1. Biological standardization and screening of drugs
Lecture 6 By
Dr. Mohamed Fahmy

2. Neuromuscular blockers (NMBs)
Drugs which block the nicotinic receptors in the neuromuscular junction Classification 1- Competitive e.g., Curare & Flaxedil 2- Depolarizing e.g., Succinylcholine & Decamethonium

3. Screening tests 1- The neuropharmacological test
When injected in mice NMBs cause decreased limb tone & grip strength
Mice become unable to remain on revolving drum
This is non specific test as it gives similar effects with CNS depressants

4. (due to initial muscle contraction)
2- Chick test
It is used to differentiate between competitive and depolarizing blockers
When injected into wing vein of chicks:
Competitive NMBs Flaccid paralysis
Depolarizing NMBs Spastic paralysis
(due to initial muscle contraction)

5. 3- Nerve muscle preparations
A- The sciatic nerve tibialis muscle of cat
This is an in situ preparations for screening and differentiation between NMBs
It consists of a cholinergic nerve in close conjunction with skeletal muscle
The cat is anaesthetized with chloralose and the nerve is exposed
Electrical stimulation of the nerve causes muscle contraction

6. Competitive NMBs block the effect of released Ach while depolarizing NMBs cause an initial contraction followed by a block
Ether, chloroform & barbiturate are not used as anesthetic for cat because they potentiate the effect of NMBs
B- The sciatic nerve gastrocnemius muscle of cat
This method of screening is similar to the cat sciatic nerve anterior tibialis muscle

7. Bioassay Methods 1- Rabbit head drop method
This method is used for bioassay of curare
Principle:
Competitive NMBs relax the neck muscle of rabbit Head drop (Quantal assay)
Procedure:
The solution of NMBs is infused into the ear vein of rabbit until the end point (head drop is reached)

8. The assay is carried out using 6 animals for S & 6 animals for T
The minimal (threshold) dose of T required for head drop is determined
Similarly, the minimal dose of S required for head drop is also determined
The assay is performed by comparing the dose of T with that of S

9. 2- The rat phrenic nerve diaphragm preparation
This preparation consists of a skeletal muscle (diaphragm) innervated by a cholinergic nerve (phrenic nerve)
This is a widely used in vitro method for bioassay of NMBs
The preparation is suspended in an isolated organ bath containing Kreb's solution
Electrical stimulation of the nerve causes contraction of the diaphragm
The dose of S & T which produces 50% inhibition in contraction caused by electrical stimulation is determined

10. Differentiation between competitive & depolarizing blockers
1- The chick test
Competitive NMBS cause flaccid paralysis
Depolarizing NMBS cause spastic paralysis
2- Action on frog rectus abdominis
Competitive NMBS cause no contraction while Depolarizing NMBS cause initial contraction
3- Action on tibialis anterior muscle
Competitive NMBS cause no contraction while Depolarizing NMBS cause rapid contraction

11. Antihistaminics Screening methods
Drugs which antagonize the action of histamine
Classification
1- H1 blockers e.g., Mepyramine
2- H2 blockers e.g., Cimetidine
Screening methods
Antihistaminics block the following actions induced by histamine:
Contraction of guinea pig ileum
Contraction of guinea pig tracheal muscle
Blood pressure reduction in anesthetized cat

12. Bioassay Methods 1- Protection against histamine aerosol Principle:
Antihistaminics prolong the pre-convulsive time caused by exposure to histamine
Procedure:
Guinea pigs are placed on a transparent box and aerosol of 2% histamine is sprayed for 5 minutes
The time from introduction of aerosol till the onset of convulsive state is determined (T1)

13. The animals are removed from the box & allowed to recover for 2 days
Animals are injected with the test antihistaminic before the exposure to histamine aerosol and the pre-convulsive time is determined again (T2)
The prolongation of the pre-convulsive time is determined (T2-T1) and compared with that of a standard antihistaminic

14. 2- Antagonism on isolated organs
The assays depend on that antihistaminics inhibit histamine-induced contraction
The degree of inhibition is proportional to the dose
The assay is carried out using the matching technique
Two isolated organs are used:
1- Isolated guinea pig ileum
2- Isolated guinea pig tracheal chain

15. Bioassy of serotonin 1- Rat uterus method Principle
Serotonin causes contraction of the rat uterus in the oestrus stage only & this contraction is proportional to the dose
Procedure
Female rats are injected with diethylstilbsterol 24 hrs before the experiment to induce oestrus stage

16. One horn of the uterus is suspended in DeJalon solution at 37°C
Atropine & mepyramine are added to remove the interference by Ach & histamine
Serotonin causes contraction which is blocked by pre-treatment with 5-HT antagonist e.g., Lysergig acid diethylamide (LSD)
The assay is performed using the matching technique

17. 2- Rat fundus method
Principle
Serotonin causes contraction of the fundus strip & this contraction is proportional to the dose
Procedure
Fasted rat is killed & the stomach is removed
The fundus is separated and made into strip by transverse cut
The strip is suspended in Kreb's solution

18. Hyoscine is added to block the action of any
Ach present
Serotonin causes contraction which is abolished by LSD
The assay is carried out following the matching technique

19. Differential assay of 2 neurotransmitters
1- Mixture of Ach & histamine
Ach Frog rectus abdominis muscle, histamine does not interfere
Histamine Guinea pig ileum using atropine to block the action of Ach
2- Mixture of Ach & NE
Ach Frog rectus abdominis muscle, NE does not interfere
NE Pithed rat BP after injection of atropine to block the action of Ach

20. 3- Mixture of Ach & 5-HT Ach Frog rectus abdominis muscle, 5-HT does not interfere 5-HT Rat fundus preparation in presence of hyoscine to block the action of Ach 4- Mixture of Histamine & 5-HT Histamine Guinea pig ileum in presence of LSD 5-HT Rat stomach fundus in presence of mepyramine

21. 5- Mixture of histamine & epinephrine Epinephrine Pithed rat BP in presence of mepyramine Histamine Guinea pig ileum after inactivation of E by making the pH of the medium Mixture of NE & 5-HT NE Pithed rat BP in presence of cyproheptadine 5-HT Rat uterus after inactivation of NE by adjusting pH at 6.5-7