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2. UNDER GUIDANCE OF:PRESENTED BY:MRS. MEENU SINGH ASST. PROFESSOR DEPT OF PHARMACOLOGY PESCP, BANGALOREMISS SAYANTI SAU I M. PHARM DEPT OF PHARMACOLOGY PESCP, BANGALORE 3. OBJECTIVE To understand the concepts behind Anxiolytics and to reproduce the concepts of screening and clinicalevaluation of anxiolytics practically as well as theoretically. 4. CONTENTS oo o oo o oo oIntroduction Physiological Vs pathological anxiety Clinical presentation Types of anxiety Classification of anxiolytics Pathophysiology of anxiety Screening methods for anxiolytics In vitro methods In vivo methods Conclusion References4 5. INTRODUCTION Anxiety is an emotional state commonly caused by the perception of real or perceived danger that threatens the security of an individual.It is normal human adaptive response to stressful events.Physiological anxiety transient in naturePathological anxiety needs treatment 5 6. PHYSIOLOGICAL AND PATHOLOGICAL ANXIETYjitter Stage-frightNervousness WorryingPanic attacks Obsessions compulsionsFlashbacks nightmares Pathological fear6 7. TYPES OF ANXIETY DISORDER GAD (Generalized Anxiety Disorder)PD (Panic Disorder)AgoraphobiaSAD (Social Anxiety Disorder)Specific PhobiaObsessive Compulsive DisorderPost Traumatic StressAcute Stress 7 8. DIAGRAMMATIC VIEW OF ANXIETY DISORDER8 9. 9 10 10. GENERALISED ANXIETY DISORDER It is chronic and fills a persons day with exaggerated worry and tension, even though there is little or nothing to provoke it, associated with worrying excessively about health, money, family, school or work. Concentration and sleep problems are also common.COGNITIVE SYMPTOMS Excessive Anxiety Worries that are difficult to control Poor concentration or mind going blankPHYSICAL SYMPTOMS Sleep disturbance Irritability Restlessness 10 11. PANIC DISORDER Unexpected panic attacks associated with physiological symptoms of autonomic nervous system without any warning or apparent reason. They cant predict when an attack will occur, and many develop intense anxiety between episodes, worrying when and where the next one will occur. Symptoms include Attacks usually last no more than about 10 minutes. Symptoms Intensive fear Phobias Dizziness Racing heart Perspiring Shortness of breath Tingling hands Fear of dying or going crazy11 12. SOCIAL ANXIETY DISORDER Characterized by an intense, irrational, persistant fear of situations, usually social or performance situations, where risk of embarrassment is present. It can disrupt normal life, interfering with school, work or social relationships. Physical symptoms often accompany the anxious feelings. Blushing Profuse sweating Trembling Nausea Shortness of breath Racing heart Difficulty talking 12 13. SPECIFIC DISORDER Is an intense fear of something that posses little or no actual danger of a object or situation. Some of the more common specific phobias are Centered around closed-in places Heights Escalators Tunnels Water Flying dogs Injuries involving blood Injection Insects14 14. OBSESSIVE-COMPULSIVE DISORDER (OCD) Obsessions (recurrent, intrusive and generally distressing thoughts, images or feelings). Compulsions(repetitive, ritualistic behaviors aimed to alleviate obsessions).14 15. POST TRAUMATIC STRESS DISORDER( PTSD)Develops subsequent to experiencing or witnessing a traumatic eventSymptoms (lasting at least 4-6 weeks )Flash-backsDisturbing memoriesNightmaresIntrusive images 15 16. Pathophysiology of anxiety oNeurotransmitters like GABA, noradrenaline, serotonin abnormalities anxiety.oAmygdala, temporal lobe, hippocampus and hypothalamus - involved in anxeity.oNeurochemical theories : 1. Noradrenaline theory 2. Serotonin theory 3. GABA receptor theoryBrain structures dealing with fear and stress16 17. Gaba receptor theory GABA inhibitory neurotransmitter Inhibitory and regulatory effects on serotonin, noradrenaline and dopamine. GABAA receptor involved in anxiety; decreases neuronal excitability. Patients suffering from anxiety disorders have less level of GABA in cortex.Mechanism When GABA binds to the GABAA receptor, the Clchannel opens, influx of Cl Causing hyper polarization of membrane & decreases nerve cell excitability. The number of GABAA receptors can change with alterations in the environment (e.g., chronic stress). The subunit expression can be altered by hormonal changes. In patients with GAD, benzodiazepine binding in left temporal lobe is reduced. 17 18 18. Serotonin theory 5-HT is an inhibitory neurotransmitter, used by neurons originating in the raphe nuclei of the brain stem and projecting diffusely throughout the brain (e.g., cortex, amygdala, hippocampus, and limbic system).Stimulation of the postsynaptic 5-HT2A receptors in the limbic system results in anxiety and avoidance behavior.Abnormalities in serotonergic functioning through release and uptake at the presynaptic auto receptors (5-HT1A/1D), the serotonin reuptake transporter site (SERT), or effect of 5-HT at the postsynaptic receptors (e.g., 5-HT1A, 5-HT2A, and 5-HT2C) may play a role in anxiety disorders.Low 5-HT activity may lead to a dysregulation of other neurotransmitters. NE may act at presynaptic 5-HT terminals to decrease 5-HT release, and its activity at postsynaptic receptors can cause increased 5-HT release.SSRIs increases serotonin levels post synaptically blocks symptoms of anxiety.18 19 19. In response to threat or fearful situations, the Locus cerulus serves as an alarm center, activating NE release and stimulating the sympathetic and parasympathetic nervous systems.Noradrenaline theoryChronic central noradrenergic overactivity down- regulates 2-adrenoreceptors in patients with GAD.Drugs with anxiogenic effects (e.g., yohimbine) stimulate LC firing & increase noradrenergic activity. NE in turn increases glutamate release. This produces subjective feelings of anxiety. 19 20 20. Classification of anxiolytic BENZODIAZEPINES Diazepam Chlordiazepoxide Oxazepam Lorazepam AlprazolamAZAPIRONES (NON-SEDATIVE) Buspirone Gepirone IpsapironeSEDATIVE ANTIHISTAMINICS HyroxyzineANTIDEPRESSANTS (useful in chronic anxiety and panic attacks) Fluoxetine-BLOCKERS Propranolol 21 21. SCREENING METHODS22 22. 2.SEROTONIN RECEPTOR BINDING1.GABAERGIC COMPOUNDS a.[3H]-GABA receptor binding b.GABAA receptor binding c.GABAB receptor binding d.Benzodiazepine receptor: [3H]-flunitrazepam binding assaya.Serotonin (5-HT1A) receptor: binding of [3H]-8-hydroxy-2-(din-propylamino) tetralin([3H]DPAT) b. Serotonin (5-HT1B) receptors in brain: binding of [3H]5hydroxytryptamine ([3H]5-HT) c. 5-HT3 receptor in rat entorhinal cortex membranes: binding of [3H]GR 656303. HISTAMINE H3 RECEPTOR BINDING IN BRAINIn-vitro methods 22 23. IN-VIVO METHODS ANTICONVULSANT ACTIVITYANTI-AGGRESSIVE ACTIVITY Pentylenetetrazole (Metrazol) induced convulsions Strychnine-induced convulsions Picrotoxin-induced convulsions Isoniazid-induced convulsions Yohimbine-induced convulsions Foot-shock induced aggression Isolation-induced aggression Resident-intruder aggression test Water competition test Maternal aggression in rats Rage reaction in catsEFFECTS ON BEHAVIOR Anti-anxiety test (light-dark model) Anticipatory anxiety in mice Social interaction in rats Elevated plus maze test Water maze test Staircase test Cork gnawing test in the rat Distress vocalization in rat pups Schedule induced polydipsia in rats Four plate test in mice Footshock induced freezing behavior in rats Experimental anxiety in mice mCPP induced anxiety in rats Acoustic startle response in rats Unconditioned conflict procedure (Vogel test) Novelty-suppressed feeding Shock probe conflict procedure Ultrasound induced defensive behavior in rats Anxiety/defense test battery in rats Marmoset human threat test Aversive brain stimulation23 24. CONDITIONED BEHAVIORAL RESPONSES Sidman avoidance paradigm Geller conflict paradigm Conditioned defensive burying in rats Taste aversion paradigmEFFECTS ON THE ENDOCRINE SYSTEM Plasma catecholamine levels during and after stress Plasma corticosterone levels influenced by psychotropic drugsBENZODIAZEPINE DEPENDENCE Benzodiazepine tolerance and dependence in rats24 25. Elevated plus maze testPRINCIPLEThe elevated plus maze test has been extensively used for the selective evaluation of anxiolytic drugs. When the animals enter in to open arm they show fear like movement, freeze and become immobile.Rodents have aversion for high and open space and prefer enclosed arm and therefore spend greater amount of time in enclosed arm.ANXIOLYTICAdvantages of this test are : 1.simple fast and less time consuming. 2.No prior training is required. For selective identification of anxiolytic and anxiogenic drugs Anxiolytics decrease anxiety increase open arm exploration time Anxiogenics decrease open arm exploration time. 25 26. REQUIREMENTS Animal : Rat(200-250 g) Drugs: Diazepam (1 mg/ kg ; i.p.) Equipment : Elevated plus maze apparatus and a stop clock26 27. Elevated plus maze apparatus ELEVATED PLUSMAZE2 open arms (16 x 5 cm for mice & 50 x 10 cm for rats)2 closed arms (16 x 5 x 12 cm for mice and 50 x 10 x 40 cm for rats)An open roof with entire maze elevated (25 cm for mice and 50 cm for rats) from the floor. Two open arms are opposite to each other.28 28. The mice housed in pairs for 10 days prior to testing; 6animals selected for each groupTest drug administered 30min prior to experimentation by i.p route.P R O C E D U R EThe mice is then placed in the centre of the maze facing one of the enclosed arms. 28 29. Parameters Measured During Next 5 minutes Time spent in the open armsEntries into the open armsTime spent in the closed armsEntries into the closed armsTotal arm entries 29 30. 30 31. Anxiolytic effect indicated parameter Increase in the proportion of time spent in open arms i.e., time in open arms/total time in open or closed armsIncrease in the proportion of entries into open arms i.e., entries into open arms/total entries into open or closed arms. 31 32. EVALUATIONCRITICAL ASESSMENT OF THE METHOD Motor activity and open arm exploratory time are registered. The values of treated groups are expressed as percentage of controls. Benzodiazepines decrease motor activity and increase open arm exploratory time. The method is rather time consuming, but can be regarded as a reliable measure of anxiolytic activity. Computerized automatic systems are available for elevated plus maze, radial maze, Y-maze, and T-maze and may help to overcome these difficulties. 32 33. LIGHT-DARK MODEL IN MICE AND RATS Rodents have exploratory activity Animals are placed in a two chambered systems, where they can freely move between a brightly lit open field and a dark corner. After the treatment with an anxiolytic they show more crossings between the two chambers and more locomotor activity. The number of crossings between the light and dark sites is recorded. 33 34. METHODS The apparatus consists of a dark and a light chamber which are divided by a photocell equipped zone. A polypropylene animal cage (44 21 21 cm) is darkened with black spray over one-third of its surface.Animal: RatsA partition containing a 13cm(l) ,5 cm (h) opening is used for separating the dark one-third of the cage.(200-250g) Drugs: Diazepam(1 mg/ kg , i.p.)This case rests on an activity monitor which counts total locomotor activity. 34 34 35. A electronic system consisting of four sets of photocells across the partition.It automatically counts movements through the partition and records the time spent in the light and dark compartments.M E TThey are treated 30 min before the test drugs or vehicle given i.p. placed in the cage and observed for 10 min.HO DExperiments are conducted on mice or rats.Groups of 6-8 animals should be used for each dose. 35 35 36. evaluation No. of crossings through the partition between the light and dark chambers compared with total activity counts during the 10 min.Anxiolytics like Diazepam increase locomotor activity and no. of crossings.Loco motor activity also monitored.Dose response curves are plotted & number of crossings through the partition between the light and the dark chamber are compared with total activity counts during the 10 min.It has been reported that anxiolytics like Diazepam and produce a dose dependent facilitatory effect whereas the non anxiolytics are not effective in this model36 36 37. advantage i.Using black and white test box studied the effects of anxiolytic agents and reported an anxiolytic effect of dopamine receptor antagonists.i.Study the interaction of optical isomers modifying rodent aversive behavior.i.Study anxiogenic and anxiolytic activity.i.Study animal models of anxiety and their relation to serotonin interacting drugs. 37 38. [3H]-GABA receptor binding PRINCIPLE Abnormalities in GABA system have been found in neurological and psychiatric diseases like anxiety, epilepsy etc.Radio labeled GABA is bound to synaptic membrane preparations of mammalian brain.Labeling of the synaptic receptor with 3H-GABA requires careful attention to possible interference from non synaptic binding since 3H-GABA can also bind nonspecifically to plasma membranes. The most prominent of which is Na dependent binding of GABA to brain membranes.Sodium-independent binding of 3H-GABA has characteristics consistent with the labeling of GABA receptors.Therefore, the sodium-independent binding of 3H-GABA provides a simple and sensitive method to evaluate compounds for GABA-mimetic properties. 38 39. PROCEDUREREAGENTS0.32 M Sucrose-109.5 g of sucrose are dissolved in distilled water and filled up to 1000 ml. The solution is stored at 4 C0.05 M Tris-maleate buffer (pH 7.1) TEST DRUGS-1mM stock solutions are initially prepared.These are serially diluted to the required concentrations prior to the addition to the incubation mixture. Final concentrations are usually from 2 108 to 1 105 M.3H-GABA(specific activity approximately 40 Ci/mmol) is made up to a concentration of 780 nmol in distilled water and 20 l is added to each test tube (yielding a final concentration of 15 nmol in the assay). Isoguvacine or muscimol is prepared by dissolving 8.35 mg of isoguvacine or 6.40 mg of muscimol in 10 ml water. 20 l of these solutions when added to 1 ml of incubation medium give a final concentration of 0.1 mM isoguvacine or muscimol.39 40. TISSUE PREPARATION Rats (100150 g) are decapitated, brain removed. Homogenized in 15 vol. of ice-cold 0.32 M sucrose.Centrifuged at 1000 g for 10 min.Discard pellet & recentrifuged supernatant, 20000 g, 20 min. Discard supernatant & pellet is resuspended in 15 vol. disttiled water using a Tekmar homogenizer.The suspension is centrifuged at 8000 g for 20 min.Collect Supernatant and resuspend the pellets soft, upper, buffy layer. Centrifuged at 48000 g for 20 min. The final pellets are resuspended (without homogenization) in 15 vol. disttiled H20 and centrifuged at 48000 g for 20min.Supernatant is discarded, and the centrifuge tubes containing pellet are capped with parafilm and 40 stored frozen at 70 C. 40 41. ASSAY PROCEDURE The standard Na-independent 3H-GABA binding assay procedure, aliquots of the previously frozen, Triton treated crude synaptic membranes are incubated in triplicate at 4 C for 5 min in 0.05 M Tris-maleate buffer (pH 7.1) containing 15 nM 3H-GABA alone or in the presence of 0.1 mM isoguvacine or muscimol, or the test drug. The procedure is as follows 1 ml of the 0.05 M Trismaleate homogenate20 l of 3H-GABA20 l of test drug or 20 ml of 0.1 mM isoguvacine or muscimolIncubate at 4 C for 5 min, the reaction is terminated by centrifugation for 15 min at 5000 rpm. Supernatant fluid is aspirated & pellet washed twice with 1ml of the Tris-maleate buffer. 2ml of liquiscint are added to each tube which is then vigorously vortexed. The contents of tubes are transferred to scintillation vials, tubes rinsed with an additional 2ml of cocktail. An additional 6 ml of liquiscint are added to each scintillation vial. The radioactivity is measured by liquid scintillation photometry. 4141 42. EVALUATION Specific 3H-GABA binding is defined as the radioactivitywhich can be displaced by a high concentration of unlabeled GABA and is obtained by subtracting from the total bound radioactivity the amount of radioactivity bound in the presence of 0.1 mM isoguvacine. Results are converted to percent of specifically bound 3H-GABA displaced by a given concentration of test drug. IC50 values with 95% confidence limits are then obtained by computer derived linear regression analysis. 42 43. SEROTONIN (5-HT1B ) RECEPTORS IN BRAIN : BINDING OF [3 H] 5-HYDROXYTRYPTAMINE ([3 H ]5-HT) PURPOSE AND RATIONALE To determine the affinity of test compounds for the serotonin (5-HT1B) receptor in brain. The existence of two populations of 5-HT1 receptors in rat brain was shown by differential sensitivity to Spiroperidol. The Spiroperidol-sensitive receptors were designated as the 5-HT1A subtype and the insensitive receptors were referred to as the 5HT1B subtype.The 5-HT1B subtype has been identified in the brain of rats and mice and can be selectively labeled by 5-HT in rat striatum when Spiroperidol is included to mask the 5HT1A and 5-HT2 receptors. The distribution of 5-HT1B sites in rat brain is similar to that of 5-HT1D sites in human brain. By comparing the results in the 5-HT1B assay with those in the5-HT1A, 5-HT2 and the 5-HT3 receptor binding assays the relative affinity of a test compound for the major subclasses of 5-HT receptors in the rat brain can be determined. 43 44. PROCEDURE REAGENTS 1. Tris buffers, pH 7.7 2. 5-Hydroxy[G-3H] tryptamine creatinine sulfate 3. Serotonin creatinine sulfate 4. Spiroperidol 5. Test compounds44 45. TISSUE PREPARATION Rats are sacrificed by decapitation.Striata are removed, weighed and homogenized in 20 vol. of 0.05 M Tris buffer, pH 7.7 The homogenate is centrifuged at 48000 g for 10 min and discard supernatant. The pellet is resuspended in an equal volume of 0.05 M Tris buffer, incubated at 37 C for 10 min and recentrifuged at 48000 g for 10 min. The final membrane pellet is resuspended in 0.05 M Tris buffer containing 4 mM CaCl2, 0.1% Ascorbic acid and 10 mM Pargyline.45 46. ASSAY 800 l tissue 80 l 0.05 M Tris+CaCl2+Pargyline+Ascorbic acid 20 l vehicle/ 5-HT/ drug 50 l [3H]5-HT 50 l Spiroperidol Tubes are incubated for 15 min at 25 C. The assay is stopped by vacuum filtration through Whatman filters which are then washed 2 times with 5 ml of ice-cold 0.05 M Tris buffer. The filters are then placed into scintillation vials with 10 ml of Liquiscint scintillation cocktail and counted. 46 47. EVALUATION Specific binding is defined as the difference between total binding and binding in the presence of 10 M 5-HT. IC50 values are calculated from the percent specific binding at each drug concentration. The Ki value may then be calculated by the ChengPrusoff equation Ki = IC50 / 1 + L /KD The KD value for [3H] 5-HT binding was found to be 16.5 nM by Scatchard analysis of a receptor saturation experiment. 47 48. conclusion Anxiety is a leading disease now a days. To understand the treatment of various types of anxiety, it is necessary to have a detailed knowledge about anxiolytics. All anxiolytics do not act similar way to understand the pharmacology and to invent more safe and potent drugs different screening models are very important . So further research on various sreening models are required. Nevertheless, the knowledge gathered from animal studies undoubtedly valuable therapeutically in the future studies. 48 49. REFFERENCES Drug Discovery and Evaluation by H. Gerhard Vogel Pharmacological Screening & Evaluation by S.K.Gupta Internet sourceDrugs The Straight Facts Anti anxiety Drugs 49 50. 50 51. 51