Principles of Pharmacological and

Pre-clinical Evaluation of Drugs

Department of Pharmaceutical
Sciences, Nagpur

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Introductio
n: Pharmacology Science of
drugs

Categories of Pharmacology
Pharmacognosy study of characteristics of natural drugs
and their sources
Pharmacodynamics study of what drugs do to the body
Pharmacokinetics what the body does to drugs
Pharmacotherapeutics study of how drugs are used to
treat disease
Toxicology study of poisonous effects of drug

Pharmacodynamics

Study of what a drug does to the body

Includes interaction between the drug and target cells or
tissues and the bodys response to that interaction
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Pharmacokinetics

The study of what the body does to a drug includes:

Absorbs converts a drug into a form the body can use
Metabolizes drug molecules are transformed into simpler
products
Distributes transporting a drug from its site of administratio
to its site of action
Excretes manner
in which a drug is eliminated from the bod
Principles
of Pharmacology
Medication safety
Medication legislation
Naming
Classifications
Schedules
Pharmacokinetics
Storage and security
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Autonomic pharmacology

Principles of Pharmacology (contd)

Metabolism and excretion
Mechanism of action
Phases of medication activity
Medication response relationships
Medication interactions
Toxicity

Pharmacotherapeutic
s- Safety is how many and what kinds of adverse effects are
associated
with the drug.
Safety and
An adverse reaction may require immediate action.
efficacy

Be alert to complaints from patient after starting new drug.

Efficacy:- Drug working as excepted

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Toxicology
Study of poisonous effect or toxicity of drugs
Toxic effects includes:
Adverse effect on a fetus or infant
Adverse reactions reported in clinical trials
Adverse effect in pediatric or elderly patients

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Pre-clinical Evaluation
of Drugs
Introduction

Before a pharmaceutical company can initiate the testing in

humans, it must conduct extensive preclinical or laboratory
research
This research typically involve years of experiment in
animals
and human cells
Definition
Research using animals to find if a drug, procedure or a
treatment is likely to be useful

Preclinical study take place before any testing in humans is

done
Objective
of preclinical study
Support for human pharmacology
Support for human toxicology
Prediction of human pharmacokinetics
Screening of new dosage form and formulation
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Preclinical Study Design

Assess pharmacology fate in relevant animal model of
disease/injury, as feasible
Assess safety/toxicology in healthy animals
pharmacology-toxicology study design
Apply the 3 Rs Reduce, Refine, Replace

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Commonly used Laboratory animal in

pharmacological research

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Selection of Animal Species

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Standard testing used in laboratory animal

Animal test are conducted all over the world
Majority of animals are used for testing are rats and mice.
Other animals used are hamster, rabbit, guinea pig, dogs, cats,
nonhuman primates, sheep and cattle
Millions of the animals listed above are locked inside cold
cages in laboratory, waiting in fear to be experimented with next
painful procedure
Every year more than 100 million animals suffer
and die from animal testing
Before a product is claimed as human friendly
hundreds of animals are poisoned, blinded and kill
every year

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Types of animal testing

1. In vivo studies
Human-based
Skin irritation
Pyrogenicity
Modular immune in vitro study
2. In vitro studies
Cell culture and tissue engineering
Molecular targets

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Preclinical Development in vivo StudiesAnimal Models:

Proof of therapeutics principle

Animal pharmacokinetic and pharmacodynamics
Toxicology
Starting dose and schedule for clinical trial

Preclinical Development in vitro Studies

Pharmacology:

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Molecular mechanisms of action

Mechanisms of drug resistance
Determinant of response
Intracellular pharmacodynamics
Molecular pharmacology
Eg: In vitro studies of anticancer agents
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Euthanasia
Euthanasia refers to the practice of intentionally
ending a life in order to relieve pain and suffering.

Classification of euthanasia
Voluntary euthanasia
Euthanasia conducted with the consent of the
patient is termed voluntary euthanasia
Non-voluntary euthanasia
Euthanasia conducted where the consent of the
patient is unavailable is termed non-voluntary
euthanasia.
Eg: Child euthanasia

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Involuntary euthanasia
Euthanasia conducted against the will of the
patient is termed involuntary euthanasia.

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Passive and active euthanasia

Voluntary, non-voluntary and involuntary euthanasia can all
be further divided into passive or active variants.
Passive euthanasia implies the withholding of common
treatments, such as antibiotics, necessary for the continuance of
life.
Active euthanasia implies the use of lethal substances, such as
administering a lethal injection, to kill.
The choice of a method will depend on the nature of
study, the species of
animal to be killed.
The method should in all cases meet the following
requirements:
1. Death, without causing anxiety, pain or distress with
minimum time lag
phase.
2.
Minimum physiological and psychological disturbances.
3.
Compatibility with the purpose of study and minimum
emotional effect on the operator.
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4.
Location should be separate from animal rooms and free

Euthanasia Of Laboratory Animals

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Precautions:

-Method should be reliable, reproducible and irreversible.

-Not in the housing environment.
-It should not produce distress vocalizations, pheromones in
anxiety.
-Confirmation by observing the cessation of the heart best,
respiration and absence of reflexes.
-Can be guaranteed by exsanguinations or decapitations.
-Physical method: Concussion- Blow on the head of the
animals.
RECENT DEVELOPMENTS
-Microwave irradiation: Use of microwave beam
-Neuropharmacological studies
-Useful for fixation of brain metabolites
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-Without destroying the anatomical areas of brain.

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Regulations For The Laboratory Animal Care And

Ethical Requirements
CPCSEA Guidelines For Laboratory Animal
Facility (committee for the purpose of control and supervision of experiments on animals, Ministry of
Environment and Forests, Government of India)
Good Laboratory Practices (GLP) for animal facilities is proposed
to assure quality maintenance and safety of animals used in
laboratory studies while performing behavioral research and
testing of products
Goal
The goal of these Guidelines is to promote the humane
care of animals used in behavioral research
And testing with the basic objective of providing
specifications that will
enhance animal well being, quality in the purpose of
advancement of biological knowledge that is relevant to
humans and animals.
GLP addresses:
Regulatory requirements
Occupational Health and Safety
Housing and Routine Care
Animal Identification
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Sex Determination

Regulatory
Requirements
To ensure the humane
treatment of laboratory
animals, animal research is regulated by two federal
agencies:
The United States Department of Agriculture (USDA) /
Animal Care enforcing the Animal Welfare Act Regulations
The National Institutes of Health

Housing and Routine

Care:
Animal Crowding

Animal crowding in a cage affects environmental quality (the

accumulation of urine, for example, leads to excess ammonia
and moisture).
Crowding can also cause newborn pups to be injured or killed.
Crowding is a special concern for multiple litters in a cage since
pups grow very quickly and rapidly increase their output of
excreta.
In particular, if the mother is about to give birth to a second
litter,
the first litter should be weaned and removed to new
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cages to prevent smothering and trauma of the newborns.

Routine Sanitation

Routine sanitation and environmental controls are

necessary for protecting animal health and for minimizing
the introduction for non-experimental variables which could
undermine the quality of research data.
Sanitation schedules vary according to the type of mouse
caging.
Based on the types of caging and bedding in use at your
facility, your institution will have a standard operating
PHYSICAL
FACILITIES
procedure (SOP)
on the sanitation
schedule for mouse
cages.
BUILDING MATERIALS should be selected to facilitate
efficient and hygienic operation of animal facilities.
CORRIDOR(S) should be wide enough to facilitate the
movement of personnel as well as equipments and should
be kept clean
UTILITIES such as water lines, drain pipes, and electrical
connections should preferably be accessible through
service panels or shafts in corridors outside the animal
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EXTERIOR WINDOWS they may be necessary to provide

alternate source of light and ventilation
FLOORS should be smooth, moisture proof,
nonabsorbent, skid-proof, resistant to wear, acid, solvents,
adverse effects of detergents and disinfectants
DRAINS To prevent high humidity, drainage must be
adequate to allow rapid removal of water and drying of
surfaces
WALLS & CEILINGS Walls should be free of cracks,
unsealed utility penetrations, or imperfect junctions with
doors, ceilings, floors and corners
STORAGE AREAS Separate storage areas should be
designed for feed, bedding, cages and materials not in use
FACILITIES FOR SANITIZING EQUIPMENT AND
SUPPLIES
An area for sanitizing cages and ancillary equipment is
essential with adequate water supply
EXPERIMENTAL AREA
All experimental procedures should be carried out in a
separate area away from the place where animals are
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housed

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Environmental Controls
The Guide for the Care and Use of Laboratory Animals
establishes standards for laboratory animal environments
in regard to:

Room temperature 18 to 29C (64.4 to 84.2F)

Humidity 30% to 70%
Ventilation
Illumination and light schedule
Noise abatement

The details of these standards are generally not a

concern to researchers who house their animals in an
institutional animal facility, because it is the animal care
staff who have the responsibility of operating the facility
in compliance with these environmental standards
Researchers should be aware of the lighting schedules
used in the rodent housing rooms (commonly 12 hr
light:12 hr dark or 14 hr light:10 hr dark)
If researchers working late turn on the lights in the
animal rooms during the dark period, the disruption of
the light schedule may cause the animals to be
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perturbed, which may have effects on breeding

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Animal Handling and Restraint

It's important to remember the following:
As a small animal, mice can be easily injured if handled
roughly. You should learn how to handle them firmly but
gently and with confidence to avoid injuring these delicate
animals
Mice are inclined to become aggressive and bite. Although
their teeth seldom break through your skin, a bite can hurt!
Develop your confidence in handling mice by learning from an
experienced mentor and practice hand restraint first on
anesthetized mice

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Hand
Restraint
There are two common hand methods for restraining
mice. Mice are picked up the following way:
If your mice are in barrier or containment housing, please
open their cages in an appropriate hood or laminar-flow
workbench
Remove the cage top if they are housed in a filter-top cage
Place the wire lid top sideways on top of the cage
Pick up a mouse by the tail (away from the tail tip) and lift
the mouse directly to the wire lid. You will find that the wire
lid is a useful area to which the mouse will want to hang on
with its front feet, allowing you the opportunity to reposition
your grasp
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Tail Wrap Method

In the tail wrap method, you transfer the tail from your thumb
and forefinger to the 4th and 5th fingers of the same hand. These
fingers press into your palm the mouse's tail, close to the animal's
rump
Then, your thumb and forefinger grasp a scruff of skin at the
occiput of the mouse
In this method, the mouse is held by two points: the occiput and
the base of the tail
To further immobilize the hindquarters, you may gently entrap
one leg along with the tail beneath your 5th finger. It is important
to provide back support to the mouse and avoid hyperextension of
its back
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Points to remember:
When picking up a mouse
Pick up the tail at the middle, not the tip. A mouse does
not need to be picked up at the base of the tail like a rat does,
because the mouse is light and its weight will not damage its
tail
Never dangle a mouse by its tail, but instead lift it directly
to the cage wirelid or some other support. When dangled,
mice appear anxious as seen by the their behavior in hyper
extending their feet
If you need a place to briefly sort and hold your mice, say
while you are rapidly administering injections to a cage of
mice, each mouse can be placed on the wirelid after its
injection
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Sex and Age

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Reproductive Data
Of Mice
Stage
Sexual maturity

Age

40-60 days

Estrous cycle

Proestrus
(12 h)
Estrus (9-15
h)
Metestrus
Gestation
(20 h)
Diestrus (6070 h)

Weaning age

Reproductive life

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4 5 days
21 days
21-30 days
8 months

Life span

1 - 3 years

Pups begin eating

dry food

12 14 days
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Types of Observations
A common approach to assessing animal appearance and
behavior is through observation of the following:
Activity Level
e.g., hypoactivity (hunched, huddled, lethargic),
hyperactivity, restlessness, lack of inquisitiveness
Attitude
e.g., arousal, depression, awareness of surroundings
Behavior, Spontaneous
e.g., vocalization, self-trauma, isolation from cage mates.
These observations are made without disturbing the animal
Behavior, Provoked
e.g., vocalization, hiding, aggressiveness, minimal
response. These observations are made when the animal is
disturbed or even prodded
Body Condition
e.g., emaciation, missing anatomy
Food and Fluid Intake
e.g., elimination of feces and urine
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Types of Observations
Fur and Skin
e.g., unkempt or greasy or dull fur; porphyrin staining
around eyes and nostrils; cyanotic, pale, or congested
mucous membranes or skin (ears, feet, tail); skin lesions;
soiled anogenital area
Eyes
e.g., clarity/condition of lens, cornea; position of globe
(e.g., sunken in orbit or protruding); condition of eyelids,
encrustation
Posture
e.g., hunched back, tucked abdomen; prostrate; head
tucked down

Locomotion

e.g., gait, ataxia, lameness, action of each limb, position of tail

when ambulating

Neurological

e.g., tremor, convulsion, circling, paralysis, head tilt, coma

Vital Signs

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respiratory distress (open mouth breathing, pronounced

chest movement)

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Physical Examination
After assessing the animals appearance and behavior, conduct a
physical exam using methods that are appropriate to the species and
experimental model

You may evaluate:

Behavior
Body weight
Surface lesions (wounds, masses)
Hydration status
Body temperature
Blood parameters (Blood collection can be difficult/stressful in
mice; may be used to confirm disease or failed treatment.)

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Acclimation

Upon arrival to your facility, your mice should have an

acclimation period before they are used in research studies
This period of time allows animals to adapt to a new
environment
Effects of transportation stress include alterations in various
blood parameters, immune cell function, food intake, and animal
behavior.
The period of time necessary for biological stabilization will
depend on the parameters to be studied
Typically, acclimation periods can range from days to over a
week, depending on the studies involved

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Ethical treatment of laboratory

animals
Justification of Research

Research should be undertaken with

a clear scientific purpose.
There should be a reasonable
explanation that the research will:
Increase knowledge of the processes
being studied
Provide results that benefit the health
and welfare of humans and other
animals
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Justification of Research
The scientific purpose of the research
should be of sufficient potential
significance to justify the use of animals.
The species chosen for study should be
best suited to answer the question(s)
posed.
Research on animals may not be
conducted until the protocol has been
reviewed by an appropriate animal care
committee, to ensure that the procedures
are appropriate and humane.
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Personnel
Animal use procedures must conform
with federal regulations regarding
personnel, supervision, record
keeping, and veterinary care.
Ensure that all individuals who use
animals under their supervision
receive instruction in experimental
methods and in the care,
maintenance, and handling of the
species being studied.
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Care and housing of animals

Animals are to be provided with
humane care and healthful
conditions during their stay in the
facility.
Researchers are encouraged to
consider enriching the environments
of their laboratory animals and
should keep current with literature on
well-being and enrichment for the
species with which they work.
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Acquisition of animals
Animals not bred in the research facility
are to be acquired lawfully.
Researchers should make every effort to
ensure that those responsible for
transporting the animals to the facility
provide adequate food, water, ventilation,
space, and impose no unnecessary stress
on the animals.
Endangered species should be used only
with full attention to required permits and
ethical concerns.
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Experimental procedures
Procedures in which the animal is
anesthetized and insensitive to pain
throughout the procedure and is
euthanized before regaining
consciousness are generally
acceptable.
Behavioral studies that involve no
aversive stimulation to, or overt sign
of distress from, the animal are
acceptable.
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Experimental procedures
Procedures involving more than
momentary or slight aversive stimulation,
which is not relieved by medication or
other acceptable methods, should be
undertaken only when the objectives of
the research cannot be achieved by other
methods.
Procedures involving the use of paralytic
agents without reduction in pain sensation
require particular prudence and humane
concern.
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Experimental procedures
Experimental procedures that require
prolonged aversive conditions or produce
tissue damage or metabolic disturbances
require greater justification and
surveillance.
An animal observed to be in a state of
severe distress or chronic pain that cannot
be alleviated and is not essential to the
purposes of the research should be
euthanized immediately.
In surgical procedures animals should be
maintained under anesthesia until the
procedure is ended.
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Experimental procedures
Animals reared in the laboratory
should not be released because, in
most cases, they cannot survive or
they may survive by disrupting the
natural ecology.
Euthanasia shall be accomplished in
a humane manner, disposal of
euthanized animals should be
accomplished in a manner that is in
accord with all relevant legislation.
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LABORATORY ANIMAL ETHICS

All scientists working with laboratory animals must have a deep ethical
consideration for the animals they are dealing with. From the ethical point of
view it is important that such considerations are taken care at the individual
level, at institutional level

TRANSGENIC ANIMALS
Transgenic animals are those animals, into whose germ line foreign gene
have been engineered, whereas knockout animals are those whose specific
gene have been disrupted leading to loss of function
These animals can be bred to establish transgenic animal strains.
Transgenic animals are used to study the biological functions of specific
genes, to develop animal models for diseases of humans or animals, to
produce
therapeutic products, vaccines and for biological screening, etc.
MAINTENANCE
Housing, feeding, ventilation, lighting, sanitation and routine management
practices for such animals are similar to those for the other animals of the
species as given in guidelines

DISPOSAL
The transgenic and knockout animals should be first enthanized and then
disposed off as prescribed in the guidelines

BREEDING AND GENETICS

For initiating a colony, the breeding stock must be procured from CPCSEA
registered
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status of animal is known

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Limitations of animal
paradigms
No site specific claim
Particular mechanism may not be
targeted
Non-selective, partial agonistantagonist creates the problem
Surgical intervention develops other
complications
Predicting therapeutic efficacy is far
from perfect
Tissue culture: Inability to replicate
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the in-vivo environment (cell-cell-

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Limitations of animal
paradigms
Phenotype: no counterpart in animals for mania,
delusion or migraine attack
Common cause: some common or complex
diseases like Alzheimers, osteoarthritis and
Parkinsons diseases.
Similarity of pathophysiology: depression and
anxiety; psychosis and reward
Treatment response: drug treatment as test of
validity for models and miss the other potential
mechanisms.
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Species differences

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