PRECLINICAL STUDIES

Presenter – Dr.Manisha Anand

Moderator – Dr. Inderpal Kaur
\
•PK/ PD studies
Lead • in vitro studies
identification/ • TOXICOLOGY
validation/ • Physico-chemical
optimization studies
• Formulation
stability
Definition

■ Testing the newly discovered compound in animals with the

objective of gaining information regarding the various aspects
of the compound with respect to the biological systems so
that the same can be extrapolated for the use of that
compound in humans
Several steps in preclinical studies

5 File for IND

Establish effective
4 and toxic doses
Screen the drug in
3 assay

Develop a
2 Bioassay
Identify a
1 lead
compounds
Benefits of conducting preclinical study

■ It is conducted to know the efficacy of a new drug or medical device

■ Knowledge of the initial safe dose for administration in humans and
how the dose can be escalated for treating the disease.
■ Knowledge of the pharmacological action of the drug.
■ What is the toxic dose of the drug, target organs for toxicity, and
whether this toxicity is reversible or not?
■ What is the best route for the administration of the drug to obtain its
maximum benefits
Screening of drugs
 Screening is the process of filtering among the large number of
compounds, the one with required biological activity
 A thorough investigation to
 Get pharmacological activity of new/ chemically undefined
substances
 Investigate the functions of endogenous mediators
 Measure/ define the toxicity and unwanted action
Types of screening
• A single test or two similar tests to find
Simple screening
substances that are active in a single way. Ex:
(is the substance active?) hypoglycemic test

• entirely new compound

Blind screening • Subjected to a series of simple tests to reveal the
(is there any biological nature of any pharmacological activity it possess
activity?) • More detailed investigations

• This employs sound physiological, biochemical,

Programmed screening pathological knowledge and identification of
(in what ways compound drug action. The compound is aimed at
act?) mitigating the derangement caused by the
disease.
 Bioassays
■ Bioassays are the procedures by which the potency or the nature of the
substance is estimated by studying its effects on living matter
■ To measure the pharmacological activity of new or chemically
unidentified substances.
■ Can be done :
 Isolated tissues or organs
 Cell cultures: The pure cells of specific lines are grown on a suitable
culture media which is used to measure the specific type of activity
 Intact animals: The whole animal is used to measure the potency of the
compound
 Bioassay of insulin using mouse or of digitalis in guinea pigs
 Selection of Animals for Preclinical Studies

■ The selection of the type and the number of animals used for the
preclinical studies is one of the most important steps.
■ The choice of the species is based on the fact that which one will give
the best correlation to the human trials
■ Experiments are generally performed on a rodent ( mouse, rat, guinea
pig, hamster, rabbit ) and then on a larger animal ( cat, dog , monkey )
 Tests on animal models of human disease
Ch
em • Involves use of some chemical that selectively destroys some cells or tissues. Ex: MPTP
ical model for Parkinsonism
mo
Ph
del
sys
ic • use of various physical factors to induce the symptoms Ex: Use of Electric shock to induce
al convulsions in rats for studying the anticonvulsant activity of a compound
m
o
Sur
de
gic
ls • Involves the surgical removal of certain tissue or organ of the body.
al
mo
del
G
sen
• Genetic manipulation done by:
et • Addition of genes - Transgenic Technology
ic • Modification of the existing genes - Gene Knockout Technology
m
o
de
 Isolated tissues or organs
ANIMAL TISSUE ACTIVITY STUDIED

Guinea pig Ileum, Heart, Trachea Spasmodic, Antispasmodic Histaminic &

Local Anaesthetic activity

Albino Brain Tissue CNS Active drugs & Analgesics, Toxicity

Mouse studies
Rabbit Heart, Aorta, Duodenum and Sympathomimetic & Sympatholytic
Ileum activity

Frog Heart, Rectus abdominis muscle, Muscarinic activity & Nerve block local
Muscle nerve preparation anesthesia

Dogs Prepared fistula & Pouches of Gastric secretory function

dogs
Selection of animals for preclinical studies
■ Based on which species is best correlated to humans
BIOLOGICAL ACTIVITY ANIMALS USED
BLOOD PRESSURE DOG and CAT
CARDIAC EFFECTS DOG
PERIPHERAL AND ANS DOG

RESPIRATORY EFFECTS DOG AND GUINEA PIG

DIURETIC ACTIVITY DOG

GASTROINTESTINAL EFFECTS RAT

CENTRAL NERVOUS SYSTEM MOUSE OR RAT

 Quantitative tests
■ Dose makes a compound a drug or a poison
■ Two types
 Graded Effect: A proportionate increase in the observed response
with a subsequent increase in the concentration
 The contraction of the smooth muscle preparation for assaying
histaminic activity of a compound
 Quantal Effect: These are based on “All or None” phenomenon
 Determine the percentage responding
 Whether a compound causes cardiac arrest or not.
 ED50
■ ED50 (mg/kg) is defined as the dose effective for producing a certain
sign in 50% of the animals of a group
■ Different for each route of administration
■ When the quantal response is death, the ED50 becomes the LD50
■ LD50 is the dose of a test substance that produces 50% death in a
given species of animals
Determination of Pharmacokinetic Parameters
■ Pharmacokinetics is the quantitative study of the drug movement in,
through and out of the body with respect to time
Absorption Parameters Distribution Parameters Excretion Parameters
Absorption of compound via • Tissue permeability of • Routes of excretion
various routes compound • Clearance
• Volume of distribution • Dose Adjustments
Mechanism of absorption • Protein and tissue binding of • Factors influencing excretion
Various factors affecting compound
absorption • Factors influencing distribution

Metabolic parameters
Pathway of metabolism
Interactions
First pass metabolism
Pharmacokinetic interactions
Factors influencing metabolism
Pharmacodynamic interactions
Enzyme induction/enzyme inhibition
TOXICITY STUDIES
An Insight into Good Laboratory
Practices(GLP) for Preclinical Studies
■ In order to safeguard the quality, integrity, and safety of preclinical
studies, FDA promulgated the Good Laboratory Practice Regulations
on 22nd December 1978.
■ OECD (Organization for Economic Co-operation and Development)
produced GLP principles of international standard in 1981.
■ In india, National GLP-compliance Monitoring Authority (NGCMA)
was established in 2002 by Department of Science and Technology
 What are the objectives of GLP

■ It encompasses a set of principles for defining a framework within which

preclinical studies are planned, performed, monitored, recorded,
reported, and stored for future reference.
■ It assures that the preclinical or lab study has utilized standard
equipments and methods to conduct and report an experiment
■ It makes sure that the data submitted to the regulatory authorities is in
conjunction to what was found in the actual study
■ It safeguards against any fraudulent activities by a study lab
■ It promotes international acceptance of test results
 Other Regulatory Bodies
Regulatory body Recommendation

Institutional Animal Ethical Ensures that all the experiments conducted on animals are rational, do
Committee not cause undue pain or suffering to animals and only minimum number
of animals are used.

International Conference Ensures more economical use of human, animal and material sources ,
on Harmonization (ICH) and the elimination of unnecessary delay in the global development and
availability of new medicines whilst maintaining safeguards on quality,
safety and efficacy and regulatory obligations to protect public health.

Organization for Economic Recommended GLP guidelines that act as standard reference tool for
Cooperation and chemical testing.
Development (OECD)
Toxicity studies
■ Study of injurious effects of the compound on the animals along
with the mechanisms of toxicity
■ FDA states that it is essential to screen new molecules for
pharmacological activity and toxicity potential in animals
■ Toxicity tests are mostly used to examine specific adverse events or
specific end points such as cancer, cardiotoxicity, and skin/eye
irritation.
■ Toxicity testing also helps calculate the No Observed Adverse Effect
Level (NOAEL) dose and is helpful for clinical studies.
Objectives
■ Identify any toxic substance prior to clinical use
■ Quantitative and qualitative assessment of drug use
■ Characterize the cumulative toxicity of the drug under study
( specially in subchronic or chronic study but not predicted by single
dose toxicity)
■ To allow a careful selection of doses for further studies (including
carcinogenicity studies )
■ Different types of dose identification can be done. For example
maximum lethal dose (MLD), Lethal dose (LD50), maximum
tolerated dose (MTD), minimum effective dose ( ED50)
■ Therapeutic index can be calculated ( LD50/ED50)
 Toxicity studies
1. Systemic toxicity studies Chronic
toxicity
Acute Repeat dose
toxicity studies
studies
2. Safety pharmacology studies
3. Special toxicity studies
■ Mutagenicity/ Genotoxicity
■ Carcinogenicity
■ Reproductive toxicity Studies
■ Neurotoxicity
■ embyotoxicity
 Acute toxicity testing
■ It evaluates the adverse effects that occur following exposure of
organisms to a single or multiple doses of a test substance within 24
hours by a known route (oral, dermal or inhalation)
■ It is carried out with 2 different animal species (one rodent and one
nonrodent).
■ The effect is observed for 14 days.
■ All mortalities caused by the product during the experimental period are
recorded
■ Morphological, biochemical, pathological, and histological changes in the
dead animals are investigated.
■ Calculate the minimum lethal dose (MLD ) , maximum tolerated dose
(MTD) and lethality dose ( LD 50) and no observed adverse effect dose
 LD 50
■ The LD50 (median lethal dose) test was introduced in 1927 to estimate the
dose of a test substance that produces 50% death in a given species of
animals.
■ It is usually the first test conducted for every chemical before further
toxicity tests are carried out.

LD50 ClassificationClassification
<5 mg/kg Extremely toxic
5–50 mg/kg Highly toxic
50–500 mg/kg Moderately toxic
500–5,000 mg/kg Slightly toxic
5000-15,000 mg/kg Practically non-toxic
>15,000 mg/kg Relatively harmless
 LD 50
■ It was used as an indicator of acute toxicity previously. The determination
of the LD50 involves large numbers of animals, and the mortality ratio is
high. Because of these limitations, modified methods were developed
■ Fixed dose procedure (FDP)
■ Acute toxic class (ATC) method
■ Up and down procedure (UDP)
 Fixed dose procedure (FDP)
■ It was introduced in 1992.
■ The test substance is given at one of the four fixed-doses (5, 50, 500,
and 2,000 mg/kg) to 5 male and 5 female animals of the same species.
■ The objective of the FDP is to identify a dose that produces clear signs
of toxicity but no mortality.
■ Depending on the results of the first test, either no further testing is
required or a higher or lower dose is tested.
■ If mortality occurs, retesting at a lower dose level is necessary except
if the original dose chosen is 5 mg/kg
 The acute toxic category (ATC) method
■ The ATC method is a sequential procedure in which 3 animals of the
same sex are used in each step
■ it is based on a sequential dosing in which one dose group 5, 50, 300 or
2,000 mg/kg body weight is used at a time
■ Depending on the mortality rate, three but not exceeding six animals is
used per dose level.
■ The result of this approach is reproducible and the number of animals
used is reduced by 40–70% compared to the traditional methods
■ In this method, death is not used as the only end point, but signs of
toxicity in its stepwise approach are also used for estimating the LD50
The up-and-down (UDP) method.
■ The UDP testing approach is also known as the staircase design.
■ This approach is most recommended method because this reduces the
number of vertebrate animals in research.
■ It involves dosing single animals sequentially at 48 h intervals.
■ Female rodents are preferable for UDP testing.
■ A dose less than the best-estimate LD50 dose is selected and
administered to an animal, and the animal is observed for 48 h.
■ If it survives, the study is continued with a higher dose (twice the
original dose); if the animal dies, testing is conducted with a lower dose
with another animal of the same sex as the original animal.
Acute toxicity testing for inhalation

■ It is performed for aerosol-like preparations

■ Rats are the most preferred animal species
■ The animal is exposed to the test substance for a minimum of 4 h,
and then it is monitored for 14 days
■ Food and water is withheld during the exposure period
■ During the observation period, the animal is observed for tremors,
convulsions, salivation, diarrhea, lethargy, sleep, and coma
 Mortality during the exposure and observation period is noted.
 Dead animals are examined for histological and pathological changes.
 At the end of the study, the animals are sacrificed, and pathological
changes are evaluated.
 Acute toxicity testing for topical preparations
■ The eye irritation test and skin irritation test are very important for
topical preparations. It can be tested using Draize tests.
■ The Draize test are used to measure the harmfulness of chemicals and
pharmaceutical substances in rabbits and guinea pigs
In the eye irritation test,
0.5 ml of a test substance is In the skin irritation test, 0.5 g of a test
administered and the animal is substance is applied to the surface of an
restrained for 4 h. Redness, animal's skin.
swelling, discharge, ulceration, During the observation period (14 days),
hemorrhage, and blindness are signs such as erythema and edema are
assessed and monitored for 14 assessed..
days.
■ At the end of the study, the animals are sacrificed and pathological
changes are evaluated.
 Repeat dose Subchronic Chronic toxicity
toxicity(28 days) (90days) (>90 days)

• The test substance is administered daily for a certain period through the
orally/ parenterally.
• Usually, a rodent and nonrodents are of any gender and age is used
• The allowable variation between the animals is in the weight is ±20%.
• Baseline parameters such as the behavioural and biochemical parameters
of the animals should be recorded.
• These will be helpful in calculating percentage changes.
• The interpretation of human safety details is essential
• Morphological, biochemical, pathological, and histological
changes in the dead animals are investigated.
• Calculate the, maximum tolerated dose (MTD) and NOAEL
 Safety pharmacology
■ Investigate the potential and undesirable pharmacodynamic effects of
drug on physiological functions in relation to exposure
■ Irrespective of the primary action of the drug, its effects on major organ
system are worked out..
CVS CNS Respiratory system
BP,HR,ECG (QT prolongation Motor activity,Behavioral changes RR,Tidal volume
Ventricular contractility Coordination,Body temperature Airway resistance, Compliance
Vascular resistance Sensory & motor reflex Pulmonary arterial pressure
Blood gases,Blood pH

GIT
Urinary system Gastric secretion
Urine volume, Specific gravity Gastric pH ,Gastric mucosal examination
Osmolality, Ph, Proteins Bile secretion, Gastric emptying time Ileocaecal
contraction
 Mutagenicity testing
■ It is used to assess submicroscopic changes in the base sequence of DNA,
chromosomal aberrations, and structural aberrations in DNA including duplications,
insertions, inversions, and translocations.
■ Certain types of mutations result in carcinogenesis
■ so the determination of the mutagenicity is essential in the drug development
process.
■ In vitro testing is carried out in 2 or 3 different bacteria and mammalian cells to
cover the end points of gene mutations, clastogenicity, and aneuploidy.
■ The test generally includes a bacterial reverse mutation assay. The choice of an
additional test depends on the chemical structure/class of the substance.
■ In vivo mutagenicity which is dose dependent is used to determine the case-by-
case basis risk assessment of the test substances.
■ Mutagenicity studies with transgenic animals are more appropriate assay
techniques to determine the toxicity of a test substance.
 Carcinogenicity testing
■ Both rodents and nonrodent animal species may be used
■ The tests are carried out over the greater portion of an animal's lifespan.
■ During and after exposure to test substances, animals are observed for
signs of toxicity and development of tumors.
■ If these are not found, a test may be terminated after 18 months in the
case of mice and hamsters and after 24 months with rats.
■ If the animals are healthy, hematological analysis is performed after the
12 months and the 18 months and the study is terminated.
■ The animals are sacrificed, and gross pathological changes are noted and
histopathological studies are carried out on all the tissues
Reproduction toxicity testing- One generation
■ The test compound is administered to both male and female animals rodents
■ Administration is for the duration of 1 complete spermatogenic cycle in male
animals and for 2 complete estrous cycles for female animals.
■ After the drug administration, the animals are allowed to mate.
■ The test compound is administered to the female animals during the period of
pregnancy and nursing.
■ The sperms of male animals are collected, and the sperm morphology and
motility are analyzed.
■ During the study period, the animals are observed for signs of toxicity.
■ Parturition, the number of offspring and their sexes are recorded.
■ The number of dead and live pups are noted
■ After the termination of the study, the animals and pups are sacrificed and
Reproduction toxicity studies- Two-generation

■ Both male and female rodents are administered the test substance.
■ The duration of administration will remain same
■ After the administration period, the animals are allowed to mate
■ Sperms are collected and the sperm morphology and motility are analyzed.
■ The test substance is administered to pregnant female animals and monitored
regularly for mortality and signs of toxicity.
■ After parturition, nursing animals are administered the test drug, and the mortality
of the pups (F1 generation) is observed.
■ From the F1 generation, one male and one female animal are selected. The same
procedure is repeated to get the F2 generation offspring.
■ Pairs without a pregnancy are evaluated for infertility.
■ Necropsies and histological examinations are carried out. At the end of the study, the
animals are sacrificed and gross histopathological examinations are carried out on all
the animals
 Neurotoxicity studies in rodents
■ The effects of a test substance on the CNS can be studied through
neurotoxicity studies.
■ Neurotoxic studies may be employed to evaluate the specific
histopathological and behavioral neurotoxicity of a chemical and are
used to characterize neurotoxic responses such as neuropathological
lesions and neurological dysfunctions (loss of memory, sensory defects,
and learning and memory dysfunctions).
■ These are carried out in adult rodents.
■ The test substance may be administered for 28 days or even more than
90 days, and neurological changes are evaluated.
 Embryotoxicity studies
■ Embyrotoxicity can be studied using both in vivo and in vitro methods.
■ Rodents are preferred for in vivo toxicity screening.
■ The compound is administered between the 8thand 14thday of
pregnancy, and embryolethal effects are studied.
■ On the 21st day of the study, a C-section is performed and parameters
such as fetuses with hemorrhagic bullae, limb malformations,
exencephaly, cleft palates, open eyelids, and tail deformities as well as
the mortality and the numbers of dead and live pups are noted.
■ Embryotoxicity studies can be performed using in vitro methods such as
the embryonic stem cell test (EST) for embryotoxicity, micromass
embryotoxicity assay, and whole rat embryo embryotoxicity assay.
 Immunotoxicity
 Immunotoxicity (adverse effects on the immune system) studies are
performed on the same animals.
 Immunotoxicological analysis is not feasible beyond the period of 14
days.
 Parameters such as
 Delayed-type hypersensitivity (DTH)
 Mitogen or antigen-stimulated lymphocyte proliferative responses,
macrophage function
 Primary antibody response to T-cell dependent antigen are assessed in
immunotoxicological studies.
 Genetic toxicity testing
■ Genetic toxicity tests are used to identify gene mutations, chromosome
changes, and alterations in the DNA sequencing.
■ These tests are usually conducted in various species including whole
animals, plants, micro-organisms, and mammalian cells.
■ In the whole animal model, rodents are preferred.
■ Genetic toxicity is assessed using the rodent chromosome assay,
dominant lethal assay, mouse-specific locus test, micronucleus test,
heritable translocation assay, and sister chromatid exchange assay
 Limitations of animal toxicity studies

■ Expensive

■ Time consuming

■ Large number of animals needed

■ Rare adverse effects unlikely to be detected

■ Humans extrapolation not completely reliable

■ ETHICAL ISSUES
Alternatives to Animal Testing in Preclinical Research

■ Currently, a strategy of 4Rs (reduction, refinement, replacement and

responsibility) is being applied for using animals in research labs.
■ Their approach motivates to use minimum number of animals in an
experiment, plan and refine experiments to minimize pain and distress
to the animals, and finally replace higher animals with alternative
methods and lower organisms.
 Responsibility
Reduction Refinement Replacement It has grown into a
• In vitro cell cultures • It is done by • Various alternatives new era of
have provided a safe decreased like in vitro cell performance-based
alternative to reduce invasiveness, cultures, computer outcomes, which
the use of animals in improved models, and new reflects integrity,
lab testing. By using instrumentation, imaging techniques honesty, and scientific
in vitro cell cultures improved control of are now being used correctness in
like human pain and improved in place of appropriate and
hepatocyte culture, control of experimental reasonable use of
embryonic stem cell techniques used for animals. Besides laboratory animals.
culture etc., you can animal research reducing the use of This ensures that
easily get relevant animals, these animal life is required
information about alternative methods and necessary for
the metabolism and are time-effective, biomedical
excretion of drugs cost-effective, and advancement
from the body. require less
manpower
Rehabilitation

“Rehabilitation” of laboratory animals is defined as “the aftercare

rendered to animals that have been
■ Bred for the purpose of experimentation
■ Subject to any form of experimentation
■ Retained in laboratory animal houses or breeding houses for the
purpose of experimentation, both for education and research,
■ with the sole intention of alleviating the pain/distress or suffering
due to the physical, physiological and psychological trauma that the
animals have been exposed to and to provide the animal a life
distinctly different from laboratory housing and care , until the point
of natural death”.
In silico approach – the computer’s role in
drug development
■ Progress in bioinformatics over the past decades has made in silico
studies attractive so that they often precede or complement in vitro
and in vivo studies.
■ In silico models are based on computer simulations and provide
information on how an investigational compound might behave in
subsequent in vitro and in vivo experiments.
■ Apart from technological requirements, these computer simulations
demand expert knowledge in biochemistry and molecular biology.
Calculation of Human Equivalent Dose (HED)

HED = Animal NOEL x (Wanimal/Whuman)1-b

where “W” is the weight in Kg

“b” is a correction factor (equal to 0.67) used to convert mg/kg to mg/m2

Calculation of Maximum Recommended Starting Dose (MRSD)

MRSD = HED/ 10
 Conclusion

■ Preclinical Studies acts as the bridge between the discovery of a newly

discovered compound and its testing is human
■ It play an inevitable role in deciding and designing clinical studies which
only gets approval based on preclinical assessments on pharmacology
and toxicology profile of the drug.
■ The data generated helps in picking up a right compound, a right
formulation and a right drug delivery system that reduces the risk of
injurious effects on humans
■ Adequate design of preclinical studies and careful choice of model
systems are vital to ensure relevant results that translate into
applicability in clinical settings.
 References
■ N.Venkatesan, M. Ramanathan Preclinical Toxicity Studies-Tool of Drug
Discovery. PVPE 2017;1:1-7
■ Parasuraman S. Toxicological screening. Journal of Pharmacology &
Pharmacotherapeutics. 2011 Jun;2(2):74.
■ Badyal DK, Desai C. Animal use in pharmacology education and
research: The changing scenario. Indian Journal of Pharmacology. 2014
May 1;46(3):257.
■ Advances in acute toxicity testing: strengths, weaknesses and
regulatory acceptance [Internet]. [cited 2020 Sep 15]. Available from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117820/
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