1. Biological Standardization of Drugs Lecture2 by Dr. Magdy M. Awny

2. Bioassay of drugs = Assay carried on a living organism
It is the quantitative determination of a new drug potency or conc. relative to standard, by measurement of the magnitude of biological response that it produces, and drawing DRC.
In vivo……In vitro……In situ
DRC = quantitative relationship between the doses of drug and magnitude of its biological response.

3. Response of the subject to various doses of stimulus
Components of
Bioassay
1-Stimulus
2-Subject
3-Response
Response of the subject to various doses of stimulus
Drug
Test sample
Animal, Tissues, cultured cells, Sub-cellular systems e.g. enzyme or receptor

4. Uses/Applications of bioassay
1-When the chemical assay is not available, too complex or insensitive to low dose
e.g., Insulin has no chemical assay
2- For Comparing the therapeutic advantages of one drug over another
in treatment of a certain disease
3- For determination of structure activity relationship (SAR) for
a group of compounds
4- For determination of biological activity of isomers
5- When the active principle of drug is unknown
6-To standardize preparations of impure drugs.

5. Disadvantages of bioassay?
1-Expensive, consume animals & lots of reagents
2-Time consuming
3- Less accurate, more difficult than chemical assay
4- Suffers from biological variability
Types of bioassay
1- Graded assay
• A gradual biological response is measured at several doses
Response may be: muscle contration, body temperature
weight of an organ
• Example: Assay of acetylcholine on the rabbit intestine

6. • The response is all or none (may happen or not)
2- Quantal assay
• The response is all or none (may happen or not)
We detect no of animal giving response e.g.
death, convulsions or writhing
• ED50: which produces a response in 50% of the animals
is used as a measure of the quantal assay
LD50: dose which kill 50% of animals
• e.g. digitalis induced Emesis in pigeon
digitalis induced cardiac arrest in guinea pigs

7. Designs of bioassay R 3- The three point assay (2+1 point assay)
1- The matching technique
2- The four point design (2 & 2 point assay) R
3- The three point assay (2+1 point assay)
DRC of St (25-75%) ……sigmoid curve log Dose
interpolation of test response (T)
4- Bracketing assay ( T )
S1 S s (T) S2
test is bracketed between 2 doses of standard which produces a smaller & a larger contraction. The mean value of St represents the amount of Ach in test solution
*simplest design that used when
-small amount of drug preparation sensitivity is variable
Most widely
used methods S2
Are inaccurate
&
not widely used S1 T
Three point assay based on assumption that log dose response is straight in the middle range of responses, approximately between 25-75% of max response . We dtect responses of 2 doses of standard in this range ( straight part) . Dose of test is then found which produces intermediate response. This assay is not vvey accurate
0.1
0.4
0.1 +o.4/2=0.25

8. Requirements for good bioassay?
1- Specificity of response
2- Reproducibility
3-Sensitivity
4-Objectivity of the end point
5-Accuracy & Precision
Most widely
used methods
Are inaccurate
&
not widely used

9. 1. Specificity of the response = end point or biological indicator
Determination of BGL reflects changes in many hormones such as adrenaline, glucocorticoids, glucagon and thyroxine, so it is not a specific test.
So biological response should be specific to ! tested drug, It may be
pharmacological action e.g. -The proliferative effect produced by progesterone
on the uterus
- salivation induced by cholinergic drugs
Side effect e.g. Emesis In pigeon for bioassay of digitalis
lethal effect e.g. Cardiostasis in guinea pig for bioassay of digitalis
Q: Bioassay of Ach on
- Guinea pig ileum is non specific?
- Frog rectus abdominis is specific?
2. Reproducibility
The response to the tested drug must be reproducible, precise and gives a good dose-response relationship

10. 4. Objectivity of the end point
3. Sensitivity
A good bioassay method can detect relatively small amounts of the drug. e.g. Guinea-pig ileum is more sensitive to histamine than rabbit intestine.
4. Objectivity of the end point
the biological response should be measured by instruments to↓ effect
of the observer opinion
Example: Estrogen is assayed by two methods:
i- Vaginal smear method, in which disappearance of
leukocyte indicates positive effect…..Subjective method
ii. Uterine weight method, in which the ↑ in rat uterus
weight indicates positive effect…......Objective method

11. 5-Accuracy and Precision:
Means that all result should be valid and lying in the same range
Accuracy: How close a result is to the actual (true) value Precision: means reproducibility of the result
i.e. how close data points are to each other
Accurate but Imprecise
precise but Inaccurate
Inaccurate and Imprecise
Accurate and Precise

12. Factors affecting the pharmacological response to drugs
1. Species difference
Variation in drug response may be qualitative or quantitative:
i-E.g. Morphine
• It is CNS depressant in rabbits, dogs and humans.
• It is CNS stimulant in mice and cats.
ii- E.g. Histamine:
• Causes fall in blood pressure in man due to capillary dilatation.
• Causes rise in blood pressure in rabbit due to capillary constriction.
Morphine causes excessive CNS stimulation in the Cat. This excitation is thought to be due to the release of dopamine and norepinephrine in the brain. Drugs that deplets, norepinephrine (renorpine, tetrabenzaine) and those that block dopamine receptors (Chlorpremazine, haloperidol) in the brain can prevent this excitation
These variations are due to:
a- The differences in drug metabolism by various species.
b- The differences in the mechanism of drug action between humans and animals
Example: The inflammation in rats depends on serotonin release rather than histamine in
humans

13. 2. Strain difference
• Animals belong to the same species but of different
strains show variation in the response of a drug.
Example: Many rabbit strains are resistant to atropine due
to presence of atropine esterase, the enzyme that
destructs atropine.
Example: Some rabbit strains are resistant to insulin due
to the presence of high level of destructive
enzyme in their livers.

14. 3. Sex differences:
• Sex differences can be attributed to the differences in
drug metabolism.
Example: Female rats sleep longer than males when treated with hexobarbital as they metabolize the drug more slowly.
4.Age:
Young human and animals are more sensitive to drugs than adult due to: a- The decrease in the activity of many enzyme
system during early life.
b- The metabolic enzymes in liver are not well developed.

15. 5. Environmental factors:
The condition in which the drug is tested on animals affects the response to the drug.
i-Example: The effect of amphetamine is increased when animals are housed together than when animals are housed individually.
ii-Example: Raising the temperature gradually increases
the convulsive action of insulin in mice

16. Screening & bioassay of Sympathomimetics
Are drugs that produce actions similar to sympathetic nerve stimulation

17. According to mechanism of action:
Classification:
According to structure
1-Catecholamines: contains the catechol nucleus e.g. E, NE, DA & isoprenaline
2-Non-Catecholamines: lack the catechol nucleus e.g. Ephedrine Amphetamine
According to mechanism of action:
direct acting agonists: e.g. Epinephrine …→ (α, β receptors),
Nor-epinephrine …→ (α receptors) &
Isoprenaline….→ (β receptors)
2. indirect acting agonists??: e.g., Amphetamine & Tyramine
Tachyphylaxis: Acute acquired tolerance due to rapid repeated administration
→depletion of Stores of NE, →up regulation of adrenergic
receptors → [denervation Super sensitivity]
Q-super-sensitivity ???
it is an enhanced response of the agonist [E,NE] due to up-regulation of
adrenergic receptor as a result to depletion of NE stores

18. 3. Mixed-acting agonists: have Dual mechanism
i.e. direct & indirect
e.g., Ephedrine & Metaraminol
Classification of adrenoreceptors:
-They are classified into α (α1 and α2) and
β (β1, β2 and β3) receptors.
-The agonists:
- endogenous………..norepinephrine and epinephrine.
- non endogenous …..isoprenaline.
NB: α-receptors are mainly excitatory except in GIT
Β receptors are mainly inhibitory except in Heart

19. α1 Receptor α2 Receptor -Main effects of their activation:
V.C., relaxation of GIT, contraction of
radial muscle of the iris →..??.
-Agonist potency: …….NA >A >isoprenaline
Selective agonist: phenylephrine
- Selective antagonist: prazocin
α2 Receptor
-Main effects of their activation: ↓NE release, inhibition of insulin
release, & platelet aggregation.
-Agonist potency: NA > A > isoprenaline
Selective agonist: clonidme
- Selective antagonist: yohimbine

20. β1 Receptor
Main effects of their activation: ↑ HR and force of contraction.
-Agonist potency: isoprenaline > A > NA
Selective agonist: dobutamine
- Selective antagonist: metoprolol
β2 Receptor
-Main effects of their activation: BD, VD, relaxation of GIT
-Agonist potency: isoprenaline > A > NA
Selective agonist: salbutamol
- Selective antagonist: butoxamine