BIOASSAY

NOOR WIJAYAHADI
 Bioassay = biological assay
a type of scientific experiment
essential in new drugs development
& monitoring pollutants
Measure the effects of a substance on a living
organism.
qualitative or quantitative
Quantitative bioassays are typically analyzed
using the methods of biostatistics
 Definition
Estimationof the conc / potency of a
substance by measuring its biological
response in living systems
i.e.Observation of pharmacological effects on
[1] living tissues, or cells
[2] microorganisms
[3] animals
 Bioassays/Assays
Whole animals
Isolated organs of vertebrates
Lower organisms e.g. fungi, bacteria,
insects, molluscs, lower plants, etc.
Cultured cells such as cancer cells and
tissues of human or animal organs
Isolated sub-cellular systems, such as
enzymes, receptors, etc
The use of bioassays include:
measurement of the pharmacological activity of
new or chemically undefined substances
investigation of the function of endogenous
mediators
determination of the side-effect profile, including
the degree of drug toxicity
measurement of the concentration of known
substances (alternatives to the use of whole
animals have made this use obsolete)
assessing the amount of pollutants being
released by a particular source, such as
wastewater or urban runoff.
 Drug discovery
Targets: New and Established
Established targets are those for which there is a
good scientific understanding
new targets are all those targets that are not
"established targets
Process Screening and Design
 Diseases- Molecular Basis
Over- and under-expression of catalytic proteins
(enzymes)
Toxins produced by microorganisms
Viruses (wild DNA/molecular organisms) cause
cancers, AIDS, influenza etc.
Mutation in DNA
Congenital diseases due to genetic malfunctions
Oxidation of biomolecules (proteins, carbohydrates,
lipids, nucleic acid), degenerative diseases and ageing
Deficiency of essential elements, vitamin, nutrients
etc.
I
 Principles of Bioassay
Active principle to be assayed should show the same
measured response in all animal species
The degree of pharmacological response produced
should be reproducible under identical conditions [Eg
Adrenaline shows same rise in BP in the same
species under identical cond: wt, age, sex, strain etc]
The reference standard must owe its activity to the
principle for which the sample is being bioassayed
Activity assayed should be the activity of interest
Individual variations must be minimised / accounted
for
Bioassay might measure a diff aspect of the same
substance compared to chemical assay [Eg
testosterone & metabolites
 Types of Bioassays?
In Silico Screenings
Non- physiological Assays
Biochemical or Mechanisms-Based Assays
In Vitro Assays
Cell based Bioassays
Tissue based Bioassays
In Vivo Bioassays
Animal-based Assays/Preclinical Studies
Human trial/Clinical Trials
 Broad Categories of Bioassays

VirtualScreenings
Primary Bioassays
Secondary Bioassays
Preclinical Trials
Clinical Trials
 Virtual Screenings

Target Selection
Data Mining (Chemical space of over 1060
conceivable compounds)
Screening of Libraries of Compounds
Virtually
lead optimization
Prediction of Structure-Activity
Relationships
 Primary Bioassay/Assays
Screenings

Non- physiological Assays

Biochemical or Mechanism-Based Assays
In Vitro Assays
Microorganism-based bioassays
Cell-based Bioassays
Tissue-based Bioassays
 Examples of Primary Assays
Antioxidant Assays
Enzyme Inhibition Assays
Cytotoxicty Bioassays
Anti-cancer Bioassays (Cancer Cell Lines)
Brine Shrimp Lethality Bioassays
In Vitro Antiparasitic Bioassays
Anti-bacterial Bioassays
Antifungal Bioassays
Insecticidal Bioassays
Phytotoxicity Bioassays
Etc.
 Salient Features of Primary
Bioassay Screenings
Predictive Potential
General in nature
Tolerant of impurities
Unbiased
High-throughput
Reproducible
Fast
Cost-effective
Compatible with DMSO
 Hit Rate of Primary Bioassay
Screenings

A hit rate of 1% or less is generally

considered a reasonable
False positive are acceptable
False negative are discouraged
 Secondary Bioassays

InVivo Bioassays
Animal-based Assays/Preclinical Studies
Predicting Drug Like Behavior- Lipinski
Rule of Five
Molecular weight about 500 a. m. u. (Optimum 350)

Number of hydrogen bond accepter ~ 10 (Optimum 5)

Number of hydrogen bond donor ~ 5 (Optimum 2)

Number of rotatable bonds ~5 (Conformational

Flexibility)

1-Octanol/water partition coefficient between 2-4 range

 In Vitro Bioassays
In Vitro: In experimental situation outside
the organisms. Biological or chemical work
done in the test tube( in vitro is Latin for in
glass) rather than in living systems
Examples include antifungal, antibacterial,
organ-based assays, cellular assays, etc
 Examples of In Vitro Bioassay
Activity Assays
DPPH assay
Xanthine oxidase inhibition assays
Superoxide scavenging assay
Antiglycation assay

Bioassays (cell-based)
DNA Level
Protein Level
RNA Level
Immunology assay

Toxicity Assays
MTT assay
Cancer cell line assays
 In Vivo Screenings or
Pharmacological Screenings
In Vivo: Test performed in a living system
such as antidiabetic assays, CNS assays,
antihypertensive assays, etc.
 Examples of In Vivo Bioassays

Animal Toxicity
Acutetoxicity
Chronic toxicity

Animals Study
Animal model with induced disease
Animal model with induced injury

Pre-Clinical Trials
Clinical Trials
Animal Models
 Classification of Animal Models

Exploratory
to understand a biological mechanism
Explanatory
to understand a complex biological problem
Predictive
to discover and quantify the impact of a
treatment
 Animal Models to Humans

Fidelity
The resemblance of the biological structure in
the animal with the corresponding structure in
humans
Discriminating ability (predictability)
The similarity between humans and model
species with respect to relevant biological
mechanism is more important than the fidelity of
the model.
 Classification of Disease Models

1. Induced (experimental) disease models

2. Spontaneous (genetic) disease models
3. Transgenic disease models
4. Negative disease models
Mechanism of resistance
5. Orphan disease models
Naturally occurs in a non-human species but has
not yet been identified in humans
 Induced (Experimental) Disease
Models
Healthy animals initially.
Condition to be investigated is experimentally
induced. For example, partial hepatectomy for
liver regeneration, streptozotocin-induced type 1
diabetes
Unfortunately, few induced models completely
mimic the etiology, course, and pathology of the
target disease in the humans.
 Spontaneous (Genetic) Disease
Models
Naturally occurring genetic variants
(mutants) in studying human diseases
For example: nude mice, Snells dwarf
mice (lacking pituitary), BB rats

Concern the compensatory

mechanisms may differ between
humans and the animal model species
 Transgenic Disease Models

The advance in genetic engineering and embryo

manipulation technology facilitates the
development of transgenic models.

Mutations induced by mutagens such as ethyl-

nitroso-urea (ENU) represent another approach
to the generation of new mutants.
 Animal Models in Drug Discovery

Pharmacodynamics
Pharmacokinetics
Toxicology
 Pharmacodynamic Primary Effect
Animal Models for Type 2 Diabetes

Genetic models
db/db mice, ob/ob mice, KK mice, fa/fa
Zucker rats
Oral Glucose Tolerance Test
Extrapolation from Animals to Humans
Extrapolation :
how data obtained from animal studies reliably
applies to the human
Pharmacodynamics
Adverse effects
Model body size and scaling
 What are the alternatives?

Reduction - fewer animals

Refinement - less painful
procedures
Replacement - alternative
techniques
 3Rs
Reduction
reduction in the number of animals used for a
particular purpose
Refinement
refinement of procedures to minimise suffering
Replacement
replacement of animal use wherever possible
 The 4th R

Responsibility
 Reduction alternatives
Good planning of studies
Rational and efficient use of animals
no wasting
pilot studies
screening tests
Proper statistical design
Use of inbred starins (for some study
types)
 Refinement alternatives
Minimized potential for pain or distress
Enhanced animal well-being
Improved housing conditions and
experimental techniques
Replacement alternatives (1)
Efficient use of existing information
In silico methods (computer simulations,
mathematical models, QSAR)
Read-across, grouping of chemicals
In vitro methods: isolated organs
tissue slices
tissue cultures
cell cultures
subcellular fractions
Lower organisms
Early stages of development
 Replacement alternatives (2)

In vitro methods are not primarily replacements of

in vivo methods. Typically these methods have
different roles in research, and they are
complementary for each others.

Depending on the objective of the study in vitro

methods may be the most appropriate methods for
certain areas of interest, because they can more
accurately provide the information required (e.g.
cellular and molecular events).
 Replacement alternatives (3)

Absolute replacement: no need to use animals (cell

lines, human or invertebrate cells and tissues)
no need to test for skin irritation if pH <2.0 or
>11.5
no need to test for eye irritation if the chemical is
a skin irritant
Relative replacement: humane killing of animals to
provide cells or tissues for in vitro studies
Partial replacement: e.g. use of non-animal methods
as prescreens in toxicity testing
 Replacement alternatives (4)

Direct replacement: e.g. human or guinea pig skin is

used in vitro to replace guinea pig tests in vivo
Indirect replacement: e.g. Limulus amoebocyte
lysate (LAL) test or a test based on whole human
blood is used to replace rabbit pyrogen tests
 Limulus amoebocyte lysate (LAL) test

Horseshoe grab (Limulus polyphemus)

 MTT cytotoxicity test in cell culture
Principle:
Formazan crystals are
formed by mitochondrial
succinate
dehydrogenase in living
and early apoptotic
cells, but not in dead
cells.
MTT
(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide)

http://www.ib.amwaw.edu.pl/home/dslado/video/mtt.html
Current use of replacement alternatives

Safety testing of chemicals

Local toxicity
Genotoxicity
Screening tests

Quality control and potency testing of

vaccines
Production of monoclonal antibodies
Skin corrosion and skin irritation tests

In vitro In vivo
Skin corrosion: artificial Corrosivity test on rabbit
human skin cultures skin
 Eye irritation tests

In vitro In vivo
Eye irritation: Draize test in rabbits eye
HET-CAM (hens egg chorio-
allantoic membrane) test

HET-CAM = hens egg chorio-allantoic membrane

BCOP (bovine corneal opacity)

test
ICE (isolated chicken eye) test
 Production of monoclonal antibodies
In vitro In vivo
Cell culture Mouse ascites method
 Pyrogen tests
In vitro In vivo
Limulus test Rabbit pyrogen test

Horseshoe grab (Limulus Limulus Amebocyte Lysate

polyphemus) Test Kit

Human whole blood test

Analysis of biologically active compounds

In vitro In vivo
Pregnancy test (immune assay) Frog pregnancy bioassay

Hormones, vitamins (HPLC, Bioassays in chicken, rats

immune assays) and mice
Insulin determination Convulsion test (mouse),
blood glucose determination
(mouse, rabbit)
Advantages of in vitro tests

Controlled testing conditions

Lack of systemic effects
Reduction of variability between experiments
Testing is fast (and cheap)
Small amount of test material is required
Limited amount of toxic waste is produced
Human cells and tissues can be used
Transgenic cells carrying human genes can be
used
Reduction of testing in animals
 Limitations of in vitro tests

General toxicity profile of a chemical cannot be

assessed
In vivo dose-responses cannot be obtained (for
human risk assessment)
Systemic effects cannot be evaluated
Interactions between tissues and organs cannot
be tested
Pharmacokinetics cannot be evaluated
Specific organ sensitivity cannot be assessed
Chronic effects cannot be tested
No relevant replacement alternatives

Pharmacokinetics / toxicokinetics
Systemic toxicity
Organ systems toxicity (CNS, respiratory,
cardiovascular, gastrointestinal etc.)
Immunotoxicity
Male and female reproduction toxicity (fertility
tests, developmental toxicity tests, peri- and
postnatal toxicity tests)
Subchronic and chronic toxicity
Carcinogenicity tests
Stepwise in vitro and in vivo testing

Skin / eye irritation and corrosivity testing (OECD 404,

405)
1. Existing data on human and laboratory animals
2. Toxicity based on physico-chemical properties +
QSAR
3. Toxicity in vitro
positive result classification +
negative or equivocal result in vivo testing
4. Toxicity in vivo
first one or few animals
positive classification +
negative more animals classification -