In Vitro Models for Permeability

Tests of Dosage Forms

Maliwat
Mamangon
Ocampo
Ordoez
Palpagan
Parojinog
Policarpio
Ramales
Ramirez

Introduction
What is permeability?
How easily liquid and gas pass
through a media without being
chemically or physically affected.

Introduction
What does in vitro mean?
A process performed or taking place
in a test tube, culture dish, or
elsewhere outside a living organism.

Introduction
Why study permeability in vitro?

Introduction
Solubility
Permeability

High Low

High

Class Class
I
III

Low

Class Class
II
IV

Introduction
High Permeability
A drug is said to have a high
permeability if the extent of oral
absorption is greater then 90%.

In Vitro Methods

Organotypic models
Intestinal cells are present which
basically simulates the intestine inside
the body
Study formulation with low solubility
and regional permeability

Everted Gut Sac

Use of rat intestine
Drug transport across epithelial cells of
the intestines
Muscularis mucosa is present;
underestimation of permeability is
expected

Everted Gut Sac

Isolated and perfused

intestinal segments
The use of isolated and perfused
organs are used to virtually simulate
true-to-life settings
They have the advantage of
predicting in vivo action as the intact
organs preserved the physiological
function as well as cell-cell contacts
and matrices
Disadvantage: short experimentation

Isolated and perfused

intestinal segments

Isolated and perfused

intestinal segments

Ussing Chambers
Technique develop
The technique is used to measure
the short-circuit current as an
indicator of netion transporttaking
place across anepithelium.
An epithelial tissue (membrane) is
put in between the two chambers
with the solution

Ussing Chambers

Cell Models
Contains only absorptive cells without
other factors such as mucus and other
factors such as the lamina propria and
muscularis mucosa.
Cell cultures
Caco-2 cells

Non Intestinal Cell Systems

MDCK (Madin Derby Canine Kidney)
Dog kidney cells
Distal renal tubule kidney cells that
differentiate to columnar epithelium
Form tight junctions
Do not express P-gp

Small-intestine cell lines from

fetal and
neonatal rats

IEC (intestinal epithelial cell) and RIE

(rat intestinal epithelial) cells
These cell lines are used to analyze
the role of growth factors in epithelial
cell physiology and their specific
factors (Effects of amino acids,
cholesterol synthesis and other
studies not involving permeability)

Caco-2 Cells
Most popular cell model
Derived from human colorectal
adenocarcinoma
Differentiate into intestinal cells with
apical brush border and tight
junctions and also express
hydrolases and microvillar
transporters
Imitates small intestine permeability
Less CYP3A than other intestinal cells

Caco-2 Cells
Exhibits P-gp activity unlike MDCK
cells
Monolayers and has an apical and
basal (serosal and luminal side)
Can be isolated to TC7 cells
TC7 cells
Exhibits CYP3A more than Caco-2 cells
Lower P-gp
Can serve as an alternative to Caco-2 cells
for drug transport studies

Caco-2 Cells

Caco-2 Cells
Relies on the transfer of support at
different times.

Caco-2 Cells
Variability might exist hence, use for
a reference drug when screening for
permeability is recommended
Caco-2 monolayer is a 2-D system
whereas the intestinal mucosa is a 3D system (villi and folds)

In vivo In vitro correlation

USP provides information about the
correlation of the two
Class I and II high correlation (low
permeability)
Class III and IV low correlation (low
permeability)

The use of reference molecules with

known Papp values is recommended.
20% Papp of the Caco-2 monolayer, it is
valid.

Rule of Five
1. There are more than five hydrogen bond
donors (expressed as the sum of hydroxyl
and primary amine groups)
2. The molecular mass is over 50
3. The Log P is over 5 (or MlogP is over 4.15)
4. There are more than ten hydrogen-bond
acceptors (expressed as the sum of
nitrogen and oxygen atoms)
5. Compound classes that are substrates for
biological transporters are exceptions to
the rule.

Misc.
P-gp
P glycoprotein or permeability
glycoprotein
An ATP dependent efflux pump that
pumps foreign substances out of the
cells.
Also transports various drug molecules
across the cell membrane

Misc.
Papp
Apparent permeability
The apparent permeability coefficient
(Papp) is determined in the apical-tobasolateral (A-B) and/or the B-A
directions across cell monolayers
cultured on 96-well polycarbonate
membrane filters.

Caco-2 Cells
A-B permeability and B-A
permeability (refer to figure 5)
In vitro assay depends on what type of
drug
P-gp assay can use both
Oral Absorption uses A-B

In Vitro Permeability Testing

Suggested Readings
For further knowledge about in vitro
methods:
In vitro models for the intestinal barrier:
ECVAM Workshop