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

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

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

(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)

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

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

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)

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

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

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

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

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)

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

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

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

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

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

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

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

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

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 10 6- 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