This is a presentation on experimental evaluation of adrenergic agents

3. Adrenergic System & Its
Receptors
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4. Receptor Agonist Antagonist
α1 type Phenylephrine Prazosin
α2 type Clonidine Yohimbine
α2A Oxymetazoline
α2B Prazosin
α2C Prazosin
β type Isoproterenol Propranolol
β1 Dobutamine Betaxolol
β2 Terbutaline Butoxamine
β3
Dopamine type Dopamine
D1 Fenoldopam
D2 Bromocriptine
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5. Current Agents
 α1 Receptor agonists are used for hypotension
and nasal decongestion
 α Receptor agonists are used for hypertension
2
and glaucoma
 α Blockers are used in treatment of hypertension
1
and BHP
 β Selective agonists are used as ionotropic
1
agents
 β2 Selective agonist are developed for use in
airway obstruction
 β Selective blockers are one of the most widely
1
5 used drugs in practice
Dr Aaditya Udupa 10/26/2011

6. Ideal Agent
Receptor selective
Organ specific
Highly potent
Good discrimination value reflected in
slope of DRC
Predictable action on various organ
6 systems
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7. Steps in Evaluation
Receptor
binding
studies
Tests in Isolated
anesthetized organ
animals preparations
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8. What To Look For?
 Is the drug an adrenergic agent?
 Is it an agonist or antagonist?
 Is it a directly or indirectly acting agent?
 Is it a selective agent or not?
 Is it more potent or efficacious than the other
standard agents?
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9. What to aim for?
 Sensitive
 Specific
 Accurate
 Reproducible
 Predictable
 Valid
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10. Receptor Binding Studies
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11. Tissue in which receptor is
predominant is chosen
Receptor Type Organ Used
Radioligand (Agonist/ Antagonist)
+ Test drug is added
α1 Rat heart ventricles
Receptor Type RadioLigand Drug
Mixture is homogenisedtissue
α2 Rat cortical and
α1 washed 3H-prazosin (antagonist)
β1 Rat forebrain (cortex)/
α2 3H-clonidine (agonist)/
Heart
Residual binding of agonist
3H-α-Yohimbine is
checked for.
(antagonist)
β1 β 2 3H-alprenolol (antagonist)
Rat lung/ cerebellum
Lower the residual radioactivity
β2 higher is the potency of test drug
3H-dihydroalprenolol
Hydrochloride (antagonist)
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12. Scintiplate System
Tissue +
Drug +
Radioligand
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13. Scintiplate System
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14. Receptor Binding Studies
 Advantages:
Laboratory procedure
Large no of drugs can be screened
 Disadvantages:
Agonist and antagonist are not differentiated
Radiation exposure
Equipment is costly
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15. Studies On Isolated Organs
And Tissues
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16. Tissues for studying
adrenoreceptors
 Postsynaptic α1 receptor
Rat isolated vas deferens (contraction)
Rabbit isolated aortic strips (contraction)
Rat isolated anococcygeous muscle (contraction)
Rabbit isolated jejunum (inhibition)
Guinea pig isolated ileum (inhibition)
Cat nictitating membrane (contraction)
 Presynaptic α2 receptor
Transmurally stimulated rat isolated vas deference
Electrically stimulated GP ileum (inhibition)
Rabbit isolated ear artery & pulmonary artery
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17. Tissues for studying
adrenoreceptors
β 1 Receptor system
Rabbit isolated perfused heart (contraction)
Rabbit isolated jejunum (inhibition)
 β 2 Receptor system
Guinea pig perfuse lung (bronchodialation)
Guinea pig isolated trachea (inhibition)
Rat isolated uterus (inhibition)
 Both α and β receptor
Mouse isolated spleen (α – contraction, β-
relaxation)
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18. Screening Tool
 Guinea pig isolated ileum:
Animal stunned and ileum removed
Tyrode solution at 37.0-37. 5°C
Oxygenated with O2 or air
Carbachol or histamine is used to induce
contractions
Adrenergic agents studied by their ability to induce
relaxation
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19. Cardiac Activity
Isolated excised rabbit heart(β
activity):
Described by LANGENDORFF
Krebs-Henselit bicarbonate buffered solution at
37.0-37. 5°C
95% Oxygen and 5%Carbondioxide
Coronary bed is perfused under pressure of 20 cm
of water
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20. Cannulation and re-establishment of
vascular perfusion
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21. Cannulation and re-establishment of
vascular perfusion
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23. Cardiac Activity
 Isolated excised rabbit heart:
Parameters:
Rate and rhythm
LVP (left ventricular pressure)
Myocardial oxygen consumption (MWO2)
Ultrasonic recording of two cardiac diameters
Applications:
Reversal of CHF by β-sympathomimetic drugs,
cardiac glycosides
β-blocking agent
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24. Cardiac Activity
 Isolated papillary muscle (β activity):
Cat heart is removed and papillary muscle from
right ventricle is obtained and fixed
 Ringer’s solution at 36°C
Stimulated electrically at 4-6 V, 2ms duration at rate
of 30/min
After 1 hour the contractions decrease
adrenaline, nor-adrenaline, isoprenaline and other
positive ionotropic agents can be studied
 Isolated auricle (both intact & as muscle
strips):
No stimulation is needed if SA Nodal tissue is
24 present in it
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26. Respiratory Activity
 Guinea pig tracheal chain (β activity):
Trachea is removed & cut into six by
Six rings are connected in series
Tyrode solution containing ascorbic
individual 34°C loops of silk thread
means of short
acid at rings
Oxygenated with 95% Oxygen and
5%Carbondioxide
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27. Smooth Muscle
 Isolated rat uterus (β activity):
De Jalons solution at 32 °C 5-HT &
Chiefly for oxytocin,
Oxygenated with 95% Oxygen and
adrenaline
5%Carbondioxide
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28. Smooth Muscle
 Isolated rat uterus:
It contains only β- receptors (highly sensitive and
specific for adrenaline)
β- Agonists cause uterine relaxation and are used
as tocolytics
To study effect on α- receptors prior treatment
with oestrogen and maintaining the water bath at
25 °C (temperature sensitive)
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29. Smooth Muscle
 Isolated rabbit jejunum (β
activity):
Adrenergic agonists 4°C 5°Cguinea pig
Tyrode solution at 37.0-37. as preparation
Stored for four days at cause relaxation
Used in same manner the
becomes increasingly sensitive and
of spontaneous95% Oxygen jejunal
Oxygenated with pendulous to inhibitory
ileum
5%Carbondioxide
action of isoprenaline
contractions
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30. Cardiovascular Activity
 Isolated rabbit aorta (α activity): :
Krebs-Henseleit solution containing
Thoracic aortic is removed &vascular
Spiral aorta strips are
11.5% glucoseis removed by gentle rubbing
endothelium at 37.5°C
prepared
95% Oxygen and 5%Carbondioxide
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31. Smooth Muscle (α activity):
Preparation PSS Oxygenation
Guinea pig seminal Ringer’s Solution at 32°C 95%O2 + 5%CO2
vesicle
High concentration of α1 receptor
Vas deference of rats Tyrode solution at 34°C 95%O2 + 5%CO2
It is highly recommended for quantitative evaluation of
adrenergic antagonists
Rat anococcygeus Krebs solution at 36°C 95%O2 + 5%CO2
muscle
Dense adrenergic innervation and contracts to NA, 5-HT but
not to histamine
To assess pre- and post- synaptic α-adrenoreceptor
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blocking activity 10/26/2011

32. Isolated perfused rabbit ear
RabbitSolutionrabbit’s detached ears veins
Vesselscannulated at both marginal
ear of from veins falls on a
andperfusedarteryTyrode solution
central counter to measure
drop with
Perfusion pressure must be ear
constriction or dilation of constant at
about 60 vessels
blood cm of water
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33. Isolated cat spleen
 Cat is anesthetized, abdomen cut open and
spleen isolated with its vascular connections.
 The splenic nerves are cut and ligated
 Placed in plethysmograph filled with liquid paraffin
 Perfused with Krebs solution at 37°C
 Oxygenated with 95% Oxygen and
5%Carbondioxide
 Dextran and heparin are added to perfusion fluid
 Electrical stimulation of splenic nerves to liberate
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NA

34. Isolated Organs and Tissues
Advantages:
Fast and simple procedure
Absence of the confounding effects of other
organs
Drug effect is tested directly without the
factors of absorption, metabolism or
excretion
Agonist and antagonist can be differentiated
Excellent test-bed for undertaking carefully
controlled dose-response studies of
metabolic or pharmacological interventions
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35. Isolated Organs and Tissues
Disadvantages:
Effect of drug in body is not known
Compensatory phenomenon cannot be
predicted
Pro-drugs cannot be studied
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36. Studies On Anesthetized
Animals
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37. “In vitro simplicitas, in vivo veritas”
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38. Anesthetized Dog Blood
Pressure
A ligature
Dog is
passed around
restrained &
Endotracheal Femoral artery carotid artery
and
intubation and vein and Vagus
anesthetized
performed cannulated nerve is
with
attached to
pentobarbitone
electrodes
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39. Anesthetized Dog Blood
Pressure
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40. Anesthetized Dog Blood
Pressure
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41. Mydriasis in mouse iris
Pupil diameter is narrowed by intense stimulation to
light
Intra-peritoneal administration of adrenergic agent
Preparation is dose responsive
Rabbits may also be used satisfactorily
Clonidine induces mydriasis via α2 adrenoreceptors
in brain.
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42. Cat Nictitating Membrane
Unanestetized cat:
 Routinely employed for initial evaluation of drugs
due to its simplicity
 Degree of relaxation measured by a ruler
 Drug can be given by various routes
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43. Cat Nictitating Membrane
Anesthetized cat:
 Cat anesthetized and its nictitating membrane
tone is measured with an isotonic lever system
 More precise
 Technique by which direct injection of drug into
the external carotid artery causing greater
concentration of drug at site
 The isolated nictitating membrane of cat has
been described but it is a highly cumbersome
procedure and rarely used
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44. Tests in Anesthetized Animals
 Advantages:
Actual response may be seen
Action of drug on various organ systems, their
interactions and the effects of physiological inputs is
seen
Some of the adverse drug reactions that may be
expected are seen
 Disadvantages:
Wastage of animals
Basic physiological differences between man and
test animals have to be remembered
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45. β 3-adrenoceptor
 The β3-adrenoreceptor has been cloned and
characterized in animals
 Receptor identification and quantification:
Chinese hamster ovary cell transfection system human
β3-adrenoreceptor
gene
Reverse transcription-polymerase chain reaction
 Bioassays:
Lipolysis and Oxygen Consumption in Brown Adipocytes
Ureteral relaxation in rats, rabbits and dogs
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Guinea pig taenia caecum

46. β 3 -adrenoceptor
However, the physiological function of the
human β3-adrenoreceptor, the significance
of animal data with β3-adrenoreceptor
agonists, and even the presence of a
putative fourth β-adrenoreceptor in human
adipose tissue are still a matter of debate
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47. Future Trends
 Non invasive assays
 Second messenger assays
 Transient transfection HTS assay
 Fluorescence-based assay technologies
 Radioligand Studies
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