
Google Images
http://www.histology.leeds.ac.uk/
http://the-aps.org
http://en.wikipedia.org
http://www.nature.com/
PubMed NCBI Articles
SlideShare and AuthorStream Presentations
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