Screening of anti anxiety drugs


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Screening of anti anxiety drugs

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  2. 2. EVALUATION SEMINAR ON Preclinicalevaluation of anxiolytics Presented to : Dr. KVSRG PRASAD Presented by: P. Bindu, M.Pharmacy 1st year Department of pharmacology 2
  3. 3. Contentso Introductiono Types of anxietyo Physiological Vs pathological anxietyo Classification of anxiolyticso Screening methods for anxiolytics In vitro methods In vivo methodso Conclusiono References 3
  4. 4. Introductiono Anxiety is an emotional state caused by the perception of real or perceived danger that threatens the security of an individual.o It is normal human adaptive response to stressful events.o Physiological anxiety – transient in natureo Pathological anxiety – needs treatment 4
  5. 5. Physiological and Pathological Anxiety jitter Panic attacks Obsessions, Stage-fright compulsions Nervousness Flashbacks, nightmares Worrying Pathological fear 5
  6. 6. Types of anxiety disordersa. Panic disorderb. Generalised anxiety disorderc. Social anxiety disorder.d. Obsessive compulsive disorder.e. Post traumatic stress disorder. 6
  7. 7. Pathophysiology of anxietyo Neurotransmitters like GABA, noradrenaline, serotonin abnormalities – anxiety.o Amygdala, temporal lobe, hippocampus and hypothalamus - involved in anxeityo Neurochemical theories : 1. Noradrenaline theory 2. Serotonin theory 3. GABA receptor theory 7
  8. 8. Noradrenaline theoryo ANS of anxious patients- hypersensitive to stimuli.o Locus cerulus – activates epinephrine releaseo Anxiogenics – stimulate locus cerulus firingo Anxiolytics- inhibits locus cerulus firing and decrease noradrenaline activity. 8
  9. 9. GABA Receptor Theoryo GABA – inhibitory neurotransmitter in brain.o Has inhibitory and regulatory effects on serotonin, noradrenaline and dopamine.o GABAA receptor involved in anxiety; decreases neuronal excitabilityo Patients suffering from anxiety disorders have less level of GABA in cortex. 9
  10. 10. Serotonin Theory• Abnormalities in serotonin function i.e., release and uptake plays role in anxiety.• Greater serotonin activity – reduces norepinephrine activity in locus cerulus.• SSRIs – increases serotonin levels post synaptically – blocks symptoms of anxiety. 10
  11. 11. Classification of anxiolytics Benzodiazepines alprazolam, clonazepam, diazepam Azapirones Buspirone, tandospirone, gapirone Sedative antihistaminics Hydroxyzine Beta blockers Propranalol carbamates meprobamate 11
  12. 12. Screeningmethods foranxiolytics 12
  13. 13. In vitro methods GABAA receptor binding GABAB receptor binding Benzodiazepine receptor: [3H]-flunitrazepam binding assay Serotonin (5-HTIA) receptor: binding of [3H]-8- hydroxy-2-(di-n-propylamino)-tetralin ([3H]- DPAT) Serotonin (5-HTIB) receptors in brain: binding of [3H]5-hydroxytryptamine ([3H]5-HT) 13
  14. 14. In vivo methods Methods based on unconditioned (spontaneous) response:o Exploratory activity elevated plus-maze light-dark model (two compartment box)o Social behaviour social interaction Isolation induced aggression 14
  15. 15.  Methods based on conditioned (learned) response:o Conflict models Vogel punished drinking/ Vogel’s lick conflict model Normal (adaptive) anxietyo Elevated plus-maze testo Social interactiono Light-dark modelo Marble burying test 15
  16. 16.  Stress-induced anxietyo Vogel lick conflict test Pathological anxietyo Neurochemically - induced anxiety  mCPP induced anxiety in rats 16
  17. 17. Elevated Plus Maze Testo Most widely used method; male mice used.o For selective identification of anxiolytic and anxiogenic drugso Anxiolytics –decrease anxiety – increase open arm exploration timeo Anxiogenics – decrease open arm exploration time. 17
  18. 18. o 2 open arms and 2 closed arms of 50 ˣ 10 ˣ 40cm dimensionso Open roof arrangemento Two open arms are opposite to each other.o Maze elevated at 50cm height. 18
  19. 19. Experimental Design• Group I : control• Group II : standard• Group III : test treated with dose x• Group IV : test treated with dose 2x …. 19
  20. 20. The rats weighing around 200g -housed in pairs for 10 days prior to testing; 6animals selected for each group Test drug administered 30min prior to experimentation by i.p route. The rat is then placed in the centre of the maze facing one of the enclosed arms. 20
  21. 21.  Parameters Measured During Next 5 minutes:o time spent in the open armso entries into the open armso time spent in the closed armso entries into the closed armso total arm entries 21
  22. 22.  Anxiolytic effect indicated by:o increase in the proportion of time spent in open arms i.e., time in open arms/total time in open or closed armso increase in the proportion of entries into open arms i.e., entries into open arms/total entries into open or closed arms. 22
  23. 23.  Evaluation of results:o Motor activity and open arm exploratory activity determined.o Values of treated groups expressed as % of control values.o Benzodiazepines and valproate – decrease motor activity and increase exploratory time. 23
  24. 24. Isolation induced aggressiono Male mice subjected to isolation develop aggressive behavior towards other animals of same sex.o Compounds tested for their ability to suppress this isolation induced aggression. 24
  25. 25. Animal used: Male NMRI strain mice (12g wt) Mice kept isolated for 6weeks & aggressive behavior tested. Male mice accustomed to live together placed in cage of isolated mice for 5minutes Isolated mice attacks intruder- aggressiveness observed Drugs given to isolated mice s.c or orally; aggressive behavior tested at 60, 120,240 minutes (oral route) If drug active- decrease in aggressiveness Attenuation of fighting reaction 25
  26. 26.  Evaluation of results:o No. of animals with complete suppression of aggressiveness.o Reaction time noted.o Graduated scale of inhibition of aggressiveness is established.o Results of test group animals is compared with the control group results. 26
  27. 27. Anti – anxiety test (light – dark model)o Rodents – have exploratory activityo Animals placed in 2 chambered systems, where they can freely move between a brightly –lit open field and a dark corner.o After treatment with anxiolytic - show more crossings between the two chambers and more locomotor activity.o Number of crossings between the light and dark sites is recorded. 27
  28. 28. Methodologyo Apparatus - a dark and a light chamber divided by a photocell equipped zone.o Polypropylene animal cage (44 ˣ 21ˣ 21 cm) is darkened with black spray over 1/3rd of its surface.o A partition containing 13cm(l) ,5 cm (h) opening is used for separating the dark one-third of the cage.o This case rests on an activity monitor which counts total locomotor activity. 28
  29. 29. o An electronic system consisting of 4 sets of photocells across the partition.o It automatically counts movements through the partition and records the time spent in the light and dark compartments.o Animals- treated 30 min before the test with drugs or vehicle given i.p. placed in the cage and observed for 10 min.o Groups of 6-8 animals used for each dose. 29
  30. 30. o No. of crossings through the partition between the light and dark chambers compared with total activity counts during the 10 min.o Loco motor activity also monitored.o anxiolytics like diazepam & meprobamate increase locomotor activity and no. of crossings.o non anxiolytics - not effective in this model. 30
  31. 31. Social Interaction In Rats 31
  32. 32. MethodologyAnimals placed in groups of 5 each in a perspex open topped box1hr before test,2 rats from separate housing treated with test compound orallyPlaced in box with 60W bulb and behavior observed for 10minutes 2types of activity – 1. social interactions like sniffing, crawling over the partner 2. exploratory behavior 32
  33. 33.  Parameters measured :o exploration, sniffing, rearing, social contacts, sexual behaviour, attack, fighting, biting ,defensive posture, immobility and climbing over the partner. Evaluation :o Values of treated partners compared with data from control animals – ANOVA and t - test used. 33
  34. 34. o mCPP - [ 1-(3-chlorphenyl) piperazine]o Metabolite of antidepressant trazodone.o mCPP induces hypophagia and hypolocomotion , inhibits social interaction, diminishes exploratory activity in light-dark box test.o Antagonism of these symptoms is used for screening of anxiolytic drugs. 34
  35. 35. Male SpragueDawley rats (200-250g) are housed ingroups of 6; exposed to 12 hourlight/dark cyclewith free access tofood and water. 35
  36. 36.  Parameters measured :o time spent in both sides (horizontal, vertical activity)o frequency of motiono number of transition Anxiolytic effect :o increase in parameters measured in the light/dark box or in number of transitions if test is active. 36
  37. 37. mCPP Induced Anxiety - Locomotion Study• Test compound or vehicle are administered orally 1h or i.p 30 min before the locomotion test.• mCPP is injected i.p. in a dose of 7 mg/kg 20 min before the test.• The animals are placed individually in an automated locomotor activity cages and locomotion is recorded for 10 min.• Anxiolytic effect : disinhibition of locomotion. 37
  38. 38. Vogel Lick-conflict (Vogel Punished Drinking) Source of anxiety: thirsty, native rats are administered shocks while licking water. Animals used: sprague dawley rats. 38
  39. 39. Methodologya water bottle with metal drinking tube is fitted to the animal housing Electric circuit is connected between drinking tube and floor of cage. i.p injection of drugs are given; 30min later rats placed in cage and allowed to drink water and shock given after 20 licks For 3minutes next shocks are given for every 12th lick No. of shocks delivered in 3min noted for each animal, no. of shocks received after treatment compared with control.. 39
  40. 40.  Parameters measured:o number of accepted punishments (electric shock) Anxiolytic effect :o statistically significant increase in the accepted shocks. 40
  41. 41. conclusion• Anxiety disorderstress, tension, feardisorder and associated with is a psychological and threat about future.• The pathophysiology of anxiety disorder is not fully understood and hence improper diagnosis leads to increase morbidity and mortality rates.• Development of the screening methods resulted in introduction of many new anxiolytic agents.• In future aspects more reliable and easy models for screening are to be developed. 41
  42. 42. References• Joseph T. Dipiro, Robert L.Talbert, Gary C.Yee; Pharmacotherapy-And-Pathophysiologic Approach; Seventh edition; 1161-1181.• Parmar N.S, Shiv prakash; Screening Methods in Pharmacology; 98-107.• Gerhard Vogel; Drug discovery and Evaluation – Pharmacological Assays; Second Edition; 401-458.• Shenoy Preclinical evaluation of anxiolytic agents: an overview; Journal of Pharmaceutical Research and Opinion; june 2011/volume 1/ issue 2/ 7-22 pgs. 42
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