Screening of sympathomimetic

And Sympatholytic drugs

By

Srota dawnM.Pharm(pharmacology)1st year, 2nd semester

Introduction: These are the drugs which mimic the

responses of epinephrine or stimulate the

sympathetic nervous system.

Secreted from :

Adrenal medulla in mammals

Sympathetic nerve activation occurs in

response to the stimuli including physical activity,

physiological stress, blood loss etc.

Sympathomimetic drugs may results in

any of the following events like i. mydriasis in the eye

ii. enhanced stroke volume

iii. acceleration of the heart rate

iv. dialation of the coronary arteries

v. constriction of the pulmonery vessels

vi. relaxation of bronchial muscles

vii. inhibition of gastric secretion

viii. constriction of gastrointestinal sphincters

ix. stimulation of uterus and

x. constriction of spleen capsule

Receptors in the ANS

• Cholinergic receptors: Inhibitory or excitatory - Nicotinic: fast - Muscarinic: slow

Adrenergic receptors: slow, excitatory or inhibitory - α 1 and 2 - β 1 and 2 (and 3)

ANS - Adrenergic Drugs-Adrenergic Receptors

Divided into 1 and 2 receptors Differentiated by their location on nerves

1-Adrenergic Receptors Located on postsynaptic effector cells

(the cell, muscle, or organ that the nerve stimulates)

2-Adrenergic Receptors Located on presynaptic nerve terminals

(the nerve that stimulates the effector cells) Control the release of neurotransmitters

Predominant -Adrenergic Agonist Responses Vasoconstriction CNS stimulation

ANS - Adrenergic Drugs-Adrenergic Receptors

• All are located on postsynaptic effector cells

– 1-adrenergic receptors—located primarily in the heart

– 2-adrenergic receptors—located in smooth muscle of the bronchioles, arterioles, and visceral organs

• -Adrenergic Agonist Response:

– Results in:

• Bronchial, GI, and uterine smooth muscle relaxation

• Glycogenolysis• Cardiac stimulation

Screening of sympathomimetics

Screening of sympathomimetics can be done by the following methods:

in vivo method:

Cat spleen method

In vitro method:

1. Cat spleen method

2. Rabbit pulmonary method

in vivo method:

Cat spleen method

Purpose:

This is one of the useful method for evaluation of

substances affecting the release and subsequent fate of the

adrenergic transmitter from the sympathetic nervous system.

After injecting sympathetic amines electrical stimulation of

the pre- and post- ganglionic nerves spleen contracts. Different

doses of the test compound that alter the release of transmitter

output of spleen are compared with the known standards.

Released amount of NMT output can be measured by

collecting spleen venous effluent and analyzing its NMT content.

This is achieved by the labeling of neuronal stores with

radioactive compound, which is taken up from the splenic arterial

circulation and released after nerve stimulation.

Procedure (cat spleen method):

7.Heparin is injected to avoid clotting of blood

9.Venous blood samples are collected by diverting effluent through a cannula by applying positive pressure

10.Blood is collected through chilled, silicon coated, calibrated centrifuge tubes

11.This method is enough for the collection of 80% of NA released.

12.The amount of NA present in the blood is estimated by `SCINTILLATION COUNTER for radioactivity measurement.

8.Abdominal cavity is filled with warm paraffin and aerated with 95% O2 & 5% CO2

In vitro method: 1.Cat spleen method:

1.This is the method for the estimation of NA content in IN VITRO PROCEDURE

The method for removal of spleen is same as before

The removed spleen is placed on a plethysmograph filled with liquid paraffin

The organ is perfused at a rate of 7-16 ml/min with modified Krebs solution_(temperature-37○c ,gassed with carbogen,dextran 3% for maintaining osmotic

pressure)

Ascorbic acid at 25 uGu/ml is added to prevent oxidation of NA

The perfusion is started and the samples are collected and estimated for NA

2. Rabbit pulmonary artery method:

Purpose: The pulmonary artery is very sensitive to sympathomimetic

agents . The artery is mainly consists of vascular smooth muscles innervated by postganglionic adrenergic sympathetic fibers . The NA is released in response to the nerve stimulation . The released adrenergic transmitter is measured by labeling the neuronal stores with radioactive NA & with the use of super fusion technique to reduce the dilution of amines.

Procedure (rabbit pulmonary artery method)

1.Rabbits(1-2kg) and sacrificed by exsanguinations followed by the removal of main pulmonary artery

2.The artery is cut transversely and spirally into vertical strips

3.The strip is suspended vertically in the organ bath (maintained at 37°c )

4.The last end of the tissue is tied to glass support, whereas the other end is connected to the strain gauge transducer

5.Resting tension- 2gm

6.Krebs bicarbonate solution(physiological solution)

7.The tissue is loaded with 3[H] NA by submerging it in 20 ml of Krebs bicarbonate solution at 37*c and gassed with 95% O2 & 5% CO2

8.Incubate for 60 min,the tissue is washed with fresh Krebs solution

9.During exp. Superfusates are collected in vials in every 3 mins.aliquots of collected samples are then assayed

10.The electric stimulations are given by the use of potassium electrodes

11.Responses to successive 2 min period of electrical stimulation are reproducible when applied at 16 min intervals

Screening of sympatholytics

Methods:

In vivo method:

1.nictitating membrane prolapse in cats2.alpha () & beta( β ) adrenergic antagonism in mouse eye

In vitro method:

1.vas deferences of the rat2.splenic strip of the cat3.pithed rats for evaluation of sympathomimetic and

sympatholytic activity4.to assess β 1 and β 2 adrenoreceptors agonism &

antagonism

1.Nictitating membrane prolapse in cats

1.Anaesthsised cats are used, drugs are administered orally

2.Sympatholytics exert relaxant effect on the nictitating membrane of cats

3.For each test drug 5 – 10 animals are used

4.The relative activity of different compounds is calculated by dividing the mean duration of the membrane prolapse of a group in hours by the dose

in mg/kg.

2.Alpha () & beta( β ) adrenergic antagonism in mouse eye

Nor epinephrine, epinephrine and isoproterenol have the property of inducing mydriasis,this effect is blocked by alpha () & beta( β ) adrenergic blockers. blockers block the mydriatic effect of nor- epinephrine, β blockers block the effect of isoproterenol and β blockers block the effect of epinephrine.

Procedure :

Mice(15-20 gm) are used

Animals are divided into groups as per requirement

Vehicle is administered in sc route (for control)

Test compounds are added to the test group

Std Nor-epinephrine given in i.v.

Pupil diameters are measured(before and after drug administration)

The mean value is compared between groups

In vitro method: 1.vas deferences of the rat

Male wister cats(275-300 gm)are used

Animals are killed by stunning

A midline abdominal incision is performed to disect out vas deferens

Tissue is suspended in an organ bath(tyrode, aerated,35*c)

Contractions are recorded(NA is administered repeatedly in concentration of 0.5,1,2,4 ug/ml

Test drug is added are the reduction in response is recorded

Phentolamine is used as standard)

2.splenic strip of the cat

Cat of either ser weighing around(1-2.8 kg) are used

The spleen is removed and a25 to 30 mm long strips of spleen are prepared

Strip is then suspended in an organ bath(krebs,38*c, aerated)

Tension used – 0.5 gm,magnification- 5-6 times

To induce contraction,NA/A is added

Test drug is then added(followed by agonist)

Phentolamine is used as standard(%reduction of activity of epinephrine or nor epinephrine is determined)

Thank you