Experiment 1B

BIOASSAY
PHA 6126 Laboratory
Faculty of Pharmacy, University of Santo Tomas

Kay Ann J. Tongol, RPh., MSc.

Compiled by: Ms. Kay Ann J. Tongol, RPh, MSc.

Course Facilitator
BIOLOGICAL ASSAY (BIOASSAY)
• methods that estimates the nature, constitution, or potency of a
material by means of reaction of the living matter.

• as estimation or determination of concentration or potency of

physical, chemical or biological agents by means of measuring and
comparing the magnitude of the response of the test with that of
standard over a suitable biological system under standard set of
conditions.

• CAN BE QUALITATIVE AND QUANTITATIVE

(Panuganti, 2015)
SYNONYMS

Biological Bio
assay metrics

Biological Bio-
standardization standardization
In vitro In vivo

Ex vivo
PRINCIPLES OF BIOASSAY
• To compare the test substance with the International
Standard preparation.

• To find out how much test substance is required to

produce the same biological effect, as produced by the
standard.

• Activity assayed should be the activity of interest.

PRINCIPLES OF BIOASSAY
• Standard & test sample - similar pharmacological effects &
mode of action.

• Both should be compared for their established

pharmacological effect using specified technique.

• Ex: *Ach – contractile response on frog rectus

PRINCIPLES OF BIOASSAY
• All procedures must be approved by IACUC
• Problem of biological variation must be minimized.
✔ Experimental conditions
✔ Animals
✔ Number of animals
✔ Isolated preparations
INDICATIONS OF BIOASSAY
1. No chemical method has been developed.
2. Chemical assay is too complex /not sensitive enough to
measure
3. To measure the pharmacological activity of new or
chemically undefined substances.
4. For biological standardization of drugs obtained from
natural sources as these cannot be obtained in pure form.
INDICATIONS OF BIOASSAY
5. To compare the strength of a drug obtained from various
sources due to different compositions

❖Chemicals with similar structure, but different biological activity

❖Chemical structure of the active principle is unknown

❖Chemical structure known; cannot be actively purified. Eg: Peptide hormones

CHARACTERISTICS OF A GOOD ASSAY
• Sensitivity
• Specificity
• Repeatability
• Reproducibility
• Precision
• Accuracy
• Stability – tissue has to stay “bioassay-fit
BROAD CATEGORIES OF BIOASSAYS
• Virtual Screenings
• Primary Bioassays
• Secondary Bioassays
• Preclinical Trials
• Clinical Trials
PRIMARY BIOASSAY SCREENING
• applied to a large number of samples - determine if any
bioactivity of the desired type is present.
• high capacity
• low cost
• provide the results quickly.
• not necessarily quantitative

PHBS 2 LABORATORY
PRIMARY BIOASSAY SCREENING
• Non- physiological Assays
• Biochemical or Mechanism-Based Assays
• Microorganism-based bioassays
• Cell-based Bioassays
• Tissue-based Bioassays
• Many other In Vitro bioassays/assays
PRIMARY BIOASSAY SCREENING
Examples:
■In Vitro Antiparasitic Bioassays
■Antioxidant Assays
■Anti-bacterial Bioassays
■Enzyme Inhibition Assays
■Antifungal Bioassays
■Cytotoxicty Bioassays
■Insecticidal Bioassays
■Anti-cancer Bioassays
■Phytotoxicity Bioassays
■Brine Shrimp Lethality Bioassays
SECONDARY SCREENING
• more detailed testing of lead compounds on a number of
model systems
• To select compounds for clinical trials.
• low capacity,
• slow
• costly assays
SECONDARY BIOASSAY SCREENING
• Animal-based assays (In Vivo)
• Toxicological Assessments in whole animals
• ADME Studies
• Behavioral Studies
• Preclinical Studies
CONSIDERATIONS IN CHOOSING TYPE OF
ASSAY SCREENING
1. Relevance of the type of therapeutic potential for which
biological activity is being tested.
- end-users
- prevalence of the disease
- economic and commercial potential
- current status of the drug in use
- contribution to Science and Technology
CONSIDERATIONS IN CHOOSING TYPE OF
ASSAY SCREENING
2. Research capacity
• laboratory facilities
• expertise of personnel
• personal professional training
3. Resource
• Funding/Institutional support
• Availability of research material
• samples
• reagents
• cells/organism/experimental animals
STRATEGY FOR BIOLOGICAL TESTING
1. Extensive literature search
What is known?

2. Based on existing knowledge, what needs to be done?

• Establish Collaboration
Consider common interest and mutual benefits
STAGES IN ADAPTING A MODEL ASSAY SYSTEM
1. Gather papers that used the model.
2. Authenticate the system using standards drugs and
vehicle controls.
3. Repeat procedures to measure reproducibility of the
system.
4. Statistical Analysis.
5. Compare data with those obtained in other laboratories.
 TYPES OF TEST SYSTEM
Whole Animal – intact animal
a) Proper choice f) Dose
b) Mode of administration g) Toxicity of the test drug (LD50)
c) Pharmacological models h) Vehicle/solvent
d) Control groups i) Statistical tool
e) Test animal/strain/sex
– male more ideal to use than female
BIOLOGICAL ASSAY (BIOASSAY)
BASED ON TARGET LIFE FORMS
1. Whole animal
2. Isolated organs of vertebrates
3. Lower organisms e.g. fungi,
bacteria, insects, molluscs, lower
plants, etc.
4. Cultured cells (such as cancer
cells) and tissues of human or
animal origin
5. Isolated subcellular systems, such
as enzymes, receptors, etc.
TYPES OF TEST SYSTEM
• Pharmacological Animal Models
1. Normal Animal Models (Normal Control)
2. Disease Model (Negative and Positive Controls)
 TYPES OF TEST SYSTEM
• Pharmacological Animal Models
1. Normal Animal Models
• General pharmacology assays
• Animal toxicity assay

Equipment: Behavior or Motor Coordination

a. activity cages
b. grip strength meter for mice/rat
c. Rota-rod Treadmill for mouse
TYPES OF TEST SYSTEM
• Pharmacological Animal Models
1. Normal Animal Models

• Equipments: For Metabolic Studies

• Metabolic cages
TYPES OF TEST SYSTEM
•Pharmacological Animal Models
1. Normal Animal Models
Test for Analgesia
1. Hot plate method
2. Tail flick method

Improvised experiment devices/alternative experiments

1. screen crib
2. maze
3. improvised metabolic cages ( glass wool)
4. abdominal stretch assay ( use acetic acid as irritant)
5. toe pinch assay ( “ vocalization”)
TYPES OF TEST SYSTEM
• Pharmacological Animal Models
2. Disease Model
a) Induce a disease state closest to the human disease.
b) Specific pharmacological action of the test drug.
c) May not clarify mechanism of action
d) Experiments are slow and technically complex
TYPES OF TEST SYSTEM
• Pharmacological Animal Models
2. Disease Model
• Inflammation – Rat paw edema/Mouse ear edema
• Acute or Chronic

Devices for edema measurement

1. caliper
2. plethysmometer
3. improvised lever
OTHER WAYS OF CLASSIFYING BIOASSAY
1. Direct Assay
• Dose of test to elicit ED50 or LD50 is measured
• Direct comparison on isolated tissue:- Interpolation
Method, Matching, Four Point Assay
2. Indirect assay
• Potency of test is estimated by comparing the Log
Dose-Response curve with a similar curve of the
standard
OTHER WAYS OF CLASSIFYING BIOASSAY
3. Quantal Assay
• Quantal response - the response is in the form of "all
or none“

4. Graded Assay
• Graded response - response is proportional to the
dose and response may lie between no response and
the maximum response.
 Experiment 1B
BIOASSAY – Toxicity Tests
PHA 6126 Laboratory
Faculty of Pharmacy, University of Santo Tomas

Kay Ann J. Tongol, RPh., MSc.

Course Facilitator
EXPERIMENTAL MODELS IN TESTING
TOXICITY
1. LD50
2. Alternatives/refinements to LD50
3. Brine shrimp lethality
4. Cytotoxicity assays
5. Genotoxicity assays
THE LD50 VALUE
• statistically defined dose that, when administration in an
Acute Toxicity Test , is expected to cause death in 50 % of
the treated animals in a given period.

• currently the basis for toxicological classification of chemical

TOXICITY CLASSIFICATION
LD50 Loomis Toxicity Category
≤ 1 mg/Kg Extremely toxic
1-50 mg/Kg BW Highly toxic
50-500 mg/Kg BW Moderately toxic
0.5 g/Kg BW Slightly toxic
5-15 g/Kg BW Practically toxic
> 15 g/Kg BW Relatively harmless
TOXICITY CLASSIFICATION: ORGANIZATION FOR ECONOMIC CO-
OPERATION AND DEVELOPMENT (OECD)

Classification Dose
Very Toxic < or =5mg/Kg BW
Toxic > 5 or < = 50mg/Kg BW
Harmful >50 or < = 500mg/Kg BW
No Label >500 or > = 2000mg/Kg BW
OBSERVE SIGNS FOR TOXIC EFFECTS
1. decrease in motor activity
2. micturition
3. diarrhea
4. tail lashing
5. tail erection
6. abdominal writhing or gripping
7. protrusion of the eyeball
OBSERVE SIGNS FOR TOXIC EFFECTS
8. drooping of the eyelid
9. fine/coarse body tremors
10.paralysis of legs and head
11.respiratory rate
12.salivation
13.color of the blood vessel of the ear
14.weight loss
15.decrease in appetite
MARGIN OF SAFETY
• the range between the minimal therapeutic dose and the
minimal toxic dose

LD50
Therapeutic Index =
ED50
REFINEMENT OF ACUTE TOXICITY ASSAY
• use of fewer animals.

• emphasize humane treatment of animals.

• use of endpoints other than lethality.

ALTERNATIVE METHODS FOR TOXICITY
1. Limit Test
2. Fixed-Dose procedure
3. Toxic Class Method
4. Up & Down Method
5. Methods using Human Endpoint
DETERMINATION OF LD50 AND ED50
DEFINITION OF TERMS
Lethal Dose
- A dose at which a given percentage of the subjects die.
Usually expressed as median lethal dose.

Median Lethal Dose (LD 50)

- The dose required to kill half member of the tested
population after a specific test duration.
DEFINITION OF TERMS
Effective Dose
- A dose at which a pharmacologic agent will exhibit
therapeutic effect

Median Effective Dose (ED50)

- The amount or dose required to produce a response in 50%
of the population
TOXICOLOGICAL TESTING
• Brine Shrimp
• OECD Fixed Dose
• OECD Acute Toxic Class
• OECD Up and Down
• Acute oral testing/ Classical LD50
BRINE SHRIMP
• A general assay to determine the LD50 of a test solution.

• Usually employed in the lethality of pesticides for

environmental toxicological testing of invertebrates.

• Provides estimation of toxicant that may potentially

contaminate aquatic ecological system.
OECD FIXED DOSE
• Aims to identify the appropriate hazard class for new chemicals
• It does not provide a point of estimate of the LD 50
• Animals are tested sequentially at one of the 4 doses (5, 50, 300 or
2000mg/kg BW)
• Once clear sign of toxicity appear, additional animals are dosed at
the same level for a total of 5 animals
• Subsequent groups of animals may receive a dose higher or lower,
depending on the outcome of the previous group
OECD ACUTE TOXIC CLASS
• Provides a range for lethality rather than a point of estimate
• Group of 3 animals receive one of the 4 or 5 doses
5,50,300 or 2000mg/kg
• Depending on the survival or mortality of the first group
of animals, 3 or more animals may receive the same or
higher or lower dose
• The number of animals that survive or dies determines
the classification decision
OECD UP AND DOWN
• Employs a sequential dosing, using a single animal at each
step
• The dosage depends on whether the previously dosed
animal lives or dies
• Provides a point of estimate of lethality and confidence
interval and can be used to evaluate lethality up to
5000mg/kg
CLASSICAL MEDIAN LETHAL DOSE
• Logarithmic expansion from a starting dose
• Starting dose is usually 10mg/kg BW
• Each dose level necessitates the use of a group of animals; if
many dose level are to be tested, then the classical LD 50
presents the following disadvantages
LINEAR REGRESSION METHOD
Dose Log Dose # Mice % Probit Value
(mg/kg BW) Responded (Y1) (Y2)
215 2.3324 0/9 0 -
454.36 2.6574 2/9 22.22 4.2345
960.16 2.9824 3/9 33.33 4.5684
2,029.55 3.3074 4/9 44.44 4.8592
4,289.43 3.6324 5/9 55.55 5.1383
9,065.67 3.9574 9/9 100 8.7190

Log Dose interval 0.325

LINEAR REGRESSION METHOD
Y = xb + a
• Equation 1 �Equation 2
4.8592 = 3.3074b + a 5.1383= 3.6324b + a
4.8592 = 3.3074b + a
-1(5.1383 = 3.6324b + a)
-0.2791 = -0.325b
-0.325 -0.325
b = 0.8588
 LINEAR REGRESSION METHOD
Y = xb + a
• Equation 1 • Equation 2
4.8592 = 3.3074(0.8588) + a 5.1383= 3.6324(0.8588) + a
4.8592 – 3.3074(0.8588) = a 5.1383 – 3.6324(0.8588) = a
4.8592 – (2.8404) = a
5.1383 – 3.1195 = a
2.0188 = a
2.0188 = a
LINEAR REGRESSION METHOD

Y = xb + a

• Equation
5 = x(0.8588) + 2.0188
5 – 2.0188 = x (0.8588)
2.9812 = x(0.8588)
0.8588 0.8588
X = 3.4714
= 2960.74mg/kg
GRAPHICAL METHOD