A SEMINAR ON

ALTERNATIVES TO ANIMAL SCREENING PROCEDURES

PRESENTED BY

MOHAMMADHUSAIN VASAYA

UNDER THE GUIDANCE OF

MD. SAIFUDDIN KHALID (Asst. Prof.)

DEPARTMENT OF PHARMACOLOGYLUQMAN OLLEG OF PHARMACY

GULBARGA-585 102

A SEMINAR ON

ALTERNATIVES TO ANIMAL SCREENING PROCEDURES

PRESENTED BY

MOHAMMADHUSAIN VASAYA

UNDER THE GUIDANCE OF

MD. SAIFUDDIN KHALID (Asst. Prof.)

DEPARTMENT OF PHARMACOLOGYLUQMAN OLLEG OF PHARMACY

GULBARGA-585 102

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE - KARNATAKA

General background:

TOXICITY studies are generally performed to determine drug-related effects that cannot be evaluated in standard pharmacology profile or occur only after repeated administration of the agent.

Theoretically, toxicity profiling in animals would be most useful if the test model responded in a fashion that was identical to the human. However, such is seldom true in practice, even when the route of administration and vehicle used are identical to the clinical use because deposition (pharmacokinetics) can vary dramatically among species, and even between strains.

Therefore, most toxicity tests are performed in two species – a rodent and a non-rodent – to ensure that any unexpected adverse effects are not overlooked before new chemical entities (NCE) are introduced into man.

The sequence of toxicity testing proceeds from the simple to the complex. This is because toxicity testing is desirable at an early stage in the

development process but large quantities of drug samples are usually not available at that time, limiting the advancement of medical science during the last 100 years which largely depended on research with animals.

Use of animals for research and testing:

Use of animals in scientific research and testing has raised controversy and criticism for long.

Use of animals in medical research has been objected to and a number of legislative initiatives have been proposed from time to time to limit animal research, or ensure proper treatment of animals. Animal protection movement began in England during the nineteenth century.

Antivivisection groups, sometimes referred to as abolitionists, opposed all forms of animal research. 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.

However, use of non-animal alternatives is also being developed wherever possible to meet the mandatory regulation of animal experimentation

BIOTECHNOLOGY: A better option..!

Biotechnology in its broadest sense is the use of living organisms (cells, microbes, plants and animals) to make useful products.

The application of biotechnological knowledge and in particular gene technology is especially important to pharmaceuticals in the manufacture of vaccines, development of more diagnostic aids and therapeutic agents, and ultimately in gene dosing and expression of genes; peptide engineering generation and use of various antibodies, mammalian cell cultures; and transgenic animal techniques.

These tools are already being used to some degree by pharmaceutical firms, but as their use expands and knowledge accumulates, many disciplines, including toxicology will undergo substantial changes.

Transgenic animals for mutagenecity testing are already on the market. Transgenic animal models of carcinogenesis, as well as a number of human

diseases are also available, and increasing use of such models will provide a powerful tool for assessing the potential toxicity of drugs and chemicals.

Animal Alternatives:

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.

In order to monitor physiological functions in conscious animals, survival surgery may be performed to implant catheters, electrodes, flow probes or other devices.

While chronically instrumented animal models can reduce the numbers of animals used per study and reduce numbers associated with acute procedures, these models are resource-intensive to prepare and maintain.

Generally instrumented animal models can be reused in major organ systems toxicology (MOST) for studies to evaluate more than one drug.

Alternative techniques: definition and overview:

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.

The concept of alternative techniques is now widespread throughout the scientific community.

This is due largely to regulations and standards which require consideration and support to alternatives.

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.

The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) was founded in 1981 and is structured to support four core programmes.

Different techniques to be used as an alternatives to animal testing….

1) Full thickness skin model (invitro method)*¹.

2) In sillico methods*².

3) cell line technique.

4) Patch clamp method.

*¹ and *² are used by Henkel and Phenion medical laboratory

In-vitro methods:

Instead of using animals, cell and tissue cultures can be used to test product ingredients.

Cell culture experiments can show, for example, the lowest concentration at which an ingredient causes damage to cells.

The results enable conclusions to be drawn about the ingredient’s compatibility with tissue.

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.

A familiar example is the Hen’s Egg Test, which scientists are now further developing so that it can be used to test for mutagenic properties as well.

Full Thickness Skin Model:

It has been developed as a robust full thickness skin model based on human cells. It can be produced to a constant and very high level of quality. 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 topicallyusing 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, make it suitable for use as an in-vitroalternative to animal testing.

Hen’s Egg Test

In-sillico methods: Substances with similar chemical

structures often have similar properties. In these cases, therefore, a 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.

It is anticipated that combinations of such calculations will make it possible to narrow down the number of substances to be tested.

Only these selected substances will then have to be tested according to the legally prescribed test methods. In sillico method

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. Cell strain do not have infinite life, as they die after some divisions. Types of cells used in cell line:

a) Precursor/ stem cells/ master cells.

b) Undifferentiated but committed precursor cells.

c) Mature differentiated cells.

Types of cell lines:

Mainly two types;

1) Finite cell line.

2) Continuous cell line.

(1) FINITE CELL LINE:

The cell line with limited culture life spans are referred to as Finite cell line.

The cell normally divide 20-100 times before extinction. The actual number of doublings depends on the species, cell

lineage differences, culture conditions etc.

(2) CONTINUOUS CELL LINE:

The continuous cell lines are transformed, immortal and tumorigenic.

A few cells in culture may acquire different morphology and get altered. Such cells are capable of growing faster resulting in an independent culture.

The progeny derived from these altered cells have unlimited life.

Comparison of properties of finite and continuous cell lines:

SERIAL NO PROPERTIES FINITE CELL LINE

CONTINEUOUS CELL LINE

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:

The Technique was developed by Erwin neher & bert Sakman.

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.

What is Patch Clamp….?

Use a pipette to pinch off a small region of membrane. Provides access to:

the inside of the cella small region of membrane

Used to measure current through ion channels.

Why to use patch clamp method..?? Provides access to the inside of the cell

Can insert an electrode into the cell Can change the intracellular fluid

Creates a seal impermeable to ion flow High electrical resistance

Allows one to measure current through ion channels vs. voltage, time, temperature.

The patch clamp consists of an electrode inside a glass pipette. The pipette, which contains a salt solution resembling the fluid normally found within the cell, is lowered to the cell membrane where a tight seal is formed.

When a little suction is applied to the pipette, the "patch" of membrane within the pipette ruptures, permitting access to the whole cell.

The electrode, which is connected to specialized circuitry, can then be used to measure the currents passing through the ion channels of the cell.

Varieties of ion channels can be studied by the patch clamp technique.

PATCH CLAMP

TYPES OF PATCH CLAMP:

A. On-cell

B. Inside Out

C. Whole Cell

D. Outside-Out

Patch clamp technique in kidney cells:

PRINCIPLE:- In the different parts of the kidney fluid is reabsorbed and

substances may be transported either from the tubule lumen to the blood side (reabsorption) or vice versa (secretion).

Besides active transport and coupled transport systems, ion channels play an important role in the function of kidney cells.

The various modes of the patch clamp technique (cell-attached, cell-excised, whole-cell mode) allow the investigation of ion channels.

PROCEDURE:

The patch clamp technique can be applied to cultured kidney cells freshly isolated kidney cells or to cells of isolated perfused kidney tubules.

Segments of late superficial proximal tubules of rabbit kidney are dissected and perfused from one end with a perfusion system.

The non-cannulated end of the tubule is freely accessible to a patch pipette the patch pipette can be moved through the open end into the tubule lumen and is brought in contact with the brush border membrane.

After slight suction of the patch electrode, gigaseals form instantaneously and single potassium or sodium channels can be recorded in the cell-attached or inside- out cell-excised modal.

In order to obtain exposed lateral cell membranes suitable to the application of the patch clamp method, pieces of the tubule are torn off by means of a glass pipette .

As to facilitate the tearing off, the tubules are incubated for about 5 min in 0.5 g/L collagenase at room temperature.

After tearing off part of the cannulated tubule, clean lateral cell membranes are exposed at the non-cannulated end.

The patch pipette can be moved to the lateral cell membrane and gigaseals can be obtained.

It was possible, to investigate potassium channels and nonselective cations channels in these membranes.

EVALUATION:

In isolated perfused renal tubules, concentration response curves of drugs which inhibit ion channels can be obtained with the patch clamp technique.

In isolated cells of the proximal tubule, the whole-cell mode of the patch clamp technique enables the investigation of the sodium-alanine cotransport system.

Applications of Patch Clamp Technique:

For the evaluation of antiarrhythmics 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.

REFERENCES:

1. Vogel H. Gerhard “Drug Discovery & Evaluation” Pharmacology assay. Second edition, Page No.83, 84, 319, 321, 480.

2. Alternatives to animal testing, Henkel and Phenion medical laboratory, www.henkel.com and www.phenion.com

3. http://www.sciencedisplay.com/patchclamp.html

4. Cell culture – wikipedia, free encyclopedia. www.wikipedia.com

5. www.google-image.com