METHODS OF DRUG UPTAKE

PRESENTED BY: FACILITATED TO:

Manjunath S L Mr. J. N. Hiremath
M. Pharm 2nd sem Asso. Professor & HOD
Dept. of Pharmaceutics Dept . of Pharmaceutics
HSK COP Bagalkot. HSK COP Bagalkot

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Contents
1. Methods of drug uptake

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METHODS OF STUDYING DRUG UPTAKE

Methods of studying drug

uptake

In-vitro In-situ In-vivo In-silico

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1. IN VITRO METHOD:

As the name suggests the “in vitro” these methods are
conducted/performed outside the living organisms
These types of methods involves the study of transport of

drug through different types of memberanes or biological

materials

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 Everted sac
Diffusion technique
cells

Segment of GIT of Everted sac

laboratory animal modification

Experiments are
conducted by using Everted ring or
slice techniques

Cell culture of
epithelium
Example:caco-2
cells

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1.a DIFFUSION CELL METHOD

Fig 1

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• Diffusion cells consist 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
compartment.

2. Receptor compartment: It contains the buffer solution.

Hence the amount of drug uptake is studied or determined by

measurement of rate of drug arrival in the receptor compartment.

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• Advantages
1. Can be predicted for almost all dosage forms.
2. Easily handled.

• Disadvantages
1. Tedious
2. Some of the semi permeable membranes used are costly.

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1.b. SEGMENTS OF GIT OF LABORATORY ANIMALS
I. EVERTED SAC TECHNIQUE:(EVERTED INTESTINE
SAC TECHNIQUE)

Fig 2 9
• Method
Laboratory animal is taken i.e., rat

About 3cm segment of small intestine of rat isolated

Then intestine is converted to Sac

is filled with small volume of drug free buffer solution

Both ends of segment are tied off

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 Sac is immerse in an flask containing a larger volume of
buffer solution that contains the drug

The flask and contents are then aereated and maintained at

37°C for a specified period of time and shaken mildly

At predetermined time intervals, the sac is removed, serosal

(internal) fluid is assayed for drug content

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Advantages:
1. The epithelial cells of the mucosal surface are exposed directly to the
oxygenated mucosal fluid.
2. Convenience and accuracy with respect to drug analysis.

Disadvantages:
1. Difficulty is obtaining more than one sample per intestinal segment.

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II). EVERTED SAC
MODIFICATION:(CRANE AND WILSON
MODIFICATION)

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Fig 3
Method:
The test animal is fasted for 20-24 hrs

Water is allowed ad libbitum(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 5 to 15cm region is selected and segments are

made

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Distal end of 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 aereated

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 15
• Advantages
1. A number of different solutions may be tested with single segment of
intestine.
2. It is simple and reproducible.
3. It determines the region of the small intestine where the absorption is
optimal.

• Disadvantge
1. Time consuming.

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III). 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 3mm , with

length 0.1 to 0.5 cm length

Then slices are placed an oxygenated isotonic drug solution at

37°C

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 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

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Advantages
1. This method is reproducible.
2. Kinetics studies can be performed.

Disadvantages
1. Process of cutting intestine into ring may expose highly permeable areas
of cut or damaged tissue the medium.

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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 Membrane with

collagen(which on incubation aids reproduction of cells which not retarding
drug permeation characterstics)

Solution of drug is placed in this layer of cultured cells

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The system in place in a bath or receptor compartment of buffer
solution

The drug that reaches the latter compartment is sample and analysed
periodically

Fig 4 21
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 segment

of GIT by drug solution and the amount of drug diffused through it.

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.

In situ

Doluisio Perfusion
method technique

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1. DOLUISIO METHOD:
peristaltic pump

Fig 5 24
Method:
Upper and lower part of the small intestine of and anesthetised and
dissected

Dissected rats are connected by means of tubing to syringes of capacity

10-30ml

The intestinal segment is washed with normal saline

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The syringe is filled with a solution of radio labelled drug and
a non-absorbable marker which is used as a 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

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2. SINGLE PASS PERFUSION TECHNIQUE:

Fig 6 27
In this method the drug solution passes through the intestinal segment once.

This is superiors to Dolusio method in that precise adjustment of hydrodynamic

conditions that can influence blood circulation and puts stress on intestinal wall
can be controlled.

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3. IN VIVO METHOD

These are actual method

Even through in situ method mimic the in vivo method, most
of the factors like gastric emptying time, intestinal mobility
and effects of the drug on the GIT are only predicted by in
vivo method

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.

DIRECT
METHOD

IN VIVO
IN DIRECT
METHOD

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INDIRECT 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.

Before conducting experiments on animal it should be preclinically

trialed

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The animal chosen should be resemblance 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.

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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

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DIRECT 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.

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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 :

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PAMPA MODEL(PARALLEL ARTIFICIAL MEMBRANE
PERMEABILITY ASSAY)

It contains, hydrophobic filter material coated with at 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.

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 REFERENCES
 Biopharmaceutics and pharmacokinetics - A treatise
by D.M.Brahmankar and Sunil.B.Jaiswal, 3rd edition published by
MK Jain for VALLABH PRAKASHAN,C-5 SMA cooperative
industrial estate, GT Karnal road, Delhi110033,Page number: 78-81.

https://www.researchgate.net/publication/247156466_Ex_vivo_and_I
n_situ_Approaches_Used_to_Study_Intestinal_Absorption

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