Udupa 21 feb experimental evaluation of adrenergic agents


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  • 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
  • Udupa 21 feb experimental evaluation of adrenergic agents

    1. 1. Adrenergic System & Its Receptors3 Dr Aaditya Udupa 10/26/2011
    2. 2. 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 Bromocriptine4 Dr Aaditya Udupa 10/26/2011
    3. 3. 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 15 used drugs in practice Dr Aaditya Udupa 10/26/2011
    4. 4. Ideal Agent Receptor selective Organ specific Highly potent Good discrimination value reflected in slope of DRC Predictable action on various organ6 systems Dr Aaditya Udupa 10/26/2011
    5. 5. Steps in Evaluation Receptor binding studies Tests in Isolated anesthetized organ animals preparations7 Dr Aaditya Udupa 10/26/2011
    6. 6. 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
    7. 7. What to aim for?  Sensitive  Specific  Accurate  Reproducible  Predictable  Valid9 Dr Aaditya Udupa 10/26/2011
    8. 8. Receptor Binding Studies10 Dr Aaditya Udupa 10/26/2011
    9. 9. 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
    10. 10. Scintiplate System Tissue + Drug + Radioligand12 Dr Aaditya Udupa 10/26/2011
    11. 11. Scintiplate System13 Dr Aaditya Udupa 10/26/2011
    12. 12. Receptor Binding Studies  Advantages: Laboratory procedure Large no of drugs can be screened  Disadvantages: Agonist and antagonist are not differentiated Radiation exposure Equipment is costly14 Dr Aaditya Udupa 10/26/2011
    13. 13. Studies On Isolated Organs And Tissues15 Dr Aaditya Udupa 10/26/2011
    14. 14. 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 artery16 Dr Aaditya Udupa 10/26/2011
    15. 15. 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
    16. 16. 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 relaxation18 Dr Aaditya Udupa 10/26/2011
    17. 17. 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 water19 Dr Aaditya Udupa 10/26/2011
    18. 18. Cannulation and re-establishment of vascular perfusion20 Dr Aaditya Udupa 10/26/2011
    19. 19. Cannulation and re-establishment of vascular perfusion21 Dr Aaditya Udupa 10/26/2011
    20. 20. 22 Dr Aaditya Udupa 10/26/2011
    21. 21. 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 agent23 Dr Aaditya Udupa 10/26/2011
    22. 22. 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 is24 present in it Dr Aaditya Udupa 10/26/2011
    23. 23. 25 Dr Aaditya Udupa 10/26/2011
    24. 24. 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%Carbondioxide26 Dr Aaditya Udupa 10/26/2011
    25. 25. 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%Carbondioxide27 Dr Aaditya Udupa 10/26/2011
    26. 26. 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
    27. 27. 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 contractions29 Dr Aaditya Udupa 10/26/2011
    28. 28. 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%Carbondioxide30 Dr Aaditya Udupa 10/26/2011
    29. 29. 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-adrenoreceptor31 Dr Aaditya Udupa blocking activity 10/26/2011
    30. 30. 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 water32 Dr Aaditya Udupa 10/26/2011
    31. 31. 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 liberate33 Dr Aaditya Udupa 10/26/2011 NA
    32. 32. 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 interventions34 Dr Aaditya Udupa 10/26/2011
    33. 33. Isolated Organs and Tissues Disadvantages: Effect of drug in body is not known Compensatory phenomenon cannot be predicted Pro-drugs cannot be studied35 Dr Aaditya Udupa 10/26/2011
    34. 34. Studies On Anesthetized Animals36 Dr Aaditya Udupa 10/26/2011
    35. 35. “In vitro simplicitas, in vivo veritas”37 Dr Aaditya Udupa 10/26/2011
    36. 36. 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 electrodes38 Dr Aaditya Udupa 10/26/2011
    37. 37. Anesthetized Dog Blood Pressure39 Dr Aaditya Udupa 10/26/2011
    38. 38. Anesthetized Dog Blood Pressure40 Dr Aaditya Udupa 10/26/2011
    39. 39. 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
    40. 40. 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 routes42 Dr Aaditya Udupa 10/26/2011
    41. 41. 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 used43 Dr Aaditya Udupa 10/26/2011
    42. 42. 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 remembered44 Dr Aaditya Udupa 10/26/2011
    43. 43. 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 dogs45 Dr Aaditya Udupa 10/26/2011 Guinea pig taenia caecum
    44. 44. 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 debate46 Dr Aaditya Udupa 10/26/2011
    45. 45. Future Trends  Non invasive assays  Second messenger assays  Transient transfection HTS assay  Fluorescence-based assay technologies  Radioligand Studies47 Dr Aaditya Udupa 10/26/2011
    46. 46. 48 Dr Aaditya Udupa 10/26/2011