Advanced Pharmacology-I [PHR5001]

[2] Use of Laboratory Animals in Preclinical Studies

Dr. Md. Hasanuzzaman Shohag

Dept. of Pharmaceutical Sciences

North South University

 List of Topics (to be covered from the chapter):
01 What is experimental pharmacology?

02 Animal experimentation: importance and goals

03 Classification of animal research

04 General principles of animal experimentation; 3Rs

05 Applications, advantages, disadvantages, and limitations of different

animal species in preclinical research
06 Regulatory and ethical requirements for use of laboratory animals.

07 Animal rights, Animal Welfare Act

08 Institutional Animal Care & Use Committee (IACUC)

09 Care & housing of laboratory animals.

 Experimental Pharmacology

▪ Experimental pharmacology involves the study of

pharmacology through bioassay, to test the efficacy, safety and
potency of a drug.

▪ Experimental pharmacology deals with the effect of various

pharmacological agents studied on different human and
nonhuman animal species. The aims of experimental
pharmacology are as follows:
➢ Find out the therapeutic agent suitable for human use as drug
➢ Find out the therapeutic window and index of new drugs
➢ Study the toxicity of the drugs
➢ Study the mechanism and site of action of drugs
 Experimental Pharmacology

▪ Experimental pharmacology explains the way the living

organisms react to chemical agents and pave the way for the
researchers so that the data through animal experimentation
can be used in the clinical settings.

▪ Experimental pharmacology is focused on the discovery of

new drugs or study the actions of existing drugs through
preclinical or clinical studies in two ways:
1) Study on whole animal – in vivo

2) Study on isolated organ, tissue or cell – in vitro

 Animal Experimentation

▪ Definition: Animal testing, (also known as animal

experimentation, animal research or in vivo testing), is the use
of non-human animals in experiments that seek to control the
variables that affect the behavior or biological system under
study.

▪ Experimental research with animals is usually conducted in

medical schools, pharmaceutical companies, defense
establishments and commercial facilities that provide animal
testing services to stake holders (contact research
organization, CRO).
The importance of animal experimentation in
biomedical research
Virtually every major medical advancement for both humans and animals has been
achieved through biomedical research using animal models to study and find a cure,
prevention for a disease and through animal testing to prove the safety and efficacy of a
new treatment.

▪ Most of the nation’s million insulin-dependent diabetics would be dead.

▪ Million would risk death from heart attacks, strokes or kidney failure – because there would be no
medicine to combat high blood pressure.

▪ Chemotherapy wouldn’t exist, and couldn’t save children who now survive acute lymphocytic
leukemia.

▪ Million would be blind in at least one eye – there would be no surgery to correct cataracts.

▪ There would be no kidney dialysis.

▪ Smallpox, which has been eradicated, would continue unchecked.

▪ Polio would kill or cripple thousands of unvaccinated adults and children each year.

▪ Surgery of any type would be rare and extremely painful because there would be no anesthesia.
 Goals of Animal Experimentation
▪ The goals of animal testing varies from-
✓ pure research, (focusing on developing fundamental knowledge
of an organism) to
✓ applied research, (which may focus on answering some
question of great practical importance, such as finding a cure for
a disease).
▪ Examples of applied research include testing disease treatments,
breeding, toxicological testing, and cosmetics testing.
▪ In education, animal testing is sometimes a component of biology or
psychology courses.
▪ Psychological research is done to determine behaviors of animals
towards certain conditions or stimuli, and seeks to answer questions
regarding human behavior.
 Classifications of animal research

➢ Animal research can be divided into several classifications

based on study type. Experimental designs and procedure
are precisely specified and implemented within each study
and the use of any animal must be justified.

➢ Animal research are of following types:

1) Basic research
2) Applied research
3) Toxicology testing
4) Xeno transplantation
[1] BASIC RESEARCH:
➢The goal is to investigate how organisms develop, function &
behave.
➢These studies include embryogenesis and developmental biology,
behavior, evolution and genetics.
➢Mice and rats account for the vast majority.

[2] APPLIED RESEARCH:

➢The primary purpose is to solve a specific or practical problem.
➢These studies may involve naturally occurring diseases, induced
animal models of human diseases or genetically modified animals.
➢ Applied research often represents the early stage in the drug
discovery process.
[3] TOXICOLOGY TESTING:

➢ This is generally performed by pharmaceutical companies or contract

research organizations to test drugs and other chemicals.

➢ Today all the new drugs must undergo rigorous animal testing prior to
licensing for human use.

➢ Cosmetic testing fall within this category, and it has become particularly
controversial in recent years.

➢ Toxicology testing's are of two types:

✓ Acute toxicity testing

✓ Chronic toxicity testing

[4] Xenotransplantation:

➢Xenotransplantation (xeno means foreign), is the transplantation

of living cells, tissues or organs from one species to another.

➢It is currently viewed as a mechanism to overcome the shortage

of human organs for organ transplantation.

➢Xenotransplantation of human tumor cells into

immunocompromised mice is a research technique frequently
used in pre-clinical oncology research.

➢Recently clinical trials evaluated the transplantation of pig insulin

secreting cells into diabetic patients.
General principles of animal experimentation
 General Principles: 3Rs

➢ Current regulations stress the need to search for and development of

procedures which cause the least intense and momentary pain or
distress.

➢ The concept of the 3 "R"s has been used when thinking about
alternatives to animal use.

➢ Most countries adhere to the three R’s: Replacement, Reduction and

Refinement as guiding principles for the use of animals in research.

➢ The 3R’s are important from a scientific, ethical and legal perspective.

➢ In an humane society, there is a clear moral obligation to minimize the

potential pain, suffering or distress of any animal to an absolute
minimum.
 Replacement

➢Non-living systems: Most preferred method of replacement refers to

the use of non-animal methods over animal methods whenever it is
possible to achieve the same scientific aim. Computer modeling,
organ-on-chip are the most promising methods.

➢Cell or tissue culture: These are in vitro method that utilize organ,
tissues or cells of different species including human.

➢Micro-organisms and invertebrates

➢Lower level animals on the phylogenic tree: One aspect of

replacement is the use of "Lower" or non-vertebrate animals in some
situations rather than a higher order animal. For example, using rabbit
instead of a dog or a mouse instead of rabbit.

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 Reduction

➢Reduction refers to gathering equivalent amount of information by

using fewer animals or increased information from the same number
of animals.

➢This is done mostly through careful experimental design, rational

selection of group size and the use of statistics.

➢Investigators must ensure that their research design is robust and

they have significant animal numbers to provide statistically relevant
data, while keeping animal numbers to a minimum.

➢In agreement with this principle, unnecessary duplication and

repetition of experiments involving animals are not allowed.
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 Refinement

➢Refinement refers to the measures taken to enhance animals’

comfort and minimize distress, suffering and potential pain.

➢The goals of refinement are to improve the quality of life for the
animals and in doing so improve the quality of science. Examples
of refinements includes-
➢During the execution of invasive techniques the use of pain
relieving measures such as anesthetics and analgesics
whenever possible.
➢After the operation use of antibiotics, antiiflammatory drugs or
sedatives to reduce distress and to enhance healing.
➢Environmental enrichment in their living place.
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Applications, advantages, disadvantages, and limitations of
different animal species in preclinical research
Regulatory and ethical requirements for use of
laboratory animals.
 Animal rights
➢ Animal rights is the idea in which some, or all, non-human
animals are entitled to the possession of their own lives and
that their most basic interests, such as, the need to avoid
suffering.

➢ Animal rights espouse the notion “Animal should no longer

be viewed as property or used as food, clothing, research
subjects, entertainment, or beasts of burden”.

➢ Multiple religious and cultural traditions around the world—

such as Islam, Hinduism, Buddhism, Animism, Taoism, and
Jainism also espouse some forms of animal rights.
 The human perception of animal rights

To perceive animals rights human being requires to accept that:

▪ non-human animals are conscious beings not machines or

objects

▪ non-human animals have interests of their own

▪ human beings should respect the interests of non-human

animals

▪ human beings should not exploit non-human animals

▪ human beings should not kill non-human animals

 Animal welfare science

➢ Animal welfare science is the scientific study of the welfare of

animals as pets, in zoos, laboratories, on farms and in the
wild.

➢ Although animal welfare has been of great concern for many

thousands of years in religion and culture, the investigation of
animal welfare using rigorous scientific methods is a relatively
recent development.

➢ The world's first Professor of Animal Welfare Science, Donald

Broom, was appointed by Cambridge University (UK) in 1986.
 Animal Welfare Act (AWA)
▪ The Animal Welfare Act (AWA) sets general standards for humane
care and treatment that must be provided for certain animals that
are- bred for commercial sale, exhibited to the public, used in
biomedical research or transported commercially.

▪ Enacted by Animal care division of USDA

▪ Reviewed by Animal and Plant Health Inspection Service (APHIS)

▪ Passed by US National Congress in 1966

▪ APHIS has published the Animal Welfare Act, known as the “Blue
Book,” as a tool to improve compliance among different
stakeholders concerning the transportation, sale, and handling of
certain animals.
 Animal Welfare Organizations

An animal welfare group is concerned with the health, safety and

psychological wellness of animals. It may be an animal rescue
group which helps individual animals in distress or may be many
animals of a kind suffering from some epidemic.
 IACUC Organogram

US Department of Agriculture (USDA)

Animal and Plant Health Inspection Service (APHIS)

Animal Care Division

Animal Welfare Animal Welfare

Act (AWA) 1966 Regulations

IACUC Blue Book

 IACUC Organogram

US Department of Health and Human Services

National Institute of Health (NIH)

Office of Laboratory Animal Welfare (OLAW)

IACUC
 Institutional Animal Care and Use Committees (IACUC)

▪ Institutional Animal Care and Use Committees (IACUCs) are

centrally important in applying laws about animal research in the
United States.

▪ In USA most research involving laboratory animals are funded by

the US National Institutes of Health (NIH) or other federal
agencies.

▪ The NIH Office of Laboratory Animal Welfare (OLAW) has been

directed by law to develop policies that describe the role of IACUC.

▪ IACUC reviews research protocols and conducts evaluations of the

institution's animal care and reports to the OLAW of NIH.
 Functions of IACUC: Protocol review

Each animal use protocol must be reviewed by full IACUC

committee each three years or more often. The protocol must
cover at least these points:

a. Identification of the species and approximate number of

animals to be used.

b. Rationale for involving animals, and for the

appropriateness of the species and numbers used.

c. A complete description of the proposed use of the

animals.
 Functions of IACUC: Protocol review
d. A description of procedures designed to assure that
discomfort and injury to animals will be limited to that which
is unavoidable in the conduction of scientifically valuable
research.

e. Analgesic, anesthetic and tranquilizing drugs will be used

where indicated and appropriate to minimize discomfort and
pain to animals.

f. A description of any euthanasia method to be used.

 Animal Welfare Assurance Guidelines
In protocol review, the IACUC is responsible to ensure that the proposed
work complies with Animal Welfare Assurance, which encompass the
following points:

1. Procedures with animals will avoid or minimize discomfort, distress

and pain to the animals, consistent with sound research design.

2. Procedures that may cause relatively lower level of pain or distress

to the animals will be performed with appropriate sedation,
analgesia, or anesthesia.

3. Animals that would experience severe or chronic pain or distress

that cannot be relieved will be painlessly killed at the end of the
procedure or, during the procedure.
 Animal Welfare Assurance Guidelines
4. The living conditions of animals will be appropriate for their species
and contribute to their health and comfort.

5. The housing, feeding, and nonmedical care of the animals will be

done by properly educated, trained and experienced person.

6. Medical care for animals will be available and provided as

necessary by a qualified veterinarian.

7. Personnel conducting procedures on animals must be appropriately

qualified and trained.

8. Methods of euthanasia will be consistent with the recommendations

of American Veterinary Medical Association (AVMA) Panel on
Euthanasia.
 Care and Housing of Laboratory Animals

➢ Suitable Micro-environment and Macro-Environment

❑ Microenvironment: The immediate physical environment surrounding

the animal (i.e., the environment in the primary enclosure such as the
cage, pen, or stall).

❑ Macroenvironment: The physical environment of the secondary

enclosure (e.g., a room, a barn, or an outdoor habitat).

Temperature, humidity, and concentrations of gases and particulate matter are

often higher in the animal microenvironment than in the macroenvironment,
while light levels are usually lower.

Microenvironmental conditions can directly affect physiologic processes and

behavior and may alter disease susceptibility.
 Care and Housing of Laboratory Animals

Comfortable room temperature:

Temperature

Animal °C °F

Mouse, rat, hamster, gerbil, guinea 20–26 68–79

pig

Rabbit 16–22 61–72

Cat, dog, nonhuman primate 18–29 64–84

Farm animals, poultry 16–27 61–81

 Care and Housing of Laboratory Animals

➢ Optimum Temperature and Humidity

➢ Factors that contribute to variation in temperature and humidity are:

▪ Housing design

▪ Construction material

▪ Enrichment devices such as shelters and nesting material

▪ Use of filter tops

▪ Number, age, type, and size of the animals in each enclosure

▪ Forced ventilation of enclosures

▪ Type and frequency of contact bedding changes

Mouse room: macroenvironment
Mouse box: microenvironment