Udupa 21 feb experimental evaluation of adrenergic agents
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The document discusses the adrenergic system and its receptors. It describes the different receptor types (α1, α2, β1, β2, β3, dopamine), their agonists and antagonists. Current drugs that target these receptors are used to treat conditions like hypertension, glaucoma, and airway obstruction. Ideal adrenergic agents would be receptor-selective, organ-specific, potent, and have predictable effects across organ systems. Such agents are evaluated through receptor binding studies, tests on isolated organs/tissues, and studies in anesthetized animals. These evaluation methods assess the drug's receptor selectivity, potency, efficacy, and safety profile.
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Udupa 21 feb experimental evaluation of adrenergic agents
- 3. Adrenergic System & Its Receptors 3 Dr Aaditya Udupa 10/26/2011
- 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 4 Dr Aaditya Udupa 10/26/2011
- 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 Dr Aaditya Udupa 10/26/2011
- 7. Steps in Evaluation Receptor binding studies Tests in Isolated anesthetized organ animals preparations 7 Dr Aaditya Udupa 10/26/2011
- 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? 8 Dr Aaditya Udupa 10/26/2011
- 9. What to aim for? Sensitive Specific Accurate Reproducible Predictable Valid 9 Dr Aaditya Udupa 10/26/2011
- 10. Receptor Binding Studies 10 Dr Aaditya Udupa 10/26/2011
- 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) 11 Dr Aaditya Udupa 10/26/2011
- 12. Scintiplate System Tissue + Drug + Radioligand 12 Dr Aaditya Udupa 10/26/2011
- 13. Scintiplate System 13 Dr Aaditya Udupa 10/26/2011
- 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 14 Dr Aaditya Udupa 10/26/2011
- 15. Studies On Isolated Organs And Tissues 15 Dr Aaditya Udupa 10/26/2011
- 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 16 Dr Aaditya Udupa 10/26/2011
- 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) 17 Dr Aaditya Udupa 10/26/2011
- 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 18 Dr Aaditya Udupa 10/26/2011
- 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 19 Dr Aaditya Udupa 10/26/2011
- 20. Cannulation and re-establishment of vascular perfusion 20 Dr Aaditya Udupa 10/26/2011
- 21. Cannulation and re-establishment of vascular perfusion 21 Dr Aaditya Udupa 10/26/2011
- 22. 22 Dr Aaditya Udupa 10/26/2011
- 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 23 Dr Aaditya Udupa 10/26/2011
- 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 Dr Aaditya Udupa 10/26/2011
- 25. 25 Dr Aaditya Udupa 10/26/2011
- 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 26 Dr Aaditya Udupa 10/26/2011
- 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 27 Dr Aaditya Udupa 10/26/2011
- 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) 28 Dr Aaditya Udupa 10/26/2011
- 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 29 Dr Aaditya Udupa 10/26/2011
- 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 30 Dr Aaditya Udupa 10/26/2011
- 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 31 Dr Aaditya Udupa 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 32 Dr Aaditya Udupa 10/26/2011
- 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 33 Dr Aaditya Udupa 10/26/2011 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 34 Dr Aaditya Udupa 10/26/2011
- 35. Isolated Organs and Tissues Disadvantages: Effect of drug in body is not known Compensatory phenomenon cannot be predicted Pro-drugs cannot be studied 35 Dr Aaditya Udupa 10/26/2011
- 36. Studies On Anesthetized Animals 36 Dr Aaditya Udupa 10/26/2011
- 37. “In vitro simplicitas, in vivo veritas” 37 Dr Aaditya Udupa 10/26/2011
- 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 38 Dr Aaditya Udupa 10/26/2011
- 39. Anesthetized Dog Blood Pressure 39 Dr Aaditya Udupa 10/26/2011
- 40. Anesthetized Dog Blood Pressure 40 Dr Aaditya Udupa 10/26/2011
- 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. 41 Dr Aaditya Udupa 10/26/2011
- 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 42 Dr Aaditya Udupa 10/26/2011
- 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 43 Dr Aaditya Udupa 10/26/2011
- 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 44 Dr Aaditya Udupa 10/26/2011
- 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 45 Dr Aaditya Udupa 10/26/2011 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 46 Dr Aaditya Udupa 10/26/2011
- 47. Future Trends Non invasive assays Second messenger assays Transient transfection HTS assay Fluorescence-based assay technologies Radioligand Studies 47 Dr Aaditya Udupa 10/26/2011
- 48. 48 Dr Aaditya Udupa 10/26/2011
Editor's Notes
- adrenaline and noradrenaline produce relaxation
- adrenaline reduces tone and frequency of contractions produced by ach or carbachol.
- adrenaline reduces tone and frequency of contractions produced by ach or carbachol.
- adrenaline and noradrenaline produce relaxation
- adrenaline and noradrenaline produce relaxation
- adrenaline and noradrenaline produce relaxation
- There are three polymorphisms that alter receptor function, which may influence the therapeutic success