PHBS 2B LABORATORY

Experiment No. 2

Bioassay measure the effect of a substance on a biological animal or

subject.

Rationale:

1. Searching for a specific type of pharmacological activity.

2. Using a battery of assay technique to discover new type of activity.
3. Using a single bioassay to predict multiple activities.
4. Using combination of variety of bioassay in order to detect specific
as well as multiple activities.

Types of Bioassay Screening Methods

A. Primary Screening assay that can be easily applied to a number

of samples to determine one activity.

Requirements:

1. Predict a certain type of the therapeutic directly or in analogy with a

standard drug.
2. Should be tolerant to the many impurities in the substance in low
concentration.
3. Should be unbiased and allow coding of known and unknown
samples.
4. Results produced should be reproducible.
5. Assay should allow the use of both crude as well as pure compounds.
6. Should not require more than 1-2 of the crude drug.
7. Should have a high throughput even if information content is low
with results easily available.
8. Should not require expensive equipment.
9. Should not require a highly trained personnel.
 10. If test animals are used, readily available, easily handled, easily
bred and resistant to infect.
11. Should be economical over an extended period of time.

A. Secondary Screening a more detailed testing of lead compounds on a

number of models systems in order to elucidates mechanism of actions and
compounds for clinical trials.

More costly
Require highly trained personnel
Low capacity
Slow
May be a contribution to Science and Technology

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

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

Extensive literature search

What is known?
- sample of interest and its relatives
- assay methods
- disease of interest

Based on existing knowledge, what needs to be done?

- adapt
- innovate

Establish Collaboration
Consider common interest and mutual benefits
- Sharing expertise
- sharing facilities
- sharing data
- sharing output

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
b. mode of administration
c. pharmacological models
d. control groups
e. test animal/strain/sex male more ideal to use than female
f. dose
g. toxicity of the test drug (LD50)
h. vehicle/solvent
i. statistical tool
Pharmacological Animal Models
1. Normal Animal Models
2. Disease Model

Normal Models
1. General pharmacology assays
-CNS/behavior activity assays
-Analgesia assay
-Metabolic assay
2. Animal toxicity assay

Gadgets: Behavior or Motor Coordination

a. activity cages
b. grip strength meter for mice/rat
c. Rota-rod Treadmill for mouse

For Metabolic Studies

Metabolic cages
- measures the amount of food and water intake
- measures urinary excretion and other waste

Test for Analgesia

1. Hot plate
2. Tail flick

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)

Disease Model

1. Include a disease state closest to the human disease

2. Specific pharmacological action of the test drug
3. may not clarify mechanism of action
4. experiments are slow and technically complex
Some Animal Disease Models

Inflammation Rat paw edema/Mouse ear edema

Acute Chronic

Devices for edema measurement

1. caliper
2. plethysmometer
3. improvised lever

Experimental Models in Testing Toxicity

1. LD50
2. alternatives/refinements to LD50
3. brine shrimp lethality
4. cytotoxicity assays
5. genotoxicity assays

Purpose of Acute Toxicity

1. To obtain information on the biological activity of a chemical and gain

weight into its mechanism of action.
2. The information on acute systemic toxicity generated by the test is
used in hazard ID and risk management in the context of product,
handling and use of chemicals

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
Acute Toxicity: Classical LD50

4-5 dose levels

8-15 pathogen-free both sexes of mice or rats/dose level
single administration per orem
number of lethality within 2-48 hours
manifestations of toxicity monitored within 2 weeks
surviving animals are sacrificed after 14 days for histological
examination
LD50 determine by graphical method or probit analysis by Fisher and
Yates
Oral LD50 and Parenteral LD50 date provide information on the
bioavailability of the tested compound.

Toxicity Classification

LD50 Loomis Toxicity Category

1 mg/Kg Extremely toxic
1-50 mg/Kg BW Highly
50-500 mg/Kg BW Moderately
0.5 g/Kg BW Slightly
5-15 g/Kg BW Practically
> 15 g/Kg BW Relatively harmless

Toxicity Classification: Organization for Economic Co-Operation and

Development (OECD)

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
Variations in LD50 Values have been attributed to differences in:
- protocol details
- animal strains
- test-chemical resources

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
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

TI = LD50
ED50

Refinement of acute Toxicity Assay

1. use of fewer animals
2. emphasize humane treatment of animals
3. 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