EVALUATION SEMINAR ON ALTERNATIVE

TO ANIMAL STUDIES

PRESENTED BY:

MR. UTTAM PAUL

1ST M. PHARM

DEPT. OF PHARMACOLOGY

FACILIATED TO:

DR. SHIVALINGE GOWDA K.P.

H.O.D, DEPARTMENT OF

PHARMACOLOGY

TABLE OF CONTENTS

OVERVIEW

DEFINITION TO ALTERNATIVE TECHNIQUES

DIFFERENT TECHNIQUES TO BE USED AS

ALTERNATIVES TO ANIMAL TESTING

1) Full thickness skin model (in vitro method)

2) In sillico methods

3) Cell line technique.

4) Patch clamp method

REFERNCE

OVERVIEW

OVERVIEW

Animal usage in scientific research and testing has raised

controversy and criticism for long.

A number of legislative initiatives have been proposed from time to

time to limit animal research, or ensure proper treatment of animals.

Animal welfare groups, or reformers, opposed various forms of

animal research due to the increased use of animals for developing

drugs and safety tests for pesticides.

ALTERNATIVE TECHNIQUES: DEFINITION:

The term ‘alternative’ is used to refer to those techniques or methods

that replace the use of laboratory animals altogether, reduce the

numbers of animals required, or refine an existing procedure or

technique to minimize the level of stress endured by the animal.

However, the field of alternatives study particularly in vitro toxicology

has evolved into a respected discipline and is attracting competent and

motivated scientists around the world.

It is not possible to replace whole animal models with in vitro

systems to evaluate drug effects on major organ systems.

However, techniques can greatly reduce the number of animals

needed, and refined protocols can improve the design efficiency

and quality of studies, and lessen stress and discomfort experienced

by lab animals.

Generally instrumented animal models can be reused in major

organ systems toxicology (MOST) for studies to evaluate more than

one drug.

DIFFERENT TECHNIQUES TO BE USED AS

ALTERNATIVES TO ANIMAL TESTING

1) Full thickness skin model (in vitro method)

2) In sillico methods

3) Cell line technique.

4) Patch clamp method.

1) In-vitro methods

Cell and tissue cultures are used to test product ingredients.

Cell cultures are now also used routinely to test substances for

mutagenic properties.

Tissue cultures are additionally used to test substances for

compatibility with mucous membranes.

Full Thickness Skin Model:

A test substance is applied to the Full Thickness Skin Model so that

its effect on the skin tissue can be systematically evaluated.

The substance, e.g. a cream formulation, is applied topically using a

brush. This can be done several times over a period of at least nine

days.

In this way, the effect of the substance on the cell layers in the skin

can be studied.

The standardized production of the model, in combination with its

special properties, makes it suitable for use as an in-vitro alternative

to animal testing

2. In-sillico methods

Substances with similar chemical structures often have similar

properties.

In these cases, therefore, knowledge of the properties of a few

representative substances is sufficient to be able to deduce the

properties of a series of similar substances.

By analogy, certain properties of these representative substances can

also be assumed to be properties of the other substances in the series.

The required calculations are performed using specially developed

computer programs.

Only these selected substances will then have to be tested according

to the legally prescribed test methods.

It is anticipated that combinations of such calculations will make it

possible to narrow down the number of substances to be tested.

3) Cell line technique

The term cell line refers to the propagation of culture after the

first subculture.

Once the primary culture is sub cultured, it becomes a cell line.

A cell line derived by selection or cloning is referred to as cell

strain.

Types of cell lines:

Finite cell line.

Continuous cell line.

SERIAL

NO

PROPERTIES FINITE CELL LINE

(with limited culture life

spans)

CONTINUOUS CELL LINE

(unlimited life )

1 Growth rate Slow Fast

2 Mode of growth Monolayer Suspension

3 Yield Low High

4 Transformation Normal Immortal,

Tumorigenic

5 Cloning efficiency Low High

6 Serum

requirement

High Low

7 Markers Tissue specific Chromosomal, antigenic.

APPLICATIONS OF CELL LINE

Screening of anti-cancer drugs

Cell based bioassays

To determine the cytotoxicity

In vitro screening of several drugs

Production of anti-viral vaccines

Cancer research, which require the study of uncontrolled cell

division in culture

Cell fusion technique, Genetic manipulation

Gene therapy, Recombinant DNA therapy

Biotechnology , Molecular biology……etc.

PATCH CLAMP TECHNIQUE

Patch clamp technique is a technique in electrophysiology that

allows the study of individual ion channels in cells.

The technique is used to study excitable cells such as neurons,

muscle fibers and the beta cells of the pancreas.

Use a pipette to pinch off a small region of membrane.

Provides access to:

the inside of the cell

a small region of membrane

TYPES Of PATCH CLAMP:

A. On-cell

B. Inside Out

C. Whole Cell

D. Outside-Out

Applications of Patch Clamp Technique:

For the evaluation of antiarrhythmic agents.

In kidney cells.

Used for isolated ventricular myocytes from Guinea pigs to

study a cardio selective inhibition of the ATP sensitive potassium

channel.

To identify multiple types of calcium channels.

To measure the effect of potassium channel openers.

Used in the molecular biology.

Voltage clamp studies on sodium channels.

Used to investigate a wide range of electrophysiological cell

properties.

Measurement of cell membrane conductance.