Methods for studying drug uptake

Presented by,
Pratiksha C Chandragirivar
M pharma 1st sem
Dept. of pharmaceutics
HSK COP Bagalkot
Facilitated to,
Mr. J.N.HIremath
Asso.Professor
Dept. of pharmaceutics
HSK COP Bagalkot
1Advanced biopharmaceutics and pharmacokinetics

CONTENTS:
1. Methods of studying drug uptake
2. References

METHODS OF STUDYING THE DRUG
UPTAKE
Methods of studying drug
uptake
In vitro In situ In vivo In silico

1. IN VITRO METHOD :
As the name suggests the “in vitro” these methods are
conducted/performed outside the living organisms.
These type of methods involves the study of transport of drug
through different types of membranes or biological materials.

Experiments are
conducted by using
Diffusion cells
Segments of GIT of
laboratory animals
Everted sac
technique
Everted sac
modification
Circulations
techniques
Everted ring or
slice techniques
Cell cultures of
epithelium
Example: Caco-2
cells

A. DIFFUSION CELL METHOD:
( ref .1)

Method :
Diffusion cells consists of two compartments:
1. Donor compartment: It contains the drug solution and the
lower end of which contains the synthetic or natural GIT
membrane that interferes with the compartment.
2. Receptor compartment: It contains the buffer solution.
Hence the amount of drug uptake is studied or determined by
the measurement of rate of drug arrival in the receptor
compartment.

Advantages:
1. Can be predicted for almost all the dosage forms.
2. Easily handled
Disadvantages:
1. Tedious
2. Some of the semi permeable membranes used are costly.

B. SEGMENTS OF GIT OF LABORATORY
ANIMALS
I ) EVERTED SAC TECHNIQUE:(EVERTED INTESTINE
SAC TECHNIQUE)
(ref
1 and
2)

Method:
laboratory animal is taken i.e., rat
About 3cm of segment of small intestine of rat isolated
Then intestine is inverted
Sac is filled with the small volume of drug free buffer solution

Both ends of the segment are tied off
Sac is immersed in an Erlenmeyer flask containing a larger
volume of buffer solution that contains the drug
The flask and contents are then oxygenated and maintained at
37℃ for a specified period of time and shaken mildly
At predetermined time intervals, the sac is removed, serosal fluid
is assayed for drug content

Advantages:
1. The epithelial cells of the mucosal surface are exposed
directly to the oxygenated mucosal fluid.
2. Prolongs the viability and integrity of the preparation after
removal from the animal.
3. Convenience and accuracy with respect to drug analysis.
Disadvantages:
1. Difficulty in obtaining more than one sample per intestinal
segment.

II). EVERTED SAC
MODIFICATION:(CRANE AND WILSON
MODIFICATION)
Here the essential
features of
simple sac
method are
retained.
The intestine is tied
to cannula from
which frequent
serosal samples
may be obtained.
Ref.2

Method:
The test animal is fasted for 20 -24 hrs
Water is allowed ad libitum (as directed or as desired)
The animal is killed by a blow on the head or anaesthetized with
ether or chloroform
The entire small intestine is everted

From intestine about 5 to 15cm region is selected and segments
are made
Distal end of the intestine is tied and proximal end is attached to
the cannula
The segment is suspended in the drug containing mucosal
solution about 40 – 100ml

The mucosal solution is aerated
The determination of rate drug transfer is done by taking the serosal
solution at each time with the help of syringe and replaced with fresh
buffer solution
The amount of drug that permeates the intestinal mucosa is plotted
against time which describes the absorption profile of drug at any
specific pH

Advantages:
1. A number of different solutions may be tested with a single
segment of intestine.
2. It is simple and reproducible.
3. It distinguishes between active and passive absorption.
4. It determines the region of the small intestine where the
absorption is optimal, particularly in the case of active
transport.
Disadvantages:
1. Permeability is less.
2. Time consuming.

III). CIRCULATION TECHNIQUES:
In this method, small intestine may or may not be everted.
This method involves the isolation of either the entire small intestine of
lab animal or a segment.
Oxygenated buffer containing drug is circulated through the lumen.
Drug free buffer solution is also circulated on the serosal side of the
intestinal membrane and oxygenated.
Absorption rates from the lumen to the outer solutions are determined by
sampling both the fluid circulating through the lumen and outside.

IV). EVERTED RING OR SLICE
TECHNIQUES:
Method:
Appropriate section of small intestine of rat is isolated and
everted
Dissected into slices or rings of thickness 1 and 3 mm ,with
length 0.1 to 0.5 cm length

Then slices are placed an oxygenated isotonic drug solution at
37℃
Incubated for a predetermined period of time with gentle shaking
After incubation ring are washed, dried and placed in pre weighed
scintillation vials
Each vial is reweighed to determine the wet tissue weight, and
then sample is analysed for drug

Advantages:
1. This method is reproducible.
2. Kinetics studies can be performed.
Disadvantages:
1. Process of cutting intestine into rings may expose highly
permeable areas of cut or damaged tissue the medium.

C. CELL CULTURES OF GUT
EPITHELIUM
EX: CACO-2 CELLS
Method:
Differentiated cells of the intestine, originating from Caco-2
cells i.e., cells of carcinoma of colon
These placed on previously treated polycarbonate with
collagen(which on incubation aids reproduction of cells
while not retarding drug permeation characteristics)

Solution of the drug is placed in this layer of cultured cells
The system is place in a bath or receptor compartment of buffer
solution
The drug that reaches the latter compartment is sample and
analyzed periodically

2. IN SITU METHOD
The term in situ refers to the methods where animal blood
supply intact.
They just mimics the in vivo models.
The rate of absorption is determined based on the
perfusion of a segment of GIT by drug solution and the
amount of drug diffused through it.
Advanced biopharmaceutics and pharmacokinetics 24

In situ
Absorption
from the
intestine
Doluisio
method
Perfusion
technique
intestinal
loop
technique
Absorption
from the
stomach

A. ABSORPTION FROM THE INTESTINE
1. DOLUISIO METHOD:
(1)

Method:
Upper and lower part of the small intestine of anesthetised and
dissected
Dissected rats are connected by means of tubing to syringes of
capacity 10-30 ml
The intestinal segment is washed with normal saline

The syringe is filled with a solution of radio labelled drug
and a non-absorbable marker which is used as indicator of
water-flux during perfusion
Part of the content of the syringe containing drug is
delivered to the intestinal segment which is then collected
in the second syringe and analysed for drug

2. SINGLE PASS PERFUSION
TECHNIQUE:
Ref.1

In this method, the drug solution passes through the intestinal
segment just once.
This is superiors to Doluisio method

3. INTESTINAL LOOP TECHNIQUE:
Method:
Adult male rats are fasted, water also being withheld for 1-2
hr before the experiment
Under anaesthesia an abdominal midline incision is made
and the small intestine is exposed
Both proximal and distal ends are closed by ligature

The drug solution is introduced in the lumen of the loop by
means of syringe which is secured by proximal ligature
After injection, syringe is removed and proximal suture is
tightened
The loop is placed into abdominal cavity and the incision is
closed
After a predetermined period of time animal is sacrificed, the
intestinal loop is excised and homogenized and the amount of
drug unabsorbed is determined

Advantages:
1. The method is relatively simple and reproducible.
Disadvantages:
1. Only one sample can be obtained from the experimental
animal.

B. ABSORPTION FROM THE STOMACH
Method:
Adult male rats which are fasted are anaesthetized
Cardiac is ligated
An incision is made in pylorus in which cannula is inserted
and ligated

The lumen is washed several times with saline and subsequently with
0.1 N HCl solution containing 0.15M NaCl
The drug solution of known concentration is introduced into the
stomach
After 1hr,the solution removed from the gastric pouch and assayed for
drug content
The percentage of drug absorbed in 1hr can be calculated

3. IN VIVO METHOD
These are the actual method.
Even though in situ method mimic the in vivo method,
most of the factors like gastric emptying, intestinal
motility and effects of the drug on the Git are only
predicted by in vivo method

DIRECT METHOD:
This method involves the study of drug uptake is done by
determining the drug present in urine or blood.
For this suitable sensitive analytical procedure are developed to
assay the intact drug in the biological fluid that is sampled.
If the drug undergoes metabolism extensively, specific assay is
developed for the one of the therapeutically active metabolite,
hence concentration of metabolite can also ne determined.
Before conducting experiments on animal it should be pre
clinically trialled.

The animal chosen should be resembalance to man in
some extent.
Most preferable is pig but it is not used because of
handling problem.
Then second preferred are dog.
At last if no choice the rabbits and rats.

METHOD:
A blank urine or blood sample is taken from the test animal before the
experiment
Then test dosage form is administered to the animal
At appropriate intervals of time, the blood or urine sample are collected
Assayed for drug content
From this data, we can determine the rate and extent of drug absorption

INDIRECT METHOD:
Here pharmacologic response is taken as the index of drug
absorption.
It is done when measurement of drug concentration in blood or
urine is difficult or not possible but sensitive method is
available to test the activity.
Assumption is made that, when a test dosage form is
administered in a body, the obtained pharmacologic response of
a drug is related to the amount of drug in the body.

LD50 appears to be dependent on the rate of absorption of
the drug and hence on the rate of dissolution.
A plot of log dose v/s response time is plotted
Slope is:
FKa
2.303
And intercept of is log d
Where, F = bioavailability
Ka = absorption rate constant
d = threshold dose

4. IN SILICO METHOD
In silico is an expression used to mean “performed on
computer or via computer simulation”.
Important model for determining the drug uptake by in
silico method is PAMPA model.

PAMPA MODEL(PARALLELARTIFICIAL
MEMBRANE PERMEABILITYASSAY)
It contains, hydrophobic filter material coated with a
mixture of lecithin/phospholipids dissolved in an inert
organic solvent such as dodecane creating an artificial
lipid membrane that mimics the intestinal epithelium.
The rate of permeation across the membrane barrier is
correlated with the extent of drug absorption in humans.

REFERENCES:
(1).Biopharmaceutics and pharmacokinetics – A treatise by
D.M.Brahmankar and Sunil.B.Jaiswal , 3rd edition published
by - M K Jain for VALLABH PRAKASHAN,C- 5 SMA
cooperative industrial estate, GT Karnal road, Delhi – 110033,
Page number: 78-81.
(2). Textbook of biopharmaceutics and pharmacokinetics by Dr.
Shobha Rani.R.Hiremath,1st edition , published by PRISM
BOOKS PVT.LTD,1865- 32nd cross BSK II stage Bangalore –
560070,
Page number: 25-31.
(3). Video was taken from, https://youtu.be/6xdaj6bOtLo

Methods for studying drug uptake

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