Screening and bioassay

of drugs

by

Dr. Magdy M. Awny

Lecture 5

1

Are drugs which mimic parasympathetic nerve actions.

They are classified into:

1-Directly acting: activate M also N receptors 2-Indirect acting (Anticholinesterase):

A-Choline ester:

Acetylcholine & Carbacol.

B-Cholinomimetic alkaloids:

pilocarpine.

A-Reversible:

neostigmine, physostigmine (Eserine)

B-irreversible:

organophosphorus compounds

Screening & Bioassay of

Parasympathomimetics

3

Screening of Parasympathomimetics

1- Primary general test:

Injection of parasympathomimetic to mice causes &hypotension

Red skin & tears,

Urination,

Miosis,

Bradycardia,

Lacrimation,

Salivation

2- Cardiovascular test: (hypotensive action)

-Small dose Ach → brief ↓ in BP (due to M action on heart, BV)

that is blocked by atropine

-If Ach given in large dose after atropine (a fully atropinized animal

i.e. M receptors are blocked) →↑BP not fall in BP,

This Ach reversal due to the nicotinic action of Ach, include:

1- Stimulation of Adrenal medulla (release of E, NE →↑BP)

2-Stimulation of sympathetic ganglia (release of NE from the nerve end →↑BP)

5

3- Miotic action:

parasympathomimetics cause miosis through stimulation of circular muscle.

4- Sialagogue action:

A dog is anesthetized, Wharton's duct is cannulated and the cannula is connected

to a drop counter.

Muscarinic agonists cause a copious flow of salivary secretion, which is potency

and dose dependant.

This effect is blocked by prior administration of atropine.

I- Methods based on Nicotinic action II- Methods based on Muscarinic action

1-The dorsal muscle of leech (hirudo medicinalis)

SK .muscle preparation

Principle:

Ach cause contraction of the muscle, which is

proportional to the dose.

Procedures:

- muscle is removed, suspended in ringer

solution at room temp.

-Dose response curve is carried out and the

relative potency is calculated using matching

technique

- Physostigmine (Eserine) is added to increase

the sensitivity of the muscle

2-Frog rectus abdominis (SK.m preparation)

Principle: Ach cause contraction of the muscle,

Which is blocked by curare (NMB)

Procedures: as above

1-Isolated rabbit intestine

Principle:

Ach stimulate M receptor of rabbit intestine;

→ contraction which is proportional to dose

Procedures:

piece of intestine is suspended in tyrode

solution at 37 C , matching technique is done

2-Isolated guinea pig ileum:

As intestine but more sensitive

3-Anesthsized cat BP:

- Cat is anesthetized with chloralose

- I.V. Inj of Ach →transient brief fall in BP

(due to hydrolysis of Ach by cholinesterase)

As a result of vasodilatation & bradycardia

- apply the matching technique

Bioassay of Parasympathomimetics

1-1ry

general test [neuropharmacological tests]

Anticholinesterase differ from other parasympathomimetics in

They have M, N actions due to accumulation of endogenous Ach.

They produce gradual effect

They have longer duration of action

More stimulant action of skeletal muscle so produce more muscle fasciculation

[twitch of a group of muscle fibers] & fibrillation [twitch of individual muscle fibers]

8

2-cardiovascular test:

When anticholinesterase injected to anesthetized cat they produce

Prolonged fall in BP

Potentiate the effect of Ach & Vagal stimulation in tetrad system

Potentiate the hypertensive effect of Ach when injected to atropinized Cat

3-cat nictitating membrane:

Anticholinesterase potentiate response of the membrane to preganglionic ES

as they protect Ach in autonomic ganglia from destruction by cholinesterase

Screening & Bioassay of

Parasympatholytics

10

1-1 ry general test [neuropharmacological tests]

Muscarinic antagonists (Atropine-like agents) →

Mydriasis, cycloplegia, ↑IOP

Bronchodilatation, tachycardia,

↓ Secretion (Lacrimation, salvation& sweating)

↓ Gut motility

Antispasmodic action

Screening of Parasympatholytics

2- Cat blood pressure:

Atropine like drugs will

Block the effect of both injected ACh and vagal stimulation on blood pressure and

heart rate in the tetrad system.

Reverse the hypotensive action of ACh when it is given in large dose after atropine ??

Bioassay of Parasympatholytics1-Mydriatic action [mouse pupil method]

Principle:

Atropine causes passive mydriasis with loss of light reflex & cycloplegia

i.e. ↑ pupil diameter which is proportional to dose

Procedures:

-The pupil diameter is measured before administration of the drug then

measured At 10, 20, 30 min after drug administration

-The mean increase in the pupil diameter for S & T is determined and

relative potency is calculated

2-Isolated organ preparation 50% 50%

• Principle: Ach atrop Ach atrop

Atropine blocks the Ach-induced contraction of rabbit intestine or G. p. ileum

which is proportional to dose

• Assay is indirect [i.e. detect dose of atropine that cause 50% reduction in response

to Ach] & done by using matching technique

A- Chorda tympani induced salivation B- Pilocarpine induced salivation

Rabbit is anesthetized, Wharton’s duct is

cannulated & chorda tympani nerve is

exposed, up on ES →↑salivary secretion,

the volume of secretion collected in 1 hr is

recorded

Atropine is injected & the nerve is ES again

↓volume of salivary secretion is the

response to the given dose of atropine

The dose of atropine that causes 50% ↓ in

salivation is determined & compared with a

standard

S.C pilocarpine use to induce salivation

Atropine given 70 min before pilocarpine

, Dose of atropine causing 50% reduction

in volume of saliva is calculated

(indirect assay)

3-Anti-sialogogue action:

Principle: Atropine ↓ volume of the salivary secretion induced by pilocarpine

or ES of chorda tympani nerve supplying salivary gland.