DR. MOHIT KULMIPG RESIDENT

DEPT. OF PHARMACOLOGY

Alternatives to Animal Experiments

1.Need for alternatives to animal testing

2.In vitro models for testing

3.Recent Advances –Stem Cells in Drug Discovery

Step 1: Defining an alternative: The word "alternative" is used to describe any change in an animal test that achieves one or more of the "three Rs" i.e., replaces animals, reduces the number of animals or refines a procedure to alleviate or minimize potential animal pain

Step 2: Developing the alternative: New cell and tissue tests, computer models and other innovative methods can be used to replace existing animal tests. These alternatives are more cost-effective and more reliable than traditional animal experimentation

Step 3: Validating the methods: The developed method is scientifically "validated," in multiple laboratories to see if its results reliably predict outcomes in humans

Step 4: Acceptance in scientific community: After validation, acceptance of the alternative is necessary for its success. The opinions of government regulators strongly influence the extent to which private companies use available alternatives

Introduction

Replacement: refers to the preferred use of non-animal methods over animal methods whenever it is possible to achieve the same scientific aim.

Reduction: refers to methods that enable researchers to obtain comparable levels of information from fewer animals, or to obtain more information from the same number of animals.

Refinement: refers to methods that alleviate or minimize potential pain, suffering, or distress, and enhance animal welfare for the animals used.

Three Rs(3Rs)first described by Russell and Burch in 1959

Reduction alternatives

Good planning of studies - Change in experimental design - Improve methods of data analysisSharing research animalsRe-designing Studies - To collect as much information as possible - Share informationAvoid duplicative testing - By improving communication and co-operation in the planning & execution of testing - Sharing data – avoids unintentional repetitions

Refinement alternatives

Modified to reduce pain & distress in animal - Providing relief (pain & distress) by giving drugs like analgesics, anesthetics, tranquilizers, sedatives. - By changing procedure 1) Small needle 2) Use non-invasive techniques like MRI, ultrasound - Use less sensitive species - Use smaller dose - Test can be ended at the earliest feasible time - Improve housing conditions

Replacement

Absolute replacement: no need to use animals (cell lines, tissue of human or invertebrate cells and tissues)

Relative replacement: humane killing of animals to provide cells or tissues for in vitro studies

What are the alternatives?

1.In vitro(test tube) test methods and models based on human cell and tissue cultures

2. Computerized patient-drug databases and virtual drug trials computer models and simulations

3. Stem cell and genetic testing methods4. Non-invasive imaging techniques such as MRIs and

CT Scans5. Microdosing (in which humans are given very low

quantities of a drug to test the effects on the body on the cellular level, without affecting the whole body system)

Computer (in silico) Modeling

Researchers have developed a wide range of sophisticated computer models that simulate human biology and the progression of developing diseases.

•Studies show that these models can accurately predict the ways that new drugs will react in the human body and replace the use of animals in exploratory research and many standard drug tests.

•Quantitative structure-activity relationships (QSARs) are computer-based techniques that can replace animal

tests by making sophisticated estimates of a substance’s likelihood of being hazardous

These are mechanistic models that aim to predict

sensitization from mechanistic knowledge and

empirical models that are aimed at predicting from a

statistical perspective

Expharm and Xcology are two computer based models available

In vitro systems

Mainly two types 1) Based on animal or human components (cell, tissue and organ culture) 2) Based on organisms not considered animals (micro-organisms and invertebrates)

Animal or human components

Cells, tissues & organs kept alive outside a living organism

Advantages: - Less distress to animals - Less number of animals are required - Human tissues – no need to extrapolate results from animal data

Disadvantage: - Unable to produce complete physiological response - Components lose their ability to perform special function - Can not determine the effect of route of administration

What to measure???

- Rate of synthesis of certain substances e.g : In response to irritation, cell produces prostaglandins, certain enzymes, proteins, antibodies - Changes in membrane permeability - Damage to some part of cell structure

e.g. Liver cells – retain most of special functions when cultured Response to toxic substances - Use of sugar as an indication of metabolic activity - Production of proteins or other substances - Uptake of amino acids as an indication of protein synthesis - Morphological changes - Reduction in viabilityBeating heart cells – to detect the effect of drugsRabbit kidney tubules – to detect substances that can cause acute renal failureRat vaginal tissue – to vaginal irritancy of contraceptives

In vitro models -Drug Discovery Process

In vitro models to animal testing

Cell Based –in vitro modelsThe closer a cultured cell system matches cells in

real human target tissues, the more likely that results obtained using such systems will be predictive of drug efficacy, safety and toxicity In vivo

Ideal Characteristic of in vitro modelThe most predictive cell system would be a

primary culture of fully differentiated cells derived from arelevant human tissue

Tissue Culture

Removal of cells, tissues, or organs from an animal or plant and their subsequent placement into an environment conducive to growth

Environment –Suitable plastic or glass culture vessel with liquid / semi solid medium + nutrients for survival and growth

Types of Tissue Culture

Organ CultureExplant CultureCell CultureOrganotypic Culture

Types of Cell Cultures

Two types of cell culture

–Primary Culture

–Cell Line Culture (finite / continuous / established / secondary / subclone/ immortalized cell culture)

Primary Cultures

Difficult to expand the population in cultureScarcity of human materialHigh costBatch to batch variationHence primary cultures are impractical as a

source of cells for high throughput screening

Human primary keratinocytes

Continuous Cell Line

A cell line that has potential to be subcultured indefinitely

–Infinite/Immortal cells line

–Cells whose growth properties have been altered

Immortalized/Transformed cell lines

During the process of immortalization cells acquire gross alterations in chromosomal

composition and develop aberrant functional characteristicsImmortalized cells often lack the ability to

achieve a fully differentiated phenotypeImmortalized cell lines are not the satisfactory

source of cells for high throughput screening

Advantages of Tissue Culture

Limitations of Tissue Culture

Non animal organism

Ranging from plants to single celled organism to invertebrates

Micro-organism - Use of bacteria and fungi to measure genotoxic effects - More easily & quickly cultivated - Simple genetic makeup - Protozoa have specialized functions e.g.- cilia of protozoans responds to smoke or phenols

InvertebratesCertain aspects of their physiology are similare.g. - Drosophila melanogaster – ‘fruit fly’ for mutagenic, teratogenic and reproductive toxicity

- Sea urchin – for basic reproductive research

Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) Test Method

Advantages:Do not have nerve cellsCompleted prior to the closing of the neural tube

Inexpensive

Does not require animal laboratory facilities

Mutagenicity

In vitro tests - Micro organism test (Ames test) - Mammalian cells

Tests using Insects

Ames test (Bacterial reverse mutation test)Biological assay

Recent Advances –Stem Cells/Organs on Chip

Stem Cells –DefinitionA cell whose job in the body is not yet determined

Every single cell in the body originates from this type of cell

Ability to proliferate for long periods

Under certain experimental conditions, they can be induced to differentiate

The Stem Cell Advantage

Skin Corrosion & Skin Irritation Studies

EpiDermTM Tissue Model A 3-dimensional, human cell–derived skin model

that replicates key traits of normal human skin. It replaces the use of guinea pigs or mice, who

would have been injected with a substance or had it applied to their shaved skin to determine an allergic response

EpiDerm™is also being used to replace rabbits in painful, prolonged experiments that have traditionally been used to evaluate chemicals for their ability to corrode or irritate the skin.

Organs-on-chips

Harvard’s Wyss Institute has created "organs-on-chips” that contain human cells grown in a state-of-the-art system to mimic the structure and function of human organs and organ systems

The chips can be used instead of animals in disease research, drug testing, and toxicity testing and have been shown to replicate human physiology, diseases, and drug responses more accurately than crude animal experiments.

Regulations-India

Conclusion

Animal testing is integral to

safe guard human health

Abolish animal experiments