screening methods for ans drugs ( sympathetics)

1. SCREENING METHOS FOR
ANS
DRUGS(SYMPATHETICS)
DINESH K
M PHARM PHARMACOLOGY
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2. CONTENS
 INTRODUCTION
 DRUGS ACTING ON ANS
 SCREENING METHODS FOR ANS DRUGS
DRUGS ACTING ON SYMPATHETIC NERVOUS SYSTEM
DRUGS ACTING ON PARASYMPATHETIC NERVOUS SYSTEM
 REFERENCES
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3. AUTONOMIC NERVOUS SYSTEM
 ANS is a control system that acts largely unconsciously and regulates bodily functions
such as the heart rate, digestion, respiratory rate, pupillary response, urination and sexual
arosal.
 This system is the primary mechanism in control of the flight – or – flight response.
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4. Drugs acting on ANS
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5. Screening Models for ANS
 The screening methods involved in autonomic nervous system based on the type of drugs
to be evaluated.
 These may on
 Drugs acting on sympathetic nervous system.
 Drugs action on parasympathetic nervous system.
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6. SYMPATHETIC SYSTEM
 The sympathetic nervous based on the action which inhibits or enhance the actions
of the nervous system. It is further classified into two types.
 Sympathomimetic drugs
 Sympatholytic drugs
Based on the types of drugs, the screening can be performed.
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7. Screening of Sympathomimetic drugs
 In vivo methods
 Cat spleen method
 The rat blood pressure invasive model
 The rat blood pressure non invasive method
 Cat model of nicilating membrane prolapse
 Mouse eye model
 Pithed rat model
 Rat heart and uterus model
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8.  In vitro methods
 Cat spleen model
 Rabbit pulmonary artery model
 Rat vas deferens model
 Rat seminal vesicle model
 Cat splenic
 Guinea pig tracheal chain model
 Guinea pig isolated heart model
 Isolated aorta model
 Rat submaxillary tissue model
 Mice metabolic stimulation model
 Murine macrophage model
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9. IN VIVO METHODS
 CAT SPLEEN METHOD
 one of the useful preparations for the evaluation of substances
affecting the release and subsequent fate of the adrenergic
transmitter from the sympathetic nervous system.
 After the injection of sympathomimetic amines or electrical
stimulation of the pre and postganglionic sympathetic nerves,
spleen contracts.
 Released nor adrenaline can be measured by collecting splee
venous effluent and analysing its noradrenaline content.
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10. CAT SPLEEN 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
analysing its NMT content.
 This is achieved by the labelling of neuronal stores with radioactive compound, which is taken
up from the splenic arterial circulation and released after nerve stimulation.
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11. Procedure (cat spleen method):
1.Healthy cats are selected and anaesthetized using chloroform
2.Abdomen is cut open (midline incision)& intestine is removed(from mid duodenum
to terminal colon)
3.Vascular connection between the spleen & omentum are being cut.
4.Spleenic nerves are also cut to avoid liberation of catecholamines from other sites.
5.Adrenal glands are also removed(to avoid the effect due to release of pressor amines.
6.A ligature is placed around the portal vein
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12. 7.Heparin is injected to avoid clotting of blood
8.Abdominal cavity is filled with warm paraffin and aerated with 95% O2& 5% CO2
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.
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13. IN VITRO METHOD
Cat spleen method:
 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_(temperature37○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
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14. 2. Rabbit pulmonary artery method:
 Purpose:
The pulmonary artery is very sensitive to sympathomimeticagents . The artery is
mainly consists of vascular smooth muscles innervated bypostganglionic
adrenergic sympathetic fibers . The NA is released in responseto the nerve
stimulation . The released adrenergic transmitter is measured bylabeling the
neuronal stores with radioactive NA & with the use of super fusiontechnique to
reduce the dilution of amines.
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15. 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)
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16. 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
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17. Screening of Sympatholytic drugs
 Methods:
In vivo method:
1.nictitating membrane prolapse in cats.
2.alpha ( ) & beta( β ) adrenergic antagonism in mouse eye.
In vitro method:
1.vas deferences of the rat
2.splenic strip of the cat
3.pithed rats for evaluation of sympathomimetic and sympatholytic activity.
4.to assess β 1 and β 2 adrenoreceptors agonism & antagonism
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18. 1.Nictitating membrane prolapse in cats
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.
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19. 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.
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20.  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
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21. 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)
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22.  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)
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23. REFERENCES
 DRUG SCREENING METHODS BY SK GUPTA
 DRUG DISCOVERY AND EVALUATION PHARMACOLOGICAL ASSAYS BY
WOLFGANG H.VOGEL
 SLIDE SHARE
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