1. Writhing test
Edited by
dr. Magdy Awny
2018
INTRODUCTION

2. Biological standardization of drugs using either intact animals or isolated organs
Bioassay:
is a quantitative determination of relationship between drug concentrations and biological response

3. Drugs actions on the body and their assay can be studied in both:
In vitro:
on isolated organs (tissues).
In vivo
on intact animals

5. Writhing test of Non-narcotic analgesics
Screening and/or Bioassay
of Non-narcotic analgesics
‘NSAID’ e.g. acetyl salicylic acid
Writhing test
Principle:
it depends on pain induction & recording animal response before & after
drug administration
Pain inducer: acetic acid; i.p……….acute inflammation
Animal response: writhing or stretching

6. Inflammation

7. Inflammation:
is a normal defense reaction to a tissue injury that ends with either complete healing or permanent destruction of the tissue.

8. Causes Immunological (antigen-antibody, cell mediated).
Physical: (trauma, heat or cold and radiation).
Chemical: (acids, bases organic poisons).
Infective: (bacteria, viruses, parasites).
Immunological (antigen-antibody, cell mediated).

9. Inflammation may be
ACUTE or CHRONIC the initial stages are known as the acute inflammatory reaction.
Where the process is prolonged the inflammation may be sub acute or chronic.

10. Signs of inflammation:
1. Vascular phase:
Capillary and arteriolar dilatation  redness and hotness.
2. Exudative phase:
Increase in capillary permeability  translocation of fluid and plasma proteins into tissue spaces “fluid exudate”  swelling and oedema.
Leucocytes and monocytes move to tissue spaces under the influence of chemotactic factors. “cellular exudate”.
3. Proliferative phase:
New connective tissues (granuloma) and capillaries are formed to replace those destroyed  fibrosis may occur and the affected area may lose its function.
N.B.
In acute inflammation vascular and exudative phases are more prominent, while in chronic states proliferative phase is more pronounced.

14. ANTI-INFLAMMATORY DRUGS
These are drugs which are used to treat different inflammatory conditions as rheumatoid arthritis.
Classification of anti-inflammatory drugs:
A] Steroidal: e.g., cortisone, dexamethasone,..etc.
B] Non steroidal (NSAIDs): Aspirin, indomethacin.

16. Writhing test for NSAID
Pain is induced by Acetic acid
conc: 1.5%
Dose: 8 ml/kg
Calculate the dose according to animal weight then inject the volume of acetic acid I.P.
The mice react with a characteristic stretching behavior called writhing

17. Calculation of drug volume to be administered
Weight of the animal (grams)
Suggested drug dose (mg/kg)
Drug concentration (%)

18. Example: X = 20 x 500/ 1000 = 1 mg Given: wt = 20 g
Suggested NSAID dose = 50mg/kg
Drug conc. = 0.5% (0.5 g / 100ml)
1) Find the amount of drug required:
50(mg) (1kg) 1000g
X (mg) g (animal wt)
X = 20 x 500/ 1000 = 1 mg

19.  Volume to be injected = 0.2 ml
2) Find the volume of the drug required:
0.5(g) ml (drug conc.)
500(mg) ml
1(mg) Y(ml)
 Volume to be injected = 0.2 ml
Y = 1 x 100/ 500 = 0.2 ml

20. Steps of writhing test (2 steps)
A-Divide animals to 2 groups
Control group
will receive Acetic acid (8 ml/kg; i.p) and observe writhing episodes for 10min
Test group
1-will receive NSAID e.g. aspirin (conc: 0.5%,
dose: 50 mg/Kg; i.p) & wait for 15 min
2-then receive acetic acid (8 ml/kg) and observe the writhing episodes for 10 min

21. B-The time of writhing onset is observed and counted within 10 min for each group (control and test)
No. of writhes
Onset of writhing
Drug injected
Acetic acid (control)
NSAID e.g. aspirin + Acetic acid
The % of inhibition is calculated as follows: -
Writhes in control
Writhes in the drug group
X 100
Writhes in control
NB: Compounds with < 70 % inhibition are considered to have minimal activity

22. THANK YOU…...